38 - PC - 1 - 1 - Jul - Sep - 2011

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Pharmacognosy Communications
An Official Publication of Pharmacognosy Network Worldwide [Phcog.Net] www.phcogcommn.org | www.phcog.net Editor-in-Chief Dr Ian Cock Biomolecular and Physical Sciences Griffith University, Nathan campus, 170 Kessels Rd, Nathan, Queensland 4111 Australia
Editorial Board Members Dr. William N. Setzer, Professor and Chair Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35899, USA Dr. Khuraman MUSTAFAYEVA Pharmaceutical faculty Azerbaijan Medical University Dr David Ruebhart HydroTox Services Melbourne Australia Dr. Omayma A. El Dahshan, Ph D Pharmacognosy Dept., Faculty of pharmacy, Ain shams University, Cairo, Egypt Dr. Micha Tomczyk Medical University of Biaystok, Faculty of Pharmacy, Department of Pharmacognosy, ul. Mickiewicza 2a, 15-089 Biaystok, Poland Prof. Ameenah Gurib-Fakim, CEPHYR Ltd (Centre for Phytotherapy and Research) 7th Floor, Cyber Tower 2 Ebene, Mauritius Dr. Philip G. Kerr PhD School of Biomedical Sciences Charles Sturt University Wagga Wagga NSW 2678 Australia Prof. Dr. Rimantas Venskutonis Department of Food Technology Kaunas University of Technology Radvilenu pl. 19, Kaunas LT-50254, Lithuania Editor - Publications Dr. Mueen Ahmed KK Aim and Scope Phcog Commn. is aimed at a broad readership, publishing articles on all aspects of pharmacognosy, and related fields. The journal aims to increase understanding of pharmacognosy as well as to direct and foster further research through the dissemination of scientific information by the publication of manuscripts. Subscription Rates for the year 2012 (4 issues) Rs. 2000 1000/year (National) | USD : 350 200 USD (International) DD/Cheque should be in favour of Phcog.Net payable at Bangalore, INDIA All correspondence regarding subscription should be addressed to the following address: Phcog.Net, 1713, 41 A Cross, 18 Main, Jayanagar 4 T blk, Bangalore 560041 INDIA. Email: journals@phcog.net
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Volume 1 | Issue 1 | Jul-Sep 2011
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Contents
Editorial
Pharmacognosy Communications: The Scope of Pharmacognosy I.E. Cock

Invited Review
Plant Drugs Used to Combat Menace of Anxiety Disorders Reecha Madaan, Suresh Kumar, Gundeep Bansal, Anupam Sharma
Review Article
Problems of Reproducibility and Efficacy of Bioassays Using Crude Extracts, with Reference to Aloe vera I.E. Cock
Research Article
52
Cassane-type diterpenoids from the genus Caesalpinia R. A. Dickson, T. C. Fleischer, P. J. Houghton Azadirachtolide: An anti-diabetic and hypolipidemic effects from Azadirachta indica leaves Dineshkumar B, Analava Mitra, Manjunatha M
Research Letter
63 78
Antimicrobial and anti-inflammatory activities of the leaves of Clerodendrum splendens leaves Fleischer, TC, Mensah, AY, Oppong, AB, Mensah, MLK, Dickson, RA, Annan, K Chemical Examination and Hair Growth studies on the Rhizomes of Hedychium spicatum Buch.-ham G. Venkateswara Rao, T. Mukhopadhyay, M. S. L. Madhavi, S. Lavakumar
World Wide Web
85 90
Inside Pharmacognosy: A Blog [Pharmanocognosy.in]

Medicinal Plant Images
94
Eucalyptus ficifolia and Chondrodendron tomentosum

Department Profile
95
Biomolecular and Physical Sciences, Griffith University, Australia.

Upcoming Events Instructions
96 99 100
(c) Copyright 2011 EManuscript Publishing Services, India
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Editorial Pharmacognosy Communications: The Scope of Pharmacognosy

I. E. Cocka,b*
a b
Biomolecular and Physical Sciences, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Brisbane, Queensland 4111, Australia. Environmental Futures Centre, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Brisbane, Queensland 4111, Australia
Pharmacognosy is the branch of pharmacology that studies drugs in their crude and/or natural states.[1] In general, when we describe pharmacognosy, we are usually referring to plant based medicinal systems. However, it is important to note that medicinal preparations may also be derived from animal sources as well as from fungi and microorganisms. Indeed, the discovery of the fungal antibiotic agent penicillin (from Penicillinum spp.)[2] is one of the most important medicinal findings to date. Many other useful medicinal products are also derived from fungi including the immunosuppressant mycophenolic acid (also from Penicillinum spp.)[3] and purgative anthraquinone emodin (from Penicillium islandicum).[4] Also, numerous hallucinogenic substances (eg. psilocin and psilocybin) are produced by Psilocybe spp. (family Tricholometaceae) of fungi.[5] Similarly, numerous medicinal agents are produced by bacteria, especially further antibiotic agents. Very early studies demonstrated the antibiotic potential of bacteria towards other bacterial species. In 1887 it was accidently discovered that prior injection of Streptococcus erysipelatis protected guinea pigs from developing cholera when injected with Vibrio cholera.[6] Furthermore, it was also shown that previous injection of either Streptococcus erysipelatis or Pseudomonas aeruginosa also prevented the development of anthrax in experimental animals injected with Bacillus anthracis[6] and that pre-injection of sterilised cultures of the protective bacteria have the same protective effect as live bacteria.[7] This discovery stimulated further studies into the antibiotic activity of bacteria, resulting in the discovery of streptomycin, chloramphenicol, chlortetracycline, tetracycline, erythromycin, neomycin and numerous other antibiotics, especially from Streptomyces spp. (family Streptomycetaceae). Other bacteria, particularly Bacillus spp., are noted for their production of antibiotic polypeptides such as actinomycin,[8] bacitracin,[9] tyrothrycin[10] and polymixin.[10] These antibiotic polypeptides were initially not widely used as they also display strong cytotoxic
*Correspondence: Tel.: +61 7 37357637; fax: +61 7 37355282 E-mail: I.Cock@griffith.edu.au (I. E. Cock). DOI: 10.5530/pc.2011.1.1
properties. More recently, there is renewed interest in their use due to their antitumor potential. Indeed, the bacterial antibiotic polypeptides doxorubicin, daunorubicin and actinomycin D are now routinely used in the treatment of a variety of cancers.[11,12] Although the number of animal derived pharmacognostical agents is small when compared to fungi, bacteria and plants, there has recently been an increase in interest in marine creatures as a source of new drugs. Marine invertebrates in particular, account for much of the recent publications describing animal pharmacognosy. Some species of sponges have been found to have antibacterial, antifungal, antimalarial, cytotoxic and anticancer bioactivities.[13] Furthermore, sponges produce interesting metabolites including bromophenols, cyclic peroxides, peroxyketals and modified sesquiterpenes which warrant further investigation.[13] The soft coral Sarcophyton glaucum produces the diterpenoids sarcophytol A and sarcophytol A, which have tumour inhibiting bioactivity.[14] Whilst marine animals are receiving much recent interest, there are also many examples of pharmacognostical agents derived from terrestrial animals. For examples, bees (Apis mellifica) provide us with multiple useful medicinal properties. The antimicrobial activity of honey produced by bees feeding on some plant species is known to be exceptionally good. Manuka honey (made by bees feeding on the Eastern Australian/New Zealand plant Leptospermum scoparium) is an especially good antimicrobial agent.[15] Additionally, beeswax and royal jelly are also reported to have therapeutic properties.[16] Toad skins contain cardioactive agents and were used to treat oedema prior to the development of more effective agents.[17] Pharmacognostic agents produced by vertebrates include lanolin from wool, gelatine and musk. In my own region of the world (Australia) there is also much interest in oils obtained from emu for its many therapeutic properties.[18] Inorganic chemicals may also have important medicinal properties. Silver is particularly well known for its antibacterial activity[19] and has been used since the times of ancient Greece. Silver nanoparticles have also been shown to have a potent cytoprotective bioactivity towards HIV infected cells.[20] Gold thiolates have been
1
Cock: The Scope of Pharmacognosy
used in the treatment of rheumatoid arthritis and as anti-tumour agents (as reviewed in Parish and Cottrill).[21] A variety of copper and iron complexes demonstrate potent cytotoxic activities against human cancer cells.[22] Recent studies have highlighted the importance of selenium in blocking the production of reactive oxygen species (ROS) and thus blocking oxidative stress and its associated disease states and medical conditions.[23] A variety of other inorganic molecules and ions also have medicinal promise, possibly also through their maintenance of cellular redox state. Despite the importance of pharmacognostic agents from fungi, microorganisms and animals, plants provide us with the greatest variety of medicinal agents and arguably hold the most promise for future drug discovery. Asian medicinal botany in particular has been especially well documented. Traditional Chinese Medicinal (TCM) systems and Indian Ayuverda are widely practiced with approximately 85% of Indians regularly using crude plant formulations for the treatment of various diseases and ailments.[24] Similarly, African and Middle Eastern medicinal ethnobotanies are also widely practiced well documented. Even allopathic/Western medicine practiced in developed countries owes much to our understanding of plant based remedies. Indeed, it has been estimated that approximately 25% of all prescription drugs currently in use are originally derived from plants.[26,27] Furthermore, approximately 75% of new anticancer drugs marketed between 1981 and 2006 are derived from plant compounds.[26] Recently, there has been an increase in interest in pharmacognosy and natural therapies due to the perception that natural therapeutics offer a safer alternative than synthetic formulations due to their organic origin. This is reflected in the dramatic increase in publications in pharmacognosy journals over the period 2005-2010.[28] It is evident that a further publication outlet is required to accommodate this expanding field. Pharmacognosy Communications is a new journal published by Pharmacognosy Network Worldwide [www.phcog.net]. We aim to publish high quality original research articles, methods, techniques and evaluation reports, critical reviews, short communications, commentaries and editorials of all aspects of pharmacognosy research. The journal is aimed at a broad readership, publishing articles on all aspects of pharmacognosy, and related fields. The journal aims to increase understanding of pharmacognosy as well as to direct and foster further research through the dissemination of scientific information by the publication of manuscripts. The submission of original contributions in all areas of pharmacognosy are welcomed. The journal aims to cater the latest outstanding developments in the field of pharmacognosy and natural products and drug design covering but not limited to the following topics: Pharmacognosy and pharmacognistic investigations Research based ethnopharmacological evaluations Biological evaluation of crude extracts, essential oils and pure isolates
2
Natural product discovery and evaluation Mechanistic studies Method and technique development and evaluation Isolation, identification and structural elucidation of natural products Synthesis and transformation studies We look forward to receiving your valuable pharmacognosy communications.
REfEREnCES
1. 2. The American Heritage Medical Dictionary, 2007, Houghton Mifflin Company, USA. Fleming A, 1928, On the antibacterial action of cultures of a Penicillium with special reference to their use in the isolation of B. Influenza. British Journal of Experimental Pathology, 10, 216-226. Florey HW, Gilliver K, Jennings MA, Sanders AG, 1946, Mycophenolic acid, an antibiotic from Penicillium brevi-campactum Dierckx. Lancet, 1, 46-49. Ghosh AC, Manmade A, Demain AL, 1977, Toxins from Penicillium islandicum Sopp. In Mycotoxins in Human and Animal Health, Edited by Rodricks JV, Hesseltine CW, Mehlman MA, Pathotox, Chicago, USA, 625-638. Hofmann A, Heim R, Brack A, Kobel H, 1958, Psilocybin ein psychotroper Wirkstoff aus dem moscikanischen Rauschpilz Psilocybe mexicana Heim. Experientia, 14, 107. Bouchard C, 1889, Influence quexerce sur la maladie charbonneuse linoculation du bacilli pyocyanique, Comptes Rendus de lAcadmie des Sciences, 108, 713-714. Woodhead GS, Wood C, 1889, De laction antidotique exerce par les liquids pyocyaniques sur le cours de la maladie charbonneuse. Comptes Rendus de lAcadmie des Sciences, 109, 985-988. Waksman SA, Woodruff HB, 1940, Bacteriostatic and bactericidal substances produced by soil actinomycetes. Proceedings of the Society for Experimental Biology and Medicine, 45, 609-614. Johnson BA, Anker H, Meleney FL, 1945, Bacitracin: a new antibiotic produced by a member of the B. Subtilise group. Science, 102, 376-377. Dubos RJ, Hotchkiss RD, 1941, The production of bactericidal substances by aerobic sporulating Bacilli. Journal of Experimental Medicine, 73, 5, 629-640. Lasek W, Giermasz A, Kuc K, Wakowicz A, Feleszko W, Golab J, Zagozdzon R, Stoklosa T, Jakobisiak M, 1996, Potential of the anti-tumor effect of actinomycin D by tumor necrosis factor in mice: Correlation between in vitro and in vivo results. International Journal of Cancer, 66, 374-379. Weiss RB, 1992, The anthracyclines: will we ever find a better doxorubicin? Seminars in Oncology, 19, 6, 670-686. Fusetani N, Matsunaga S, 1993, Bioactive sponge peptides. Chemistry Reviews, 93, 1793-1806. Wei H, Frenkel K, 1992, Suppression of tumor promoter-induced oxidative events and DNA damage in vivo by sarcophytol A: A possible mechanism of antipromotion. Cancer Research, 52, 2298-2303. Brophy JJ, Goldsack RJ, Bean AR, Forster PI, Lepschi BJ, 1991, Leaf essential oils of the genus Leptospermun (Mytaceae) in Eastern Australia. Part 5, Leptospermum continentale and its allies. Flavour and Fragrance Journal, 14, 98-104. Fujii A, 1995, Pharmacological effect of royal jelly. Honeybee Science, 16, 97-104. Chen KK, Kovarikov A, 1967, Pharmacology and toxicology of toad venom. Journal of Pharmaceutical Sciences, 56, 12, 1535-1541. Whitehouse MW, Turner Ag, Davis CKC, Roberts MS, 1998, Emu oil(s): A source of non-toxic transdermal anti-inflammatory agents in Aboriginal medicine, Inflammopharmacology, 6, 1-8. Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO, 2000, A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus, Journal of Biomedical Materials Research, 52, 662-668. Sun RWY, Chen R, Chung NPY, Ho CM, Lin CLS, Che CM, 2005, Silver nanoparticles fabricated in Hepes buffer exhibit cytoprotective activities towards HIV-1 infected cells. Chemistry Communications, 40, 5059-5061. Parish RV, Cottrill SM, 1987, Medicinal gold compounds, Gold Bulletin, 20, 3-12. Easmon J, Prstinger G, Heinisch G, Roth T, Fiebig HH, Holzer W, Jger W, Jenny M, Hofmann J, 2001, Synthesis, cytotoxicity, and antitumor activity of copper(II)
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12. 13. 14.
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Cock: The Scope of Pharmacognosy
23.
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and iron(II) complexes of 4N-azabiclo[3.2.2]nonane thiosemicarbazones derived from acyl diazines, Journal of Medicinal Chemistry, 44, 13, 2164-2171. Venardos K, Harrison G, Headrick J, Perkins A, 2004, Effects of dietary selenium on glutathione peroxidise and thioredoxin reductase activity and recovery from cardiac ischemia-reperfusion, Journal of Trace Elements in Medicine and Biology, 18, 1, 81-88. Kamboj VP 2000, Herbal medicine. Current Science, 78, 35-39. ,
25. 26.
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Newman DJ, Cragg GM, Snader KM, 2000, The influence of natural products on drug discovery. Natural Product Reports, 17, 215-234. Hostettmann K, Hamburger M, 1993, Search for new lead compounds of natural origin. In Perspectives in Medical Chemistry, Testa B, Kyburz E, Fuhrer W, Giger R (eds), Verlag Helvitica Acta, Basel. Ahmed MKK, 2011, New challenges in the new year for Pharmacog Mag.: 5 years of quality publication. Pharmacognosy Magazine, 7, 25, 1-3.
AbouT jouRnAl
Pharmacognosy Communications [Phcog Commn.] www.phcogcommn. org is a new journal published by Pharmacognosy Network Worldwide [www.phcog.net]. It is a peer reviewed journal aiming to publish high quality original research articles, methods, techniques and evaluation reports, critical reviews, short communications, commentaries and editorials of all aspects of medicinal plant research. The journal is aimed at a broad readership, publishing articles on all aspects of pharmacognosy, and related fields. The journal aims to increase understanding of pharmacognosy as well as to direct and foster further research through the dissemination of scientific information by the publication of manuscripts. The submission of original contributions in all areas of pharmacognosy are welcome. The journal aims to cater the latest outstanding developments in the field of pharmacognosy and natural products and drug design covering but not limited to the following topics: Pharmacognosy and pharmacognistic investigations Research based ethnopharmacological evaluations Biological evaluation of crude extracts, essential oils and pure isolates Natural product discovery and evaluation Mechanistic studies Method and technique development and evaluation Isolation, identification and structural elucidation of natural products Synthesis and transformation studies
www.phcogcommn.org
Invited Review Plant Drugs Used to Combat Menace of Anxiety Disorders

Reecha Madaan*1, Suresh Kumar2, Gundeep Bansal2, Anupam Sharma3
Chitkara College of Pharmacy, Chitkara University, Rajpura, Punjab, India (reechashirin@yahoo.com). 2Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala- 147 002, Punjab, India (thakur_pu@yahoo.com). 3Pharmacognosy Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160 014, India (ans1959@rediffmail.com)
1
ABSTRACT: In present era, a sudden holocaust of mental disorders, and recognition of severe side effects and addiction liabilities associated with long term administration of widely prescribed synthetic drugs have aroused the attention of researchers towards natural resources. This review includes 351 references, and emphasizes pharmacological reports on anxiolytic plant products and formulations. Various chemical constituents (with structures), isolated from different plants, responsible for antianxiety activity, and their possible mechanism of actions have been incorporated in this review.The review has been compiled using references from major databases like Chemical Abstracts, Medicinal and Aromatic Plants Abstracts, PubMed, Scirus, Science Direct and Online Journals. It has been concluded that preliminary antianxiety activity studies have been carried out on crude extracts of most of traditonally used and clinically potential plants. Such plantsneed to be explored properly with a view to isolate anxiolytic constituents, and to evaluate their possible mode of actions. KEY WORDS: Antianxiety activity, chemical constituents, mechanism of action, pharmacology
INTRODUCTION
Global scenario of persons afflicted by mental disorders is alarming.[1] About 500 million people suffer from neurotic, stress related and somatoform problems, 200 million from mood disorders, 83 million from mental retardation, 30 million from epilepsy, 22 million from dementia, and 16 million from schizophrenia. Anxiety disorders are serious medical illnesses that have affected 1/8th of total population worldwide irrespective of gender, age, religion, nationality and profession.[2] Anxiety Disorders Association of America (ADAA) described anxiety disorders as the most common mental illness in the US, that have affected 19.1 million (13.3%) of the adult (18-54 years) US population.[3] A study commissioned by ADAA on The Economic Burden of Anxiety Disorders revealed that anxiety disorders cost the US more than $42 billion a year, almost one-third of the $148 billion total mental health bill for the US. In India, prevalence rate for all mental disorders is 65.4 per 1000 population, and that for anxiety neurosis is 18.5 per 1000 population.[4] The Global Research on Anxiety and Depression (GRAD) network, a consortium of worlds leading psychiatric epidemiologists and clinical researchers, during the 154th annual meeting of American
*Correspondence: thakur_pu@yahoo.com; reechashirin@yahoo.com Tel.: +91-9872981142, +91-9815916142 DOI: 10.5530/pc.2011.1.2
Anxiety Disorders: An Overview
Psychiatric Association (APA) has observed that, a significant number of worlds population is plagued by chronic and excessive anxiety, also known as generalized anxiety disorder (GAD), which is more serious than those of lung disease, sleep disorders and major depression, and affects more than 5% of the world population.[5] Following is the categories of anxiety disorders.[3,6] 1. Panic disorder (PD) is characterized by panic attacks, sudden feeling of terror that strike repeatedly and without warning. Physical symptoms include chest pain, heart palpitations, sweating, trembling, shortness of breath, dizziness, abdominal discomfort, fear of losing control, fear of dying, tingling sensations, and hot flushes. Panic disorders have affected 6 million (2.7%) adult US population. Women are twice more likely to be afflicted than men. 2. Obsessivecompulsive disorder (OCD) is characterized by uncontrollable obsessions (recurring thoughts or impulses that are intrusive or inappropriate and cause the sufferer anxiety) and compulsions (repetitive behaviours or rituals). It has affected 2.2 million (1%) adult US population. It is equally common among men and women. 3. Post-traumatic stress disorder (PTSD) is characterized by persistent symptoms (nightmares, flashbacks, numbing of emotions, depression, feeling angry and irritable) that occur after experiencing a traumatic event such as war, rape, child abuse and natural disaster. It has affected 7.7 million (3.5%) adult US population. Women are more likely to be afflicted by this disorder.
Madaan, et. al.: Plant Drugs Used to Combat Menace of Anxiety Disorders
4. Social phobia or Social anxiety disorder (SAD) is characterized by an intense fear of situations where embarrassment may occur. Physical symptoms include palpitations, tremors, sweating, diarrhoea, confusion and blushing. It has affected 15 million (6.8%) US adult population. It is equally common among men and women. 5. Specific phobia (SP) is characterized by the excessive fear of an object or a situation, exposure to which causes an anxious response. Specific phobias affect an estimated 19 million (8.7%) US adult population and are twice as common in women as in men. 6. Generalized anxiety disorders (GAD) are characterized by chronic, exaggerated worry about everyday routine life events and activities, lasting at least six months. Physical symptoms include fatigue, trembling, muscle tension, headache or nausea. It has affected an estimated 6.8 million (3.1%) US adult population and is twice as common in women as in men. Though, GAD is the most frequent anxiety disorder, yet only 20% of patients receive proper treatment.[7] GAD results loss of 6 for every 30 work-impairment days.
Causes of Anxiety Disorders
Thought patterns
Negative thoughts can actually create physical symptoms of anxiety. Such a horrid emergence of mental disorders has attracted the attention of researchers towards various pharmacotherapeutic approaches for the management of these modernization borne diseases.[10] Barbiturates, benzodiazepines (BZDs), azaspirones, norepinephrine and serotonin-reuptake inhibitors, monoamine oxidase inhibitors and phenothiazines are some of the commonly used psychotropic drugs.[10] Among these, BZDs are the most widely prescribed synthetic chemical drugs for the treatment of anxiety, insomnia, epilepsy, and stress. Regular use of BZDs causes deterioration of cognitive functioning, addiction, physical dependence and tolerance.[10-12] Abrupt cessation of chronic treatment with BZDs causes the appearance of withdrawal effects comprising re-bound anxiety, restlessness, epilepsy, and motor agitation.[13,14] In the light of adverse effects associated with the synthetic drugs, researchers have been exploring natural resources to find out safer and effective drugs. Investigating plants, based on their use in traditional systems of medicine, is a sound, viable and cost effective strategy to develop new drugs.[15] Plants like Valeriana officinalis, Nardostachys jatamansi, Withania somnifera and Panax ginseng have been used extensively in various traditional systems of therapy because of their adaptogenic and psychotropic properties. Inclusion of these well-established CNS affecting plants in the arsenal of modern therapeutics has revived the faith of researchers in the plants.[16] With anxiety, various brain neurotransmitters and hormones levels change immediately. In particular, monoamines, such as norepinephrine, serotonin and dopamine, are involved in mood, stress and other physical homeostasis.[17] Serotonin and norepinephrine mainly regulate stress and negative mood in the mammalian brain, and their dysfunctions cause various mood disorders, such as social anxiety disorder and depression.[18] Dopamine also regulates mood and emotion-related behaviors and has a motivation/reward function and conditional fear responses.[19,20] Various anxiolytics and antidepressants aim at monoamine neurocircuitry, such as their receptors and transporters.[21] The 5-hydroxytryptamine 1A (5-HT1A) receptor is viewed as a relevant target for the treatment of psychiatric disorders, notably anxiety and depression.[22] 5-HT1A receptors are located at the presynaptic and postsynaptic sites.[23] The somatodendritic autoreceptor, when activated by systemic stimulation, is believed to exert anxiolytic-like effects and to reduce 5-HT release both in the cell body and in the terminal regions of the serotonergic neurons.[24] The other 5-HT1A receptor is localized postsynaptically to the serotonergic neurons in the hippocampus, septum, amygdala, and cortex, where it increases signal transfer, which leads to an inhibition of the firing activity.[25]
5 Targets for Treatment of Anxiety Management of anxiety disorders
Various factors causing anxiety disorders are described below.[8-9]

Heredity/Genetic factors
Anxiety disorders (PD and OCD) tend to run in families. Studies have shown that if one of the twins has an anxiety disorder, the second is more likely to have an anxiety disorder.
Brain chemistry
The symptoms of long term social anxiety disorder can be attributed to the improper chemical balance in the brain. Several neurotransmitters namely serotonin, norepinephrine, gammaamino butyric acid (GABA), which are produced in the brain, directly affect ones feelings about a given situation. Thus brain, too, appears to play a role in the onset of anxiety disorders because symptoms of anxiety disorders are often relieved by medications that alter the level of chemicals in the brain.
Personality
People with low self-esteem and poor coping skills are more prone to anxiety disorders. Conversely, an anxiety disorder that begins in childhood may itself contribute to the development of low self-esteem.
Life experiences
Long term exposure to abuse, violence, poverty or stressful experiences (the early death of a parent, bad marital or family relationships, or traumatic experiences) may affect individuals susceptibility to anxiety disorders.
Stress overload/Lifestyle factors
Excessive stress over time, and poor lifestyle habits such as overwork, lack of sleep, poor diet and lack of regular exercise promote anxiety.
GABA is a major inhibitory transmitter in the central nervous system. The -aminobutyric acid type A (GABAA) receptor, the chloride ion channel complex and the central benzodiazepine receptors located on the neuronal membranes within this complex have been suggested to play an important role in the regulation of the stress and anxiety states.[26,27] The benzodiazepine binding site and GABAA receptor are structurally and functionally coupled.[28] Benzodiazepines (BZDs) have become the primary pharmacological treatment for generalized anxiety disorder. However, BZDs are often associated with tolerance development and withdrawal symptoms, which pose a risk of relapse upon discontinuation.[29,30] Monoamine oxidase (MAO) catalyzes the oxidative deamination of a variety of monoamines such as dopamine, norepinephrine and serotonin. The MAO reaction yields aldehydes and hydrogen peroxide (H2O2), which induces apoptosis.[31] Increased endogenous MAO inhibitory activity (tribulin activity) is associated with conditions associated with stress and anxiety, both in animals and in man.[32] Rat brain tribulin activity is significantly augmented by anxiogenic agents like pentylenetetrazole, and this effect can be prevented by anxiolytic agents.[33] Inhibition of MAO and subsequent H2O2 generation effectively prevents depression and various oxidative stresses in the brain.[34] The presence of plant-derived MAO inhibitors suggests that such plant extracts could be useful as potential neuroprotectants in the treatment or prevention of depression.[35]
In brain, Nitric oxide synthase (NOS) has been localized in regions involved with anxiety, such as hypothalamus, amygdala and hippocampus.[36,37] Inhibition of NOS by nonselective or by relatively selective inhibitors of nNOS produced antianxietylike effect. Neurosteroids can rapidly alter the excitability of central nervous system by modulating neurotransmittergated ion channels such as GABAA and N-methyl-D-aspartate receptors.[38] Anxiolytic, anticonvulsant and anaesthetic effects of neuroactive steroids are mediated by their capacity to positively modulate GABAA receptor. 5-alpha reductase, the enzyme that converts into 5-alpha-reduced metabolites like the GABAA positive neuroactive steroid 3-alpha-hydroxy-5-alphapregnan-20-one, thus, few drugs exhibits anxiolytic action via an indirect activation of the GABA-ergic system through neuroactive steroids.[39]
PLANTS HAVING ANTIANXIETY ACTIVITY

Antianxiety activity reports of various plants, and plant constituents and formulations have been presented in tables 1 and 2. Various patented formulations of anxiolytic plant drugs have been depicted in table 3. Various review articles published on anxiolytic plants are shown in table 4.
O O O
O
O
OH
(1)
(2)
H N
R2
O
R1 (3) (4) OH H R2 OH H
R1
O
(5)
OH
OH HO O
HO O
OH
OH OH O
(6)
OH O
OH
(7)
NH2 HO N O
(8)
H
OH H
HO
(9)
CH3 H3C O HO HO HOH2C CH3 CH2OH O OH O

HO O
C CH3 CH2 O OH OH OH O
OH CH3 OHOH
OH
OH
(10)
(11)
CH2OOCCH2CH(CH3)2
CHO
(12)
O O O R O
(13), R = -Gentiobiosyl
OH HO O
OH OH
O
OH
(14)
(15)
CH3 H3CO
HO
OH
H3CO
OCH3 O OH
(17)
HO
(16)
R' CH3O N CH3O
RO
(19) (20) (21) R CH3 H H R OH H OH
O
(18)
RO N H 3CO
NH2 N N HO
H 3CO
R (22) (23) H CH3
N N O
HO
(24)
OH
H 3CO 2C H H
OH
OH
O O R 3O
R1 (25) (26) H H R2 H OH R3 H H
R 1 C(CH ) 3 3 OH O O O O
O OH
R1
OR 2
(27)
R2 O
OCH3
H 3C OH O
R1 (28) H
OH O
R2 H
OH
N
(29)
OCH3
HO HO
HO HO R CH3
CH2OH O OH O
HO HO
OH
OH
(30), R=CH3 (31), R =CH2OH
OH
O
(32)
R2 HO O
OH
OH R1 OH
R1 (33) -Glc (34) H R2 H -Glc
CH3O HO O
O O O O
(35)
H2C
OH
OH
OH
OH
CH2
(36)
H3CO
(37)
N O HO H
CH3
OH O
OCH3
HO
H H3C N OCH3 OCH3
OH
O
(38)
(39)
OH OH O O HO OH
HO
OH
HO OH H OH HO O H O H H O O OH HO OH
(40)
O O H
OH
HO
10
HO OH
OH O HO HO OR
R (41) (42) D-glucose H
OH
HO
HO
OH
OH
O
(43)
OH O O O OH OH
OH
(44)
CH
CH
COOH
HO OH
OH OH
(45)
(46)
OCH 3 R5 R1
11 10 9 14 7 12 13 8 6 4 5 3
N O
R2
R4
R3
(47) (48) (49) (50) (51) (52) R1 H H H H H H R2 R3 OCH 2OH OCH 2OH H H H H H H OCH 3 H R4 H H H H H H R5 C5-C6 C7-C8 = H H H H H = = = H H
O O O
(53)
11
OCH3 R5 R1
11 10 9 14 8 6 7 5 4 3
R2
12 13
R4
R3
R1 (54) (55) (56) (57) (58) (59) (60) R2 R3 H H H H H H H R4 H OCH3 H H OCH3 H H R5 H H OCH3 H H H H C5-C6 C7-C8 = = = = = = =
OCH2O OCH2O OCH2O OCH2O OCH2O OCH2O OCH3O CH3
H
= = = OCH3
HO
H
(61)
H
OH
H HO H
(62)
HOOC OH OH
OH
OH
(63)
O H O O O H H O O
O O
(64)
OCH3 HO O
(65)
COOH H OH H OH H O H OH O H O
HO OH (67) O
OH
(66)
12
HO
HO OH O
(68)
HO
(69)
OH
OH
O O O
OCH3
HO
H3 C
O HO OH
H3C OH O
(70)
OH OH O O HO HO O OH O O OCH3
HO OH
OH OH OH O
(71)
HO H3C
CH3
CH3
OH O
CH3 COOH
(73)
(72)
O O O HN O N CH3 N H N H
CH3
O
(75)
(74)
13
CH3 O
CH3 CH3
(76)
(77)
CH3
H2C
H CH3 CH3
(78)
(79)
OH
OH
(80)
(81)
N N H O
OH
H H
(82)
(83)
CONCLUSION
In present era, a sudden holocaust of mental disorders, and recognition of severe side effects and addiction liabilities associated with long term administration of widely prescribed synthetic drugs have aroused the attention of researchers towards natural resources. Plants like Valeriana officinalis, Nardostachys jatamansi, Withania somnifera and Panax ginseng have been used extensively in various traditional systems of therapy because of their adaptogenic and psychotropic properties. Inclusion of these well-established CNS affecting plants in the arsenal of
14
modern therapeutics has revived the faith of researchers in the plants. In present review article, amongst 143 plants reported to possess antianxiety activity (Table 1): (a) only 07 plants have been tested clinically, (b) preliminary antianxiety activity screening on crude extracts has been carried out on 90 plants. Such plants need to be explored with a view to isolate active constituents and their mode of actions,
Table 1: List of various plants reported to possess antianxiety activity.

Dose Animal/ Human being Wistar rats Elevated plus maze (EPM), Open field test (OFT), Elevated zero maze (EZM) Conflict behaviour Anxiolytic Anxiolytic [40] Experimental model/ Assessment of clinical parameters Mechanism of action Activity Ref.
S. No. 50 and 100 mg/kg, orally once daily for 3 days 12 mg/kg, p.o. Female Wistar rats
Biological source
Extract/Fraction/Isolate
01
Abies pindrow Royle (Pinaceae) Talispatra, Silver Fir, Pindrow Fir
Ethanol extract of leaves
02
Achillea millefolium Linn. (Compositae) Yarrow, Milfoil 500 mg TDS for 6 weeks 81 Patients suffering from anxiety disorder Electrophysiological parameters like EEG, ECG
Aqueous extract of flowers
[41]
03
Acorus calamus Linn. (Araceae) Bach/Bacopa monnieri Linn. (Scrophulariaceae) Brahmi
Powder of whole plant
Improvement in nervousness, restlessness, irritability, poor concentration, sleep and loss of appetite Anxiolytic Anxiolytic
[42]
04 (a) 100 mg/kg, p.o. (b) 50 mg/kg, p.o. 200 mg/kg, p.o. Male Sprague Dawley rats Laca mice EPM
Flavonoidal moiety
2 mg/kg, p.o.
Laca mice
EPM
[43] [44]
Actaea spicata Linn. (Apiaceae) Baneberry, Grapewort
(a) Methanol extract (b) Polyphenol fraction
05
Adiantum tetraphyllum Humb. & Bonpl. ex Willd. (Adiantaceae) Fourleaf maidenhair 20 mg/kg, p.o. Swiss albino mice
Ethanol extract (95%) of leaves
OFT, EPM, Acoustic startle response test
Anxiolytic
[45]
06
Aethusa cynapium Linn. (Apiaceae) Fools Parsley 100 and 200 mg/kg, p.o. 200 mg/kg, p.o. for seven days 25 or 50 mg/ kg, p.o. Male SD rats Male SD rats
Fatty acid: trideca7,9,11-trienoic acid(1) isolated from methanol extract of aerial parts
[1-(3-chlorphenyl)piperazine] induced hypolocomotion test
Anxiolytic
[46]
07
Albizzia julibrissin Durazz. (Fabaceae) Silktree, Mimosa, Nemunoki
Aqueous extract of stem bark
EPM EPM
Serotonergic system Interaction with 5-HT1Areceptor
Anxiolytic Anxiolytic
[47] [48]
Aqueous extract of bark
08
Albizzia lebbeck Benth. (Fabaceae) Siris tree, Albizia
Saponins rich n-butanolic fraction of petroleum ether extract from leaves
Albino Swiss mice
EPM
Inhibition of GABAergic transmission
Anxiolytic and nootropic
[49]
15
16
Dose Animal/ Human being Female Sprague Dawley rats Swiss albino male mice EPM Other mechanism than BZD-bs modulation at the GABAA receptors EPM, Forced Swimming Test (FST) Experimental model/ Assessment of clinical parameters Mechanism of action Activity Ref. 1.56 to 50 mg/ kg, i.p. 1.0,10.0 and 100.0 mg/kg, p.o. Inhalation 3.5 mg/L air Male ICR mice Light/Dark model (LDM) , OFT, EPM Anxiolytic and antidepressant Anxiolytic without sedative effects Anxiolytic [50] [51] [52] 30.0 mg/kg, p.o. 21 mg/kg, p.o. IC50 = 0.36 microM In vitro Male Wistar rats Male Swiss mice EPM, LDM Social interaction in rats (SI), Hole Board Test (HBT) BZD receptors agonist Anxiolytic Anxiolytic Anxiolytic [53] [54] [55]
Table 1: Continued
S. No.
Biological source
09
Aloysia polystachya Griseb. (Verbenaceae) Burrito
Hydro-alcoholic extract (60% ethanol) of leaves
Ethanol extract of aerial parts
10
Alpinia zerumbet(Pers.) Burtt & RM (Zingiberaceae) Shell flower, Pink porcelain lily
Essential oil from leaves
11
Angelica
Essential oil
Essential oil
12
Angelica dahurica (Fisch. ex Hoffm.) Benth. (Apiaceae) Dahurian angelica 25 and 50 mg/ kg, i.p. 25 and 50 mg/ kg, i.p. 25 and 50 mg/ kg, p.o. Male Swiss mice Male Swiss mice Male Swiss mice
Furanocoumarin Phellopterin(2) isolated from methanol extract of roots
13
Aniba riparia (Nees) Mez (Lauraceae) Rosewood
Riparin III(3) isolated from unripe fruits
EPM, FST EPM, OFT, HBT OFT, EPM, HBT
Anxiolytic, antidepressant Anxiolytic Anxiolytic but devoid of sedative activity
[56] [57] [58]
Riparin I (4) isolated from unripe fruits
Riparin- III (3) isolated from unripe fruits
14
Annona cherimolia Mill. (Annonaceae) Cherimoya, Custard apple
Hexane extract of leaves
6.25, 12.5, 25.0 and 50.0 mg/kg, p.o. 0.3, 1, 3, 10 and 30 mg/kg i.p.
Albino mice
Mouse avoidance exploratory behavior, Marble burying test (MBT) Albino mice EPM
GABA/BZD receptor complex
Anxiolytic
[59]
15
Annona diversifolia Saff. (Annonaceae) Llama, Anona blanca
Palmitone(5) isolated from hexane extract of leaves
Anxiolytic
[60]
16
Apocynum venetum Linn. (Apocynaceae) Dogbane 30 and 125 mg/kg, p.o. >0.02 mg/kg, p.o. Male BL6/C57J mice EPM BZD receptor interaction Anxiolytic [62] Male C75 BL/6 mice EPM Involvement of GABAergic system Anxiolytic [61]
Kaempferol(6) isolated from hydro-alcoholic extract (70% ethanol) of leaves 5 and 10 ml/ kg, p.o. 10, 20, 50, 100 and 200 mg/ kg, p.o. 500 mg/kg/ day 15 days Male Charles-Foster albino rats OFT and Morris water maze Increase in ascorbic acid level of brain which falls during brain ischemia Wistar rats EPM, OFT Anxiolytic Wistar rats SI, OFT Anxiolytic
17
Aronia melanocarpa Michx. (Rosaceae) Black chokeberry
Fruit juice
[63]
18
Azadirachta indica A. Juss. (Meliaceae) Neem tree
Aqueous extract from leaves
[64]
Aqueous extract from leaves
Anxiolytic
[65]
19
Baphia nitida Lodd. (Fabaceae) African sandalwood, Barwood 100-400 mg/ kg, p.o. Adult albino mice of either sex EPM, Y maze 200 and 400 mg/kg, p.o. Albino mice of either sex Hexobarbitone induced sleeping time, Y -maze, EPM, HBT
Ethyl acetate extract of leaves
Anxiolytic
[66]
20
Byrsocarpus coccineus Schurn. and Thonn. (Connaraceae) Kimbar mahalba 41g/mg In vitro
Aqueous extract of leaves
Anxiolytic and sedative
[67]
21
Calluna vulgaris Linn. (Hull) (Ericaceae) Heather 250 and 500 mg/kg, p.o. Albino rats of either sex
Quercetin(7) isolated from methanol extract of aerial parts
Inhibition of MAO-A
Anxiolytic
[68]
22
Calotropis gigantea (L.) Dryand. (Apocynaceae) Giant Milkweed, Crown Flower, Aak 10 mg/kg, p.o.
Alcoholic extract of peeled roots
EPM, Hot plate method, Acetic acid induced writhing, Assessment of locomotor activity, rota rod and PTZinduced convulsions EPM
Anxiolytic, anticonvulsant, analgesic and sedative Increase in dopamine levels but not GABAA receptor interaction Anxiolytic
[69]
23
Camellia sinensis (L.) O. Kuntze (Theaceae) Green tea
L-theanine(8)
Sprague Dawley rats
[70]
17
18
Dose Animal/ Human being Male Wistar rats EPM, Vogel conflict test Cannabidiol interaction with 5HT1A receptors in dIPAG in brain Anxiolytic Experimental model/ Assessment of clinical parameters Mechanism of action Activity Ref. 15, 30 and 60 nmol, intra-dlPAG (Dorsolateral peri aqueductal gray) 15, 30 and 60 nmol, intra-BNST bilateral injections 25 and 35 mg/ kg, i.p. 40, 80, 160, and 320 mg/ kg, p.o. in mice, or 1.56, 3.12, 6.25,12.5 and 50 mg/kg, i.p. in rats 795 and 1000 mg/kg, p.o. Wistar rats EPM, OFT, HBT Male and female SpragueDawley rats Wistar rats EPM, OFT Spontaneous motor activity, EPM, FST, HBT, MBT Male Wistar rats EPM, Vogel conflict test Facilitates local 5-HT1A receptormediated neurotransmission [71] Anxiolytic [72] Anxiolytic Anxiolytic, antidepressant and sedative [73] [74]
Table 1: Continued
S. No.
Biological source
24
Cannabis sativa Linn. (Cannabaceae) Bhang
Cannabidiol(9)
Cannabidiol(9)
25
Casimiroa edulis Llave & Lex. (Rutaceae) White Sapote, Zapote blanco
Aqueous extract of Leaves
Hydro-alcoholic (60% ethanol) extract of leaves
26
Casimiroa pringlei (S. Watson) Engl. (Rutaceae) Pringles Zapote 10 mg/kg, i.p. Male wistar rats
[75]
27
Cassia siamea Lam. (Fabaceae) Kasod, Siamese cassia (a) 0.5 and 1.0 g/kg, p.o. (b) 25-100 mg/kg, p.o. 3.2 g/kg/day for 5 days 1 and 1.5 g/kg, i.p.
Barakol (10)
EPM
Anxiolytic
[76, 77]
28
Cecropia glazioui Sneth (Urticaceae) Embauba, Yarumo
(a) Aqueous extract of leaves (b) Butanolic fraction of aqueous extract of leaves
Male adult Swiss mice
EPM
Anxiolytic
[78]
29
Petroleum ether extract of seeds
Albino mice Wistar rats
Behavioural disinhibition model OFT, EPM, Thirsty rat conflict paradigm
Serotonergic mechanism
[79] [80]
Celastrus paniculatus Willd. (Celastraceae) Jyotishmati, Maak kangni
Oil of seeds
30
Centella asiatica (L.) Urb. (Umbelliferae) Gotu Kola 12 g/day, p.o. Double-blind, placebocontrolled study in 20 subjects Male Sprague-Dawley (SD) rats EPM, OFT, SI, locomotor activity, punished drinking, novel cage test Anxiolytic [82] Significantly attenuated the peak of acoustic startle response amplitude Anxiolytic [81] (a) 500 mg/kg, p.o. (b) 3047 mg/ kg, p.o. (c) 111 mg/kg, p.o. (d) 3 mg/kg, p.o. 5 mg/kg, p.o. Wistar rats Inhibition of orientation reflexes and motor activity Anxiolytic
Powdered drug
(a) Marketed formulations (b) Methanol extract (c) Ethyl acetate extract (d) Asiaticoside(11)
31
Centranthus ruber (L.) DC (Valerianaceae) Red valerian Pods - 12.17 ng and Leaves - 18.7 ng diazepam equivalent 750 mg/kg, p.o. Male ICR mice EPM In vitro BZD receptor interaction
Valepotriate valtrate(12)
[83]
32
Ceratonia siliqua Linn. (Fabaceae) Carob tree
Methanol extract of leaves and pods
Anxiolytic
[84]
33
Cinnamomum cassia Blume. (Lauraceae) Cassia Bark, Chinese cinnamon 300, 600 and 1000 mg/kg, i.p. 1 g/kg, p.o. 0.5 and 1.0 g/ kg, p.o. 100, 200 and 400 l Wistar male rats Male Swiss mice Male Swiss mice EPM, OFT LDM, MBT EPM, LDM Male and female Swiss albino mice
50% Ethanol extract from stem barks
Regulation of 5-HT1A and GABA receptor system
Anxiolytic
[85]
34
Cissus sicyoides Linn. (Vitaceae) Possum grape vine, Princess vine
EPM, HBT, MBT, Sodium Pentobarbital-induced sleeping time, PTZ-induced convulsion
Anxiolytic, anticonvulsant
[86]
35
Citrus aurantium Linn. (Rutaceae) Bitter Orange
Essential oil from peel (EOP) of leaves
Anxiolytic Anxiolytic Anxiolytic
[87] [88] [89]
Essential oil from fruits
36
Citrus sinesis Linn. (Rutaceae) Sweet Orange, Blood Orange 100-400 mg/ kg, p.o. 100 mg/kg, p.o.
Essential oil
37
Clitoria ternatea Linn. (Papilionaceae) Butterfly pea
Methanol extract of roots
Male Swiss albino mice and Wistar rats Sprague-Dawley rats and Swiss albino mice
EPM, LDM
Anxiolytic
[90]
38
Convulvulus pluricaulis Choisy. (Convolvulaceae) Shankhpuspi
Ethyl acetate fraction of ethanol extract of the aerial parts
EPM, OFT and rotarod performance
Anxiolytic
[91]
39
Copaifera reticulata Ducke (Leguminosae) Brazilian copaiba
Essential oil
100, 400 and 800 mg/kg, i.p.
Wistar rats
EPM
Anxiolytic
[92]
19
20
Dose Animal/ Human being Male albino mice EPM Anxiolytic [93] Experimental model/ Assessment of clinical parameters Mechanism of action Activity Ref. 100 mg/kg, p.o. 50 mg/kg, i.p. Wistar rats LDM Anxiolytic [94] (a) 56, 80, 320 and 560 mg/ kg, i.p. (b) 50, 200 and 600 mg/ kg, i.p. (c) 0.05, 0.15 and 0.35 ml/ kg, i.p. 3 mg/kg, i.p. Wistar rats EPM Razi male mice EPM, OFT, Pentobarbital sleeping time, Rotarod test Anxiolytic (At lower dose), hypnotic (At higher dose) [95] Anxiolytic [96] 1, 3 and 10 l/100 g, p.o. Male Wistar rats OFT, SI, EPM, HBT, FST Antidepressant and mild anxiolytic Involvement of inducible NOS OFT, Rota-rod test, Spontaneous motor activity, Barbiturate sleeping-time, Transcorneal electroshock, PTZ-induced convulsions, Punished response test EPM, LDM EPM, OFT Anxiolytic [97] 20 mg/kg, i.p. Swiss albino mice EPM, OFT, LDM, SI [98]
Table 1: Continued
S. No.
Biological source
40
Coriandrum sativum Linn. (Umbelliferae) Coriander, Dhaniya
Aqueous extract of seeds
41
Crocus sativus Linn. (Liliaceae) Saffron, Autumn crocus
Crocin(13) isolated from aqueous extract of red dried stigmas
(a) Aqueous extract of stigmas (b) Crocin(13) (c) Safranal (14)
42
Croton celtidifolius Baill. (Euphorbiaceae) Sangue-de-adave
Proanthocyanidin(15) rich fraction isolated from aqueous extract of bark
43
Croton zehntneri Pax & Hoffman (Euphorbiaceae) Canela de Cunha
Methyl eugenol(16) from essential oil
44
Curcuma longa Linn. (Zingiberaceae) Curcuma, Turmeric 200 mg/kg, i.p. Male albino Swiss mice
Curcumin(17)
45
Cymbopogon citratus (DC.) Stapf (Poaceae) Lemongrass, Ginger grass 0.5 and 1.0 g/ kg, i.p. 15 mg/kg, p.o. Swiss male mice Male Wistar rats
Citral (18) or tea abafado
Central Nervous depressant
[99]
Essential oil
[100] [101]
46
Davilla rugosa Poiret (Dilleniaceae) Cipo-Caboclo, Fire vine 100 mg/kg, p.o.
Hydro-alcoholic extract (70% ethanol) of stems
47
Drymaria cordata (L.) Willd. ex Roem. & Schult. (Caryophyllaceae) Tropical chickweed
Swiss albino mice
EPM, LDM, OFT, HBT
Anxiolytic
[102]
48
Ducrosia anethifolia Boiss. (Apiaceae) Hazza, Hazzaz 25, 50, 100, 200 and 400 mg/kg, p.o. 3-7 mg/kg, p.o. Male Wistar rats EPM, SI, shock induced social avoidance test, OFT Only extract (d) showed anxiolytic activity [104] Swiss albino mice EPM, Spontaneous motor activity, Ketamine-induced sleep time Anxiolytic but not sedative [103]
Essential oil
49
Echinacea purpurea (L.) Moench. (Asteraceae) Cone flower
(a) E. purpurea root extract (ethanol 4% v/v; Echinacoside 4%) (b) E. purpurea herb extract (ethanol 60% m/m; total phenols 4%) (c) E. angustifolia root extract (ethanol 85% v/v; Echinacoside 4%) (d) E. purpurea root extract (ethanol 70% v/v) 5, 10, 30, 62.5, 80 and 125 mg/kg, i.p. 50 mg/kg, i.p. Male TO mice EPM Male NMRI albino mice EPM Anxiolytic
50
[105]
Echium amoenum Fisch. Et Mey. (Boraginaceae) Vipers bugloss, Red feathers
Hydro-ethanol extract (80%) of the plant flowers (a) 150 and 300 mg/kg, p.o. (b) 30 mg/kg, p.o. Acute (200 mg/kg, p.o.) chronic (50 mg/kg, p.o. for 7 days) Acute study 200 and 400 mg/kg, p.o. and chronic study for 21 days, 50 and 200 mg/kg, p.o. 3 and 10 mg/ kg, p.o. Male Swiss Mice Male Wistar rats ETM Male Wistar rats Wistar rats Locomotor activity, EPM, HBT, Cold restraint induced gastric ulcer and white blood cell count in the milk induced leukocytosis challenge Elevated T maze (ETM), LDM, Cat odor test
Anxiolytic
[106]
51
Eclipta alba Linn. (Asteraceae) Bhringaraj, False daisy
(a) Aqueous, hydroalcoholic extracts (b) Hydrolyzed fraction obtained from whole plant
Nootropic, sedative, anxiolytic and antistress Anxiolytic
[107]
52
Erythrina mulungu Mart. (Papilionaceae) Mulungu, Corticeira
Hydro-alcoholic extract (70% ethanol) from the inflorescence
[108, 109]
Water : Alcohol (7:3) extract of inflorescence
Anxiolytic
[110]
Erythrinian alkaloids i.e (+)- hydroxyersotrine(19), erythravine(20) and (+)-11-hydroxy erythravine(21) isolated from hydro-alcoholic extract of flowers
EPM, LDM
Anxiolytic
[111]
21
22
Dose Animal/ Human being Male Swiss mice T-maze, Locomotor activity test Anxiolytic [112] Experimental model/ Assessment of clinical parameters Mechanism of action Activity Ref. 3-10 mg/kg, p.o. CE (50, 100, 200 and 400 mg/kg, p.o.) 3 and 10 mg/ kg, p.o. Male albino mice EPM, LDM Anxiolytic [113] Acute study - 200 and 400 mg/kg, p.o., and chronic study - 50 and 200 mg/kg, p.o. 50 and 100 mg/kg, p.o. for 23-26 days 25 mg/kg, i.p. Male Swiss mice LDM Adult male Swiss albino mice EPM Male Wistar rats ETM Anxiolytic [110] Anxiolytic [114] BZD receptor interaction CCl4 induced neuropathic pain, hot plate and carrageenan induced pain Stair case test, LDM Anxiolytic [115]
Table 1: Continued
S. No.
Biological source
Crude extract (CE), Erythrinian alkaloids: (+)- hydroxyersotrine(19), erythravine(20) and (+)-11-hydroxy erythravine(21) isolated from hydro-alcoholic extract of flowers
53
Erythrina suberosa Roxb. (Fabaceae) Coral tree
Alkaloids Erysodine(22) and erysothrine(23) isolated from hydro-alcoholic extract of flowers
54
Erythrina velutina Willd. (Fabaceae) Bico-De-Papagaio
Water : Alcohol (7:3) extract of stem bark
Hydro-ethanol extract of stem bark
55
Hydro-alcoholic extract (60% ethanol) of aerial parts 100 to 300 mg/kg, i.p. Male Wistar rats
Eschscholzia californica Cham. (Papaveraceae) California poppy, Gold poppy
70% ethanol extract of aerial parts
Anxiolytic and antineuropathic pain Anxiolytic
[116]
56
Euphorbia hirta Linn. (Euphorbiaceae) Asthma weed 12.5 and 25 mg/kg, i.p. (a) 2 g/kg, s.c. (b) 30 mg/kg, s.c. 400 mg/kg, p.o.
Aqueous extract of whole plant
Swiss albino mice
[117]
57
Euphoria longana Lamarck (Sapindaceae) Longan Arillus
(a) Methanol extract (b) adenosine(24) isolated from pulp or flesh
Male ddY mice
Vogel type anti-conflict method
Anxiolytic
[118]
58
Euphorbia nerrifolia Linn. (Euphorbiaceae) Indian spurge tree, Oleander spurge
Hydro-alcoholic (50% ethanol) extract of leaves
Swiss albino mice
EPM
Anxiolytic
[119]
59
Eurycoma longifolia Jack (Simaroubaceae) Tongkat ali, Penawar bias 0.3 g/kg, p.o. for 5 days twice daily Albino mice EPM, OFT, Foot shock induced flighting behaviour Anxiolytic [120] 100 mg/kg, p.o. Sprague-Dawley rats and Swiss albino mice EPM, OFT and rotarod performance Anxiolytic, neuromuscular coordination and antioxidant Anxiolytic [91]
Chloroform, n-butyl alcohol and water fractions obtained from methanol extract of roots
60
Evolvulus alsinoides Linn. (Convolvulaceae) Shankhpushpi 15 mg/kg, i.p. Male ICR mice EPM
Ethyl acetate fraction of ethanol extract of the aerial parts
61
Galphimia glauca Cav. (Malpighiaceae) Calderona amarilla
Galphimine B(25), galphimine A(26) and galphimine rich fractions (GRFs) obtained from methanol extract of aerial parts 125, 250, 500, 1000 and 2000 mg/kg, p.o. 310 mg twice daily for 4 weeks 50-200 mg/kg, p.o. Male ddY mice SI A controlled randomized double blind clinical trial HAMA scale, the clinical global impression scale and patient global evaluation ICR albino mice EPM, LDM, FST
[121]
Methanol extract of aerial parts
Anxiolytic and antidepressant
[122]
Capsules containing 310 mg of aqueous extract of aerial parts
Anxiolytic
[123]
62
Gardenia jasminoides Ellis (Rubiaceae) Cape jasmine (a) 400 mg/kg, p.o. (b) 50 and 100 mg/kg, i.p. Male ICR mice EPM
Kamishoyosan
Anxiolytic
[124]
63
Gastrodia elata Blume (Orchidaceae) Tian ma (China); Gastrodia Tuber(English name) 150 mg/kg, p.o. 5C, 9C and 30C dilutions Swiss albino mice ICR-CD1 male mice EPM
(a) Aqueous extract of rhizomes (b) Phenolic constituents: 4-hydroxyl-benzyl alcohol, and benzaldehyde and its phenolic constituents
Interaction with 5-HT(1A) receptor
Anxiolytic
[125]
64
Methanol extract of roots and rhizomes
LDM, OFT
[126] [127]
Gelsemium sempervirens (L.) Ait. (Loganiaceae) Carolina yellow Jasmine
Centesimal dilutions of hydro-alcoholic extract of plant as in homeopathic system
23
24
Dose Animal/ Human being In vitro using rat brain mitocondrial extract Inhibition of monoamine oxidase (MAO A and B) GABA/ BZD/ Cl- channel receptor interaction Anxiolytic Experimental model/ Assessment of clinical parameters Mechanism of action Activity Ref. 5 and 10 mg equivalent [128] 8-16 mg/kg, i.p. Wistar AF rats SI Anxiolytic [129] 0.6 mg/kg, p.o. Charles Foster rats EPM, OFT, novelty-induced feeding latency and SI Anxiolytic [130] 0.5 and 1.0 g/ kg, p.o. for 7 days; 1 and 2 mg/kg, p.o. for five days 10-300 mg/kg, i.p. 100 mg/kg, i.p. Male Swiss albino mice Swiss albino mice EPM, foot shock induced aggression EPM, OFT, Barbiturate-induced sleeping time test Male ddY mice EPM Other mechanism but not through GABA/ BZD/ Cl- channel receptor interaction Anxiolytic [131] Anxiolytic [132]
Table 1: Continued
S. No.
Biological source
65
Ginkgo biloba Linn. (Ginkgoaceae) Ginkgo, Maidenhair tree
Aqueous and ethanol extracts of leaves
Ginkgo biloba extract (EGb-761)
Ginkgolic acid(27) conjugates (GAC) isolated from chloroform: methanol extract (2:1) of the leaves
G. biloba extract (GBE), standardized to contain 24% ginkgoflavoglycosides and 6% ginkgo-terpenoid lactones or ginkgolide A(28)
66
Glycyrrhiza glabra Linn. (Leguminosae) Licorice, Mulethi
Hydro-alcoholic extract of roots and rhizomes
67
Hedyosmum brasiliense Mart. (Chloranthaceae) Cha de bugre 350 mg/kg, p.o. DBA/2J mice
[133]
68
Heteropterys glabra Hook. & Arn. (Malpighiacae) Redwing 300 mg/kg, p.o. Wistar rats
Ethanol extract of fruits
Sleep wakefulness cycle, electroencephalogram (EEG) and visual evoked potentials (VEP) EPM, ketamine- induced sleep
[134]
69
Hibiscus sabdariffa Linn. (Malvaceae) Jamaica sorrel, Red sorrel Anxiolytic and sedative (1-10 mg/kg, i.p.), anticonvulsant (30 and 60, mg/kg, i.p.)
Aqueous, hydroalcoholic, and ethanol extract of calyxes of plant
Anxiolytic and sedative (at multiple doses) Swiss albino mice EPM, Sodium pentobarbitalinduced sleep, PTZ-provoked convulsions, FST Anxiolytic, mild sedative and anticonvulsant but not antidepressant
[135]
70
Hippeastrum vittatum (LHerit) Herbert (Amaryllidaceae) Amaryllis
Isoquinoline alkaloid: Montanine(29) isolated from ethanol extract of bulbs
[136]
71 2778 and 1852 mg/kg, p.o. Male SpragueDawley rats OFT, LDM Inhibitory influence on glutamatergic transmission mediated by NMDA receptors Affect monoamines concentration in rats brain Anxiolytic Anxiolytic [139] [140] Anxiolytic [138]
Hypericum perforatum Linn. (Guttiferae) St Johns wort Anxiolytic [137]
H. perforatum extract LI60
In vitro
receptor activation
Standardized extract of the whole plant, containing 0.54% total hypericins [0.11% hypericin(30) and 0.43% pseudohypericin(31)]and 0.09% protoforms 5 mg/kg, p.o. 100 or 200 mg/kg, p.o. OD for 3 days 300 mg/kg, p.o. for 21 days 300 mg/kg, p.o 380 mg/kg/ day chronic administration 150 and 300 mg/kg, p.o. 200-400 mg/ kg, p.o. 40 mg/kg, s.c. Male ddY mice Male Laca mice Swiss albino mice MBT, FST Mirrored chamber, EPM, EZM Vogel type Anticonflict effect in mice C57BL/6J Mice OFT, LDM, FST Male albino Swiss mice ETM Male albino Swiss mice Mouse defense test battery Wistar rats EPM, OFT, EZM, novelty-induced suppressed feeding latency, SI Male albino Swiss mice EPM
Lyophilized aqueous extract
Hydro-alcoholic extract of whole plant
H. perforatum extract LI 160
Anxiolytic
[141]
Anxiolytic Anxiolytic and antidepressant Anxiolytic and antidepressant Anxiolytic Anxiolytic
[142] [143]
[144] [145] [146]
Hydro-alcoholic extract of whole plant
72
Jatropha ciliata M. Arg. (Euphorbiaceae) Huanarpo 120 mg/kg/ day, p.o. Male Wistar rats
Vitexin(32), isoorientin(33) and orientin(34) from methanol extract of Stems
73
Kielmeyera coriacea Mart. (Clusiaceae) Pu santo
EPM
Anxiolytic
[147]
25
26
Dose Animal/ Human being Adult male SpragueDawley albino rats Open field behavior test Lavender oil potentiates the responses of GABA receptors at low concentrations and inhibits responses of GABA receptors at high concentrations in vitro Interaction with GABAA/BZD receptor Experimental model/ Assessment of clinical parameters Mechanism of action Activity Ref. Inhalation 0.1-1.0 ml Anxiolytic [148, 149] I ml/100 g, i.p. (a) 250 mg/kg, p.o. (b) IC50values of 2.1 microM (1), 45 microM (2), 3.3 microM (3) and 40 microM (4) EO1 and EO3 (100 mg/kg, i.p.) and EO2 (25 mg/kg, i.p.) 1.8, 3.7, 7.5 and 15.0 mg/ kg, i.p. Male Swiss Mice (a) Rats (b) In vitro radio receptor assay with [3H] Flunitrazepam Mature male and female gerbils EPM Locomotion study Male ICR Mice Galler type conflict test Anxiolytic Anxiolytic Anxiolytic [150] [151] [152, 153]
Table 1: Continued
S. No.
Biological source
74
Lavandula angustifolia Miller (Lamiaceae) English Lavender
Lavender oil
Lavender odour
75
Leptospermum scoparium J.R. et G. Forst. (Myrtaceae) Manuka or Tea tree
(a) Hydro-alcoholic extract (70% ethanol) (b) 5,7-dimethoxyflavone (1), 5,7-dimethoxy-6methylflavone (2), 5-hydroxy-7-methoxy-6methylflavone (3) and 5-hydroxy-7-methoxy6,8-dimethylflavone (4)
76
Lippia alba (Mill.) N.E. Brown (Verbenaceae) Cidreira, Bushy matgrass
Three chemotypes of essential oil (EO1, EO2, EO3) from leaves
EPM, OFT and rotarod
Anxiolytic and myorelaxant
[154]
77
Loeselia mexicana Brand (Polemoniaceae) Mexican false calico, Espinosilla 100 and 300 mg/kg, p.o.
Daphnoretin(35) isolated from hydro-alcoholic extract (60% ethanol) of whole plant
Male ICR mice
OFT, EPM
Anxiolytic
[155]
78
Magnolia dealbata Zucc. (Magnoliaceae) Eloxochiti
Male Swiss albino mice
EPM, HBT, exploratory rearings, Sodium pentobarbital-induced hypnosis, PTZ-induced seizures
Anxiolytic, sedative and anticonvulsant
[156]
79
Honokiol (36) 0.2-1 mg/kg, p.o. for seven days 0.2 mg/kg, seven days, p.o. 0.2, 0.5 and 1.0 mg/kg, p.o. ICR male mice EPM, HBT GABA-BZDreceptors / Cl- channel activation Interaction with GABAA/BZD receptor Interaction with GABAA/BZD receptor Inhibits GABA-T (transaminase) activity and increase GABA level in brain Interaction with GABA receptors GABAA agonist Anxiolytic Male mice of the ddY strain EPM Anxiolytic [158] Male mice of the ddY strain EPM Anxiolytic [157]
Magnolia Obavata Thunb. (Magnoliaceae) Japanese bigleaf magnolia
Honokiol (36)
Obovatol (37)isolated from leaves
[159]
80
Apigenin(38) isolated from aqueous extract of branchlets with flowers 30 mM In vitro, Rats
3 mg/kg, i.p.
Male C Fl mice
EPM, HBT, Locomotor activity test, Horizontal-wire test, Seizure testing
Anxiolytic and mild sedative at 10 times dose Anxiolytic
[160]
Marticaria chamomila Linn. or Matricaria recutita (Asteraceae) German chamomile, Amerale 120, 240 and 360 mg/kg, p.o. for 15 days 200 and 400 mg/kg, p.o. (a) IC50 22.8 g/ml (b) IC50 27.2 g/ml 80 and 160 mg/kg, i.p. Adult Swiss male mice In vitro Swiss albino mice EPM, MBT C57Bl/6Jico mice EPM
Apigenin(38)
[161163] Anxiolytic [164]
81
Melissa officinalis Linn. (Lamiaceae) Lemon balm, Common balm
Cyracos : hydro-alcoholic (30% ethanol) extract of aerial parts
82
Mitragyna parvifolia Roxb. (Rubiaceae) Kaim, Gulikadam
Methanol, ethyl acetate and alkaloid rich fraction of stem bark
Anxiolytic
[165]
83
Morinda citrifolia Linn. (Rubiaceae) Noni, Indian mulberry
(a) Methanol extract of fruits (b) Butanolic fraction
Anxiolytic
[166]
84
Nauclea latifolia J.E.Smith (Rubiaceae) Negro peach, African peach 100 mg/kg, i.p. Male ICR mice
Decoction from bark of the roots
EPM, Diazepam-induced sleep, MES-, Strychnine-, PTZ-induced convulsions test, OFT
Anxiolytic
[167]
85
Nelumbo nucifera Gaertner (Nymphyaeaceae) Sacred water lilly, Pink lotus 50 mg/kg, i.p.
Neferine(39) isolated from methanol extract of embryos of the seeds
EPM
Anxiolytic
[168]
86
Nepeta persica Boiss. (Lamiaceae) Catmint
Hydro-alcoholic extract (80% ethanol) of aerial parts
Male NMRI mice
EPM
Anxiolytic
[169]
87
Ocimum gratissimum Linn. (Lamiaceae) Vana Tulsi
Methanol extract of leaves
200 and 400 mg/kg, p.o.
Swiss albino mice
OFT, PTZ-induced seizure test
Anxiolytic and anticonvulsant
[170]
27
28
Dose Animal/ Human being Swiss Mice EPM, Passive avoidance paradigm, Scopolamine and diazepam induced amnesia Hamiltons brief psychiatric rating scale (BPRS) Anxiolytic and nootropic effects Anxiolytic Experimental model/ Assessment of clinical parameters Mechanism of action Activity Ref. 200 mg/kg, p.o. 500 mg/ capsule twice daily after meal 150 mg/kg, p.o. Swiss albino mice Staircase test, EPM, aggressive behavior, Pentobarbitone induced sleeping time, locomotor activity, rotorod test 35 male and female human beings [171] [172] Sedative, antianxiety muscle relaxant and antiaggressive activity Decrease MAO activity in brain Anxiolytic Anxiolytic [173] 100 mg/kg, p.o. 20 and 50 mg/ kg, p.o. twice daily for 5 days RG (100 mg/ kg, p.o.) and SG (25 and 50 mg/kg, p.o.) (a) 300, 600 and 1200 mg/ kg, p.o (b) 50, 100, and 200 mg/ kg, p.o (c) 2.5, 5 and 10 mg/kg, i.p (a) 50 and 100 mg/kg, p.o. (b) 5, 10 and 25 mg/kg, p.o. once daily for 3 days 50 and 100 mg/kg, p.o. Male ICR mice Male ICR albino mice Albino mice EPM Male Wistar strain albino rats and albino mice Wistar rats Vogel conflict procedure EPM, OFT, conflict behavior in thirsty rats, foot shock induced fighting in paired mice [174] [175] Anxiolytic [176]
Table 1: Continued
S. No.
Biological source
88
Ocimum sanctum Linn. (Lamiaceae) Tulsi, Holy basil
89
Pachyrhizus erosus Linn. (Leguminosae) Bangkwang, Jicama
Ethanol extract of seeds
90
Ginseng extract G-115
Panax ginseng C.A.Meyer (Araliaceae) Chinese, Japanese, Korean ginseng, Ninjin
Aqueous extract of white and red roots powder
Butanol fractions of roots of red (RG) and sun ginseng (SG)
(a) Ginseng root powder (b) Crude saponin ginseng fraction (c) Ginsenoside Rb1(40)
EPM
Anxiolytic
[177]
(a) Ginseng aqueous extract (b) Ginsenosides Rg3(41) and Rh2(42) from roots
EPM
Interaction with GABA/BZD receptors
Anxiolytic
[178]
91
Panax quinquefolius Linn. (Araliaceae) American ginseng
Saponins
Male Swiss albino mice
EPM, LDM, HBT
Anxiolytic
[179]
92
Passiflora actinia Hooker (Passifloraceae) Wild bell apple, maracuj-do-mato HE (300 and 600 mg/kg, p.o.) ME (100 and 300 mg/kg, p.o.) Anxiolytic [180] 50,100 or 150 mg/kg, i.p. 400 and 800 mg/ kg, p.o. 50 and 100 mg/kg, i.p. 1 mg/kg, i.p. Male CF1 mice EPM, HBT Interaction with BZD receptors Wistar rats EPM Adult male Wistar rats EPM Anxiolytic Adult female Wistar rats EPM Anxiolytic [181] [182] Male albino-Swiss mice EPM GABAA receptor interaction
Hydro-ethanol (HE) and Methanol (ME) Extract from leaves
93
Passiflora alata Dryander (Passifloraceae) Winged-stem Passion flower Anxiolytic Anxiolytic
Spray dried powder of aqueous extract of leaves
Aqueous extract of leaves
[183, 184] [185, 186]
94
Passiflora coerulea Linn. (Passifloraceae) Blue Passion flower 50, 100 and 150 mg/kg, i.p. (a) 230 mg/kg, p.o. (b) 100 mg/kg, p.o. (c) 30 mg/kg, p.o. Adult male Swiss mice EPM, MBT Adult female Wistar rats EPM
Chrysin (43)
95
[181] [187]
Passiflora edulis Sims (Passifloraceae) Bat-Leaved Passion flower
(a) Aqueous extract (b) Total flavonoid fraction (c) Luteolin-7-O-(2rhamnosyl glucoside) (44) from total flavonoid fraction of aqueous extract of leaves 400 and 800 mg/ kg, p.o. 50, 100 and 150 mg/kg, i.p. 100 and 300 mg/kg, p.o. Adult male Swiss mice Wistar rats Adult male Wistar rats EPM
Spray dried powder of aqueous extract of leaves
Anxiolytic
[182]
Aqueous extract
EPM LDM, Ethyl etherinduced hypnosis, PTZ-induced convulsions HAMA scores
Anxiolytic Anxiolytic and sedative but not anticonvulsant Anxiolytic
[183, 184] [188]
Aqueous extract of mature fruits and its butanolic fraction 45 drops/day for 4 weeks 125 mg/kg, p.o. 100 mg/kg, p.o. 10 mg/kg, p.o.
96
Aqueous extract (PassipayTM, Iran Darouk)
A double blind randomized trial on 36 patient with GAD Swiss Albino mice Swiss Albino mice Swiss Albino mice
[189]
Passiflora incarnata Linn. (Passifloraceae) Passion flower, Maypop
EPM EPM EPM
[190193] [194] [195]
Homoeopathic formulations
Benzoflavone nucleus as basic moiety compound from methanol extract
29
30
Dose Animal/ Human being Male Sprague-Dawley rats EPM Interaction with GABA/BZDreceptors Experimental model/ Assessment of clinical parameters Mechanism of action Activity Ref. 2 mg/kg, i.p. Anxiolytic [196, 197] Anxiolytic [198] 500 mg, p.o. A double blind placebocontrolled study on 60 patients with anxiety Male BL6/C57 J mice EPM Numerical rating scale, Trieger dot test and the digit-symbol substitution test 375 mg/kg, p.o. (a) 150 and 300 mg/kg, p.o. (b) 2.1 and 4.2 mg/kg, p.o. (c) 0.17 and 0.34 mg/kg, p.o. 250 and 500 mg/kg, p.o. Adult male Wistar rats and Swiss mice EPM, OFT, HBT Male C57BL/6J mice EPM Interaction with GABA receptors Anxiolytic [199, 200] Anxiolytic [201]
Table 1: Continued
S. No.
Biological source
Methanol extract of aerial parts and Chrysin
Tablet containing 1.01 mg benzoflavone (BZF)
Hydro-ethanol extract (50% ethanol) of aerial parts
(a) Hydro-alcoholic extract (50% ethanol) of aerial parts (b) Butanol fraction (c) Chloroform extract
97
Passiflora quadrangularis Linn. (Passifloraceae) Giant granadilla 10 mg/kg, p.o. Albino mice
Aqueous and ethanol extract of leaves
Anxiolytic
[202]
98
Perilla frutescens (L.) Britton (Lamiaceae) Purple Perilla, Wild red basil
Rosmarinic acid (45)and caffeic acid(46) isolated from hydro-alcoholic extract of leaves
FST
Modulation of the 1A- adrenoceptormediated signal transductions and also attenuates the down regulation of BDNF transcription Female Swiss mice EPM, OFT
Anxiolytic
[203]
99
Petiveria alliacea Linn. (Phytolaccaceae) Guinea hen weed
Hexane, hydro-alcoholic, and precipitated hydro-alcoholic extract (50%) of roots
100 and 200 mg/kg, i.p. and p.o. 300 and 900 mg/kg, p.o.
Anxiolytic
[204]
Whole plant extract
Male albino Swiss mice
EPM, OFT
Anxiolytic
[205]
100
Piper methysticum Forst. (Piperaceae) Kava, Kawa 50 mg, p.o. 25-week multicenter randomized placebocontrolled double-blind trial on 121 outpatients suffering from anxiety of non-psychotic origin Controlled clinical trial on a 37-year-old female outpatient with GAD, SP and SAD Patients suffering with GAD A randomized double-blind placebo-controlled clinical trial on 37 patient with DSM-IV GAD Wistar rats A randomized double-blind placebo-controlled clinical trial on 129 patients suffering from GAD i.p. injections of different concentrations Chick social separation procedure HAMA Scale and Boerner Anxiety Scale (BoEAS), CGI, a sleep questionnaire (sf-13), and a quality of life questionnaire EPM HAMA Scale, Hospital Anxiety and Depression Scale (HADS), Self- Assessment of Resilience and Anxiety (SARA) Baroreflex control of heart rate (BRC) and respiratory sinus arrhythmia (RSA) Anxiolytic CGI scale, AMDP module, HAMA, Hamilton depression scale, Beck anxiety inventory, Speilberger trait anxiety inventory Anxiolytic [207] HAMA, somatic and psychic anxiety, Clinical Global Impression (CGI), Self-Report Symptom Inventory-90 Items revised, and Adjective Mood Scale Anxiolytic [206]
WS 1490 extract
Kava extract LI 150
3 tablets daily equivalent to 135 mg kava pyrones daily for 12 weeks 280 mg/day for 4 weeks 50 mg/day for 4 weeks
Kava extract standardized to 30% kavalactones
[208]
Kava-Kava special extract WS 1490
Anxiolytic
[209]
Hydro-alcoholic extract of roots 400 mg/day
120-240 mg/ kg, p.o.
[210] [211]
Kava Kava LI150 extract
Samples containing 12.8-100% total kavalactones, and fractions containing kavalactones 1-6(47-52) in varying concentration (0.1-67.5%) 125 mg/kg and 88 mg/kg, i.p. 50 mg/day for 4 weeks Swiss albino mice
Cockerels (Gallus gallus; strain W36)
Anxiolytic
[212]
Ethanol extract of the aerial parts
Mirrored chamber avoidance assay and EPM The total score of the Anxiety Status Inventory (ASI) observer rating scale, structured well-being self-rating scale (Bf-S) and CGI HAMA Scale, subjective well- being scale (Bf-s), Erlanger Anxiety, Tension, Aggression Scale (EAAS), CGI, The Brief Personality Structure Scale and The Adjective Checklist
Anxiolytic
[213]
A randomized double-blind placebo-controlled clinical trial on 141 patients suffering from neurotic anxiety A randomized double-blind placebo-controlled clinical trial on 230 patients suffering from neurotic anxiety Swiss male mice
Anxiolytic
[214]
50-300 mg/ day for 4 weeks
Anxiolytic
[215, 216]
101
Piper solmsianum C. DC. (Piperaceae) Pariparoba
Emulsion of the essential oil from aerial parts
5 or 10% v/v
EPM
Anxiolytic
[217]
31
32
Dose Animal/ Human being Swiss male mice EPM, OFT Anxiolytic Experimental model/ Assessment of clinical parameters Mechanism of action Activity Ref. 50 and 100 mg/kg, i.p [218] HE, fractions (250, 500 and 1000 mg/kg), p.o., Dihydrostyryl2-pyrones and styryl-2pyrones (0.3 fmol25 pmol, i.c.v.) 0.3 fmol25 pmol, i.c.v. Male adult Swiss mice EPM Male adult Swiss mice EPM, Pentobarbital-and ethyl ether-induced hypnosis, PTZ-induced convulsions, Rota-rod test Hypnotic, anticonvulsant and anxiolytic [219] BZD receptor interaction Anxiolytic [220] 40, 80 and 160 mg/kg, p.o. 10, 25 and 50 mg/kg i.p. or p.o. Male Swiss mice Male adult Swiss mice EPM, OFT, HBT Anxiolytic [221]
Table 1: Continued
S. No.
Biological source
102
Piper tuberculatum Jacq. (Piperaceae) Pimenta darta and Pimenta Longa
Piplartine (53) amide alkaloid isolated from roots
103
Polygala sabulosa A.W. Bennett (Polygalaceae) Timutu-pinheirinho
Three dihydrostyryl-2pyrones I-III (54-56) and four styryl-2-pyrones I-IV (57-60) isolated from ethyl acetate fraction of hydro-ethanol (HE) extract of whole plant
Dihydrostyryl-2pyrones(54-56) and styryl-2- pyrones(57-60) isolated from ethyl acetate fraction of hydro-ethanol (HE) extract of whole plant
104
Polygala tenuifolia Willd. (Polygalaceae) Yuan Zhi
Polygala saponins
105
Protium heptaphyllum (Aubl.) March. (Burseraceae) Brasil resintree 20 mg/kg, i.p.
and amyrin (61-62) pentacyclic triterpenes isolated from stem bark resin
EPM, OFT
BZD receptor interaction
Anxiolytic
[222]
106
Prunus domestica Linn. (Pleuronectidae) Mirabelle, Plum, Alu bukhara 200 mg/kg, p.o.
Chlorogenic acid(63) isolated from fruits
Swiss albino male mice
EPM, LDM, free exploratory test
Anxiolytic
[223]
107
Pulsatilla nigricans Stoerck (Ranunculaceae) Pasqueflower, Windflower, Meadow anemone
Laca mice
EPM
Anxiolytic
[224]
108
Punica granatum Linn. (Punicaceae) Pomegranate, Granada 100, 250, and 500 mg/kg, p.o. 15 mg/kg, p.o. Male CD1 mice LDM Anxiolytic [226] Young and old male Swiss albino mice EPM, Pentobarbital-induced sleeping time, FST, tail flick and hot plate test Anxiolytic, antidepressant and antinoceceptive [225]
109
Rhodiola rosea Linn. (Rhizophoraceae) Arctic root, Golden root, Roseroot 1.62 to 6.25 mg/kg, p.o. Albino mice Avoidance exploratory behavior paradigm GABA/BZD receptors interaction Interaction with GABAA receptor Anxiolytic
Hydro-alcoholic extract (contains 3% rosavin and 1% salidroside)
110
Rollinia mucosa (Jacq.) Baill. (Annonaceae) Wild sugar apple 150 mg/kg, per gavage 300 mg/kg, p.o. Male Swiss albino mice PTZ-induced seizures, sodium pentobarbital-induced hypnosis, exploratory activity, anxiety by unfamiliar environment and nociception EPM Male Wistar rats and Swiss mice EPM
Hexane extract of leaves
[227]
111
Rubus brasiliensis Martius (Rosaceae) Amora branca
Anxiolytic
[228, 229] Anxiolytic, anticonvulsant, sedative, antinociceptive [230]
112
Ruta chalepensis Linn. (Rutaceae) Fringed rue, herb-of-grace 100 mg/kg, i.p. Male NMRI mice
113
Salix aegyptiaca Linn. (Salicaceae) Egyptian muskwillow 10 mg/kg, p.o. Albino mice EPM, FST
Anxiolytic
[231]
114
Salvia cinnabarina M.Martens & Galeotti (Lamiaceae) Sage, Wild Kus 0.001-1000 g/kg, s.c. Adult male Sprague-Dawley rats
A diterpenoid CMP I
Anxiolytic
[232]
115
Salvia divinorum Epling & Jtiva (Lamiaceae) Diviners sage 125, 250, 500, 1000 and 2000 mg/kg, p.o. 12.5 mg/kg, i.p. 10-60 mg/kg, p.o. Male ICR mice
Salvinorin-A(64)
EPM, FST, Spontaneous motor activity in mice, Tail suspension test EPM, LDM, OFT
k-opioid and endocannabinoid systems
Anxiolytic, antidepressant
[233]
116
Salvia elegans Vahl. (Lamiaceae) Scarlet pineapple
Hydro-alcoholic (60% ethanol) extract of leaves and flower
Anxiolytic
[234]
60% ethanol extract of leaves
Sprague Dawley rats Albino mice
EPM, FST Four plate test
BZD receptor interaction
Psychotropic Anxiolytic
[235] [236]
117
Salvia miltiorrhiza Bge. (Lamiaceae) Red sage 100 mg/ kg, i.p.
Diterpene quinine Miltirone(65) isolated from ethereal extract of roots
118
Salvia reuterana Boiss. (Lamiaceae) Sage
Hydro-alcoholic extract (80% ethanol) of aerial parts
Male Syrian mice
EPM, Spontaneous locomotor activity
Anxiolytic
[237]
33
34
Dose Animal/ Human being Albino mice EPM, Y -maze, HBT, Actophotometer, MBT GABAergic transmission Anxiolytic [238] Experimental model/ Assessment of clinical parameters Mechanism of action Activity Ref. 200 and 400 mg/kg, p.o. Inhalation Anxiety in a woman in labour Behavioural responses Anxiolytic [239] 7.5, 15 and 30 mg/kg, p.o. Male ICR mice EPM Interaction with GABA/ BZD receptor Interaction with GABAA / BZD receptor (a) Interaction with GABAA / BZD receptor (agonist) (b) Interaction with GABAA / BZD receptor (selective antagonist) Interaction with BZD binding site of GABAA receptors EPM Interaction with GABAA / BZD receptor EPM, SI Interaction with GABAA / BZD receptor Anxiolytic [240] IC50 values 0.008 to 100 M Radio receptor BZD-S assay 6.05 mM In vitro Radio receptor BZD-S assay In vitro, Forebrains of Sprague-Dawley rats Anxiolytic [241] Anxiolytic [242, 243] Baicalein (10 mg/kg, i.p.) and baicalin (20 mg/kg, i.p.) 100 mg/ml, orally (a) 40 mg/g, p.o. (b) 33 mg/g, p.o. (c) 1.6 mg/g, p.o. (d) 31 mg/g, p.o. 100-400 mg/ kg, p.o. Male Sprague-Dawley rats Male ICR mice Vogel shock conflict test Anxiolytic [244]
Table 1: Continued
S. No.
Biological source
119
Sapindus mukorossi Gaertn. (Sapindaceae) Soapberry,Ritha
Methanol extract of seeds and fruits
120
Saussurea lappa C.B. Clarke (Asteraceae) Kuth, Kustha
Essential oil
121
Scutellaria baicalensis Georgi (Labiatae) Huangqin
A monoflavonoid Wogonin(66), isolated from dichloromethane extract of roots
Only 2-OH flavones isolated from dichloromethane, water extracts of roots
(a) 5,7,2-trihydroxy-6,8dimethoxy flavones (b) 5,7-dihydroxy-6methoxyflavone isolated from dichloromethane extract of roots
Flavonoid baicalin(67) and its aglycone baicalein(68)
122
Scutellaria lateriflora Linn. (Labiatae) Blue skullcap, Hoodwort
Aqueous extract of roots
Anxiolytic
[245]
(a) Flavonoid baicalin(67) in ethanol extract of roots (b) baicalein(68) in ethanol extract of roots (c) Ethanol extract of roots (d) glutamine in water extract of roots
Adult male SpragueDawley rats
Anxiolytic
[245]
123
Securidaca longepedunculata Fresen (Polygalaceae) Violet tree
Aqueous roots extract
Albino mice of either sex
EPM, Y -maze, Strychnine- and Picrotoxin-induced seizure, Hexobarbitone- induced sleep test, Exploratory activity
Anxiolytic, anticonvulsant, sedative
[246]
124
Sesbania grandiflora (L.) Poir. (Fabaceae) Agati 100 and 200 mg/kg, p.o. Male Sprague-Dawley rats and male mice (NIN strain) EPM, MES-, PTZ-, Strychnine-, Lithium-pilocarpine-and electrically induced seizures, Pentobarbital-induced sleep, Amphetamine antagonism EPM Anxiolytic [248] Increase in the brain content of GABA and 5-HT Anxiolytic and anticonvulsant [247]
Benzene:ethyl acetate (BE) fraction of the acetone soluble part of petroleum ether extract of leaves 30-300 mg/kg, p.o. Adult male Swiss mice
125
Sonchus oleraceus Linn. (Asteraceae) Sow thistle, Milky tassel PE (10 mg/kg, i.p.), EE (10 mg/kg, i.p.), WE (30 mg/kg, i.p.) 100 mg/kg, p.o. Albino Wistar mice and rats of either sex EPM Swiss albino male mice EPM, OFT, Foot shock induced aggression Anxiolytic
Hydro-alcoholic (50% ethanol) and dichloromethane extract from aerial parts
126
Sphaeranthus indicus Linn. (Asteraceae) Mundi
Petroleum ether extract (PE), 90% ethanol extract (EE), Water extract (WE) of flowers
[249]
Hydro-alcoholic Extract (50% ethanol) from fully grown flowering herb 12.5 to 100 mg/kg, i.p. Albino wistar rats and Swiss mice Muricidal action of rats, Porsolts FST
Anxiolytic
[250]
127
Spondias mombin Linn. (Anacardiaceae) Hog plum, Jobo, Yellow mombin 100 mg/kg, i.p. Male TO mice EPM
GABAA receptor
[251]
128
Stachys lavandulifolia Vahl. (Lamiaceae) Lavendelblaetrige and Wood betony PF (25 and 50 mg/kg, i.p.) EF (25 and 50 mg/kg, i.p.) AF (50 mg/kg, i.p.) 100 mg/100g, o.s. Male Wistar strain rats ETM Male Syrian mice EPM
Hydro-alcoholic extract (80% ethanol) and essential oil of aerial parts
Anxiolytic
[252]
Petroleuum ether (PF), ethyl acetate (EF) and water (AF) fractions of hydro-alcoholic extract (80% ethanol) of aerial parts
Anxiolytic
[253]
129
Theobroma cacao Linn. (Sterculiaceae) Cacao, Chocolate tree, Kakao
Mass or cake
Conditional fear relating behaviour, but did not affect the concentration of brain monoamines such as norepinephrine, serotonin and dopamine
Anxiolytic
[254]
35
36
Dose Animal/ Human being Male Swiss albino mice EPM, HBT, sodium pentobarbital-induced hypnosis and ambulatory activity EPM Anxiolytic Anxiolytic and Sedative at higher dose (30 mg/kg, i.p.) Experimental model/ Assessment of clinical parameters Mechanism of action Activity Ref. 1 to 10 mg/kg, i.p. [255, 256] 25-100 mg/kg, p.o. Albino ICR mice [257] 10-300 mg/kg, p.o. Male Swiss albino mice EPM, HBT, sodium pentobarbital (SP)-induced hypnosis potentiation, ambulatory Activity EPM, HBT Anxiolytic and sedative [258] Dose equivalent to 1 g of plant material 80 mg Women with postmenopausal anxiety Laca mice Laca mice Laca mice EPM EPM EPM Swiss albino Mice Interaction with BZD receptors Anxiolytic [259] HADS, Zungs self- rating depression scale Anxiolytic and antidepressant Anxiolytic Anxiolytic Anxiolytic [260] 25 mg/kg, p.o. 50 mg/kg, p.o. 2 mg/kg, p.o. [261] [262] [263]
Table 1: Continued
S. No.
Biological source
130
Tilia americana Linn.var. mexicana (Malvaceae) American basswood, American linden
-sitosterol(69) isolated from hexane extract of inflorescences
Methanol extract from bracts and flowers [Tiliroside(70) main constituent]
Aqueous extract of inflorescences [Quercetin(7) and kaempferol(6) may be active constituents]
131
Tilia tomentosa Moench (Malvaceae) Silver Lime
Hydro-alcoholic extract (70% ethanol) of Inflorescences and butanol fraction
132
Trifolium pratense Linn. (Fabaceae) Red clover
Isofavones (MF11RCE)
133
Turnera aphrodisiaca Ward (Turneraceae) Damiana
Homoeopathic formulations
Apigenin(38) isolated from methanol extract of aerial parts 200 mg/kg/ day p.o for 7 days 0.61.0 and 2.63.2 mg/ kg/day, p.o.
134
Uncaria rhynchophylla (Miq.) Jacks (Rubiaceae) Cats Claw herb
Male SD rats and male ICR mice
EPM, HBT
Serotonergic nervous system
Anxiolytic
[264]
135
Vaccinium ashei Reade (Ericaceae) Blueberry
Anthocyanin fraction from 96% ethanol extract of berries
Adult male Swiss mice (aged 3 months)
EPM, OFT, Inhibitory avoidance
Memoryenhancing, anxiolytic and locomotion increasing effects Male ICR mice PTZ-induced seizures, Exploratory rearing, Rotarod Anxiolytic, anticonvulsant and myorelaxant
[265]
136
Valeriana edulisssp. Procera Nutt. ex Torr. (Valerianaceae) Tobacco root
Hydro-alcoholic (70% ethanol) extract of roots
100, 300 and 1000 mg/kg, p.o.
[266]
137
Valeriana glechomifolia Meyer (Valerianaceae) Valerian 10 mg/kg, p.o. Male albino Swiss mice EPM, OFT Anxiolytic [267] Valdan drops o.s. daily for 15 days 83.1 mg per day 36 patients with GAD DSM III-R Psychic factor of HAMA Significant reduction in the psychic factor of HAMA Interaction with GABAA / BZD receptor Anxiolytic Anxiolytic Female albino mice Behavioural studies in locomotion test and FST Anxiolytic [268]
Valepotriate fraction
138
Valeriana officinalis Linn. (Valerianaceae) Valerian
Hydro-alcoholic extract called valdan drops
Valepotriates
[269]
6-methylapigenin (70) and hesperidin(71), isolated from roots and rhizomes Hesperidin (4 mg/kg, i.p.), 6-methylapigenin (1 mg/kg, i.p.) 4 and 7 mg/kg, i.p. 0.2 g/kg, p.o. 3 mg/kg, i.p. Female hooded rats EPM Male Wistar rats EPM Albino mice HBT GABA(A) ergic system Adult male Wistar rats EPM , HBT
[270, 271]
Flavonoid linarin(72)
[272] [273] [274, 275] Anxiolytic [276]
Dichloromethane extract of roots
Valerenic acid(73) isolated from hydroalcoholic extract of roots 100 and 200 mg/kg, p.o. Swiss albino mice EPM, LDM
139
Vitex negundu Linn. (Verbenaceae) Five-leaved chaste tree 20 and 50 mg/ kg, p.o. for 5 days 50, 200 and 500 mg/kg 15 and 30 mg/ kg, i.p. Wistar rats Male Sprague-Dawley rats Wistar rats
Ethanol extract of roots
140
Withania somnifera (Linn.) Dunal (Solanaceae) Ashwagandha
Glycowithanolides isolated from the roots
SI, Novelty-induced suppressed feeding latency, EPM EPM EPM
Anxiolytic
[277]
[278] [279]
141
Zingiber officinale Linn. (Zingiberaceae) Ginger 0.5 g/kg, p.o. 320 mg/kg, p.o. thyroid tablet for nine days 2.0 mg/kg, p.o. Male ICR mice
Benzene fraction of acetone soluble part of petroleum ether extract of dried rhizomes
142
[280] [281]
Ziziphus jujuba Miller (Rhamnaceae) Desi Ber
Alcoholic extract of seeds
Yin deficiency mice
Black/white test, EPM and ambulatory behaviour test EPM, LDM
Sanjoinine-A(74) from alkaloidal fraction of seeds
Male ICR mice
EPM, OFT, HBT
Increase the GABA and expression of GABAA GABAergic transmission
Anxiolytic
[282]
37
38
Dose 2001600 mg/kg, i.p. Male ICR mice Geller conflict test and Vogels conflict test Other mechanism but not through GABA/BZD Only rose oil exhibited anxiolytic activity Animal/Human beings Experimental model/Clinical studies parameters Mechanism of action Activity Ref. [283] 1600 mg/kg, i.p. 1 ml/100 g, i.p. Male ICR mice (a) Anticonflict (b) Increased ambulatory effect (c) Increased ambulatory effect Dopamine receptor involvement Male ICR mice Geller type conflict test Anxiolytic Anxiolytic [284] [285] 1 ml/100 g, i.p. Inhalation (1.0, 2.5 or 5.0% w/w) Inhalation 1 ml ICR strain mice EPM, FST, OFT Adult Wistar male rats EPM Male ICR mice Geller conflict test and Vogels conflict test 5-HTnergic pathway and the suppression of DA activity related to enhanced 5-HTnergic neurons GABAergic transmission OFT, EPM, HBT, Tail suspension and FST LDM Extracellular and whole cell patch clamp recordings on CA1 pyramidal neurons Male rats and patients with arterial hypertension I - II degree, accompanied by psycho-emotional disturbances Behavioural parameters Hetero-geneous with calcium antagonism Guinea pigs and Long-Evans rats Anxiolytic Anxiolytic Anxiolytic and antidepressant [286] [287] [288] Inhalation 100 l 12.5, 25, 50 mg/ kg, p.o. 25, 50 mg/kg, i.p. 1 mg/kg, i.p. 100- 250 mg/l, i.p. Male Swiss mice Male Swiss mice Male Swiss mice Gerbils Locomotor activity, FST EPM Anxiolytic Anxiolytic Anxiolytic and antidepressant Anxiolytic Anxiolytic and antiepileptic Anxiolytic [289] [290] [291] [292] [293]
Table 2: List of various anxiolytic formulations and compounds.
S. No.
Formulation/Extract/Fraction/Isolate
01
Oils of rose, ylang-ylang, and Chamomile extracted from flowers of Rosa sp., Cananga odorata, and Anthenis nobilis (or Matricaria chamomilla), respectively. orange oil extracted from the rind of Citrus sp..
Lavender oil
Essential oils:
(a) Lavender oil 2-phenethyl alcohol, citronellal(75) (b) Rose oil 1,8 cineole(76), menthone(77), pulegone(78), methyl alcohol, caryophyllene(79) (c) Peppermint - menthol(80)
Rose oil
Rose oil
Lemon oil
Neroli essential oil
02
Monoterpenic phenol Carvacrol(81) from essential oil fraction of Oregano and Thyme
03
Monoterpene alcohol Isopulegone(82)
04
Indole alkaloid alstonine(83)
05
Aswal
06
Iridol containing compounds iridoids
10 mg/kg, p.o.
[294]
07
Kavosporal forte (Standardized extract of Kava)
150 mg for a week
Clinical trial (20 patients with situationally induced anxiety) Double blind, placebo controlled, randomized cross over, 24 healthy volunteers Male Wistar rats One-trial step-through avoidance task EPM and FST Decrease serotonergic activity Anxiolytic Defined Intensity Stressor Simulation (DISS) and Cognitive performance Ameliorated the negative effects of the DISS on ratings of anxiety [296]
Two self-rated scale and one observer rated scale
Amelioration of anxiety arises in connection with mammary biopsy
[295]
08
Standardized product containing Melissa officinalis and Valeriana officinalis 600 mg
09
Zingicomb, a preparation consisting of Z. officinale and G. biloba extracts 50, 100, 300 mg/ kg, p.o. 5g Human beings Male Swiss mice
0.5, 1, 10 or 100 mg/kg, intragastrically
[297]
10
Polyherbal formulation
Antianxiety and antidepressant Anxiolytic
[298] [299, 300]
11
Suanzaorentang, Chinese medicine (Semen Ziziphi Spinosae, Rhizoma Chuanxiong, Poria, Rhizoma Anemarrhenae, Radix et Rhizoma Glycyrrhizae) SK (10% solutions for 7 days) Male mice of the ddY strain EPM
12
Sho-ju-sen (SK), a Japanese herbal medicine, contains a water extract of Sasa kurinensis Makino et Sibata (Kumazasa; Poaceae) leaves (SS), ethanol extract of Pinus densiflora Siebold et Zucearini (Japanese red pine; Pinaceae) (PN) and Panax ginseng C.A. Meyer (Ginseng; Araliaceae) (PX) in the ratio of 8:1:1 25-100 mg/kg, p.o. Albino mice SI
Anxiolytic
[301]
13
Kami-Shoya-San (TJ-24) is one of the traditional Chinese herbal medicine: Bupleurum scorzoneraefollium Willd. (Bupleuri Radix; Bupleuraceae), Paeonia lactiflora Pallas (Paeoniae Radix; Paeonaceae), Atractylodes lancera (Thunb.) DC. (Actractylodis Lanceae Rhizoma; Compositae), Archangelica officinalis Hoffm. (Angelicae Radix; Umbelliferae), Poria cocos (Schw.) Wolf (Hoelen; Polyporaceae), Gardenia jasminoides Ellis (Gardeniae Fructus; Rubiaceae), Paeonia suffruticosa Andr. (Moutan Cortex; Paeonaceae), G. glabra (Glycyrrhizae Radix; Leguminosae), Z. officinale (Zingiberis Rhizoma; Zingiberaceae), Mentha arvensis Malinvaud (Menthae Herba; Labiatae)
5-reductase inhibitor, involvement of neurosteroid synthesis followed by GABA receptor stimulation
Anxiolytic
[302]
39
40
Dose (a) 28 and 56 mg/ kg, i.p. (b) 12.5 and 25 mg/kg, i.p. (a) IC50 0.35 mg/ml (b) 1 mg/ml (c) 0.11-0.65 mg/ ml In vitro (a) inhibit GABA transaminase (b) stimulate glutamic acid decarboxylase (c) inhibit glutamic acid decarboxylase EPM Anxiolytic Anxiolytic Chick Chick social separation-stress procedure Anxiolytic [303] Animal/Human beings Experimental model/Clinical studies parameters Mechanism of action Activity Ref. [304] Phytoestrogenrich Phyto-600 diet LongEvans males and females rats [305] Composition of formulation Activity Ref. [306] [307] [308] [309] [310] [311] [312] [313] [314] Antianxiety Antianxiety Antianxiety [315] [316] [317]
Table 2: Continued
S. No.
Formulation/Extract/Fraction/Isolate
14
Botanical extracts
(a) Aqueous extract of the Rutaceae family (b) hydro-alcoholic extract of Alchemilla erythropoda Juz. (Ladies Mantle; Rosaceae)
(a) Aq. Extract of Melissa officinalis (b) Aq. extracts of Centella asiatica and Valeriana officinalis (c) Aq. extracts of Maricaria recutita and Humulus lupulus
15
Dietary products: Dietary soy phytoestrogens
Table 3: List of anxiolytic patented formulations.
S. No.
01
02 03 04
05 06 07 08
L-tryptophan, linseed oil, thyme oil, aqueous extracts of St-Johns wort, Arenaria blossom, Valerian, Elecampane Theanine, green tea, red ginseng, Sasamorpha purpurascens extracts Water, aq.-alcoholic and CO2 extract of Forget-me-not (Myosotis) Tablet, pill or granule comprising Bupleuri Radix, Rhizoma Anemarrhenae, saponin component isolated from Semen Ziziphi spinosae Extracts of plants containing betulinic acid and its derivatives Cassia tora aq. Extract Rosamarinic acid isolated from Perilla extract Hydro-alcoholic extract of Piper methysticum leaves
09
Management of stress including sleep disturbances, aggresiveness, instability of temper and state of anxiety Management of anxiety and stress Anxiolytic, nootropic, anticonvulsant and cerebroprotective activity Treatment of acute anxiety (Panic anxiety) and chronic anxiety (Generalized anxiety) Anxiolytic Anxiolytic Antianxiety and antidepressant Anxiolytic, anticonvulsant, muscle relaxant, analgesic, sleep inducing, antiinflammatory and neuroprotective Antianxiety
10 11
12
Homoeopathic complex comprising Aconite, Avena sativa, Passiflora incarnata, Scutellaria laterifolia, Stramonium and Valeriana Tablet, granule, capsules or oral liquid of methanol extract of Rumex madaio Lozenges containing Citrus pectin, trisodium citrate, raw sugar, water, glucose fructose syrup, and mixture of lavender oil, extracts of Melissa, hop and oat Phenolic compounds having phenolic molecule covalently linked an oxygen containing group, a nitogen or another oxygen containg group and C1-C4 alkoxy group obtained from monocotyledon plants like corn
Table 4: List of review articles published on anxiolytic plants, and their constituents and formulations.
Plant drugs Valeriana officinalis, Melissa officinalis, Passiflora incarnata, Humulus lupulus, Lavendula officinalis, Piper methysticum, Tilia platyphyllos, Leonurus cordiaca, Hypericum perforatum Anti-anxiety and antidepressant Catha edulis, Cola species, Datura species, Pausinystalia yohimbe, Tabernanthe iboga Psychoactive (a) Ephedra species, Paullinia species, Catha edulis (b) Cannabis sativa, Tabernanthe iboga, Psychotria viridis, Banisteriopsis (c) Passiflora incarnata, Valeriana, Piper methysticum (a) Adaptogen (b) Hallucinogenic (c) Analgesic and anxiolyitc Piper methysticum, Ginkgo biloba, Galphimia glauca, Matricaria recutita, Passiflora incarnata, Valeriana officinalis Ginkgo biloba, Hypericum perforatum, Valeriana officinalis, Panax ginseng Anxiolytic Anxiolytic, Generalized anxiety disorders [318] Phytoconstituents reported Therapeutic activity reported Mode of action reported Ref.
S. No.
Information available
01
Pharmacological reports
02
Traditional uses, chemical constituents, pharmacological reports
[319]
03
[320]
04
Clinical reports
[321]
05
Pharmacological reports, Mode of action
Interaction with receptors of CNS (-aminobutyric acid, glutamate, dopamine, muscarinic and adenosine receptors) Anxiolytic
[322]
06
Kava, Skullcap, Lemon balm (Melissa officinalis), Valeriana officinalis, Passiflora, Dietary supplements Hawthorn and California Poppy, Immature Oat Seed, Passionflower, Lemonbalm, Vervain, Lavender and Linden Piper methysticum, Bacopa monniera, Kava Herbal drugs / Herbal constituents Brazilian plants (39-anxiolytic and 28- hypnotic) Foods
[323]
Pharmacological reports and Clinical reports
Nervine, anxiolytic
[324]
07
Clinical reports
Flavonoids, essential oils, phenolic acids, alkaloids
Anxiolytic Psychiatric disorders Anxiolytic and hypnotic
[325] [326] [327]
08
Phytoconstituents, Pharmacological reports and Mechanism of action
09
Phytochemical reports, Pharmacological reports
10
[328]
41
42
Plant drugs Brazilian plants (a) Flavonoids (b) Alkaloids (c) Essential oil (d) Lignans (e) Tannins (f) Triterpene and saponins Psychotherapeutic activity Anxiolytic, antidepressant, neuroleptic, antidementia In vitro radioligand receptor binding and enzyme assays such as acetylcholine esterase, choline acetyl transferase, monoamine oxidase A and B. Selectively on GABAA, NMDA and MAO receptors Ethanol extracts of 31 traditional plants Natural remedies such as St Johns Wart, Kava Kava, Passion flower, Inositol, Valerian root, Melatonin, Omega-3-fatty acids, s-adenosyl-Lmethionine (a) Kava kava roots (b) Ginkgo extract Hypericum perforatum Anxiolytic and antiepileptic Anxiolytic GABAA BZD receptor, Inhibition of GABA transaminase [332] (a) Analgesic, antipyretic, antianxiety, hypnotic (b) Hallucinogen, stimulant (c) Antipyretic, antianxiety (d) Hallucinogen (e) Antianxiety (f) Hypnotic [329] Phytoconstituents reported Therapeutic activity reported Mode of action reported Ref. Eighty five herbal drugs Methanol extracts of traditional plants [330] [331] [333] (a) Anxiolytic (b) Nootropic Antidepressant, anxiolytic, nootropic, sedative, analgesic, anticonvulsant, antischizophrenic, alcohol, nicotine and caffeine deaddiction Anxiolytic Anxiolytic Anxiolytic (a) Analgesic, anesthetic (b) Anxiolytic Side effects- Skin rash and kava dermopathy [334] [335]
Table 4: Continued
S. No.
Information available
11
12
13
14
15
Clinical reports
16
17
Pharmacological reports, Clinical reports
18 Kava Kava Kava lactones
Efficacy, Safety Profile, Pharmacological reports
Kava
(a) Non-opiate pathway (b) GABA receptor binding
[336] [337] [338341] [342]
19
Safety profile
20
Clinical reports
21
Pharmacological reports, Side effects, Mode of action
22
Matricaria recutita Flavonoids, phenolic compounds, essential oil Antioxidant, antimicrobial, antiplatelet, antiinflammatory, antimutagenic, antispasmodic, anxiolytic, cholesterol lowering Anxiolytic, sedative, hypnotic, aphrodisiac, anticancer, hypotensive, antiinflammatory Narcotic-anxiolytic, hallucinogenic Sedative, hypnotic, anxiolytic, antidepressant, antipsychotic, anticonvulsant Anxiolytic [344] [343]
23
Zizyphus jujuba
24
Phytoconstituents, Pharmacological reports Cannabinoids, 9tetrahydrocannabinol, cannabidiol Flavonoids chrysin, apigenin and semisynthetic derivatives of flavone Flavonoids Food proteins -opioid peptides, gluten, exorphins, rubiscolins Terpenoids: Monoterpenoids (linalool, -thujone, borneol, valepotriates); Sesquiterpenoids (valerenic acid, artemisinin); Diterpenoids (ginkgolides, forskolin, salvinorine A); Triterpenoids (ginsenosides); Meroterpenoids (cannabinoids) Wogonin (Flavonoid) Anxiolytic Antinociceptive, memory enhancing, anxiolytic Sedative, anxiolytic, antinociceptive, anticonvulsant, hallucinogenic
Alkaloids in Sceletium and Mesembryanthamaceae
[345]
25
Phytoconstituents, Pharmacological reports
[346]
26
[347]
27
Phytoconstituents, Pharmacological reports, Mode of action
BZD site on GABAA
[348] [349]
28
29
[350]
30
Anxiolytic
BZD binding site of GABAA and modulation of receptor activity
[351]
43
(c) chemical constituents responsible for antianxiety activity have been reported in 53 plants and (d) possible mechanism of action has been reported in 41 plants. Seven formulations of plant drugs, 02 well known classes of phytoconstitunts and 03 pure constituents present in various plants, and nutraceuticals reported to possess antianxiety activity in battery of experimental models of anxiety have been compiled in present work (Table 2). Twelve anxiolytic formulations containing plants have been patented (Table 3). A survey of literature revealed that 30 review articles have been published on anxiolytic plant formulations and specific plant covering broad aspects as phytochemistry, pharmacology, clinical studies, toxicology and safety profiles (Table 4). This review article would of immense help to natural product researchers to select traditionally used and clinically potential plants for their future research work.
14. 15.
16. 17. 18. 19.
20. 21. 22. 23.
AKNOWLEDGEMENT
Authors are grateful to Mr Rakesh Chawla, Lecturer, S.D. College of Pharmacy, Barnala for providing necessary full research articles for compilation of this review.
24.
25.
26. 27.
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44
42.
43. 44. 45.
46. 47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
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ABBREVIATIONS
ADAA American Psychiatric Association APA Anxiety Disorders Association of America ASI Anxiety Status Inventory BRC Baroreflex control of heart rate BZD Benzodiazepines BoEAS Boerner Anxiety Scale CNS Central nervous system CGI Clinical Global Impression dlPAG Dorsolateral peri aqueductal ECG Electrocardiogram EEG Electroencephalographic EPM Elevated plus maze EZM Elevated zero maze EAAS Erlanger Anxiety, Tension, Aggression Scale FRA Federal Regulatory Authorities FST Forced swimming test GABA Gamma-amino butyric acid GAD Generalized anxiety disorder GRAD Global Research on Anxiety and Depression HAMA Hamilton Anxiety Scale HBT HADS 5-HT1A i.p. LDM MBT MAO NOS OCD OFT PD PTZ p.o. PTSD SARA SI SAD SP s.c. TDS Hole board test Hospital Anxiety and Depression Scale 5-hydroxytryptamine 1A Intraperitoneally Light / Dark model Marble burying test Monoamine oxidase Nitric oxide synthase Obsessivecompulsive disorder Open field test Panic disorder Pentylenetetrazole Per oral Post-traumatic stress disorder Self Assessment of Resilience and Anxiety Social interaction Social phobia or Social anxiety disorder Specific phobia Subcutaneously Thrice daily
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Review Article Problems of Reproducibility and Efficacy of Bioassays Using Crude Extracts, with reference to Aloe vera
I. E. Cocka,b*
a Biomolecular and Physical Sciences, Nathan Campus, Griffith University, 170 Kessels Road, Nathan, Queensland 4111, Australia. bEnvironmental Futures Centre, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Queensland 4111,Australia
ABSTRACT: Aloe vera has a long history of medicinal usage and its biological activities have been well documented in a variety of bioassays. However, isolated Aloe vera leaf components generally do not display the same bioactivities, or have lower efficacies than crude juice/extracts. It is likely that several components work in a synergistic manner in the crude mixture, resulting in increased bioactivities. Furthermore, different laboratories often report varying bioactivities using the same extraction procedure on plant material from the same species. Individual Aloe vera cultivars may have widely varying levels of the bioactive phytochemicals. Due to the structure and chemical nature of many of the Aloe vera phytochemicals, it is likely that many of its reported medicinal properties are due to anti-oxidant or pro-oxidant effects. The anti-oxidant/prooxidant activities of many of Aloe veras phytochemicals is dependent not only on their individual levels, but also on the ratios of various components, and on their individual redox states. Therefore, discrepancies between bioactivity studies are likely when using different crude mixtures. The potential differences between these crude mixtures need to be taken into account when analysing the reproducibility and efficacy of bioassays of crude extracts. KEY WORDS: Aloe barbadensis Miller, Aloe vera, anti-oxidant, pro-oxidant, medicinal plant, crude extracts.
IntRodUCtIon
Plants have a long history of usage as medicinal agents and were the main source of medicines prior to the advances of modern medicine. In many developing countries, herbal medicinal systems remain important in the treatment of many ailments. Ayuvedic medicine is still commonly practiced within India with an estimated 85% of Indians still using crude plant preparations for the treatment of a wide variety of diseases and ailments.[1] Traditional Chinese medicine (TCM) and African medicinal systems also account for a major portion of health care in these regions. Even in countries where allopathic/Western medicine is dominant, much is also owed to plant medicinal systems. Furthermore, many users are returning to herbal medicinal systems due to the perception that natural medicines are often safer than allopathic drugs, as well as seeking treatments to diseases for which modern medicine does not yet have solutions. Many of the prescription drugs currently marketed for a wide variety of ailments were originally isolated from plants or are
semi-synthetic analogues of phytochemicals. It has been estimated that approximately 25% of all prescription drugs currently in use are of plant origin.[2,3] Furthermore, approximately 75% of new anticancer drugs marketed between 1981 and 2006 were derived from plant compounds.[3] Traditionally, plant based medicines have been used as crude formulations such as infusions, tinctures and extracts, essential oils, powders, poultices and other herbal preparations. The current trend is to isolate and characterise the individual phytochemical components with the aim of producing an analogue of increased bioactivity/bioavailability. Such studies have given rise to many useful drugs such as quinine (from Cinchona spp.) and digoxin (from Digitalis spp.) as well as the anticancer drugs vincristine and vinblastine (from Vinca rosea). However, the bioactivities seen for crude extracts are often much enhanced, or even totally different to those seen for the individual components.[4,5] Crude plant extracts may contain hundreds, or even thousands of different chemical constituents that interact in complex ways. Often it is not known how an extract works, even when its therapeutic benefit is well established. The study of crude extracts is itself fraught with difficulties. Plants grown under varied conditions will often produce different phytochemical profiles, or at least different quantities of the individual components.[6,7] Similarly, different cultivars within a
*Correspondence: Tel.: +61 7 37357637; fax: +61 7 37355282. E-mail: I.Cock@griffith.edu.au (I. E. Cock). DOI: 10.5530/pc.2011.1.3
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Cock: Problems of Reproducibility and Efficacy of Bioassays
species may also produce different levels of other bioactive components or other constituents which enhance/counteract their medicinal activities.[8] Therefore, the bioactivity of crude extracts may be reliant on the conditions in which the plant grows, the season, and the individual plant itself. Other contributing factors may even include induced chemical defences against predators or pathogens. The extraction procedure, treatment and handling of crude plant extracts may also affect the condition and therefore the bioactivity/efficacy of the phytochemical components. Most plant extracts contain a complex mixture of terpenes, phenolic compounds and alkaloids, many of which can undergo oxidation/reduction processes.[6] The alteration of the redox state may change the behaviour of phytochemicals. Indeed, the maintenance of cellular redox state has been associated with the treatment and prevention of many diseases and ailments including atherosclerosis, inflammatory injury and cancer,[9,10] cardiovascular disease[11] and neurological degenerative disorders.[12] Redox control is also linked with diabetes/anti-diabetic bioactivities[13] and has been associated with the reduction of obesity.[14] Antioxidants can directly scavenge free radicals, protecting cells against oxidative stress related damage to proteins, lipids and nucleic acids.[15] The following discussion will examine some problems associated with reproducibility and efficacy of using crude extracts in bioassays, with reference to the well characterised medicinal plant, Aloe vera.
anti-diabetes[23] bioactivities. However, many studies examining the therapeutic potential of Aloe extracts report conflicting results, showing either a lack of therapeutic bioactivity for some Aloe species,[24] or even toxicity associated with some Aloe vera preparations.[25-27] It is well known that plant age is an important determinant of Aloe vera bioactivities. With respect to anti-oxidant potential, the bioactivity has been shown to fluctuate within a given cultivar in relation to the age of the plant, with highest anti-oxidant levels reported for 3 year old plants.[28] This is complicated further as the relative levels of a plants anti-oxidant phytochemicals also fluctuates seasonally.[29] Furthermore, the phytochemical profiles of individual plants will change, dependent on a variety of other environmental and growth conditions.[6,7] Plants may produce a wide variety of secondary metabolites which have no apparent role in primary plant growth or development processes. These molecules are often unique to plants from a single species and increase during times of high stress such as drought, fire and bacterial infection.[6] Therefore, whilst Aloe vera plant growth may be optimal during times of good growth conditions, it is likely that the level of useful bioactive phytochemicals will be elevated in conditions which stress the plant. Many of these secondary metabolites may exhibit anti-microbial, antioxidant, cytotoxic and other medicinally useful properties.[6]
VARIABIlIty In BIoACtIVIty And EffICACy of CRUdE AloE VERA ExtRACts

Aloe barbadensis Miller (commonly known as Aloe vera) has a long history of usage as a food, cosmetic and as a medicinal agent. Amongst its noted therapeutic activities, Aloe vera has been reported to have anti-bacterial,[16,17] anti-fungal,[16] antiviral,[18,19] immune-stimulatory,[20] anti-inflammatory[21,22] and
A. bArbAdensis PhytoChEmIstRy
Many bioactive phytochemical components have been isolated from Aloe vera leaves and their bioactivities extensively examined. In particular, the anthraquinones, anthrones and chromones have been particularly well studied and have been shown to be effective at counteracting various disease states.[21,30] The anthraquinones aloe emodin (Figure 1a) and aloin (Figure 1b)
Anthraquinones
Figure 1: Chemical structures for the anthraquinones (a) aloe emodin and (b) aloin, the chromone (c) aloesin, (d) anthrone, (e) cinnamic acid and (f) -sitosterol.
53
are thought to exert their reported therapeutic potentials via an anti-oxidant mechanism. For example, aloe emodin has high inhibitory free radical scavenging activity and has been shown to act as an anti-oxidant by inhibiting lipid peroxidation.[31] Interestingly, aloe emodin and aloin have been shown to be capable of behaving as either an anti-oxidant or as a pro-oxidant, with their action being dependent upon their concentration.[32] Aloe emodin exerts anti-oxidant behaviour at lower concentrations, yet acts as a pro-oxidant at high concentrations. In contrast, aloin has an anti-oxidant effect at higher concentrations, yet a pro-oxidant effect at low concentrations. Thus, the variable effects reported for crude Aloe vera extracts in various studies may be due to differing level of aloe emodin and/or aloin present in the extract. Similar pro-oxidant effects have been reported for other antioxidant phytochemicals including flavonoids,[33] tannins[34] and curcumin.[35] Previous studies have shown that transition metal ions, such as copper or iron, can enhance the conversion of the anti-oxidant to the pro-oxidant state.[36,37] The pro-oxidant/antioxidant effect of plant extracts is due to a balance between the free radical scavenging activities and reducing power of their phytochemical components. This can be explained using the anti-oxidant vitamin ascorbic acid as an example. Although ascorbic acid has well characterised anti-oxidant bioactivities, it is also known to act as a pro-oxidant at high concentrations.[38] This is due to the greater reducing power of ascorbic acid compared to its free radical scavenging activity. In the presence of transition metal ions, ascorbic acid will function as a reducing agent, reducing the metal ions. In the process, it is converted to a pro-oxidant. Therefore, high dietary intake of ascorbic acid in individuals with high iron levels (e.g. premature infants) may result in unexpected negative health effects due to the induction of oxidative damage to susceptible biomolecules.[39-41] The anti-oxidant activity of aloesin (Figure 1c) and other chromones has also been extensively described.[42,43] In contrast, a literature search did not reveal any studies examining the potential pro-oxidant activity of these compounds. One study reported several chromones to have higher reducing power than ascorbic acid.[42] The relatively high reducing power of ascorbic acid is believed to be responsible for its ability to function as a pro-oxidant.[38] It is therefore possible that aloesin and other Aloe vera chromones may have a similar anti-oxidant/pro-oxidant profile to ascorbic acid (i.e. anti-oxidant activity at lower concentrations and pro-oxidant activity at higher concentrations). However, it must be emphasised that this possibility is based on the reported higher reducing power of the chromones compared to their free radical scavenging activity[42] and has not been adequately tested. Cinnamic acid (Figure 1e) and its derivatives are phenolic molecules which are present in many fruits, vegetables and whole grains, as well as in Aloe vera leaves. Studies indicate that cinnamic
54 Other phenolic Aloe vera constituents
acid derivatives also have concentration dependent anti-oxidant/ pro-oxidant activities. Cinnamic acid derivatives behave as antioxidants at lower concentrations, but convert to pro-oxidants at concentrations above 5 M.[44] In contrast, Yen et al. (2000) demonstrated that the chemical structure of anthrone (Figure 1d) predisposes it to function as an electron acceptor (electrophile), hence as a strong anti-oxidant, independent of its concentration within an extract.[31] It therefore remains possible that Aloe vera extracts with high concentrations of anthrone may maintain anti-oxidant potential, even under conditions which would otherwise predispose the extract to function as a pro-oxidant. For example, Aloe vera extracts containing high aloe emodin and low aloin concentrations (both of which favour pro-oxidant bioactivity) may still function as an anti-oxidant if high enough levels of anthrone are present to maintain the redox state of these anthraquinones. Conversely, low levels of anthrone may predispose an extract to display pro-oxidant activities. It is therefore likely that the redox character of an extract is not only dependent on the levels of the different phytochemicals present, but also on the ratios of several important components within the mixture. Aloe vera leaves also contains a number of other medicinally important phytochemicals including -sitosterol (Figure 1f) and -sitosterol glucosides. These phytosterols have been shown to promote arterial endothelial cell proliferation.[45] They also promote the expression of proteins involved in angiogenesis and thus have potential applications in the management of chronic wounds. Recently, -sitosterol has also been trialled for the treatment of breast cancer[46] and diabetes,[47] although the efficacy is still under investigation. It appears that these therapeutic bioactivities may be due, at least in part, to their redox state of the molecule. A recent study has indicated that -sitosterol treatment results in glutathione reduction as well as maintaining the anti-oxidant enzymes superoxide dismutase and glutathione peroxidise in a reduced state.[48] This bioactivity in turn is related to the redox state of the sterol. Interactions between the various components within the crude extracts may also play a role in converting otherwise anti-oxidant molecules into pro-oxidants in the extract or vice versa. Other phytochemical components of Aloe vera leaf extracts include acemannan (Figure 2), a long chain polymer of (14) linked galactomannan saccharides.[49,50] Acemannan has been reported to accelerate wound healing,[51-54] activate macrophages[55,56] and have synergistic anti-viral activity in combination with azidothymidine and acyclovir.[19] It has been reported that acemannan also has anti-oxidant properties and that these properties may be responsible for its therapeutic activities.[57] Furthermore, the anti-oxidant potential of Aloe vera polysaccharides is dependent upon the concentration of the molecule and the degree of acetylation of the monomeric units.[58] High polysaccharide concentrations (>8 mg mL-1) were found
Non-phenolic components
Figure 2: The structure of acemannan (a major polysaccharide component of Aloe vera leaves) consists of a polymer of (14) linked galactomannan sugars.
to be necessary for Aloe vera polysaccharides to display antioxidant activity. The same study also showed that increased acetylation enhances the anti-oxidant activity of Aloe vera polysaccharides.[58] However, the polysaccharide components within Aloe vera leaves are not constant. Instead, the composition and concentration of the polysaccharides fluctuate with changes in the growing environment and conditions. Aloe vera leaves also contain inorganic minerals in variable concentrations (e.g. calcium, magnesium, zinc, iron and copper). As previously discussed, the redox state of many Aloe vera phytochemicals is affected by the presence of metal ions, converting otherwise anti-oxidant components into pro-oxidants. Thus, Aloes growing in soil containing elevated levels of metallic ions would be expected to have higher concentrations of metal ions, and thus tend towards pro-oxidant rather than anti-oxidant bioactivities. Other molecules (such as vitamins, amino acids and proteins) may also have an effect on the redox state of the phytochemical components.
problems with reproducibility when analysing crude extracts by bioassay due to differences in the levels of specific phytochemicals, their redox state, and their ratio to other components.
Anti-inflammatory Activity
Inflammation is a complex response by the body to injury. It typically follows a variety of insults including burns, wounds, bites and stings etc. It is characterised by a wide variety of symptoms[59] including: Swelling. Injury may result in increased capillary permeability which allows leukocyte migration and fluid accumulation in the damaged tissue. This accumulation results in the swelling characteristic of inflammation. Redness and heat are caused by vasodilation, reducing blood pressure and increasing circulation. Pain is a complex reaction resulting from the release of short peptides and prostaglandins. These inflammatory processes require the cellular release of several classes of molecules. Vasoactive substances (e.g. bradykinin, prostaglandins and vasoactive amines) are required to dilate blood vessels, opening junctions between cells to allow leukocytes to pass through capillaries. Any compound capable of blocking these vasoactive substances would potentially have a therapeutic effect on the symptoms of inflammation. -sitosterol is the most abundant phytosterol in Aloe vera extracts. -sitosterol stimulates smooth muscle cells to release of prostacyclin (PGI2).[60] However, -sitosterol treatment blocks the release of PGI2 and prostaglandin E2 (PGE2) from macrophages.[60] Thus, -sitosterol treatment would be expected to affect vasodilation and, therefore, have a therapeutic effect on inflammation. The Aloe vera leaf chromone aloesin, and its derivatives, inhibit cyclooxygenase-2 and thromboxane A2 synthesis through their anti-oxidant activities.[61,62] Thus, Aloe vera chromones produce anti-inflammatory effects. In contrast, anthraquinones have been shown to stimulate PGE2 release[63] and would, therefore, be expected to promote pro-inflammatory activity.
55
mEdICInAl EffECts of AloE VERA REqUIRIng mUltIPlE PhytoChEmICAls

The multitude of phytochemicals present in an Aloe vera crude extract not only affect each others redox state and ability to function as an anti-oxidant/pro-oxidant, but several phytochemicals may also be required for different aspects of the same therapeutic effect. Some of the medicinal properties associated with plant extracts require the concerted action of several bioactivities. The following discussion examines several therapeutic properties of Aloe vera extracts that require the synergistic action of several bioactivities, each of which may be reliant on multiple phytochemicals. This is by no means a complete examination of the therapeutic properties of Aloe vera extracts, but instead serves to illustrate the difficulties of assigning a therapeutic effect to a single component. Similarly, it further illustrates the
The peptidase bradykinase has been isolated from Aloe vera leaves and has been shown to break down the vasoactive peptide bradykinin.[64,65] As bradykinin treatment results in vasodilation, hydrolysing this protein would result in decreased vasodilation and, therefore, inhibit leukocyte passage and fluid leakage from the capillaries into the surrounding tissue. Aloe vera leaf bradykinase would, therefore, be expected to contribute to the therapeutic effects on the symptoms of inflammation. Chemotactic factors, including several proteins and peptides, are required to increase cell motility, especially the motility of leukocytes during inflammation. Blocking these chemotactic factors, or blocking their effects, prevents inflammatory swelling. Several compounds in Aloe vera extracts have been shown to be capable of blocking chemotaxis. Anthraquinones suppress cytolytic T-lymphocytes in favour of suppressor cells.[66,67] Furthermore, anthraquinones decrease cytokine production and IL-2 mRNA expression in activated T lymphocytes,[68] thereby decreasing chemotaxis. More recent studies have demonstrated that the anthraquinone emodin decreases plasma levels of the cytokines IL-2 and TNF-, whilst increasing IL-10 (which itself down-regulates IL-2 and TNF- cytokine activity).[69] None of these studies, however, examined the relationship of the redox state of the anthraquinones with these effects. Furthermore, these studies have not rigorously examined the effects of a range of doses of these phytochemicals. In contrast, Aloe vera polysaccharides (including acemannan) have a stimulatory effect on chemotaxis. Acemannan exposure stimulates cytokine production and activates lymphocytes.[70,71] Specifically, pure acemannan isolated from Aloe vera leaves has been shown to stimulate macrophages to release IL-1, IL-6, interferon, GM-CSF and TNF- in vitro.[72] Similarly, Aloe vera lectins stimulate cytokine production. Aloctin A, the best characterised of the Aloe lectins, has been shown to stimulate the production of IL-2[73] and to enhance the production and activation of macrophages.[73] Therefore, Aloe vera extracts contain both chemotactic stimulatory and inhibitory compounds. The chemotactic effect of Aloe vera extracts would, therefore, be dependent on the levels and ratios of the factors affecting chemotaxis as well as their redox state. Aloe vera extracts contain multiple active phytochemicals. It is likely that several of these may be required to address different aspects of the inflammatory process. Failure to consider this is likely to be responsible for past ambiguities about the efficacy of Aloe extracts in relation to its anti-inflammatory activity.
Antiseptic activity
Aloe vera leaf extracts have been previously shown to display good anti-bacterial[16,74,75] and anti-fungal bioactivities.[16,76] Early anti-bacterial studies of Aloe vera extracts have provided confounding and even contradictory results. Some of these studies indicate that the bioactive agent(s) are anthraquinones,[77,78] whilst other studies found Aloe vera anthraquinones to be inactive as anti-bacterial agents.[79] Numerous subsequent studies have demonstrated the anti-bacterial activity of isolated anthraquinones from Aloes[80,81] and various other plant species.[82-84] Whilst the mechanism of anti-bacterial activity is still subject to investigation, it has been suggested that aloe emodin and aloesin function by inducing bacterial membrane disruption.[80] This study also determined that the form of aloe emodin and aloesin tested also affects their anti-bacterial activity. It was demonstrated that anthraquinone loaded liposomes had strong anti-bacterial activity, whilst the purified free anthraquinones did not. It is, therefore, possible that some of the observed differences in the anti-bacterial activities of anthraquinones and Aloe vera extracts may be due to the form of anthraquinones that the bacteria were tested against. Whilst this study showed that anti-bacterial activity is dependent on the form of anthraquinone tested, the effect of concentration was not extensively examined. MIC values were determined by testing across a range of concentrations, although only relatively low concentrations were tested. It is possible that higher concentrations may have a very different effect, analogous to the concentration effects already described for anthraquinone anti-oxidant/pro-oxidant activity. Other Aloe vera components have also been implicated in the antibacterial activity of leaf extracts. A recent study tested anthraquinone free leaf extracts and isolated components.[85] This study showed that cinnamic acid, coumaric acid, ascorbic acid and pyrocatechol purified from Aloe vera gel all display good anti-bacterial activity, especially towards Gram-positive bacteria. It was postulated that the phenolic anti-bacterial agents functioned by disrupting bacterial cell membranes, as well as by denaturing bacterial proteins. Furthermore, cinnamic acid is known to block bacterial glucose uptake and ATP production,[86] therefore, inhibiting bacterial growth. Coumaric acid has been shown to inhibit bacterial enzymatic activity.[87] A number of other phenolic components were also found to have low to moderate anti-bacterial activity. In addition to direct inhibitory effects on bacteria, Aloe vera components may also function by selectively modulating the cells of the immune system (described in detail in section 4.4). Furthermore, acemannan also inhibits bacteria adhering to epithelial cells and establishing an infection.[88] It is likely that the anti-bacterial activity of Aloe vera extracts in vivo is due to the synergistic effects of multiple bioactive components, functioning through several mechanisms. Anti-fungal activity has received less attention, although some studies have demonstrated the ability of Aloe vera extracts to
The interruption of the external epidermal barrier by a wound, burn or other such event allows microbes to enter and infect the wound. The invasion of microorganisms may cause or intensify inflammation (described in section 4.1.) and may hinder wound healing (described in section 4.3) and/or cause disease.
56
inhibit fungal growth.[16,76,89] Anthraquinones, especially aloe emodin and aloesin, were implicated in this anti-fungal activity,[76] however, the identity of anti-fungal components and their mechanisms of action have not been extensively examined. Similarly, the anti-viral activity of Aloe vera leaf extracts has been demonstrated,[18,90] although detailed purification, identification and mechanistic studies are required.
Wound Healing
Whilst anti-inflammatory and anti-microbial bioactivities are complex processes requiring the synergistic action of several bioactivities, wound healing is more so. Wound healing, a relatively well studied therapeutic property of Aloe vera, is the result of several bioactivities including: Inflammation, which has summarised in section 4.1. Antiseptic bioactivity, which has summarised in section 4.2. Cell growth and proliferation Matrix remodelling
Other Aloe vera phenolic compounds have also been implicated in the wound healing effects of Aloe vera extracts. -sitosterol and -sitosterol glucosides promote endothelial cell proliferation and angiogenesis,[45] although their activity appears to be dependent on its redox state.[48] The reduced sterol has anti-oxidant activity and stimulates wound healing processes, whilst oxidised sterols are pro-oxidants and induce cell death. -sitosterol and -sitosterol glucosides, therefore, have potential applications in wound management in their reduced state. The Aloe vera chromone aloesin has also been reported to stimulate cellular proliferation.[51,61,99] It is possible that the proliferative effect of aloesin is due to its high anti-oxidant activity.[42,43] In contrast, cinnamic acid has been shown to down-regulate expression of cell proliferation and anti-apoptotic gene products, although the affects of both high and low concentrations were not examined.[100,101] The redox environment affects cellular signal transduction, DNA and RNA synthesis, protein synthesis, enzyme activation, regulation of the cell cycle, ligand binding, DNA binding and nuclear translocation, and therefore ultimately cell proliferation/ death.[102,103] Transcription factors are active in their reduced form and their translocation to the nucleus is also redox dependent.[104] A reducing environment favours cellular proliferation whilst an oxidising environment results in an increase in reactive oxygen species, initiating cell death.[105,106] Therefore, extract conditions favouring anti-oxidant activity (e.g. low aloe emodin, high aloin, low cinnamic acid, low ascorbic acid, low transition metal and high anthrone concentrations) would be expected to favour cellular proliferation whilst conditions favouring pro-oxidant activity (e.g. high aloe emodin, low aloin, high cinnamic acid, high ascorbic acid, high transition metal and low anthrone concentrations) would favour cell death. The non-phenolic components, particularly acemannan, have also been shown to have a role in wound healing. For example, the stimulation of gingival fibroblast proliferation has been demonstrated when treating oral wounds with high doses of acemannan.[107] This stimulatory effect was found to be due to an induction in expression of the growth factors KGF-1, VEGF and an increase in collagen expression. This study only examined the effects of relatively high concentrations of acemannan, in the range that would correlate to anti-oxidant activity. As lower concentrations may correlate to pro-oxidant activities, it is possible that the induction of fibroblast proliferation may not be seen at these concentrations. Indeed, as lower concentrations of acemannan correspond to pro-oxidant effects, it is possible that at lower concentrations, cell death may be induced. The concentration dependent redox effect of acemannan may also contribute to the discrepancies seen between proliferative studies of Aloe vera extracts. As well as requiring cellular growth and proliferation, wound healing also requires matrix remodelling. Aloe vera gel extracts have been shown to stimulate and speed up the production of hyaluronic acid and dermatan sulphate.[52] Activities of the enzymes
57
The growth of endothelial, epithelial and fibroblast cells are critical in wound healing. As a first step in wound healing, a fibrin clot is formed as a temporary repair. This step is vital as it helps avoid microbial infection which may retard the healing process. The wound is subsequently invaded by a variety of cell types, some of which stimulate an inflammatory response, and others which are directly involved in the repair mechanism. [91] The effects of Aloe vera extract components on inflammation processes and chemotaxis have already been summarised in section 4.1. Wound repair itself occurs in three phases: the migration of epithelial cells and fibroblasts to the wound site, proliferation of cells and cellular maturation. It is likely that the wound healing effect of Aloe vera extracts involves the synergistic action of multiple components on several pathways. Aloe vera anthraquinones reportedly possess contradictory effects on cell growth and proliferation. For instance, Aloe emodin has been shown to stimulate a 2.5 fold increase in rat hepatocyte DNA synthesis with a corresponding increase in cell growth.[92] Additionally, aloe emodin has been shown to protect hepatocytes from apoptosis.[69] In contrast, other studies have shown aloe emodin to induce apoptosis in pro-myeloleukemic HL-60 cells[93] and human lung squamous cell carcinoma,[94,95] and to inhibit human neuroectodermal tumour growth.[96] Some studies have postulated that the pro-apoptotic effect of aloe emodin is due to an induction of caspase 3 activity, together with a decrease in the levels of the anti-apoptotic protein Mcl-1.[93] Another study has implicated caspase 8 mediated cleavage in the apoptotic activity of emodin.[97] Studies into the pro-apoptotic mechanism of aloe emodin are ongoing. Similarly, anthrones have also been shown to induce cell death. In a recent study, an anthrone from the Ethiopian medicinal plant Kniphofia foliosa was shown to induce rapid death in mouse and human cancer cells via necrosis.[98]
-glucuronidase and N-acetyl glucosaminidase are increased during wound healing, resulting in increased carbohydrate turnover at the site of the wound. Other studies also demonstrated that wounded diabetic rats treated with Aloe vera gel show increased collagen formation[53] and cross linking.[54] It is evident that a synergistic action is required by several Aloe vera extract components on multiple wound healing associated bioactivities. The reported discrepancies between different studies may be due to differences in concentrations, ratios and redox states of these components. Manipulation of the immune system has therapeutic potential in the treatment of a variety of diseases. Aloe vera leaf extracts have been reported to have both good immuno-stimulatory[20] and immune-suppressive activities (as reviewed in Boudreay and Beland[108]); however, rigorous scientific examination of these effects is limited. Much of the studies into the immune-modulatory potential of Aloe vera extracts have focused on the immunestimulatory effects, particularly of the polysaccharide components. Whilst numerous Aloe vera polysaccharide components have been shown to have immune-modulatory effects,[109-111] acemannan has been particularly well studied. The immune-modulatory effects of acemannan are thought to be due to activation of macrophage cells and antigen processing. The activated macrophages secrete cytokines including IL-1, IL-6, interferon, GM-CSF and TNF- in vitro.[72] The release of these cytokines is itself associated with further pathology through the induction of inflammation. Acemannan also enhances macrophage sensitivity to IFN-, inducing apoptosis.[20] Neither acemannan nor IFN- was capable of inducing apoptosis alone. Instead, a synergistic effect is required and this effect appears to function through the inhibition of the expression of Bcl-2 proteins.[20] Studies have also highlighted the immune-modulatory properties of the smaller phenolic components of Aloe vera leaves. Aloe emodin and other anthraquinone derivatives have been shown to have an immune-suppressive effect by blocking lymphocyte proliferation.[66,67] Emodin also reduced IL-1, IL-2 and IL-2 receptor expression.[66] It was suggested that emodin suppresses both macrophages and lymphocytes. Further studies have identified 37 other anthraquinones with the ability to block cytolytic T lymphocyte induction and the ability to prevent antibody production.[67] The effect of concentration and the ratio between anthraquinones were not tested in these studies. It has been postulated that Aloe vera extracts may exert immunemodulatory effects through their functioning as anti-oxidants, inhibiting/stimulating the production of free radicals.[28] Treating streptozotocin induced diabetic[112] or gamma-irradiated rats[113] with Aloe vera leaf extracts reduces lipid peroxidation and the formation of hydroperoxides whilst increasing the levels of anti-oxidant enzymes (e.g. reduced glutathione, glutathione peroxidise, glutathione-S-transferase, catalase, superoxide dismutase) in the liver, lungs and kidney. Similarly, Aloe vera gel has been shown to inhibit ROS production in colorectal mucosa
58 Immunomodulation
cells.[114] Interestingly, this study found the Aloe gel extract lacks this activity at either higher or lower concentrations, indicating a concentration dependence similar to that reported for the redox effects of Aloe vera components.[32] It is, therefore, possible that the variable immune-modulatory effects reported for Aloe vera extracts in different studies may be due to the concentrations, ratios and redox states of several important compounds in the tested extracts, with extract conditions favouring anti-oxidant bioactivity resulting in immune-stimulation. Conversely, conditions favouring pro-oxidant activity would be expected to result in immune-suppression, although this has not been extensively tested. Diabetes mellitus refers to a group of metabolic disorders that result in increased blood glucose concentrations, either because the pancreas does not produce enough functional insulin (type 1 diabetes), or because cells do not respond to the insulin which is produced (type 2 diabetes). The causes of diabetes mellitus include the auto-immune destruction of pancreatic cells,[115] viral infections,[116] genetic and environmental factors,[117] insulin or insulin receptor gene mutations[118] and altered pancreatic prostaglandin metabolism.[119] Diabetes has significant health effects, impacting on the quality of life and life expectancy of those suffering with it. A number of studies have indicated the beneficial effects of Aloe vera extracts in diabetic patients.[23,120] Administration of Aloe vera extracts to streptozotocin-induced diabetic rats resulted in a decrease in blood glucose and a corresponding increase in liver glycogen.[120] The maintenance of glucose homeostasis by Aloe vera extracts in diabetic rats was shown to involve a number of mechanisms. Aloe vera extract treatment altered the activities of multiple enzymes: glycogen phosphorylase activity was decreased and glycogen synthetase increased, resulting in increased hepatic glycogen stores.[120] Hexokinase activity and mRNA levels were decreased in diabetic rats,[121] yet treatment with Aloe vera extract returned these parameters towards normal levels.[120] Similarly, increased lactate dehydrogenase, glucose-6-phosphatase and fructose-1, 6-bisphosphatase activities were seen in diabetic rats.[122] Aloe vera extract treatment significantly restored these enzyme activities.[120] Glycosylation of blood proteins including haemoglobin, albumin and lipoproteins is also characteristic of diabetes mellitus.[123] Under the hyperglycaemic conditions of diabetes mellitus, blood glucose interacts with specific amino acids on the proteins surface, forming glycosylated protein products which may undergo a series of further chemical modifications resulting in the production of advanced glycation end products (AGE).[124] The binding of AGEs to their receptors results in altered cell signalling which in turn results in free radical production.[125] Indeed, diabetes mellitus has been shown experimentally to be associated with an increase in free radical formation and an associated decrease in anti-oxidant potential.[126,127] Studies have directly linked oxidative stress with
Anti-Diabetic activity
the impaired maintenance of glucose homeostasis and the enhanced lipid peroxidation seen in diabetes mellitus.[127] Furthermore, increased total anti-oxidant levels have been measured in the blood and saliva of diabetic patients, further supporting the proposed role of oxidative stress in diabetes mellitus.[128] Oxidative stress induction has also been suggested to be the common link between the diverse medical complications (including cardiovascular disease, renal and neural degeneration, impaired vision and erectile dysfunction) seen in diabetes mellitus.[129,130] Therefore, treatment with anti-oxidants would be expected to counteract many of these complications. Aloe vera has a number of compounds (both phenolics and non-phenolic compounds) that can act as anti-oxidants (as described in section 3. - A. barbadensis phytochemistry). As many of these compounds can potentially behave as either anti-oxidant or pro-oxidant dependant on their concentration, redox state and ratio between compounds, it is not surprising that studies using Aloe vera crude extracts to treat diabetes mellitus have had mixed success.
Anti- Cancer activity
that average tumour weights increased in severe combined immune-deficient (SCID) mouse tumour xenografts from cells over expressing catalase or thioredoxin.[137] Tumours from both transfectants contained fewer apoptotic cells but mitotic cell numbers were similar. This suggested that anti-oxidant over expression resulted in increased tumour size due to a decrease in apoptosis. The cell proliferation/apoptosis inducing abilities of Aloe vera extracts and isolated components have been described in Section 4.3. Briefly, ROS based tumour therapy may induce regression in apoptosis/oxidatant sensitive tumour cells. Thus, if Aloe vera components were present in concentrations and ratios consistent with pro-oxidant activity, the extract would induce apoptosis and, therefore, would have anti-cancer activity. If the levels of components were consistent with a reducing environment, anti-oxidant activity would result and the extract would not have anti-cancer activity. Conversely, should the protocol be repeated on a tumour with apoptotic resistant/ oxidant resistant cells, the converse would apply and tumour progression would be likely.
The growth and development of healthy cells depends on fine regulation of growth promoting and inhibiting pathways. Protooncogenes and tumour suppressor genes are responsible for encoding proteins that regulate cell division/cell cycle, as well as for the repair of damaged DNA and cell programmed death by apoptosis. Mutations within these genes have been implicated in the onset of cancer.[131] Such mutations result in cells that no longer require external signals to proliferate. Furthermore, these cells fail to recognise signals that restrict cell division, resulting in uncontrolled cell growth. In tumour genesis, multiple genes may be altered and transmitted to daughter cells, which subsequently escape normal growth restraints and form a tumour, which may be benign or malignant. The induction of oxidative stress has been linked with several types of cancer.[132,133] Chromosome instability is also a common feature of many of the cancers that have been linked with oxidative stress, suggesting that increased oxidative stress may contribute to development of genetic instability. Oxidative stress leading to genetic instability may result in the emergence of new tumour phenotypes. In such populations, a decrease in apoptosis but an increase in tumour growth and subsequent tumour progression is observable. Currently used anti-cancer agents (e.g. doxorubicin, daunorubicin, mitomycin C, etoposide, cisplatin, arsenic trioxide, ionising radiation, photodynamic therapy) depend exclusively, or in part, on the production of ROS for cytotoxicity. Sensitivity of tumour cells to oxidative stress and/or apoptosis may affect treatment success.[134,135] Studies indicated that WEHI7.2 mouse thymoma cells over expressing catalase (CAT38) or thioredoxin (THX) were resistant to glucocorticoid-induced apoptosis in vitro.[136,137] This suggested that glucocorticoid-induced apoptosis occurred by a ROS dependant/independent mechanism. It was observed
ConClUsIons
The problems associated with reproducibility and efficacy of bioassays using Aloe vera juice and/or crude extracts illustrates some of the difficulties encountered in natural products research. Individual extract batches may vary widely with regards to individual phytochemical profiles, ratios between various components, and the redox state of these components. These variances may have profound effects on the reported bioactivities and are likely to account for the reported discrepancies between different studies bioassaying crude mixtures. Despite these difficulties, the use of crude extracts is often necessary as the individual components often do not show the same bioactivities, or have different efficacy to crude extracts. This is true for Aloe vera. Aloe vera juice, or Aloe vera crude extracts, often display higher efficacy than the purified components. It is likely that the biological activity of Aloe vera is a synergistic and perhaps additive action of the different classes of compounds found within the plant, rather than a single constituent or just a few compounds. Furthermore, these compounds are required in the correct levels/ ratios/redox states for bioactivity to be observed.
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Research Article Cassane-type diterpenoids from the genus Caesalpinia

R. A. Dickson1*, T. C. Fleischer1, P. J. Houghton2
1
Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical, KNUST Kumasi, Ghana. 2Pharmacognosy Research Laboratories, Pharmaceutical Sciences Research Division, Kings College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK
ABSTRACT: Medicinal plants belonging to the Caesalpinia (Ceasalpiniaceae) genus are widely distributed in most tropical countries and have been frequently employed in folkloric medicine worldwide in the treatment of various ailments including skin diseases, malaria, cancer, infections, erectile dysfunction, pain and wounds. Interest in this genus has increased considerably over the years and the biological properties of different phytoconstituents, such as the cassane-type diterpenoid isolates, have been studied. Over the past 60 years, a number of cassane-type diterpenoids have been isolated from species of this genus and some of them possess interesting biological activities. Recently, three novel cassane-type diterpenoids, benthaminin 1, 2 and 3, which demonstrate antimicrobial and antioxidant properties, have been isolated from Caesalpinia benthamiana growing in Ghana. This review seeks among other things to collate all these isolated compounds, recognising their diversity and commenting on their relevance as bioactive compounds. KEY WORDS: Cassane-type diterpenoids, Caesalpinia, Ceasalpiniaceae, Biological activity.
INTRODUCTION
Caesalpinia is the name of a genus of enormous size and of ancient origin. It is named after the Italian naturalist, Andreas Caesalpino, of Arezzo (1519-1603). He was also a botanical collector, systematist and philosopher, chief physician to Pope Clement VIII, and a professor of medicine and botany in Pisa and Rome.[1] Caesalpinia consists of about 200 species, consisting of shrubs, tall climbers, small and tall trees, mostly armed with spines and curves, hooked, sharp thorns and rarely unarmed. Their leaves are bipinnate, lacy, and attractive, while the leaflets are few to many, opposite, rarely alternate, small or large, herbaceous or leathery. The flowers are yellow, red, or variegated, showy, handsome, medium to large and are multiflowered. Finally, the pods are variable, often prickly, flat, straight or beaked.[2,3]
into 8 informal generic groups: the Gleditsia group (2 genera), the Acrocarpus group (monogeneric), the Sclerolobium group (3 genera), the Peltophorum group (13 genera), the Caesalpinia group (16 genera), the Poeppigia and Pterogyne groups (both monogeneric) and the Dimorphandria group (10 genera). These authors stated that the tribe is a remarkable mixture of relics and complexes of relatively recent speciation, providing many pitfalls for formal systematics and biogeographical interpretations.[4] Since 1980, several studies have cast new light on intergeneric relationships within the Caesalpinieae, necessitating the restructuring of some of the nine informal generic groups.[5,6] Without doubt, the genus with the greatest taxonomic and nomenclatural complexity within the Caesalpinieae is the genus Caesalpinia, which in its broadest sense comprises about 140 species and contains 25 generic names in synonymy.[4]
THE CAESALPINACEAE FAMILY

The leguminous trees fall under the sub-families Caesalpinaceae, Fabiaceae and Mimosaceae. The trees of Caesalpinaceae are by far the most scenic, exhibiting many-coloured splendour. The leguminosae family is currently divided into three subfamilies and 36 tribes. Subfamily Caesalpinioideae comprises of four tribes and 2,250 species, subfamily Mimosoideae four tribes and 3,270 species, and subfamily Papilionoideae 28 tribes and 13,800 species.[2,3] Polhill and Vidal,[4] divided the Caesalpinieae
*Correspondence: ritadickson2000@yahoo.co.uk; +233 204620000 DOI: 10.5530/pc.2011.1.4
GEOGRAPHICAL DISTRIBUTION OF THE GENUS CAESALPINIA

Members of Caesalpinia are widely distributed throughout the tropics and subtropics, primarily in America and Asia, and extending to Australia, Polynesia Madagascar and Africa[7,8] (Table 1). In Africa it is widespread in the western and southern areas with C. benthamiana being the most common. About 25 species are found in the Caribbean, 10 in Cuba and the Bahamas, 2-3 species in Mexico and a few extending to Central America.[4] C. pulcherrima is found in the Philippines and the Caribbean where it is an ornamental plant. It is red with yellow margins, and this variety is the national flower of Barbados, known as Pride of Barbados.[9]
63
Dickson, et. al.: Review on C. major
Caesalpinia is also found in Colombia, Ecuador, Peru, Paraguay and Argentina and is very popular in Brazil. Indeed, the name Brazil, had its origin in the Portuguese word Bresil or brazil which means bright red and resembling glowing coals, and was used to describe the colour of Caesalpinia wood abundant in this area.[10] Nine species are widespread in Asia with about two confined to China.[11] C. major is widely distributed in Southeast Asia.[12] The genus Caesalpinia is also popular in Thailand and Indonesia[13] and C. minax, is found in China.[14] Apart from their medicinal importance, Caesalpinia species may also serve as garden ornamentals and hedge plants. The beauty of C. pulcherrima, whose showy red flowers are borne in long spikes, is reflected in its common names: pride of Barbados, paradise flower, Spanish carnation, peacocks crest, flower tree, and others. This semi-drought-resistant species flowers in favourable habitats when only 8 months old.[10]
Republic.[21] In addition, plant part decoctions are employed in folk medicine to treat intermittent fever and as an abortifacient, emmenagogue, and as a general tonic. Bonducin, an amorphous, white bitter glycoside, is abundant in the seed cotyledons of C. bonduc Roxb., C. bonducella Flem., and C. crista L.[22,23] It is sometimes referred to as poor mans. quinine because it is used as a substitute for quinine in the treatment of intermittent fever. The seeds of C. bonducella are grey, round, smooth and stony. The buoyancy of the seed accounts, in part, for this species being widely dispersed tropically by ocean currents. They are used as talismans and beads, and also by children as marbles. They yield oils for cosmetics and use in medical preparations. It is a shrubby tree of Argentina and Chile, exuding a golden yellow gum that contains approximately 80% arabin. It is completely soluble in water and is an acceptable substitute for gum Arabic.[24] C. echinata Lam. is the national tree of Brazil. The name Brazil, had its origin in the Portuguese words Bresil or brazil which means bright red, resembling glowing coals and were used to describe the colour of caesalpinia wood abundant in this area.
ETHNOPHARMACOLOGICAL USES
The roots of C. benthamiana are considered to be an effective dysentery remedy in Ghana.[15] The powdered roots are mixed with shea butter or palm kennel oil to treat skin diseases and wounds.[15] An infusion of the dried root is consumed or used in bathing for general malaise in Senegal.[16] In the Philippines and the Caribbean, decoctions of the leaves, bark and roots of C. pulcherrima are used traditionally to treat liver disorders, ulcers of the mouth and throat. It also reduces fevers, acts as an abortificient, and alleviates fungal infections. The fruit is also used to check bleeding and prevent diarrhoea and dysentery.[17] The flowers have also been used to combat oxidative stress by eliminating free radicals from the system.[18] Within southeast Asia, C. major has traditionally been implemented as a tonic, anthelmintic, and for rheumatism and back-ache.[19] In Thailand, the seeds of this plant are used as an expectorant and antitussive agent.[19] C. minax finds use in Chinese folk medicine in the treatment of common colds, fever and dysentery.[20] Also in folk medicine in Kagoshima in Japan, C. decapetala is used in the treatment of neuralgia. A decoction from the pods of C spinosa is used in eye washes in the Callera district of the Czech
Table 1: Distribution of Caesalpinia species
Selected species C. benthamiana (Baill.) Herend. & Zarucchi C. brevifolia Baill. C. coriaria (Jacq.) Willd. C. crista L. C. decapetala (Roth) Alst. C. japonica S. & Z. C. percherrima (L.) Sw. C. sappan L. C. spinosa (Mol.) Ktze.
BIOLOGICAL ASPECTS OF THE GENUS CAESALPINIA

The genus Caesalpinia (Ceasalpiniaceae) has been associated with a number of biological activities. Plants in this group have been employed globally in folkloric medicine in the treatment of numerous diseases. Various investigations have been carried out on plants belonging to the genus Caesalpinia in order to validate the folkloric uses of these plants to determine its antimicrobial, antimalarial and anticancer properties, among others. Extracts and compounds from the seeds have been screened against pathogenic organisms that include viruses, bacteria and fungi. Cassane furanoditerpenes, designated as caesalmin C-G, were evaluated for their effects on the proliferation of the Para 3 virus. The tetracyclic furanoditerpenoid isolates showed significant activity against the Para 3 virus, with IC50 values ranging between 7.8 and 14.8 g/mL. However, caesalmin G, which is the only furanoditerpenoid lactone, is highly toxic, with a therapeutic index (TI) value of 3.0.[25] It is noteworthy that the
Antimicrobial Activity
Geographical location W. Africa France Philippines Hawaii, USA Zimbabwe S. Africa Japan Philippines Hawaii, USA S. Africa
References Irvine (1961), Burkill (1994) Naudin (1894) Banados & Fernandez (1954) Allen & Allen (1936b) Corby (1974) Grobbelaar & Clarke (1974) Asai (1944) Banados & Fernandez (1954) Allen & Allen (1936b) Grobbelaar & Clarke (1974)
64
therapeutic index (TI) value of caesalmin C is almost the same as that of ribavirin (an inhibitor of DNA and RNA viruses), which serves as a positive control in the bioassay. It can be concluded that the anti-Para3 virus activity of tetracyclic furanoditerpenoids is better than that of the furanditerpenoid lactone.[25] Since the major components of the seed of C. minax possess such potent activity, it may be feasible to develop a new antiviral agent from this medicinal plant. Furthermore, macrocaesalmin, a cassane furanoditerpenoid lactone from the seeds of C. minax, was evaluated for antiviral activities against RSV, Para-3 and influenza Type A viruses according to an established protocol which showed inhibitory activity against the RSV (IC50 = 24.2g/mL, TC 50 = 138.3g/mL and SI = 5.7) in cell culture, and the corresponding values for the positive control (ribarivin) were 3.4, 60.6 and 17.8g/mL, respectively. The antiviral activity of the compound was less than the positive control; however, the selectivity index for natural products was considered significant (SI > 4). However, it is inactive against the para-3 virus (IC 50 = 51.9g/mL, TC 50 = 137.5g/mL and SI = 2.6) with the corresponding values for the positive control being 2.7 g/mL, 62.5g/mL and 23.1g/mL, respectively. Similarly, macrocaesalmin was inactive against the influenza Type A virus. Respiratory viral infections have long been recognized as important contributors to morbidity and mortality in young children and older adults, and the search for natural products as antiviral agents against respiratory viruses has attracted considerable attention in recent years. The isolation of macrocaesalmin and the evaluation of its efficacy on three major respiratory pathogens thus provide useful clues in the search for antiviral drugs against RSV infection.[26] Four new cassane-type furanoditerpenoids possessed antimicrobial activities against several bacteria including S. aureus, E. coli, P. aeruginosa and B. subtilis and fungi (C. albicans and T. mentagrophytes have been isolated from the air-dried leaves of C. pulcherrima. A cassane-type diterpene ester, pulcherralpin, isolated from the stems of this plant has potential fertility regulating and antitumor activities.[27] Two antitubercular cassane furanoditerpenoids, namely 6 -benzoyl-7 -hydroxyvouacapen-5 -ol and 6 cinnamoyl7-hydroxyvouacapen-5 -ol, have been isolated from the root of Caesalpinia pulcherrima. It was observed that 6-cinnamoyl7-hydroxyvouacapen-5 -ol possessed stronger antitubercular activity demonstrating a minimum inhibitory concentration (MIC) of 6.25 g/mL, while the benzoyl analogue 6 -benzoyl-7 -hydroxyvouacapen-5 -ol was less active (MIC 25 g/mL). Both compounds exhibited moderate cytotoxic activity towards KB (human oral carcinonoid cancer), BC (human breast cancer) and NCl-H187 (small cell lung cancer) cell lines.[28] According to Mexican folklore, plants belonging to the genus Caesalpinia have found use in the treatment of kidney ache, cystitis, urethritis, prostate inflammation, fever, tooth-ache and
abdominal cramps.[29] In the Philippines, decoctions of the leaves, bark and roots are used to manage liver bleeding and prevent diarrhoea and dysentery, whilst the flowers are utilized to combat oxidative stress.[30] These plants are used in the treatment of common cold, fever and dysentery in China.[31] In South Sulawesi of Indonesia, the seed kernel of the plant has been traditionally used as an anthelminthic and antimalarial.[32] The seeds of plants belonging to this genus are used as expectorant and antitussive agents in the herbal medicine practice of Thailand.[33] Their usefulness in the treatment of rheumatism and back-ache and as a tonic has also been reported in Indonesia.[34,35] Antiviral and anticancer activities from these plants have also been reported.[36,37] In Caribbean folk medicine, plants of this genus have been employed extensively.[38] Medicinal plants belonging to this group have been used in traditional medicine in the management of diseases in African countries including Senegal, Nigeria, Sudan and Liberia.[39] Ghana is not an exception in this regard as plants belonging to the genus are extensively employed in folkloric practice in the treatment of various ailments such as skin diseases and wounds, gonorrhoea, sleeping sickness and constipation.[40] The taxonomy of the family Ceasalpiniaceae has been the subject of much debate. It was previously referred to as Fabaceae, and prior to this was known as Leguminosae.
Antimalarial Activity
Forty four cassane-and norcassane-type diterpenes isolated from Caesalpinia crista of Myammar and Indonesia were evaluated for their antimalarial activity against the malaria parasite Plasmodium falciparum (FCR 3/A2 clone in vitro). Most of the tested diterpenes displayed antimalarial activity, and norcaesalpinin E showed the most potent activity with an IC50 value of 0.090M, a greater potency than the clinically used drug chloroquine (IC50, 0.29M).[41] Ten new cassane diterpenes including caesalpinins H-P and norcaesalpinin F were tested for their inhibitory activities on the growth of Plasmodium falciparum (FCR 3/A2 in vitro. All displayed activity in a dose dependent manner. Among the newly isolated compounds, caesalpinin K and norcaesalpinin F showed the most potent inhibitory activity with an IC50 value of 120 and 140nM respectively, which is lower than the value reported for the well-characterizedantimalarial drug, chloroquine (IC50, 282 291nM).[42] Three new cassane furanoditerpenoids (1-3) exhibiting antimalarial activity against the multidrug-resistant K1 strain of Plasmodium falciparum have been isolated from kernels of Caesalpinia bonduc.
Anticancer Activity
Caesaldekarin J possesses inhibitory activity against glutathione S-transferase, an enzyme that has been implicated in resistances during treatment of cancer and parasitic infections, and can be isolated from the ethanolic extract of Caesalpinia bonduc bark.[43] Two new cassane butenolides, caesalpinolide A (1) and B (2), epimeric at the hemiketal position, were isolated from the marine
65
creeper Caesalpinia bonduc. They exhibited inhibitory effects on MCF-7 breast cancer cell lines, with IC50 values of 12.8 and 6.1 (M), respectively, and also inhibited endometrial and cervical cancer cell lines.[44] Similarly, Phanginin I, a cassane-type diterpenoid isolated from the seeds of Caesalpinia sappan exhibited cytotoxic effects against KB cell line with IC50 value of 4.4g/ml.[45] Plants belonging to the genus Caesalpinia have proven to be a rich source of cassane-type diterpenoids.[46,47-51] These cassane diterpenoids are characterized by a molecular skeleton constructed from the fusion of three cyclohexane rings A, B and C and a furan ring (1) .Ring C may sometimes be aromatic as in compounds 24, 38, 59-62. The existence of an exocyclic methylene group at position 14 is a characteristic of some of these cassane-type diterpenoid compounds (see 62, 76, 81, 83, 88). Generally, these diterpenoids give a red colour with Ehrlich reagent, suggesting the presence of a furan ring in their molecular structure. However, not all cassane-type diterpenes have this furan ring (e.g 5, 6) and therefore will not respond to this test. The isolation of the cassane-type diterpenoids may have begun in the mid 1950s from other sources other than the genus Caesalpinia. However, Jiang et al (2001), isolated pulcherrimin A (3) and -caesalpin (4) from Caesalpinia pulcherrima.[52] In 1992, the roots of Caesalpinia decapetala yielded caesaljapin (9), a cassane diterpenoid.[53] Caesaldekarin A (15), C (16), D (16i) and E (16ii) were also isolated from the roots of Caesalpinia major.[54] Caesalpinin 1 (17), a cassane furanoditerpene, has been isolated from Caesalpinia bonducella roots.[48] From the seeds of the same
Cassane-type diterpenoids from the genus Caesalpinia Basic Molecular Skeleton of Cassane-type Diterpenoids
plant, two cassane diterpenes neocaesalpin A (18) and B (19) were isolated.[55] From the roots of the same plant in the following year, caesaldekarin F (20) and G (21), were isolated.[56] In the same year, and again from the roots of this plant, seven cassane diterpenoids that included caesaldekarin A (15), H, I, J, K and L (22-26) and demethylcaesaldekarin C (27) were isolated.[57] Additional diterpenoid lactone compounds, caesalmins A, B, C, D, E, F and G (28-34) were isolated from the seeds of C. minax.[58] Four novel diterpenoid compounds (35-38), possessing both antibacterial and antifungal activities, have been isolated from the leaves of C. pulcherrima.[59] A novel diterpenoid named macrocaesalmin (39), together with caesalmin B, D and H possessing antiviral and anticancer activities were isolated from the seeds of C. minax.[31,60] This was followed in the following year by the isolation of cassane diterpenoid compounds (+)-vouacapenic and (+)- vouacapenate (2, 7) from Vouacapoua americana belonging to the Leguminosae.[61] Novel norcassane-type diterpenes norcaesalpinin A, B and C (44-48), have also been obtained from the seed kernels of C. crista.[61] In the following year, five new cassane-type diterpenes, caesalpinins MA-ME 1-5 (49-53) and three new norcassane-type diterpenes, norcaesalpinins MA-MC (54-56) together with known cassane-type diterpenes ( 29, 30, 32 and 51) were isolated from C. crista.[62] Nine new cassane-type diterpenes taepeenin A-I
O H H CH3 O O O O
O
11 1 2 3 4 10 9 8 7 6 12 14
16
HO
15
HOOC
OH
13
17
Figure 1: Carbon skeleton of cassane-type diterpenoids
Figure 2: Pulcherrimin A
O OAc AcO H OH
Figure 3: e-caesalpin
O HOOC H MeOOC
Figure 4: Caesaljapin
H OH
66
O H
O H
OH OAc
Figure 5: Caesaldekarin A
MeOOC
Figure 6: Caesaldekarin C
OH
O O OH O
Figure 7: Caesalpinin 1
O OAc AcO H OH
Figure 8: Neocaesalpin A
HO
O H OH
OAc OAc
O OAc AcO H OH
Figure 9: Neocaesalpin B
HO
O H H
MeOOC
Figure 10: Caesaldekarin F
O H H OH
OH O
H
O H H AcO
Figure 12: Caesaldekarin H
H MeOOC
Figure 11: Caesaldekarin G
OH
OH CH2
67
O H H OH CH2OH
Figure 13: Caesaldekarin I
OH
MeOOC
Figure 14: Caesaldekarin J
OH
O H H MeOOC
Figure 15: Caesaldekarin A
O
H HO HOCH 2
Figure 16: Caesaldekarin B
OH
OH
OH
O H H HOOC OH
Figure 18: Caesalmin A
O OH H H OH H OAc H O
Figure 17: Demethylcaesaldekarin C
O OAc H H OH H
Figure 19: Caesalmin B
O OAc H H OH OAc
Figure 20: Caesalmin C
H H
OAc
O OAc H OH OAc
Figure 21: Caesalmin D
O OH OAc H OAc OH OAc

Figure 22: Caesalmin E
H OH OAc
68
O OAc H OH OAc
Figure 23: Caesalmin F
O OH H H OH
Figure 24: Caesaldekarin G
H OMe OAc
H H
OAc
O H H OH O O
H OH O
O H OH
Figure 25: Isovouacapenol A
Figure 26: Isovouacapenol B
O H H OH O H OH O
OH O
Figure 27: Isovouacapenol C
Figure 28: Isovouacapenol D
O O H O
Figure 29: Macrocaesalmin
H O O
OAc H OH
Figure 30: Caesalmin H
H H OH
H OAc
69
O OH H OH H OAc
Figure 31: Bonducellpin D Figure 32: Norcaesalpinin A
O OAc H H OH O
H O
AcO
O OAc O OH
Figure 33: Norcaesalpinin B Figure 34: Norcaesalpinin C
OH OAc
O H
OH
O AcO H H AcO
Figure 35: Caesalpinin MA
O AcO CH3 OH
Figure 36: Caesalpinin MB
H H
COOCH3 H
OH
O AcO Me AcO
Figure 37: Caesalpinin MC
O AcO AcO Me OH OAc

Figure 38: Caesalpinin MD 4
OH
OH
H H AcO H OAc
Figure 39: Caesalpinin ME
O H
O O
AcO
OH
Figure 40: Norcaesalpinin MA
70
OH AcO
O AcO H H O OAc OAc

Figure 42: Norcaesalpinin MC
OH
Figure 41: Norcaesalpinin MB
OH
MeOOC
Figure 43: Taepeenin A
HOOC
Figure 44: Taepeenin B
MeOOC
Figure 45: Taepeenin C
H OH
MeOOC
Figure 46: Taepeenin D
H COOMe
O O
MeOOC
Figure 47: Taepeenin E
H CHO
Figure 48: Taepeenin F
H COOMe
O H
OH
H H H
Figure 49: Taepeenin G
H MeOOC
Figure 50: Taepeenin H
H CHO
71
O H H MeOOC
Figure 51: Taepeenin I
O H H H MeOOC
Figure 52: Nortaepeenin A
H CH2OH
O H H H OH MeOOC
Figure 53: Nortaepeenin B
O OAc
O
AcO
Figure 54: Caesaldekarin e
OH
O OAc AcO AcO AcO HO OAc
OH
OH
Figure 55: 2-Acetoxycaesaldekarin e
Figure 56: 2-Acetoxy-3-deacetocaesaldekarin e
O OAc
O OAc H H
OH OAc
Figure 57: 6-Acetoxy-3-deacetoxycaesaldekarin e
OH
Figure 58: 14 (17)-Dehydrocaesalmin F
O O H OH OAc
Figure 59: Bonducellpin B Figure 60: Bonducellpin C
O OAc
COOMe OH
H H
COOMe H OH
OH H
72
O OAc H OH H
Figure 61: 7-Acetotoxybonducellpin C
O COOMe H OAc OH OAc

Figure 62: 1-Deacetoxy-1-oxocaesalmin C
O H
OAc
O O H OH OH
Figure 63: S-Caesalpin
O O H
OAc
OH
Figure 64: 1-Deacetylcaesalmin C
OAc OAc
O OAc H H AcO
Figure 65: Caesalpinin C
O OAc H O H OH OAc
Figure 66: Caesalpinin D
O OAc
OH OAc
O OAc AcO H AcO

Figure 67: Caesalpinin E
O O
O OH OAc
Figure 68: Caesalpinin F
OAc
OH
O OH H O H OH
Figure 69: Caesaldekarin H
O O H O H OH
Figure 70: Caesaldekarin I
OAc
OAc
73
O O H H OH OAc
Figure 71: Caesalpinin J Figure 72: Caesalpinin K
O OAc H H OH H OH
COOMe OAc
O OAc H OH OH
Figure 73: Caesalpinin M Figure 74: Caesalpinin N
COOMe H OAc
OAc H OH
CHO H OH
O OAc O O OH
Figure 75: Caesalpinin O
O OAc AcO H OH
Figure 76: Caesalpinin P
OH
O OAc H OAc
Figure 77: Caesalpinin MF
O
OAc
COOMe H
OH H
COOMe H OAc
OH
OAc
Figure 78: Caesalpinin MG
O OAc H OH OAc
Figure 79: Caesalpinin MH Figure 80: Caesalpinin MI
COOH H OH
OH H
OH
74
O OAc H H OH
Figure 81: Caesalpinin MJ
O O H
OAc
Figure 82: Caesalpinin MO
OH OAc
OAc
O O H OH OAc
Figure 83: Norcaesalpinin MD
O O H H AcO
Figure 84: Norcaesalpin D
H O OAc
AcO
OH
O O H OH
Figure 85: Norcaesalpin E
O O H H OH OAc
Figure 86: Norcaesalpin F
O OH
O OH
O H H
MeOOC
Figure 87: Benthaminin 1
MeOOC
O H H H MeOOC
(57-65) and two new norcassane-type diterpenes nortaepeenin A-B (66-67) were also isolated from the stems and roots of C. crista.[63] From the seed kernels of the same plant, known cassane and norcassane-type diterpenes including compounds 29, 30, 32, 34, 46-49, 54, 68-78 and new cassane-type diterpenes, namely caesalpinins C-K (65-68), M-P (73-76), caesalpinins MFMJ (77-81), MO (82) and norcaesalpins MD (83), D-F (84-86) possessing antimalarial activity, have been isolated. Norcaesalpinin E (85), displayed the most potent antimalarial activity.[62]
75
CONCLUSION
Cassane-type diterpenoids continue to be isolated from medicinal plants. Three novel cassane-type diterpenoids- benthaminin 1 (87), 2 (88) and 3 (89) possessing antimicrobial and antioxidant properties have been isolated from Caesalpinia benthamiana.[51] Similarly, two novel cassane-type diterpenoids designated magnicaesalpin and neocaesalpin O together with three known ones named caesalmin D and E and neocaesalpin L have been isolated from the seeds of Caesalpinia magnifoliolata.[52] A number of these cassane-type furanoditerpenoids have been found to manifest various biological activities including antibacterial, antifungal,[29] anti-inflammatory, anti-analgesic,[52,53] antiviral and anticancer,[31] antimalarial[54] and antituberculosis activities.[36] Thus, these cassane diterpenoids are of interest due to their structural diversity and their broad spectrum of biological activities. Further studies should be performed to indicate which of these isolated bioactive chemical constituents may serve as lead compounds in the synthesis of biomolecules to tackle the numerous global health challenges due to the emerging and ongoing drug resistance associated with long term use of conventional medicines used in the treatment and management of infectious diseases.
16. 17. 18. 19.
20. 21.
22. 23. 24. 25. 26.
27. 28. 29.
ACKNOWLEDGEMENTS
R. A. Dickson is grateful to the Commonwealth Scholarship Commission, UK and the Kwame Nkrumah University of Science and Technology, Ghana for sponsorship.
30. 31. 32.
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Research Article Azadirachtolide: An anti-diabetic and hypolipidemic effects from Azadirachta indica leaves
Dinesh kumar B1, Analava Mitra2*, Manjunatha M2
1
Department of Pharmaceutics, PSG College of Pharmacy, Coimbatore-641 004, India. 2School of Medical Science and Technology, Indian Institute of Technology, Kharagpur -721302, West Bengal, India
ABSTRACT: Introduction: Azadirachta indica (Meliaceae) leaves are used traditionally in the Indian Ayurvedic medicinal system to treat diabetes. The aim of the present study is to investigate the effect of azadirachtolide (tetranortriterpenoid from Azadirachta indica leaves) on blood glucose and serum lipid profiles on streptozotocin-induced diabetic rats. Methods: Streptozotocin-induced diabetic rats were used for the study. Azadirachtolide (at a dose 50 and 100 mg/kg) was administrated intra-peritoneally in diabetic rats once a week for 30 days. Biochemical parameters notably fasting blood sugar, total cholesterol, triglycerides, low-density lipoprotein, very low-density lipoprotein and high-density lipoprotein were determined. The in vitro alpha amylase and alpha glucosidase inhibitory effects of azadirachtolide were measured and IC50 values were determined. Results: Azadirachtolide exhibited significant (P < 0.05) anti-diabetic as well as hypolipidemic effects by lowering FBS, TC, TG, LDL, and VLDL levels; but also with elevation of HDL level in diabetic rats. Azadirachtolide showed appreciable alpha amylase (IC50 value of 55.80 1.7 g/ml) and alpha glucosidase inhibitory effects (IC50 value of 47.85 1.4 g/ml) compared with acarbose (IC50 value of 83.33 1.8 g/ml). Conclusion: The present study indicated that azadirachtolide possesses anti-hyperglycemic and anti-lipidemic effects. Thus, results suggested azadirachtolide has a beneficial effect in the management of diabetes associated with abnormal lipid profile and related cardiovascular complications. KEYWORDS: Azadirachta indica, Azadirachtolide, Anti-diabetic, Hypolipidemic
IntRoductIon
Diabetes is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion or insulin action, or both.[1] Broad research on diabetes has resulted in the development of a number of oral hypoglycemic agents including biguanides, sulphonylureas and thiozolidinediones which are available commercially for the management of diabetes. However, these drugs also produce nondesirable side effects.[2] Hence, there is a need to develop alternative anti-diabetes medicines. The herbal medicines are widely used for the treatment of disease because of their effectiveness, safety, affordability and acceptability. [3] Medicinal plants including their phyto-compounds have been used in the Indian traditional systems of medicine for treatment of diabetic populace all around the world with less known scientific basis of their functioning.[4-7] Hence, phyto-products from medicinal plants need to be investigated by scientific methods for their anti-diabetic activity. Various medicinal effects have been reported for anti-inflammatory, anti-arthritic, antipyretic,
*Correspondence: +913222-282220/282657; Fax: +913222-282221; Email: analavamitra@gmail.com, amitra@adm.iitkgp.ernet.in DOI: 10.5530/pc.2011.1.5
antifungal, anti-bacterial, diuretic, immunomodulatory and anti-tumor properties. Phyto-compounds such as azadirachtins, nimocinol, isomeldenin, 2, 3-dehydrosalanol gedunin, nimbin, nimolicinol from Azadirachta indica have been reported in the leaves.[8] Tetranortriterpenoids has been reported for anticancer, antiviral, anti-allergic and anti-inflammatory activities.[9-12] There is no report on azadirachtolide (tetranortriterpenoid from Azadirachta indica leaves) for antidiabetic and hypolipidemic activities. Therefore, the effect of azadirachtolide (tetranortriterpenoid from Azadirachta indica leaves) on blood glucose and serum lipid profiles on streptozotocin-induced diabetic rats was investigated. Further, in vitro alpha amylase and alpha glucosidase an inhibitory effect of azadirachtolide was evaluated.
MAteRIAls And Methods

Streptozotocin, starch azure, porcine pancreatic amylase, alpha glucosidase from yeast Saccharomyces cerevisiae, para-nitrophenyl gluco-pyanoside and Tris-HCl buffer were procured from Sigma Chemicals, USA. Dimethyl sulfoxide, acetic acid, calcium chloride, ethanol, chloroform, petroleum ether, potassium bromide,
Chemicals and reagents
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Kumar, et. al.: Azadirachtolide: An anti-diabetic and hypolipidemic effects from Azadirachta indica leaves
deuterated chloroform, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, Whatmann filter paper and sodium carbonate were purchased from Merck, India. Thin layer chromatography plates were obtained from Merck (silica gel 60 F254 grade, Germany). Diagnostic kits and reagents for fasting blood sugar, total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein and very low density lipoprotein were obtained from Merck, India. Acarbose was gifted by Zota Pharmaceutical Pvt. Ltd., Chennai. Glibenclamide (Aventis Pharma- Mumbai) was procured from local medical market. Azadirachta indica leaves (Rutaceae) were collected from the locality of IIT Kharagpur campus, West Bengal, India in the month of September and October 2007. The leaves were inspected to be healthy and botanically identified and authenticated by Dr. M. Senthilkumar, Plant Biotechnologist, Prathyusha Institute of Technology and Management, Chennai. The herbarium Azadirachta indica leaves was deposited in the Prathyusha Institute of Technology and Management (PITAM) against voucher no. PITAM/ CH/00015/ 2007. Azadirachta indica leaves after collection were dried at room temperature (27-30C) for 25-30 days. After complete drying (inspection), the dried materials were ground into fine powder using a domestic electric grinder (Product: GX 21, Bajaj appliances, Mumbai, India) and used for extraction. Dried plant powder of Azadirachta indica leaves (500 g) was extracted with ethanol (1 L) at room temperature. Then extract was filtered (Whatmann filter paper, 110mm, Cat. no 1001 110). The filtrate was evaporated by rotary evaporation (Buchi Rotavapor R-210) to get a dark greenish solid residue. These greenish solid residues (15 g) was successively extracted with petroleum ether (3.5 g) and chloroform (5.2 g) and subjected for in vitro alpha amylase inhibitory activity. The chloroform fraction showed appreciable alpha amylase inhibitory compared to petroleum ether fraction. The chloroform fraction was subsequently subjected to column chromatography using gradient elution using acetone and chloroform as solvents (10% acetone in chloroform for 15 mins, 20% acetone in chloroform for 15 mins and 30% acetone in chloroform for 15 mins). The fractions obtained with 20% acetone in chloroform afforded compound-I (10 mg). These fractions were subjected to preparative TLC with mobile phase hexane: ethyl acetate (8.5:1.5) for isolation of compound-I. Compound-I was identified as azadirachtolide by comparing its FTIR, ESI-MS and NMR with previously published literature (Ragasa et al., 1997).
General experimental procedure Extraction and isolation Plant materials
twin-trough glass chamber previously saturated with mobile phase vapor for 20 min. After developing the plate, it was dried at 105C for 15 min and then it was scanned using Scanner 3 (CAMAG, Switzerland) at 254nm using WinCATS 4 software. IR spectrum was recorded using a Thermo Nicolet Nexus 870 FT-IR Spectrophotometer using potassium bromide pellets. Mass spectrum was recorded on Electro-Spray Ionization Mass Spectroscopy (Waters, UK). NMR spectra were recorded in CDCl3 in a Bruker 400 MHZ spectrometer using Topspin software. The assay was carried out following the standard protocol with slight modifications.[13] Starch azure (2 mg) was suspended in a tube containing 0.2ml of 0.5 M Tris-Hcl buffer (pH 6.9) containing 0.01 M calcium chloride (substrate). The tube was boiled for 5 min and then pre-incubated at 37 C for 5 min. Azadirachtolide was dissolved in 0.1% of dimethyl sulfoxide in order to obtain concentrations of 10, 20, 40, 60, 80 and 100 g/ml. Then 0.2 ml of azadirachtolide of a particular concentration was put in the tube containing the substrate solution. 0.1 ml of porcine pancreatic amylase in Tris-Hcl buffer (2units/ml) was added to the tube containing the azadirachtolide and substrate solution. The reaction was carried out at 37 C for 10 min. The reaction was stopped by adding 0.5 ml of 50% acetic acid in each tube. The reaction mixture was then centrifuged (Eppendorf -5804 R) at 3000 rpm for 5 min at 4C. The absorbance of resulting supernatant was measured at 595 nm (Perkin Elmer Lambda 25 UV-VIS). The concentration of the azadirachtolide required to inhibit 50% of alpha amylase activity under the conditions was defined as the IC50 value. The experiments were repeated thrice with the same protocol. The alpha amylase inhibitory activity was calculated as follows: Alpha amylase inhibitory activity = (Ac+) (Ac) (AsAb) 100 (Ac+) (Ac) Where, Ac+, Ac, As, Ab are defined as the absorbance of 100% enzyme activity (solvent with enzyme alone), 0% enzyme activity (solvent without enzyme), a test sample (with enzyme) and a blank (a test sample without enzyme) respectively.
In vitro alpha glucosidase inhibitory assay In vitro alpha amylase inhibitory assay
HPTLC (CAMAG, Switzerland) analyzes was performed using silica gel 60 F254 TLC plate. All collected fractions were spotted (10 l) on a silica gel 60 F254 (Merck, Darmstadt, Germany) TLC plate. The plate was air dried and then developed using the solvent system hexane: ethyl acetate (8.5:1.5) in a CAMAG-
The assay was performed using a standard protocol.[14] Alpha glucosidase (2U/ml) was premixed with 20 l of azadirachtolide at various concentrations (10, 20, 40, 60, 80 and 100 g/ml) and incubated for 5 min at 37C. 1mM para-nitrophenyl glucopyanoside (20 l) in 50mM of phosphate buffer (pH 6.8) was added to initiate the reaction. The mixture was further incubated at 37C for 20 min. The reaction was terminated by addition of 50 l of 1 M sodium carbonate and the final volume was made up to 150 l. Alpha glucosidase activity was determined spectrophotometrically at 405nm on a Biorad microplate reader
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by measuring the quantity of para-nitrophenol released from pNPG. The assay was performed in triplicate. The concentration of azadirachtolide required to inhibit 50% of alpha glucosidase activity under the conditions was defined as the IC50 value. The experiments were repeated thrice with same protocol.
Animal studies
Statistical analysis
Adult male Wistar Rats (weighing 150-200 g) were used for this investigation. The animals were acclimatized to the laboratory conditions for a period of 2 weeks prior to the experiment. They were maintained at an ambient temperature (25 2 C) and relative humidity (40-60%), with 12/12 h of light/dark cycle. The animals were maintained on balance diet and water ad libitum. Institutional Animal Ethical Committee (IAEC) approved the study and all the experiments were carried out by following the guidelines of CPCSEA, India.
Induction of diabetes and blood sample collection
All values were expressed mean standard deviation. Statistical analysis of in vivo results were performed by one-way analysis of variance (ANOVA) followed by Students t-test. P < 0.05 was considered statistically significant. In vitro inhibitory assay statistical difference and linear regression analysis were performed using Graphpad prism 5 statistical software.
Results
Azadirachtolide (10 mg) was isolated from 500 g of dried leaves of Azadirachta indica (Figure 1). HPTLC analyzes indicated that F2 contained azadirachtolide and the retention factor (Rf) values of azadirachtolide was found to be 0.31 (Figure 2). The F2 fractions were subjected to preparative TLC with the solvent system hexane: ethyl acetate (8.5:1.5) to get the compound-1 (azadirachtolide). FTIR (KBr disc) is shown in Figure 3: peak at 3444 cm-1 indicated presence of OH group, peak at 2925 cm-1, 2854 cm-1 was due to presence of C-H, peak at 1370 cm-1 showed C-H bending, peak at 1736 cm-1 indicated presence of ester carbonyl group, peak at 1666 cm-1 showed presence of C-O group and peak at 1458 cm-1 indicated presence of CH-CH bending (Figure 3). ESI-mass spectroscopy showed the presence of a molecular weight peak of azadirachtolide at 593. ESI-MS (m/z, % intensity): m/z 593 [M-H]-. Proton NMR (CDCl3 solvent) showed senecioyloxy subtitutent 1.88 (3H), 2.20 (3H), 5.70 (1H). an acetate 1.97 (3H). Four additional methyl singlet 0.8, 1.25, 1.28, 1.30, two olefinic hydrogen 5.57, 5.71, methylene hydrogen bonded to oxygenated carbons 4.15 (1H), 3.81 (1H), 3.68 (1H), 3.59 (1H) and methine hydrogen bonded to oxygenated carbons 4.12 (1H), 4.15 (1H), 5.30 (1H), 5.47 (1H). Azadirachtolide showed appreciable alpha amylase (IC50 value of 55.80 1.7g/ml) and alpha glucosidase inhibitory effects (IC50 value of 47.85 1.4g/ml) as compared with acarbose (IC50 value of 83.33 1.8g/ml) (Figure 4). The body weight was slightly increased in normal control rats compared to initial body weight whereas streptozotocin-induced diabetic rats showed loss of body weight (172.6 2.05 g) after 30 days as compared with initially weight of diabetic rats (186.6 1.24 g). However, body weight of diabetic rats was restored by treating with
A freshly prepared solution of streptozotocin (45mg/kg) in 0.1M citrate buffer pH 4.5 was injected intra-peritoneally in overnight fasted rats. After 3 days, blood was collected from the tail vein of overnight fasting rats under the supervision of a veterinary surgeon using aseptic conditions. The FBS level of blood was checked regularly up to the stable hyperglycemia stage, usually one week after streptozotocin injection. Animals with marked hyperglycemia (FBS 250 mg/dl) were selected for the study.[15]
Experimental design
Group I - Normal control Group II - Diabetic control Group III - Diabetic +50 mg/kg (i.p.) azadirachtolide Group IV Diabetic +100 mg/kg (i.p.) azadirachtolide Group V - Diabetic + 0.5 mg/kg (i.p.) glibenclamide The experiment was carried on five groups (I, II, III, IV and V) of six rats each. Group-I served as normal control. Group-II served as diabetic control. Group III-diabetic + 50 mg/kg (i.p.) of azadirachtolide. Group IV-diabetic + 100 mg/kg (i.p.) of azadirachtolide. Group V-diabetic + 0.5 mg/kg (i.p.) of glibenclamide and served as positive control. The azadirachtolide was suspended in 0.3% w/v sodium carboxy methyl cellulose (Sodium CMC) as a vehicle and injected intra-peritoneally into rats once a week for one month with a dose of 50 mg/kg and 100 mg/kg body weight. The blood samples were collected from each rat by retro-orbital vein-puncture. Biochemical parameters were estimated at the beginning and after 30 days of experiment.
Biochemical parameters
Biochemical parameters notably fasting blood sugar (FBS), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL) levels and highdensity lipoprotein (HDL) level in blood serum were measured spectrophotometrically (Semi-Autoanalyzer, Microlab 300, Merck) as per the manufacturers instructions using diagnostic kits and reagents obtained from Merck, India.
80
Figure 1: Structure of azadirachtolide.
Figure 2: HPTLC peaks of collected column fractions CE-Crude extract (Pink peak), F1-10% acetone in chloroform (Violet peak), F2-20% acetone in chloroform (Green peak), F3-30% acetone in chloroform (Orange peak).
Figure 3: FTIR Spectrum of azadirachtolide.
glibenclamide (0.5 mg/kg) and azadirachtolide (at a dose of 50 mg/kg and 100 mg/kg, i.p.) for 30 days (Table 1). Streptozotocin treatment resulted in elevation of fasting blood glucose, triglycerides, total cholesterol, low density lipoproteins, very low density lipoproteins and a reduction in high densitylipoprotein levels as compared to the normal control rats (Table 2). Intra-peritoneal administration of azadirachtolide (at a dose of 50 mg/kg and 100 mg/kg, once a week for 30 days) exhibited significant (P < 0.05) reduction in fasting blood sugar levels (204.0 2.94 and 198.3 2.86 mg/dl in diabetic rats. Diabetic
rats treated with azadirachtolide (at a dose of 50 mg/kg and 100 mg/kg, i.p.) once a week for 30 days on being compared with diabetic rats exhibited significant (P < 0.05) reduction in fasting blood sugar levels (204.0 2.94 and 198.3 2.86 mg/dl respectively). The standard glibenclamide (0.5 mg/kg, i.p.) also showed anti-diabetic activity with reduction of fasting blood sugar level (215.0 2.18 mg/dl) on 30 days as compared to the diabetic control. There was a significant (P < 0.05) reduction in triglycerides, total cholesterol, low density lipoprotein and very low density lipoprotein levels of diabetic rats treated with azadirachtolide (50 and 100 mg/kg, i.p.) on being compared with diabetic control. Also, there was a significant (P < 0.05) elevation of HDL level in azadirachtolide (50 and 100 mg/kg, i.p.) treated diabetic rats.
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Figure 4: Alpha amylase and alpha glucosidase inhibitory effects of azadirachtolide.
Table 1: Body weights of streptozotocin-induced diabetic rats after treatment with azadirachtolide.
Group Normal control Diabetic control 50 mg/kg of azadirachtolide 100 mg/kg of azadirachtolide 0.5 mg/kg of glibenclamide Initial body weight 191.0 0.81 186.6 1.24 183.0 1.60 182.0 1.63 180.3 0.47 Final body weight 200.0 0.63 172.6 2.05 178.3 1.69* 177.6 1.69* 176.3 1.24*
role in occurrence of premature and severe atherosclerosis, which affects patients with diabetes.[20] In the present study, an increase in blood sugar levels in diabetic rats was observed after the induction of diabetes by streptozotocin. This was prevented by treating diabetic rats with azadirachtolide (at a dose 50 and 100 mg/kg, i.p.) once a week for 30 days. The standard drug glibenclamide has been used to treat diabetes, which stimulate insulin secretion from pancreatic beta cells, it may be suggested that the mechanism of action of azadirachtolide is similar to glibenclamide. The azadirachtolide (at a dose 50 and 100 mg/kg, i.p.) treated diabetic rats showed a significant reduction in both fasting blood sugar levels and some lipid parameters (TC, TG, LDL, and VLDL). Some biological active compounds such as mimbidin, sodium nimbidate, nimbin, nimbolide, gedunin, azadirachtin, mahmoodin, gallic acid, catechin, margoone, isomargolone, cyclic trisulphide, cyclic tetrasulphide and polysacharides were isolated from leaves and seeds of Azadirachta indica.[21] Aqueous extract of neem leaf significantly reduced the blood glucose level of male albino rats of Wistar strains.[22] The combined ethanolic extracts of Azadirachta indica and Vernonia amygdalina leaf extracts showed anti-hyperglycemic effect on alloxan induced albino wistar rats.[23] The weight loss in diabetic rats may be associated with lipid lowering activity of azadirachtolide or due to its influence on various lipid regulation systems. Treatment with azadirachtolide (at a dose 50 mg/kg and 100 mg/kg body weight) in diabetic rats may have potential role to prevent formation of atherosclerosis and coronary heart disease. The present in vivo study showed that intra-peritoneal
*(P <0.05) compared with treated diabetic groups Vs Diabetic control. n = 6/group. Values are expressed as mean S.D
dIscussIon
The present study was designed to explore the effect of azadirachtolide (tetranortriterpenoid from Azadirachta indica leaves) on blood glucose and serum lipid profiles on streptozotocininduced diabetic rats. A comparison of the FTIR, ESI-MS, NMR spectra of isolated fraction showed significant similarity with previously reported azadirachtolide data.[16] Intra-peritoneal administration of 50mg/kg and 100 mg/kg of azadirachtolide once a week for 30 days showed anti-diabetic and hypolipidemic effects in diabetic rats. Lipid abnormalities associated with atherosclerosis is the major cause of cardiovascular disease in diabetes. High level of TC and LDL are major coronary risk factors.[17] Further, several studies suggested that TG itself is interdentally related to coronary heart disease.[18,19] The abnormalities in lipid metabolism lead to elevation in the levels of serum lipid and lipoprotein that in turn play an important
82
Table 2: Effect of azadirachtolide on biochemical parameters in normal and diabetic rats.

BBP (mg/dl) FBS TC TG HDL LDL VLDL Days 0 30 0 30 0 30 0 30 0 30 0 30 Group 1 75.6 1.60 76.6 1.69 93.6 1.75 94.6 1.24 68.5 1.47 69.0 2.94 31.6 1.69 33.6 1.24 51.7 1.28 54.1 1.30 18.3 1.30 19.6 1.69 Group 2 271.0 2.9 294.3 3.29# 200.3 1.36 224.3 3.09# 182.3 2.05 200.0 1.63# 25.3 2.05 21.1 0.62# 110.0 1.63 117.8 2.24# 40.7 1.25 48.6 0.69# Group 3 278.3 1.24 204.0 2.94* 201.5 1.47 127.3 2.05* 176.3 1.24 133.3 2.05* 22.5 1.08 30.3 1.69* 114.6 2.05 81.3 0.94* 37.0 1.50 25.9 1.96* Group 4 280.0 3.77 198.3 2.86* 201.7 1.83 125.3 1.24* 170.0 1.63 121.6 1.69* 21.7 0.98 31.6 1.24* 113.0 2.16 77.3 1.69* 33.5 1.08 21.6 1.24* Group 5 286.0 1.41 215.0 2.18* 200.8 1.92 126.6 1.24* 159.6 1.24 115.6 1.69* 21.1 1.04 32.0 1.63* 112.6 2.86 83.5 1.22* 30.2 1.20 20.8 0.47*
Values are expressed as mean S.D. n = 6/group. Group -1 (rats treated with 0.3% w/v sodium carboxy methyl cellulose (i.p) - served as normal control), Group-2 (rats treated with (45mg/kg) of streptozotocin (i.p) - served as diabetic control), Group 3 - (diabetic+50 mg/kg (i.p.) of azadirachtolide), Group 4- (Diabetic+100 mg/kg (i.p.) of azadirachtolide), Group 5 - (diabetic+0.5 mg/kg (i.p.) of glibenclamide (positive control). BBP-Biochemical parameters, FBS-fasting blood sugar, TC-total cholesterol, TG-triglycerides, LDL-low-density lipoprotein, VLDL-very low-density lipoprotein, HDL-high-density lipoprotein. #P < 0.05, Group 1 vs. Group 2. *P < 0.05, treated diabetic groups vs. diabetic control group.
administration of azadirachtolide (at a dose 50 and 100 mg/kg) exhibited anti-diabetic and hypolipidemic effects in streptozotocininduced diabetic rats. One of the therapeutic approaches for type 2 diabetes is to reduce the post-prandial hyperglycemia. Alpha amylase and alpha glucosidase are the enzymes involved in the metabolism of carbohydrates. Alpha amylase degrades complex dietary carbohydrates to oligosaccharides and disaccharides, which are ultimately converted into monosaccharide. Liberated glucose is then absorbed by the gut and results in postprandial hyperglycemia. Inhibition of alpha amylase and alpha glucosidase limits postprandial glucose levels by delaying the process of carbohydrate hydrolysis and absorption.[24] The plant based alpha amylase and alpha glucosidase inhibitor offers a prospective therapeutic approach for the management of post-prandial hyperglycemia.[25] In this study, azadirachtolide showed appreciable alpha amylase and alpha glucosidase inhibitory effects compared with acarbose.
RefeRences
1. Mitra A. Some salient points in dietary and life style of rural Bengal particularly tribal populace in relation to rural diabetic prevalence. Studies on Ethno-Med. 2008; 2:51-56. Hermans MP Buysschaertm M. Pharmacological treatment of type 2 diabetes, , Acta clinica belgica. 2004; 2:59-66. Dineshkumar B, Mitra A, Manjunatha M. A comparative study of alpha amylase inhibitory activities of common anti-diabetic plants at Kharagpur 1 Block. Int J Green Pharmacy. 2010; 4:115-121. Dineshkumar B, Mitra, A, Manjunatha M. In vitro and in vivo studies of antidiabetic Indian medicinal plants: A review. J Herbal Med Toxicol. 2009; 3:9-14. Franco OL, Rigden DJ, Melo FR, Grossi-de-sa MF Plant -amylase inhibitors and . their interaction with insect -amylase structure, function and potential for crop protection. Eur J Biochem. 2002; 269:397-412. Patwardhan B, Vaidya ADB, Chorghade M. Ayurveda and natural products drug discovery. Curr Sci. 2004; 86:789-799. Said O, Fulder S, Khalil K, Azaizeh H, Kassis E, Saad B. Maintaining a physiological blood glucose level with Glucolevel, a combination of four anti-diabetes plants in the traditional Arab herbal medicine. Evid Based Complement Alternat Med. 2007; 5:421-428. Atawodi SE, Atawodi JC. Azadirachta indica (neem): a plant of multiple biological and pharmacological activities. Phytochem Rev. 2009; 8:601-620. Kishore CK, Vijayalakshmi K, Bibha C, Mridula N, Gopal GR, Sathees RC. Methyl angolensate, a natural tetranortriterpenoid induces intrinsicapoptotic pathway in leukemic cells. FEBS Lett. 2008; 582:4066-4076. Bueno CA, Barquro AA, Consoli H, Dimaier MS, Alche LE. A natural tetranortriterpenoid with immunomodulation properties as a potential anti-HSV agent. Virus Res. 2009; 141:47-54. Penido C, Costa KA, Pennaforte RJ, Costa MFS, Pereira JFG, Siani AC, Henriques MGMO, Anti-allergic effects of natural tetranortriterpenoids isolated from Carapa guianensis Aublet on allergen-induced vascular permeability and hyperalgesia. Inflamm Res. 2005; 54:295-303. Penido C, Conte FP Chagas MSS, Rodrigues CAB, Pereira JFG, Henriques , MGMO. Antiinflammatory effects of natural tetranortriterpenoids isolated from Carapa guianensis Aublet on zymosan-induced arthritis in mice. Inflamm Res, 2006; 55:457-464. Hansawasdi C, Kawabata J, Kasai T. - amylase inhibitors from Roselle (Hibiscus sabdariffa Linn.) tea. Biosci Biotechnol Biochem. 2000; 64:1041-1043. Pistia-Brueggeman G, Hollingsworth RI. A preparation and screening strategy for glycosidase inhibitors. Tetrahedron. 2007; 57:8773-8778. Gupta RK, Kesari AN, Murthy PS, Chandra R, Tandon V, Watal G. Hypoglycemic and antidiabetic effect of ethanolic extract of leaves of Annona squamosa L. in experimental animals. J Ethnopharmacol. 2005; 99:75-81. Ragasa CY, Nacpil ZD, Natividad GM, Tada M, Coll JC, Rideout JA. Tetranortriterpenoids from Azadirachta indica. Phytochem. 1997; 46:555-558. Temme Eh, Vaqn HPG, Schouten EG, Kesteloot H. Effect of plant sterol-enriched spread on serum lipids and lipoproteins in mildly hypercholesterolaemic subjects. Acta Cardiol. 2002; 57:111-115.
2. 3.
4. 5.
6. 7.
8. 9.
conclusIon
The present study indicated that azadirachtolide (at a dose 50mg/kg and 100 mg/kg body weight) exhibited anti-diabetic and hypolipidemic effects in streptozotocin-induced diabetic rats. Therefore, azadirachtolide could be used as anti-diabetic agent in the management of diabetes associated with abnormalities of lipid profiles.
10.
11.
12.
13.
AcknowledgMents
Authors would like to acknowledge Prof. P.K. Dutta, Head, School of Medical Science and Technology, IIT Kharagpur and for his valuable support in the research work. The authors would like to acknowledge the Central Research Facility (CRF) of Indian Institute of Technology, Kharagpur for providing the facility of FTIR, ESI-MS and NMR.
14. 15.
16. 17.
83
18.
19. 20.
21.
Bainton D, Miller NE, Botton CH, Yarnell JWG, Suretman PM, Baker IA. Plasma triglycerides and high density lipoprotein cholesterol as predictors of ischemic heart disease in British man. Br Heart J. 1992; 68:60-66. EI-harzmi MA, Warsy AS. Evaluation of serum cholesterol and triglyceride level in 1-6- year -old Saudi children. J Trop Pediatr. 2001; 47:181-185. Ravi K, Rajasekaran S, Subramanian S. Anti-hyperlipidemia effect of Eugenia jambolana seed kernel on streptozotocin-induced diabetes in rats. Food Chem Toxicol. 2005; 43:1433-1439. Biswas K, Chattopadhyay I, Banarjee RK, Bandyopadyay U. Biological activities and medicinal properties of neem (Azadirachta indica). Curr sci. 2002; 82:1136-1345.
22. 23.
24. 25.
Bajaj S, Srinivasan BP Investigations into the anti-diabetic activity of Azadirachta . indica. Indian J Pharmacol. 1999; 31:138-141. Ebong PE, Atangwho IJ, Eyong EU, Egbung GE. The anti-diabetic efficacy of combined extracts from two continental plants: Azadirachta indica (A.Juss) (Neem) and Vernonia amygdalina (Del.) (African bitter leaf). Am J Biochem Biotechnol. 2008; 4: 239-244. Bell DS. Type 2 diabetes mellitus: What is the optimal treatment regimen? Am J Med. 2004; 116:23-29. McCue P Vattem D, Shetty K. Inhibitory effect of clonal oregano extracts against , porcine pancreatic amylase in vitro. Asia Pac J Clin Nutr. 2004; 13:401-408.
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Research Letter Antimicrobial and anti-inflammatory activities of the leaves of Clerodendrum splendens leaves
Fleischer, TC1#, Mensah, AY2, Oppong, AB2, Mensah, MLK1, Dickson, RA2, Annan, K2
1
Department of Herbal Medicine, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana. 2Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana
ABSTRACT: Clerodendrum splendens is a West African climbing shrub used in traditional medicine for wounds and infectious conditions. The petroleum ether, ethyl acetate, and 70% ethanolic extracts of the leaves obtained by successive Soxhlet extraction, inhibited the growth of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Eschericia coli and Candida albican. The ethyl acetate extract was the most active. Again, all the extracts dose-dependently inhibited carrageenaninduced foot paw oedema in 7-day old chicks. Again, the ethyl acetate extract showed the greatest inhibition. The results of this study provide scientific evidence for the ethnomedicinal use of the leaves of C. splendens. KEY WORDS: Clerodendrum splendens, Antimicrobial, Anti-inflammatory activity.
INTRODUCTION
Clerodendrum splendens G. Don (Family: Verbenaceae) also known as the Flaming Glory - bower is a woody or semi-woody evergreen vine which grows in the tropical and subtropical regions of the world. In ethnomedicine, the plant is used to treat wounds and burns,[1] haemorrhoids, diarrhoea and dysentery.[2] The leaves have been found to contain reducing sugars, glycosides, unsaturated sterols, triterpenoids and flavonoids.[3] Recently the plant has been reported to show wound healing, antioxidant and antimicrobial properties.[4] Various species of Clerodendrum, including C. trichotomum, C. indicum and C. serratum which are used traditionally in the management of inflammatory conditions, have been shown to possess potent anti-inflammatory activities. [5] We have investigated the antimicrobial and anti-inflammatory activities of the leaves of C. splendens and in this report provide further support for its ethnomedicinal uses.
by Mr. Ntim-Gyakari, the curator of the Herbarium of the Forestry Commission in Kumasi and a voucher specimen (KNUST/HM1/2010/L033) has been deposited at the herbarium of the Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology (KNUST) Kumasi, Ghana. The leaves were air dried for four days and ground into a coarse powder. The powder (0.5 kg) was serially extracted using petroleum ether, ethyl acetate and 70% ethanol. The various extracts were evaporated under reduced pressure using a rotary evaporator until a viscous extract of each was produced. The petroleum ether extract gave a yield of 9.19 %w/w, that of ethyl acetate extract was 9.59 %w/w and 13.45 %w/w for the ethanolic extract. Phytochemical screening of the powdered leaves using methods described by Sofowora[6] and Harborne[7] showed the presence of flavonoids, tannins and alkaloids. The microorganisms used in this study were obtained from the stocks of the Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, KNUST, Kumasi. They included; Staphylococcus aureus (NCTC 10788), Bacillus subtilis (ATCC 6633), Pseudomonas aeruginosa (NCTC 10662), Eschericia coli (ATCC 25922) and Candida albicans (ATCC 102321). A 24 hour broth culture of the organisms was used. The media used was Nutrient agar (MERCK) for the bacteria and Sabouraud agar (MERCK) for Candida.
85 Test organisms Extraction of plant material
MATERIALS AND METHODS

Plant material
The leaves of C. splendens were collected from Asokore Mampong in Kumasi in May, 2008. The plant material was authenticated
#
Correspondence: +233-3220-60359; +233-244597464; Email: tcfleischer.pharm@knust.edu.gh DOI: 10.5530/pc.2011.1.6
Fleischer, et. al.: Antimicrobial and anti-inflammatory activities of the leaves of Clerodendrum splendens leaves
Materials used in Anti-inflammatory Studies
Day old post-hatched Cockerels (Gallus gallus; strain Shaver 579) were obtained from Akropong Farms, a commercial breeder, in Kumasi. The chicks were housed in standard environmental conditions at the Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, KNUST. The standard drugs used for the positive control were diclofenac sodium and dexamethasone. Carrageenan sodium (Sigma - Aldrich Inc., St Louis, MO, USA) was used to induce oedema in the chicks. Extracts of C. splendens (10 mg/ml) were prepared in Dimethyl sulphoxide (DMSO) for the antimicrobial assay. Ciprofloxacin and Ketoconazole were used as the positive controls at a concentration of 0.5 mg/ml each. The inocula were prepared by inoculating the test organisms in nutrient broth and incubating them for 24 hours at 37C for the bacteria, while for Candida albicans in Sabourauds dextrose broth was incubated for 48 hours. One milliliter of the diluted cultures was inoculated into a sterile molten nutrient agar at 45C and poured into a sterile petri dish. Similarly, 1 ml of the diluted fungal suspension was poured into sterile Sabourauds dextrose agar plates. These were swirled gently and allowed to solidify. Wells were bored into the solidified inoculated nutrient agar plates using cork borer number 6. The wells were filled with equal volume of 0.1 ml of each extract. One hour was allowed for the extract to diffuse into the agar after which the plates were incubated overnight at 37C and 25C for fungi and bacteria respectively. At the end of the incubation period, the diameter of inhibition zone(s) were measured with a ruler and recorded. The extracts and standard antibiotics were tested in triplicate and mean zones of inhibition were calculated for each extract and the standard antibiotics.
Anti inflammatory Assay Agar well diffusion bioassay Preparation of extracts
intervals over the next 6 hours post carrageenan injection. The right footpads of the chicks were injected intraplantar with carrageenan (10 l of a 1% solution in saline). The change in foot thickness for the various groups was recorded hourly for six hours by means of a digital caliper. The oedema component of inflammation was quantified by measuring the foot thickness before carrageenan injection and at the various time points.
Statistical analysis of data
The extracts were tested against test organisms in triplicates and the results were presented as the mean the standard error of means (SEM). Raw scores for the right foot thickness were individually normalized as percentage of change from their values at time 0 and then averaged for each treatment group. The timecourse curves for foot thickness were subjected to two-way (treatment time) repeated measures analysis of variance with Bonferronis post hoc t test. Total foot thickness for each treatment was calculated in arbitrary units as the area under the curve (AUC) and to determine the percentage inhibition for each treatment, the following equation was used. % Inhibition of oedema = AUC control AUC treatment 100 AUC control
RESULTS AND DISCUSSION

Undeniably, plants have played very important roles in the lives of humans for centuries. C. splendens enjoys traditional use as antiinflammatory and antimicrobial agents. This study was conducted on the leaves of C. splendens to validate these folkloric uses. All the extracts showed some level of antimicrobial activity against Staph. aureus, B. subtilis, P. aeruginosa, E. coli and C. albicans in vitro, with the ethyl acetate extract exhibiting the highest activity (Table 1). The zones of inhibition ranged from 4.00 0.5 4 mm to 9.0 0.16 mm. The activity of the petroleum ether and ethanolic extracts ranged between 3.50 0.50 mm to 4.67 0.33 mm and 3.33 0.47 mm to 4.3 0.47 mm respectively. The least susceptible organism to the extracts was P. aeruginosa. Staph. aureus which generally causes infections that are very difficult to combat due to their multi drug resistance[9,10] was found to be susceptible to all extracts. Generally, the activities of the extracts were weak compared to the activities of the standard antibiotics used in the study. The anti-inflammatory activity of the leaves of C. splendens was established using the carrageenan-induced oedema in chicks, a common experimental animal model used to evaluate NSAIDs.[11] It is believed to act in a biphasic manner. The initial phase of inflammation (0-2 h) has been attributed to the release of histamine and kinins, followed by a late phase (2.5-6 h) mainly sustained by release of prostaglandins.[12] The second phase is sensitive to most clinically effective anti-inflammatory drugs.[13] In this study, the time course curves revealed a dose-dependent effect of the extracts on oedema (Figure 1). Furthermore, when
The anti-inflammatory properties of the extracts were evaluated using the carrageenan-induced foot oedema in 7-day old chicks as described by Roach and Sufka[8] with some modifications. The experiment was performed to evaluate the prophylactic effects of the petroleum ether, ethyl acetate and 70% ethanolic extracts on the oedema component of inflammation. Dexamethasone, a steroidal anti-inflammatory drug and diclofenac, a non-steroidal anti-inflammatory drug (NSAID) were used as positive controls. In this method, chicks were randomly selected, grouped (5 per group) and fasted for 24 hours before the experiment. Water was available ad libitum. The test samples were prepared by dissolving the fluid extracts in 2% tragacanth in distilled water. Doses of 30, 100 and 300 mg/kg were prepared and given orally (p.o) 1h before the carrageenan challenge and for the diclofenac (10, 30 and 100 mg/kg) and dexamethasone (0.1, 1.0 and 3 mg/kg) were given intraperitoneally (i.p) 30 minutes before the carrageenan challenge. The foot thickness of each chick was measured before carrageenan injection (baseline measurement) and then at hourly
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Table 1: Antimicrobial Activities of C. splendens extracts

Extracts E.coli Pet ether Ethyl acetate 70% ethanol Ciprofloxacina Ketoconazoleb 2% DMSO 4.2 0.17 9.0 0.17 3.7 0.33 20 0.67 0 B. subtilis 4.3 0.67 7.0 0.17 4.0 0.56 24 0.50 0 Mean Zones of Inhibition (mm) Staph aureus 4.2 0.17 5.2 0.44 4.3 0.33 17.5 0.71 0 P. aeruginosa 3.5 0.50 4.0 0.50 3.3 0.33 23.5 0.43 0 C. albicans 4.7 0.33 6.3 1.17 3.7 0.33 18 0.30 0
; no assay performed, the data are shown as mean Standard Error of the Mean (SEM), a,b ; positive controls
pet-ether % increase in foot volume

50 40 30 20 10 0
pet ether auc Total edema (calculated as. AUC) control pet-ether 30 pet-ether 100 pet-ether 300
200 150 100 50 0
4 Time /hrs
control
30 100 Dose mg / kg
300
ethyl acetate % increase in foot volume

50 40 30 20 10 0
ethyl acetate auc Total edema (calculated as. AUC) control ethylacetate 30 ethylacetate 100 ethyl acetate 300
200 150 100 50 0
***
*** ***
4 Time /hrs
control
30 100 Dose mg / kg
300
ethanol % Increase in foot volume

50 40 30 20 10 0
ethanol auc control ethanol 30 ethanol 100 ethanol 300 Total edema (calculated as. AUC)
200 150 100 50 0
**
4 Time /hrs
control
30 100 Dose mg / kg
300
Figure1: Time course effects of Petroleum Ether, Ethyl acetate and Ethanol Extracts (10-300 mg kg-1 p.o), in the prophylactic protocol on carrageenan induced foot oedema in the chick and their respective total oedema responses for 6 h [defined as the area under the time course curve (AUC)]. Each point on the column represents the Mean S.E.M. (n = 5). ***P < 0.001, **P < 0.01, *P < 0.05.
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total oedema over the period of the experiment was represented arbitrarily as AUC of the time course curves, all the extracts significantly reduced total oedema with a maximal inhibitory effect of 47.29 8.65%, 66.09 13.13% and 45.19 5.09% respectively at 300 mg/kg (Table 2). Diclofenac (10-100 mg/kg, i.p) also showed significant effect on the time course curve and total oedema with maximal inhibitory effect of 79.56 18.24% at 100 mg/kg as seen in Figure 2. Similarly, treatment with dexamethasone, a steroidal anti-inflammatory agent, (0.3-3 mg/kg, i.p) exhibited a significant effect on the time course curve of carrageenan-induced oedema (Figure. 2) with a maximal inhibitory effect of 78.69 3.91% at 3 mg/kg. Thus, all the extracts inhibited oedema from the second hour (Figure. 1). The extracts may therefore be acting in the late phase of the inflammation by
inhibiting chemical mediators such as prostaglandins. The ethyl acetate extract exhibited the highest inhibitory effect in a dosedependent manner at all doses with a maximal effect of 66.1 3.67% at 300 mg/kg body weight. The extent of inhibition of the foot oedema by the extracts was less than the standard antiinflammatory drugs, diclofenac and dexamethasone. Phytochemical screening revealed the presence of tannins, alkaloids, flavonoids, glycosides and sterols in the leaves. Some of these metabolites have been reported to possess antimicrobial activity.[10] Our results agree with that observed by Gbedema et al.[4] and lend further support to the use of the leaves of C. splendens for the treatment of wounds and microbial infections in traditional medicine. The results again provide support for the ethnomedicinal use of C. splendens in the treatment of inflammatory diseases.
Table 2: Inhibitory effects of Petroleum ether, ethyl acetate and 70% ethanolic extract on carrageenan-induced oedema on 7-day old chicks.
Extract Pet ether Ethyl acetate 70% Ethanol Diclofenac (100 mg/kg) Dexamethasone (3 mg/kg)
Values are mean S.E.M (n = 5), P < 0.001
300 mg/kg 47.29 8.65% % 66.1 3.67% 45.19 5.09% 79.56 18.24% 78.69 3.91%
100 mg/kg 46.43 2.98 50.57 0.67% 19 5.34%
30 mg/kg 24.41 3.97% 44.65 4.77% 11.11 9.77%
diclo auc Total edema (calculated as. AUC) 150
diclo % Increase in foot volume

40 30 20 10 0 10
control diclo 10mg/kg diclo 30mg/kg diclo 100mg/kg
100 * 50 ***
4 Time / hrs
CONTROL
10 30 Dose mg/kg dexa auc
100
dexa % increase in foot volume 60 control dexa 3mg/kg dexa 1mg/kg dexa 0.3 mg/kg
Total edema (calculated as. AUC)
250 200 *** 150 100 50 0 *** ***
40
20
4 Time /hrs
control
0.3 1 Dose mg/kg
Figure 2: Time course effects of Diclofenac (10-100 mg kg-1, i.p) and Dexamethasone (0.3-3.0 mg kg-1 i.p) in the prophylactic protocol on carrageenan induced foot oedema in the chick and the total oedema response for 6 h). Each point and column represents the mean S.E.M. (n = 5) ***P < 0.001, **P < 0.01, *P < 0.05
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CONCLUSION
The present study demonstrates a weak antimicrobial activity compared to standard antibiotics and a good anti-inflammatory activity in chicks. Of the various extracts tested, the medium polar EtOAc extract showed the highest activity. The results support the wound healing activities of earlier reports, and provide the rationale for the ethnomedicinal use of the leaves of C. splendens in the management of inflammatory disorders. Flavonoids, tannins and alkaloids which were found present in the leaves of the plant may be responsible for these antimicrobial and anti-inflammatory activities.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
REFERENCES
1. Mshana, N. R., Abbiw, D. K., Addae- Mensah, I., Adjanouhoum, E., Ahyi, M. R. A., Odunlami, H., et al. Traditional Medicine and Pharmacopoeia; Contribution to the
Revision of Ethnobotanical and Floristic Studies in Ghana. Accra: Science and Technology Press, CSIR, 2000:587 Burkhill HM. Useful Plants of West Africa. Kew: Royal Botanic Gardens, 1985:130. Shehata AH, Yousif MF Soliman GA. Egypt J. Biomed. Sci. 2001; 7:145. , Gbedema, S. Y., Kisseih, E., Adu, F Annan, K. and Woode, E. Pharmacognosy ., Research 2010; 2:63. Jung-Ho C, Wan-Kyun W, and Hong-Jin K. Arch. Pharmaceut. Res. 2003; 27:189. Sofowora A. Medicinal plants and Traditional medicine in Africa. Ibadan: Spectrum Books Ltd, 1993 Harborne JB. Phytochemical methods: A guide to modern techniques of plant analysis. London: Chapman and Hall, 1973. Roach JT, Sufka KJ. Brain Res. 2003; 994:215 Afolayan AJ, Aliero AA. Afr. J. Biotechnol. 2006; 5:369. Cowan MM. Clin. Microbiol. Rev.1999; 12:564. Di Rosa M, Willoughby DA. J. Pharm. Pharmacol. 1971; 23:297. Di Rosa M. J. Pharm. Pharmacol. 1972; 24:89. Vinegar R, Schreiber W, Hugo R. J. Pharmacol Exp. Ther. 1969; 66:96.
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Research Letter Chemical Examination and Hair Growth studies on the Rhizomes of Hedychium spicatum Buch.-ham
G. Venkateswara Rao*, T. Mukhopadhyay, M. S. L. Madhavi, S. Lavakumar
M/s. CavinKare Research Centre, 12, Ekkattuthangal, Chennai-600 032, India
ABSTRACT: The hexane extract of the rhizomes of H. spicatum yielded two known compounds, pentadecane, and ethyl p-methoxycinnamate. The structures of these compounds were established by spectroscopic data (UV, IR, GC, 1H and 13C NMR, Mass) and comparison with an authentic compounds. The crude extract, fractions and one of the isolated compounds showed hair growth property. KEYWORDS: Hedychium spicatum, rhizomes, pentadecane, hair growth activity.
INTRODUCTION
Hedychium spicatum (Zingiberaceae), also known as spiked Ginger Lily is employed in the preparation of Abir, a fragrant coloured powder used during the Holi festival. The rhizomes possess strong aromatic odour and bitter camphoraceous smell. The rhizomes of the plant have been used in the preparation of cosmetic powders used for promoting hair growth. The rhizomes are also considered to have insect-repelling properties and are used for preservating clothes. The rhizomes are stomachic, carminative, stimulant and tonic, and are used in dyspepsia in the form of powder or decoction.[1] The rhizomes are much used in veterinary medicine.[2] The prior literature on Hedychium spicatum reveals that the cosmetic composition containing this plant extract regulates the firmness, tone or structure of skin or regulate wrinkles.[3] The compositions containing extract of Hedychium spicatum are useful for treating Tinea infections by topical application.[4] The ethanolic extract of rhizomes of H. spicatum possessed anti-inflammatory and analgesic activity. The anti-inflammatory activity was found in the hexane fraction and the compound hedychienone was found responsible for such activity and the analgesic activity was found in benzene fraction. [5] The cinnamic acid ester, obtained from the extracts of H. spicatum and Alpinia galanga and the same has been patented for natural sunscreen property.[6] The essential oil extracted from the rhizomes of H. spicatum was evaluated for in-vitro pediculicidal
activity at 1, 5 and 25% level. At all the three concentrations, the essential oil showed more significant activity than 1% permethrin based product.[7] Previous reports on this plant occurring in different regions yielded, furanoditerpenoids,[8] terpenoids,[9-10] steroids[11] and aromatic esters.[1] However, no information was available on the preparation of an appropriate selective extract or fraction of the plant and its efficacy directed towards promoting hair growth or retarding hair fall or isolation of hair growth active compounds based on bioassay. In continuation of our interest on the isolation of biologically active molecules from medicinal plants for personal care applications,[12-21] we have undertaken the chemical examination of the rhizomes of H. spicatum. The present study describes the isolation of two known compounds, pentadecane (1) and an aromatic ester, ethyl p-methoxycinnmate (2) and hair growth studies of crude hexane extract, fractions and active compound.
MATERIALS AND METHODS

Melting points reported are uncorrected. UV spectra were recorded on Shimadzu UV spectrophotometer. IR spectra were recorded on a Shimadzu IR prestige 21. GC spectra were recorded in Shimadzu GC-17A capillary GC. 1H and 13C NMR spectra were recorded on a Bruker AMX 400 in CDCl3 with TMS an internal standard and the chemical shifts being represented in parts per million (ppm, d values). GC-MS mass spectrum on a Jeol SX 102/DA 6000 mass spectrometer. Column chromatography was performed on silica gel (100-200 mesh, Acme synthetic chemicals, Mumbai, India). Fractions and purity of the compounds were monitored by analytical thin layer chromatography (TLC) and the spots were visualized by exposure to iodine vapour or 5% sulphuric acid in methanol followed by heating the plate at
General
*Correspondence: Dr. G. Venkateswara Rao, Principal Scientist, CavinKare Research Centre, Chennai - 600 032. E-mail: rao.gv@cavinkare.com DOI: 10.5530/pc.2011.1.7
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Rao, et. al.: Chemical Examination and Hair Growth studies on the Rhizomes of Hedychium spicatum Buch.-ham
110C for 5 min. The TLC was performed on pre-coated silica gel plates (aluminium sheets 20X20 cm, silica gel 60 F254 plates of Merck KGaA, Germany). All solvents and reagents used were of analytical grade obtained from Merck. Pentadecane was obtained from M/s. Sigma aldrich, USA.
Plant material
food and water ad libitum. The floor mat husk in each cage was removed and laid afresh on daily basis.
Hair growth activity in vivo
The rhizomes of Hedychium spicatum were obtained from bazaar in December, 2007 and was authenticated by Dr. P. Santhan, botanist, M/s. Durva Herbal Centre, Chennai. A voucher specimen was deposited in M/s. CavinKare Research Centre, Chennai. The air dried and finely powdered rhizomes (2.2 kg) were extracted with hexane through soxhlet apparatus for 8 hrs. The dilute extract was filtered and evaporated to dryness in vacuo using a rotary evaporator at 40oC to get crude hexane extract (33g). The crude hexane extract was submitted for hair growth studies and found to shown good hair growth. Part of the crude hexane extract (30g) was subjected to column chromatography eluted with hexane, hexane: chloroform (1:1, 1:3) and chloroform to get corresponding fractions 4.3g (Fr. I), 15.5g (Fr. II) and 8.8g (Fr. III), respectively. All three fractions were submitted for hair growth studies along with crude hexane extract. Out of three, fraction I showed good hair growth promotion activity. Part of the fraction I, 1.0g was subjected to normal silica gel chromatography followed by repeated silver nitrate impregnated column chromatography with hexane: chloroform (95:5) yielded colorless compound 1 (130mg). Compound 2 (1.6 g) obtained from fraction II as colorless solid which was further crystallized in hexane to afford colorless crystalline compound. Compound 1: Colorless oil; 1H NMR (400 MHz, CDCl3): d 0.86 (6H, s), 1.26 (26H, s). 13C NMR (100 MHz, CDCl3): d 14.3 (C-1,15), 22.9 (C-2,14), 29.3 (C-3,13), 29.6 (C- 4 to12). Compound 2: Colorless crystals; mp = 49-50oC; IR (KBr): 2931, 1711, 1605, 1512, 1250, 1150, 830 cm1; 1H NMR (400 MHz, CDCl3): d 7.62 (1H, d, J = 16.0 Hz), 7.45 (1H, d, J = 8.8Hz), 6.88 (1H, d, J = 8.8Hz), 6.29 (1H, d, J = 16.0Hz), 4.25 (2H, q, J = 7.1Hz), 3.82 (3H, s), 1.32 (3H, t, J = 7.1Hz) ; 13C NMR (100 MHz, CDCl3): d167.5, 161.1, 144.2, 129.8, 129.4, 127.3, 115.8, 114.4, 114.4, 60.5, 55.4, 14.3.
Hair growth promotion activity Extraction and isolation
The hair on the dorsal portion of the body of each animal was depilated using a standard, commercially available depilatory cream. After removal of the hair, the skin was cleaned with distilled water and wiped with surgical spirit. Four centimeter square area in the depilated dorsal skin was marked with permanent ink marker. The animals which showed any skin irritant response to the depilatory were removed from the experiment and new animal was replaced. The rats were divided into 3 groups of 6 animals each. Group 1 animals were served as negative control without any treatment. The negative control comprised of the vehicle for application (only) without having any active extract/fraction/compound. Group 2 animals were applied 50 micro liters of commercial 2% Minoxidil solution in the pre defined area. The group 3 animals were applied samples (extract/fractions/compounds) prepared in liquid paraffin at 2%. The quantity of the solution used for the experiment was 50 micro liters per 4 cm sq area per animal. The application of the Minoxidil and the test samples were continued for 30 days. The observations such as hair growth initiation time in days and hair growth completion time in days were recorded for all the animals on daily basis. The hair growth initiation time was defined as the presence of new hair in the treated site of 4 cm sq area. The hair growth completion time was defined as complete filling of hair in the treated site of 4 cm sq area in each animal which become indistinguishable from the adjacent untreated portion of the body. The average of hair growth initiation time and hair growth completion time was calculated for each group along with control animals. The untreated control for hair growth initiation time (HGIT) is 10 days and hair growth completion time (HGCT) is 30 days. The percentage reduction in hair growth completion time (% Reduction in HGCT) for the treatment is calculated by the formula given below. The results of hair growth activity are shown in [Table 1]. Calculation = HGCT in untreated control HGCT in test sample 100 HGCT in untreated control
Table 1: Comparison of in-vivo hair growth promotion activity

Extract/ Fraction/ Compound Hexane extract Fraction 1 Pentadecane Minoxidil Untreated control Hair growth initiation Time (HGIT in days) 8 8 7 6 10 Hair growth completion Time (HGCT in days) 20 20 21 16 30 % Reduction in time 33 33 30 47 0
The hair growth promotion activity was studied by using in vivo animal model[15],[22]. Animals: Female Wistar rats weighing 120-150 g, from Dr. MGR Janaki College, Chennai were used for hair growth study. Based on the guidelines of the ethical committee of the college, the animals were maintained in a clean cage and were provided with
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RESULTS AND DISCUSSIONS

The initial screening of the hexane extract of the rhizomes of H. spicatum showed positive response in hair growth promotion activity. The bioassay guided purification of the hexane fractions of the rhizomes of H. spicatum repeated chromatography with a silica gel and re-crystallization with solvents furnished pentadecane and ethyl p-methoxycinnamate. The structure of the compounds were elucidated on the basis of UV, IR, GC, 1H and 13C NMR and Mass spectral data and comparison with an authentic samples. The hair growth promotion activity of pentadecane showed good reduction in hair growth time, where as minoxidil, a positive control showed an excellent activity in the standard method but it had other side effects[23]. Even though the plant is being used in the preparation of hair oils, so far no reports on the compounds responsible for hair growth promotion activity. The compound 1 was readily recognized as hydrocarbon by its preliminary spectral data. Its molecular formula was established as C15H32 by GC-MS, M+ 212. Its IR and UV spectra showed no characteristic peaks. Its proton spectrum showed only two peaks: methyl at d 0.86 (s) and methylene at d 1.26 (s). Its carbon spectrum showed the presence of four signals. It showed methyl and methylene carbons. Its mass spectrum showed m/z value 212. Based on the spectral data the compound has been identified as pentadecane.[24] To confirm further the compound, pentadecane has been purchased from M/s. Sigma-Aldrich, USA and analyzed by GC along with compound 1. The retention time of both the compounds were exactly matching with each other. Thus, the compound 1 has been established as pentadecane. The compound 2 was identified as colorless crystals from hexane: chloroform, mp:49-50oC. It was readily recognized as aromatic acid ester based on its preliminary spectral data. Its molecular formula was established as C12H14O3 by GC-MS, M+ 206. The IR spectrum showed the presence of an ester peak at 1711cm-1 in the molecule. Its proton spectrum showed the presence of four aromatic protons at d 7.45 (2H, d, J = 8.8Hz) and 6.88 (2H, d, J = 8.8Hz), one aromatic methoxyl group at d 3.82 (3H, s), two double bond protons each showed as doublet at d 7.62 and 6.29 (J = 16.0Hz), one oxymethylene group at d 4.25 (2H, q, J = 7.1Hz), one methyl at d 1.32 (3H, t, J = 7.1Hz). Based on the aromatic proton integration, the molecule has 1,4 di-substitution patteren. The two olefinic protons showed large coupling constant indicates that these two protons are in trans position. The carbon spectrum showed total of 12 carbons including ester carbonyl at d 167.5. Out of twelve, eight double bond carbons at d 161.1, 144.2, 129.8, 129.4, 127.3, 115.8, 114.4, 114.4, of which six aromatic and two olefinic carbons. It also showed one methoxy carbon at d 55.4, one oxy methylene carbon at d 60.5 and one methyl carbon at d 14.3. By revealing the literature, the spectral data of the compound 2 is exactly matching with those of previously reported values. So, the compound 2 has been identified as ethyl p-methoxycinnamate.[24-25]
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1 O O MeO 2 CH3
Figure 1: Compounds from H. spicatum
The results of hair growth promotion (Table 1) showed that crude hexane extract was required less time than pure compound, pentadecane. It is worth mentioning that many crude extracts or active fractions are showing better activity than individual compounds.
CONCLUSION
To our best knowledge, the present study is the first report of the isolation of active compound from Hedychium spicatum for hair growth studies.
ACKNOWLEDGEMENT
We thank Mr. C.K. Ranganathan, CMD and of CavinKare Pvt. Ltd., Chennai for his interest, constant encouragement and providing necessary facilities. We are also thankful to Dr. K. S. Rao for isolating the compounds.
REFERENCES
1. 2. 3. 4. 5. The Wealth of India, CSIR, New Delhi, 2001, vol. 5, 11, Tayal JN and Dutt S. Proc Nat Acad Sci India 1940; 10A:47-51. Martin KM and Saliou C. Compositions containing H. spicatum and use thereof. PCT Int Appl.WO 2002; 02056859. Chuahan VS, Satyan KS and Kadam KP Herbal compositions for Tinea infections. . US patent 2009:7635493. Srimal RC, Sharma SC and Tandon JS. Anti-inflammatory and other pharmacological effects of Hedychium spicatum (Buch-Hem). Ind J Pharmacol 1984; 16:143-147 Mitra SK, Babu UV and Ranganna MV. Natural sunscreen compositions and processes for producing the same. US Patent 2007; 7311896. Varsha J, Anagha K and Kadam VJ. In-vitro pediculidical activity of H. spicatum essential oil. Fitoterapia 2007; 78:470-473. Sharma SC,Tandon JS and Dhar MM. 7-Hydroxyhedychenone, a furanoditerpene from H. spicatum. Phytochem 1976; 15:827-828. Joshi S, Chanotiya CS, Agrwal G, Prakash O, Pant AK and Mathela CS. Terpenoid compositions and antioxidant and antimicrobial properties of the rhizomes essential oils of different Hedychium species. Chemistry and Biodiversity 2008; 5:299-309. Botini AT, Garfagnoli DJ, Delgado LS, Dev V, Duong ST, Kelley CG, Keyer R, Raffel R, Joshi P and Mathela CS. Sesquiterpene alcohols form H. spicatum var. acuminatum. J Nat Prod 1987; 50:732-734. Shekhar CS, Shukla YN and Tandaon JS. Alkaloid and terpenoids of Ancitrocladus heyneanus, Sagittaria sagitifolia, Lyonia Formosa and H. spicatum. Phytochem 1975; 15:578-579. Rao GV, Annamalai T, Mukhopadhyay T and Madhavi MSL. Chemical constituents and melanin promotion activity of stems of Cissus quadrasngularis Linn. Res J Chem Sci 2011; 1, 24-28.
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Rao GV, Annamalai T and Mukhopadhyay T. Chemical examination and biological studies on the bark of Crataeva nurvala Buch.-Ham. Pharmcog J 2011; 3:1-4. Rao GV, Mukhopadhyay T, Annamalai T, Radhakrishnan N and Sahoo MR. Chemical examination and biological studies of Origanum vulgare Linn.. Phar. Res 2011; 000 Rao GV, Annamalai T and Mukhopadhyay T. Phytochemical investigation and hair growth studies on the rhizomes of Nardostachys jatamansi DC. Pharm. Mag 2011; 7:142-146 Rao GV, Mukhopadhyay T and Radhakrishnan N. Artoindonesianin F a potent , tyrosinase inhibitor from the roots of Artocarpus heterophyllus Lam. Ind J Chem 2010; 49B:1264-1266. Rao GV, Rao KS, Annamalai T, Radhakrishnan and Mukhopadhyay T. Chemical Constituents and Mushroom Tyrosinase Inhibition Activity of Chloroxylon swietenia Leaves. Turk J Chem 2009; 33:521-526. 18. Rao GV, Rao KS, Annamalai T and Mukhopadhyay T. A new coumarin derivative from the leaves of Chloroxylon swietenia DC. Ind J Chem 2009; 48B: 1041-1044. Rao GV. Chemical constituents of Adiantum genus: A review. Indian Drugs 2008; 45:837-858.
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Rao GV, Annamlai T and Mukhopadhyay T. Nardal, a new sesquiterpene aldehyde from the plant, Nardostachys jatamansi DC. Ind J Chem 2008; 47B: 163-165. Rao GV. Chemical constituents and biological studies of Chloroxylon swietenia DC: A review. Indian Drugs 2008; 45:5-15. Adirajan N, Ravikumar T, Shanmugasundaram N and Mary B. In vivo and in vitro evaluation of hair growth potential of Hibiscus rosa-sinensis Linn.J.Ethnopharmacol 2003; 88:235-238. Semalty M, Semalty A, Joshi GP and Rawat MS. Development and in vivo studies of Herbal hair oil for hair growth promotion. Indian Drugs 2010; 47:28-32. Yu J, Yu D, Sun L, Zhang S, Zheng C and Chen Y. The chemical constituents of diterpenoids from Kaempferia marginata Carey. J Chinese Pharm Bull 2010; 10:61-64. Benjamin L, Arno D, Maria THF Andreas J and Ramon RT. A practical, efficient , and atom economic alternative to the Wittig and Horner-Wadsword-emmons reactions for the synthesis of (E)- ,-unsaturated esters from aldehydes. Tetrahedron 2006; 62:476-482.
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World Wide Web Inside Pharmacognosy A Blog

I.E. Cocka,b*
Editor in Chief, Pharmacognosy Communications
a b
Biomolecular and Physical Sciences, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Brisbane, Queensland 4111, Australia. Environmental Futures Centre, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Brisbane, Queensland 4111, Australia.
Pharmacognosy Network Worldwide (www.phcog.net) has established a blog for researchers interested in Pharmacognosy and medicinal plant research. The Inside Pharmacognosy A Blog website can be accessed at http:// www.pharmacognosy.in/. The blog combines reviews of new publications related to this expanding field with profiles of international departments and institutes that are engaged in Pharmacognosy research. A recent visit to the website showed 14 reviews of Pharmacognosy related books and texts, 4 profiles of Pharmacognosy research departments and details of an independent pharmacognosy consulting service, for the month of May 2011 alone. The blog began in October 2010 and has been steadily growing since, to reach its current size.
Archives of previous posts are also readily available for access by readers of the blog. The blog also serves to notify readers of the publication of new issues of journals under the Pharmacognosy Network umbrella. The names, scope and contacts of other related journals within the pharmacognosy/natural product/plant science fields are also provided in the blog. This is a valuable resource for researchers deciding for which journal their research is best suited. The blog is well set out and easy to use. The reader can either read the latest posts or search for relevant articles under the categories of books, databases, departments worldwide, journals, organisations/associations and resources. Whether you are involved in phytochemical studies, bioactivity investigations or ethnobotanical research, Inside Pharmacognosy A Blog is worth visiting, bookmarking and/or signing up for the blog newsletter (this is a free notification service). I encourage all researchers in pharmacognosy and related fields to read this site and submit relevant articles. I reviewed this site on 3rd June 2011.
*Correspondence: Tel.: +61 7 37357637; fax: +61 7 37355282 E-mail: editor@phcogcommn.org, I.Cock@griffith.edu.au DOI: 10.5530/pc.2011.1.8
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Medicinal Plant Images Eucalyptus ficifolia and Xanthorrhoea johnsonii
Figure 1: Eucalypts are the most iconic Australian medicinal plants and are possibly the most useful commercially for their medicinal properties (including antimicrobial, insect repellent, pesticidal, anticough and decongestant bioactivities). Eucalyptus is a diverse genus of trees in the family Myrtaceae. Of the more than 700 species that comprise this genus, most are endemic to Australia. A smaller number are also native to New Guinea, Indonesia and the Philippines. Pictured is the red flowering species Eucalyptus ficifolia (also known as Corymbia ficifolia). Photograph taken in Brisbane Australia by Dr Ian Cock.
Figure 2: The genus Xanthorrhoea (Australian grasstrees) is a small genus of slow growing and very long living plants endemic to Australia. The leaves of Xanthorrhoea johnsonii (pictured) have recently been shown to have an anaesthetic effect (Cock and Kalt, 2010) similar to the effects previously described for tubocurarine, dimethyltubocurarine and alcuronium (collectively known as curare, a South American arrow poison) from Chondrodendron tomentosum. Photograph taken in Toohey Forest, Brisbane Australia by Dr Ian Cock.
Cock IE, Kalt FR, Toxicity evaluation of Xanthorrhoea johnsonii leaf methanolic extract using the Aretemia franciscana bioassay. Phcog Mag 2010; 6, 23: 166-171.
DOI: 10.5530/pc.2011.1.9
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Department Profile Biomolecular and Physical Sciences, Griffith University, Australia

I.E. Cocka,b*
Editor in Chief, Pharmacognosy Communications
a b
Biomolecular and Physical Sciences, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Brisbane, Queensland 4111, Australia. Environmental Futures Centre, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Brisbane, Queensland 4111, Australia
Upcoming issues of Pharmacognosy Communications will be feature departmental profiles from the authors and readers of Pharmacognosy Communications. To begin, I have included a profile of my own department, Biomolecular and Physical Sciences, at Griffith University, Australia. We welcome departmental profile contributions from all regions of the world where pharmacognosy research and studies occur. Griffith University consists of five main campuses in the Brisbane and Gold Coast region of southeastern Queensland, Australia. The university has diverse and unique settings, with two campuses sitting in a bushland/nature conservation area (Nathan and Mt Gravatt campuses), one campus in a rural setting bordered by farmland and a golf course (Logan campus), one campus in an urbanised coastal region (Gold Coast campus) and another campus in the central business district (CBD) of Brisbane (Southbank campus). The University currently has approximately 40,000 students and 4,000 full time equivalent staff. Science, Environment, Engineering and Technology (SEET) is one of four main academic groups/ faculties that comprise the university. SEET is further divided into individual schools. The School of Biomolecular and Physical
*Correspondence: Tel.: +61 7 37357637; fax: +61 7 37355282. E-mail: editor@phcogcommn.org, I.Cock@griffith.edu.au DOI: 10.5530/pc.2011.1.10
Sciences (BPS) is one of four schools that comprise SEET (the others being Engineering, Environment and Information and Communication Technology). BPS offers degree programs and postgraduate studies in diverse fields including the physical sciences, biomolecular and biomedical sciences, medical science, forensic sciences and aviation. Both traditional and emerging science disciplines are taught. The facilities include modern research facilities, with access to most modern technologies. BPS researchers undertake a diverse range of research with projects including but not limited to: Medicinal agents discovery from Australian and international plants and fungi. Mechanistic studies into the toxicity of Australian native plants. Cancer drug discovery and cancer therapies. Molecular modelling for drug discovery and design. Novel antimicrobial agents, antimicrobial mechanisms and antimicrobial therapies. Antimalarial drugs and antimalarial therapies. Ataxia telangiectasia. Neurodegenerative disorders, treating Parkinsons and Alzheimers Diseases. Stem cell research and stem cell therapies. Cytotoxic natural products from marine invertebrates. Ecosystem restoration. Drug design with novel target proteins to fight parasitic diseases. The molecular basis of symbiosis in insects.
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Cock: Biomolecular and Physical Sciences, Griffith University, Australia
The replicative mechanisms of thermophilic bacteria. Investigation of metal based small molecule drug targets. Molecular probes for pancreatic cancer. Novel antimicrobial agents from bacteriophage proteins that interfere with DNA replication. Novel therapeutics for Human African trypanosomiasis. Protein engineering of variants of the Green Fluorescent Protein (GFP). Regulation of apoptosis (programmed cell death).
Regulation of cell surface sialylation by targeting the CMPsialic acid transporter: towards the development of antimetastatic agents. The role of Semaphorins in the immune system, neuronal development and cancer development. The use of natural product scaffolds in the generation of novel chemical libraries. Transcriptional control of gametocytogenesis. Wolbachias role in nematodes. Natural compounds from traditional Chinese medicine (TCM). This is by no means a complete listing of the research projects undertaken in BPS at Griffith University. For a more comprehensive and up to date listing, see the Griffith University web site.[1] New projects will be listed on this site as they become available.
Figure 2: Research postgraduate students in a Biomolecular and Physical Sciences research laboratory at Nathan campus.
Figure 1: The diversity of Griffith Universitys campuses: (a) Nathan campus, (b) and (c) the unique bushland setting of Nathan campus, (d) Mt Gravatt campus, (e) Mt Gravatt campus surrounded by Toohey Forest, (f) Logan campus, (g) the rural setting of farmland adjoining Logan campus, (h) Southbank campus, (i) the river side setting of Southbank campus, (j) Gold coast campus and (k) the coastal setting surrounding Gold Coast campus.
Figure 3: Dr Derek Kennedy of Biomolecular and Physical Sciences instructing a student in a laboratory on Gold Coast campus, Griffith University.
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Cock: Biomolecular and Physical Sciences, Griffith University, Australia
Griffith University in general and BPS specifically provides an outstanding research environment for its staff. Recently, Excellence in Research for Australia (ERA) ranked the university in the top eight research universities in Australia.[2] Forty five research disciplines within the university were regarded as performing above world standard with some research fields (including the physical sciences) awarded the highest possible ranking for outstanding research. Indeed, 93% of the universitys researchers have been assessed by ERA as being world standard or better. Furthermore, recent Nature rankings (based on the number of primary research articles published in the Nature family of journals in a one year period) ranked Griffith University seventh
in Australia and 30th in the Asia-Pacific region for research outputs.[3] The university is currently experiencing rapid growth and whilst it already outperforms many larger universities, the universitys administration is predicting further improvement in Griffith Universitys ranking in future years.
RefeRenceS
1. 2. 3. http://www.griffith.edu.au/science-aviation/school-biomolecular-physicalsciences/research/research-projects http://www3.griffith.edu.au/03/ertiki/tiki-read_article.php?articleId=28602 http://www3.griffith.edu.au/03/ertiki/tiki-read_article.php?articleId=29462
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Upcoming Events
August 11-14, 2011 8th Brazilian Symposium of Pharmacognosy and 11th International Symposium of the Brazilian Society of Pharmacognosy, Brazil. http://www.unb.br/fs/farmacognosia/ 7th European Conference on Marine Natural Products, Strmstad, Sweden. http://fkogserver.fkog.uu.se/7ecmnp/ International Symposium on Medicinal and Aromtaic Plants, Flores Petn, Guatemala. http://www.imaps2001-peten.org/ 59th International Congress and Annual Meeting of the Society for Medicinal Plant and Natural Producy research, (GA2011), Antalya, Turkey. http://www.ga2011.org/ 42nd International Symposium on Essential Oils (IESO 2011), Antalya, Turkey. http://www.iseo2011.org
August 14-18, 2011 August 17-19, 2011 September 4-9, 2011
September 11, 2011
September 27-30, 2011 PSE Conference Bari: Phytochemicals in Nutrition and Health, Giovinazzo (Bari), Italy. http://www.phytochemicalsociety.org/bari October 17-20, 2011 November 3-6, 2011 5th InternationaL Conference on Polyphenols and Health (ICPH), Barcelona, Spain. http://www.icph2011barcelona.com/ BITs 9th annual Congress of International Drug Discovery Science and Technology (IDDST), Shenzen, China. http://www.iddst.com/iddst2011-06-01
December 10-15, 2011 Phytochemical Society of North America, 50th annual meeting, Fairmont Orchid, Kohala Coast Hawaii. http://psna.uhhconferencecenter.com/
DOI: 10.5530/pc.2011.1.11
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figures are accepted provided they are of high quality. Please note that graphs produced by many statistical packages are rarely adequate. In particular, letter quality on axes and captions are often poor. Such figures should be exported into an accepted graphics package and lettering rendered using a text function. Authors should note that .dot, .bmp, and .pat fills should be avoided. Do not use postscript fill patterns. When filling illustrations, use fills such as lines, tints or solids. Line width minimum is 0.25 pt (0.09 mm). Also avoid the use of bitmap scans to render text and detail. Text should be saved as text at a minimum text size of 6 pt (2.1 mm). Submit line art as Corel Draw, Adobe Illustrator, or EPS files. These must be at a minimum resolution of 800 DPI at publication size. High resolution may be necessary where fine line detail is present. For graphs, Excel graphs are also acceptable. Note that vertical axes must all be at the same scale especially when the paper compares them. Otherwise they should be produced as separate figures. Avoid 3D plots when presenting 2D data. All tables and figures must be placed in appropriate places in the manuscript and when this is not possible, appropriate place must be indicated in the manuscript. Please note, good quality figures must be submitted as separate files as outlined above, apart from presenting a copy of the same at appropriate places in the manuscript. Figures, tables or other materials copied verbatim or adopted from previously published materials, the author must have written permission from the the copyright holder of that material (publisher and/or authors) for reproduction in your article. A copy of the permission release must be submitted with the manuscript. It is the authors responsibility to obtain permission.
new lead anti-malarial compounds, several research groups screen plant extracts to detect secondary metabolites with relevant biological activities that could serve as templates for the development of new drugs. Flavonoids have been isolated and characterized from many medicinal plants used in malaria endemic areas.[1-2] However, controversial data have been obtained regarding their antiplasmodial activity, probably because of their structural diversity.[3,5,6] More recently, several flavonoids have been isolated from Artemisia afra[7] and Artemisia indica[8] two plants related to Artemisia annua, the famous traditional Chinese medicinal plant from which artemisinin is isolated.
rEFErEncE stylE
Journal References 1. Standard journal article
Single/Multiple Authors: List the first six authors followed by et al. (Note: NLM now lists all authors.)Halpern SD, Ubel PA, Caplan AL. Solid-organ transplantation in HIV-infected patients. N Engl J Med. 2002 Jul 25; 347(4):284-7. As an option, if a journal carries continuous pagination throughout a volume (as many medical journals do) the month and issue number may be omitted. Halpern SD, Ubel PA, Caplan AL. Solid-organ transplantation in HIV-infected patients. N Engl J Med. 2002; 347:284-7. More than six authors: Rose ME, Huerbin MB, Melick J, Marion DW, Palmer AM, Schiding JK, et al. Regulation of interstitial excitatory amino acid concentrations after cortical contusion injury. Brain Res. 2002; 935(1-2):40-6. Organization as author: Diabetes Prevention Program Research Group. Hypertension, insulin, and proinsulin in participants with impaired glucose tolerance. Hypertension. 2002; 40(5):679-86. Both personal authors and an organization as author: Vallancien G, Emberton M, Harving N, van Moorselaar RJ; Alf-One Study Group. Sexual dysfunction in 1,274 European men suffering from lower urinary tract symptoms. J Urol. 2003; 169(6):2257-61.
2. Journal article on the Internet
tAblE And FIgurE cAPtIons

Figure captions/legends should be single spaced and typed in the journal format. Explanations should be brief and authors should keep in mind that captions/legends will be placed below figures. Tables are to be incorporated at the end of Manuscript.
AcKnowlEdgEMEnts
Those who have helped the authors carry out the study and/or prepare the manuscript but have not made significant intellectual contribution to deserve authorship must be acknowledged. Mention all applicable grants and other funding that supported the work.
rEFErEncEs
In-text citation Correct/Acceptable Format
Saraswathy A, Shakila R, Sunilkumar KN; Phcog.Net. HPTLC Fingerprint Profile Of Some Cinnamomum Species Pharmacognosy Journal [Phcog J]. Pharmacognosy Journal. 2010 April; 2(8):211-215. Available from: http://phcogj.com/content/ hptlc-fingerprint-profile-some-cinnamomum-species. Hussain A, Mohammed S, Rizvi A, Wahab S; Phcog.Net. Pharmacognostical Standardization of Stem Bark of Adenanthera
Natural products have proven to be a great source of new biologically active compounds. Thus, in an effort to discover
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pavonina L. Pharmacognosy Journal. 2010 April; 2(8):240-246. Available from: http://phcogj.com/content/pharmacognosticalstandardization-stem-bark-ad.... Abood S. Quality improvement initiative in nursing homes: the ANA acts in an advisory role. American Journal of Nursery [serial on the Internet]. 2002 June [cited 2002 Aug 12]; 102(6): [about 3 p.]. Available from:http://www.nursingworld.org/ AJN/2002/june/Wawatch.htm
3. Book author(s)
8. Conference proceedings
Harnden P, Joffe JK, Jones WG, Editors. Germ cell tumours V. Proceedings of the 5th Germ Cell Tumour Conference; 2001 Sep 13-15; Leeds, UK. New York: Springer; 2002.
9. Thesis
Senol FS. Pharmacognosic research on some Salvia species growing in Turkey. M.Sc. Thesis, Institute of Health Sciences, Gazi University, Ankara, Turkey, 2009. Website information Cancer-Pain.org [homepage on the Internet]. New York: Association of Cancer Online Resources, Inc.; c2000-01 [updated 2002 May 16; cited 2002 Jul 9]. Available from: http://www.cancer-pain.org/. Manuscript Submission Manuscripts may be submitted electronically through the online submission at the journals web site (http://phcogcommn.org/ home). Alternately, manuscripts may be submitted by email submissions@phcogcommn.org. All submissions are peer reviewed by the editorial board and a select group of reviewers. Please make sure that all guidelines are followed carefully. All the accepted articles will be queued for publication and will appear in the futures issues based on the priorities set by the editorial board.
10. Websites
Murray PR, Rosenthal KS, Kobayashi GS, Pfaller MA. Medical microbiology. 4th ed. St. Louis: Mosby; 2002.
4. Editor(s), compiler(s) as author
Gilstrap LC 3rd, Cunningham FG, VanDorsten JP, Editors. Operative obstetrics. 2nd Ed. New York: McGraw-Hill; 2002.
5. Author(s) and editor(s)
Breedlove GK, Schorfheide AM. Adolescent pregnancy. 2nd Ed. Wieczorek RR, Editor. White Plains (NY): March of Dimes Education Services; 2001.
6. Organization(s) as author
Royal Adelaide Hospital; University of Adelaide, Department of Clinical Nursing. Compendium of nursing research and practice development, 1999-2000. Adelaide (Australia): Adelaide University; 2001.
7. Chapter in a book
contActs
Editor-in-Chief
Meltzer PS, Kallioniemi A, Trent JM. Chromosome alterations in human solid tumors. In: Vogelstein B, Kinzler KW, Editors. The genetic basis of human cancer. New York: McGraw-Hill, p. 93-113; 2002.
Dr. Ian Cock editor@phcogcommn.org

Phcog.Net
mueen.ahmed@phcog.net
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Pharmacognosy Communications: The Scope of Pharmacognosy I.E. Cock

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Biomolecular and Physical Sciences, Griffith University, Australia.

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Editorial Pharmacognosy Communications: The Scope of Pharmacognosy

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Cock: The Scope of Pharmacognosy

Invited Review Plant Drugs Used to Combat Menace of Anxiety Disorders

Anxiety Disorders: An Overview

(c) Copyright 2011 EManuscript Publishing Services, India

Various factors causing anxiety disorders are described below.[8-9]

PLANTS HAVING ANTIANXIETY ACTIVITY

CH3 H3C O HO HO HOH2C CH3 CH2OH O OH O

R' CH3O N CH3O

(30), R=CH3 (31), R =CH2OH

H H3C N OCH3 OCH3

OCH2O OCH2O OCH2O OCH2O OCH2O OCH2O OCH3O CH3

Table 1: List of various plants reported to possess antianxiety activity.

Abies pindrow Royle (Pinaceae) Talispatra, Silver Fir, Pindrow Fir

Ethanol extract of leaves

Aqueous extract of flowers

Acorus calamus Linn. (Araceae) Bach/Bacopa monnieri Linn. (Scrophulariaceae) Brahmi

Powder of whole plant

Actaea spicata Linn. (Apiaceae) Baneberry, Grapewort

(a) Methanol extract (b) Polyphenol fraction

Ethanol extract (95%) of leaves

OFT, EPM, Acoustic startle response test

[1-(3-chlorphenyl)piperazine] induced hypolocomotion test

Albizzia julibrissin Durazz. (Fabaceae) Silktree, Mimosa, Nemunoki

Aqueous extract of stem bark

Serotonergic system Interaction with 5-HT1Areceptor

Aqueous extract of bark

Albizzia lebbeck Benth. (Fabaceae) Siris tree, Albizia

Saponins rich n-butanolic fraction of petroleum ether extract from leaves

Albino Swiss mice

Inhibition of GABAergic transmission

Anxiolytic and nootropic

Aloysia polystachya Griseb. (Verbenaceae) Burrito

Hydro-alcoholic extract (60% ethanol) of leaves

Ethanol extract of aerial parts

Essential oil from leaves

Furanocoumarin Phellopterin(2) isolated from methanol extract of roots

Aniba riparia (Nees) Mez (Lauraceae) Rosewood

Riparin III(3) isolated from unripe fruits

EPM, FST EPM, OFT, HBT OFT, EPM, HBT

Anxiolytic, antidepressant Anxiolytic Anxiolytic but devoid of sedative activity

[56] [57] [58]

Riparin I (4) isolated from unripe fruits

Riparin- III (3) isolated from unripe fruits

Annona cherimolia Mill. (Annonaceae) Cherimoya, Custard apple

Hexane extract of leaves

GABA/BZD receptor complex

Annona diversifolia Saff. (Annonaceae) Llama, Anona blanca

Palmitone(5) isolated from hexane extract of leaves

Ethanol extract of leaves

Aronia melanocarpa Michx. (Rosaceae) Black chokeberry

Azadirachta indica A. Juss. (Meliaceae) Neem tree