ANTI INFLAMMATORY PROSPECTIVE STUDY OF COMMIPHORA MUKUL (GUGGUL) ON WISTAR ALBINO RATS

4/27/2012

KRISHNAMRAJU NALIMELA

ANTI INFLAMMATORY PROSPECTIVE STUDY OF ETHANOLIC HERBAL EXTRACT OF COMMIPHORA MUKUL (GUGGUL) BARK BY ADOPTING CARRAGEENAN INDUCED PAW OEDEMA IN WISTAR ALBINO RATS
A Dissertation Submitted to the JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY HYDERABAD.

In partial fulfillment of the requirements for the award of degree of BACHELOR OF PHARMACY By N.KRISHNAM RAJU ( 08Y41R0030 ) Under the guidance of Mr. V.S. GIRI PRASAD M.Pharm Assistant Professor

R.G.R.SIDDHANTHI COLLEGE OF PHARMACY SECUNDRABAD-500034(2011-2012)

DECLARATION OF AUTHORSHIP

This research work on” Anti Inflammatory Prospective Study Of Herbal Ethanolic Extract Of Commiphora Mukul (Guggul) Bark By Adopting Carrageenan Induced Paw Oedema In Wistar Albino Rates “ is carried out by us in “RGR SIDDHANTHI COLLEGE OF PHARMACY Secundrabad, Andhra Pradesh Submitted to Jawaharlal Nehru Technological University, Hyderabad for the award of the degree Bachelor of pharmacy. We solemnly declare that this thesis either in part or in full has not been submitted to any other University or Institution for the award of any Degree or Diploma.

KRISHNAM RAJU (08Y41R0030)

Date: 27-04-2012 Place: Secundrabad

Ref:

CERTIFICATE BY THE PRINCIPAL

This is to certify that the dissertation work entitled “Anti Inflammatory Prospective Study Of Herbal Ethanolic Extract Of Commiphora Mukul (Guggul) Bark By Adopting Carrageenan Induced Paw Oedema In Wistar Albino Rats” submitted to Jawaharlal Nehru Technological University in partial fulfillment for the award of the degree of bachelor of Pharmacy, has been successfully carried out by below mentioned students during the academic year 2011-2012 under the guidance of Mr.V.S. GIRI PRASD M.Pharm.Department of Pharmacology.

KRISHNAM RAJU (08Y41R0030)

Forwarded by Mrs.Dr. K. VIJAYA, M.Pharm, PhD Principal, RGR Siddhanthi College of pharmacy Secundrabad -500003.

Ref:

THESIS CERTIFICATE BY THE RESEARCH GUIDE

This is to certify that the dissertation work entitled “Anti Inflammatory Prospective Study Of Herbal Ethanolic Extract Of Commiphora Mukul (Guggul) Bark By Adopting Carrageenan Induced Paw Oedema In Wistar Albino Rats” submitted to Jawaharlal Nehru Technological University in partial fulfillment for the award of the degree of bachelor of Pharmacy, has been successfully carried out by below mentioned students during the academic year 2011-2012 under the guidance of Mr.V.S. GIRI PRASD M.Pharm.Department of Pharmacology. KRISHNAM RAJU (08Y41R0030)

Forwarded by Mr.V.S. GIRI PRASAD Assistant Professor, Department of pharmacology, RGR Siddhanthi College of pharmacy Secundrabad -500003.

BONAFIDE CERTIFICATE

This is to certify that the project titled Anti Inflammatory Prospective Study Of Herbal Ethanolic Herbal Extract Of Commiphora Mukul (Guggul) Bark By Adopting Carrageenan Induced Paw Oedema In Wistar Albino Rates is a bonafide record of the work done by KRISHNAM RAJU (08Y41R0030) In partial fulfillment of the requirements for the award of the degree of HYDERABAD, during the year 2011-2012. Project Viva-voce held on _____________________________ Bachelor of

Pharmacy of the JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY,

Internal Examiner
Mr.V.S. GIRI PRASAD Assistant Professor, Department of pharmacology, RGR Siddhanthi College of pharmacy Secundrabad -500003. Date: 27-04-2012

External Examiner

Place: Secundrabad

ACKNOWLEDGEMENT
I am thankful to the Almighty for blessings in successful completion of this dissertation. The satisfaction that accompanies the successful completion of any task would be incomplete without mention of the people who made it possible with constant guidance, support and encouragement that crows all effort with success. I would like to express my appreciation for all the efforts to everyone who have directly or indirectly contributed their ideas and energies in successful completion of my project. I take this golden opportunity to express my humble gratitude and respect to my research guide Mr. V.S. GIRI PRASAD M.Pharm. Asst.Professor, Department of Pharmacology, RGR Siddhanthi College of pharmacy, A.P. Ranjit Kumar M.Pharm Department of Pharmacology, RGR Siddhanthi College of pharmacy, for their constant invaluable guidance, encouragement and support throughout the project work. I am proud to say that it has been the most fruitful and enjoyable experience to work under their untiring guidance. Scrupulousness discipline, principle, and provision of fearless work environment will be cherished in all walks of my life. I express my gratitude and sincere thanks to Principal Dr. K. Vijaya M.Pharm PhD, RGR Siddhanthi College of pharmacy for providing facilities which enabled me to complete this work successfully. Then with deep gratitude I would like to thank Sri. P.L. Srinivas our beloved secretary of RGR Siddhanthi institutions who gave me admission into B.Pharmacy course there by making my dream of higher education possible. KRISHNAM RAJU (08Y41R0030)

DEDICATION TO

We are lovingly dedicate this project to our PARENTS GIRI and my research guide Mr. V.S. M.Pharm. Asst.Professor,

PRASAD

Department of Pharmacology, RGR Siddhanthi College of pharmacy.

ABBREVATIONS

COX NSAID ImSAIDs WHO BSA COX-1 COX-2 PGE UNIDO IL RBC WBC CSM

: : : : : : : : : : : : :

Cyclooxygenase Non-Steroidal Anti-Inflammatory Drug Immune Selective Anti-Inflammatory Derivatives World Health Organization Bovine serum albumin Cyclooxygenase 1 Cyclooxygenase 2 Prostaglandin E United Nations Industrial Development Organization. Interleukin Red Blood Corpuscle White Blood Cells committee on safety of medicines

Table of Contents
Table of Contents.................................................................................................10 ABSTRACT............................................................................................................ 12 INTRODUCTION.....................................................................................................14 TYPES OF INFLAMATION:....................................................................................15 CAUSES :...........................................................................................................16 SYMPTOMS:....................................................................................................... 17 INFLAMMATORY DISORDERS:.............................................................................17 THE MECHANISM OF ACTION OF ANTI-INFLAMMATORY DRUGS:.........................18 ANTI INFLAMMATORY DRUGS:...........................................................................19 steroids..........................................................................................................19 Classification of NSAIDs According to mechanism of action............................19 Herbs :........................................................................................................... 21 Adverse effects of NSAIDs..............................................................................21 LITRERATURE REVIEW..........................................................................................23 PLANT PROFILE.....................................................................................................27 CHEMICAL COMPOSITION / KEY ACTIVE CONSTITUENTS.....................................29 REMEDIES FOR :................................................................................................29 PHARMACOLOGY:..............................................................................................29 DOSAGE............................................................................................................ 29 USES & BENEFITS OF GUGGUL...........................................................................30 CAUTION........................................................................................................... 31 AIMS AND OBJECTIVES..........................................................................................32 AIM:................................................................................................................... 32 OBJECTIVES:......................................................................................................33 MATERIALS AND METHODS...................................................................................34 MATERIALS:....................................................................................................... 34 Soxhlet extractor :..........................................................................................35 Collection of plant materials :........................................................................36 Experimental animals: ..................................................................................37 Inflammation inducers:...................................................................................37

lab materials(plethysmo meter):....................................................................38 METHODS:......................................................................................................... 38 Preparation of extracts :.................................................................................39 Extraction process:.........................................................................................39 Liebermann – burchard test :.........................................................................41 Carrageenan-induced inflammation in Rats :..................................................41 Test for Anti inflammatory Activity:................................................................42 EVALUATION OF ANTI-INFLAMMATORY ACTIVITY................................................45 DISCUSSION:........................................................................................................ 48 CONCLUSION:.......................................................................................................50 BIBLIOGRAPHY......................................................................................................52

ABSTRACT

ABSTRACT

Guggul, the gum resin from Commiphora mukul, is one of the components of various formulations of traditional Ayurvedic medicine to treat inflammation, obesity, and lipid disorders. In most preparations of Ayurvedic medicine in India.

The Ethanolic extract of Guggul, consist of guggulusterone derivatives. The structures of these compounds were confirmed by spectroscopic methods. These compounds were assayed for cyclooxygenase (COX) enzyme inhibitory activities and inhibit the PGE synthesis also. so these presence may be the cause in curing the inflammation

COX enzyme (COX-1 and COX-2) inhibitory activities exhibited by compounds isolated from C. mukul may substantiate its use in traditional medicine. Injection of carrageenan in the rat paw causes oedema. The increase in paw volume was highest at 1hr after carrageenan injection. Anti-inflammatory agents such as guggulusterone derivatives from Commiphora mukul were administered orally at a daily dose of 200,400 and 600 mg/kg, respectively, for a period of 2 hrs.

The significant difference was seen in rats treated with Ethanolic extract of Commiphora mukul. The extract decreased the oedema of the rat paw during the course of drug treatment. Anti-inflammatory activity of the Commiphora mukul was assessed by using Plethysmograph.

These results indicate the beneficial role of gum Guggul in experimental anti inflammatory activity.

INTRODUCTION

INTRODUCTION
Inflammation is the first response of the immune system to infection or irritation and Inflammation a localized reaction that produces redness, warmth, swelling, and pain as a result of infection, irritation, or injury. Inflammation can be external or internal. Inflammation is not a synonym for infection, even in cases where inflammation is caused by infection. Although infection is caused by a microorganism, inflammation is one of the

responses of the organism to the pathogen. However it is considered as a mechanism of innate immunity, as compared to adaptive immunity, which is specific for each pathogen.

TYPES OF INFLAMATION:
Inflammation can be classified as acute inflammation and chronic inflammation 1) Acute inflammation: can lead to chronic inflammation, but the latter can also exist by itself. Acute forms that are common include: headaches, stiff joints, back pain and acne. Acute inflammation will become chronic if the immune system is unable to rid the body of the offending foreign agent or if the agent is constantly able to re-enter the body. Acute inflammation is the initial tissue reaction to a wide range of injuries or insults and may last from a few hours to a few days. The acute inflammatory response is similar whatever the causative agent. • • • • Short term (minutesàdays) Exudation of fluid, plasma, proteins,and leukocytes (neutrophils). Phagocytosis and enzymatic release occurs. Activation of neutrophils and macrophages--digest foreign material--involves recognition, attachment, engulfment, and degradation. • Recognition and attachment is enhanced when serum factors, Opsonins present immunoglobin G (Ig G), complement activated fragment C3b can adsorb to biomaterials. • Neutrophils and macrophages have receptors for these proteins.

2) chronic inflammation:The word 'chronic' applied to any process implies that the process has extended over a long period of time. This is usually the case in chronic inflammation, but here the term 'chronic' takes on a much more specific meaning, in that the type of cellular reaction differs from that seen in acute inflammation. Chronic inflammation may be defined as an inflammatory process in which lymphocytes, plasma cells and macrophages predominate, and which is usually accompanied by the

formation of granulation tissue, resulting in fibrosis. Chronic inflammation is usually primary, but does occasionally follow acute inflammation. • • • • • Long term (≥≥ days). Characterized by the presence of macrophages, monocytes, and mononuclear cells including lymphocytes and plasma cells. Accompanied by the proliferation of blood vessels and connective tissue. Lymphocytes and plasma cells are involved in the immune reactions- mediate antibody production. Macrophages process and deliver antigen to immunocompetent cells— mediate immune reactions.
Comparison between acute and chronic inflammation:

Acute

Chronic Persistent acute inflammation due to non-degradable

Causative agent

Bacterial Pathogens, injured tissues

pathogens,viral infection, persistent foreign bodies, or autoimmune reactions

neutrophils (primarily), basophils (inflammatory response), and Major cells involved eosinophils (response to helminth worms and parasites), mononuclear cells (monocytes, macrophages) IFN-γ and other cytokines, Primary mediators Onset Vasoactive amines, eicosanoids Immediate growth factors, reactive oxygen species, hydrolytic enzymes Delayed Mononuclear cells (monocytes, macrophages, lymphocytes, plasma cells), fibroblasts

CAUSES :
  

Burns Chemical irritants Toxins

     

Infection by pathogens Physical injury, or penetrating Immune reactions due to hypersensitivity Ionizing radiation Foreign bodies, including splinters, dirt and debris Trauma

SYMPTOMS:
• • • • • • • • • Pain Bruising Redness Swollen Fever Stuffy nose and head Breathing problems (asthma) Fluid retention Blood clots.

INFLAMMATORY DISORDERS:
Examples of disorders associated with inflammation include:
 

Asthma Autoimmune diseases

     

Glomerulo nephritis Hypersensitivities Inflammatory bowel diseases Pelvic inflammatory disease Rheumatoid arthritis Transplant rejection

THE MECHANISM OF ACTION OF ANTI-INFLAMMATORY DRUGS:
When there is injury to any part of the body, the arterioles (minute blood vessels) in the surrounding tissue dilate (widen). This allows an increased blood flow to the area (redness). Vaso active substances also increase the permeability (increase pore size) of these arterioles which allows blood cells, chemical mediators, blood proteins and fluid to accumulate in the area. This fluid accumulation causes swelling and may compress nerves in the area resulting in pain. Furthermore, the main chemical mediators of inflammation like prostaglandins, which are produced by cells, may also cause ‘irritation’ of the nerves and further contribute to pain. Inflammation is caused by release of chemicals from tissues and migrating cells. Most strongly implicated are the prostaglandins (PGs), leukotrienes (LTs), histamine, bradykinin, and, more recently, platelet- activating factor (PAF) and interleukin-1.

ANTI INFLAMMATORY DRUGS:
steroids • •

glucocorticoids corticosteroids

Classification of NSAIDs According to mechanism of action 1) COX-1 selective inhibitors - Acetylsalicylic acid at low dosage 2) Non selective COX inhibitors - Acetylsalicylic acid at high dosage - Diclofenac - Ibuprofen - Indomethacin - Piroxicam

- Naproxen 3) COX-2 selective inhibitors -Nimesulid - Meloxicam - Nabumeton

Therapeutic classification of NSAID s A: Analgesics Aspirin, paracetamol B: Anti-inflammatory Indomethacin, naproxen, ibuorofen C: Anti-coagulants Aspirin D: Anti-pyretics Aspirin, paracetamol, indomethacin, celecoxicv, ibuprofen E: Anti-cancer drugs Methotraxate F: Anti-malarial Chloroquine, hydroxychloroquine G: Tissue transplantation Cyclosporine H: Anti-gout drugs Indomethacin, ibuprofen

Herbs : • • • • •

Harpagophytum, hyssop, ginger, turmeric, Arnica montana

Adverse effects of NSAIDs

• • • • • • •

Gastrointestinal effects: abdominal pain, gastric and duodenal ulcer, diarrhea, pancreatis Gastrointestinal hemorrhage, hepatotoxicity Renal effect Disturbances of renal function with water and sodium retention Inhibition of platelet aggregation central symptoms: headache, decreased hearing, tinnitus, dizziness, confusion, depression Allergic reactions: asthma, rashes, photosensitivity

LITRERATURE REVIEW

LITRERATURE REVIEW
1. Department of Pharmacology and University of Pittsburgh Cancer Institute on zGuggulsterone, a constituent of Ayurvedic medicinal plant Commiphora mukul, inhibits angiogenesis in vitro and in vivo: Our previous studies have shown that z- guggulusterone, a constituent of Indian Ayurvedic medicinal plant Commiphora mukul, inhibits the growth of human prostate cancer cells by causing apoptosis. We now report a novel response to z-guggulusterone involving the inhibition of angiogenesis in vitro and in vivo. The z-guggulusterone treatment inhibited capillary-like tube formation (in vitro neovascularization) by human umbilical vein endothelial cells (HUVEC) and migration by HUVEC and DU145 human prostate cancer cells in a concentration- and time-dependent manner. The z- and E-isomers of guggulusterone seemed equipotent as inhibitors of HUVEC tube formation. The z- guggulusterone mediated inhibition of angiogenesis in vitro correlated with the suppression of secretion of proangiogenic growth factors, down-regulation of VEGF receptor 2 (VEGF-R2) protein level, and inactivation of Akt. The z-guggulusterone–mediated suppression of DU145 cell migration was increased by knockdown of VEGF-R2 protein level. Ectopic expression of constitutively

active Akt in DU145 cells conferred protection against z-guggulusterone mediated inhibition of cell migration. Oral gavage of 1 mg z-guggulusterone (five times/wk) to male nude mice inhibited in vivo angiogenesis in DU145-Matrigel plug assay as evidenced by a statistically significant decrease in tumor burden, microvessel area (staining for angiogenic markers factor VIII and CD31), and VEGF-R2 protein expression. In conclusion, the present study reveals that z-guggulusterone inhibits angiogenesis by suppressing the VEGF–VEGF-R2–Akt signaling axis. Together, our results provide compelling rationale for further preclinical and clinical investigation of z-guggulusterone for its efficacy against prostate cancer. 2. Mutsuhiro Ikuma, MD, Ph.D., FACP, AGAF, First Department of Medicine, Hamamatsu University School of Medicine on Guggulusterone Suppresses Bile Acidinduced and Constitutive Caudal-related Homeobox 2 Expression in Gut-derived Adenocarcinoma Cells:

Guggulusterone, a plant polyphenol guggulipid, has several antitumour effects and acts as an antagonist for the farnesoid X receptor. Although bile acids induce caudal-related homeobox 2 (CdX2), a transcription factor essential for intestinal development and gut tumourigenesis, the effects of guggulusterone on regulation of CdX2 in the gut are unknown. Materials and Methods: Regulation of CdX2 expression by treatment with bile acids and/or guggulsterone was analysed by immunoblot analysis in human gut-derived adenocarcinoma, Bic-1 cells. Nuclear factor-κB (NF-κB) activity and the cell cycle distribution were also examined. Results: Chenodeoxycholic acid and deoxycholic acid increased CdX2 expression in Bic-1 cells. Guggulsterone reduced bile acid-induced and constitutive CdX2 expression at 5μM. Guggulsterone (up to 5μM) did not affect cell viability or the cell cycle and did not attenuate bile acid-induced or constitutive NF-κB activation. Conclusion: Guggulsterone may be used as a novel drug to target CdX2 expression in certain gut adenocarcinomas. 3. lipid Lowering Activity of Guggulsterone from Commiphora mukul in Hyperlipaemic Rats by Ramesh Chander, A. K. Khanna, N. K. Kapoor The lipid lowering action of guggulusterone, the active constituent of guggulipid, has been studied in triton and cholesterol fed hyperlipaemic rats. Serum lipids were found to be lowered by guggulsterone (50 mg/kg, b.w.) in triton WR-1339 induced hyperlipaemia. Chronic feeding of this drug (5 mg/kg, b.w.) in animals simultaneously fed with cholesterol (25 mg/kg, b.w.) for 30 days, caused lowering in the lipid and apoprotein levels of very low density and low density lipoproteins in experimental animals. Guggulsterone activates lipolytic enzymes in plasma and liver as well as stimulated receptor mediated catabolism of low density lipoprotein. The hypolipidaemic activity of this drug is mediated through

inhibition of hepatic cholesterol biosynthesis, increased faecal bile acid excretion and enhanced plasma lecithin: cholesterol acyltransferase activity 4 .Rhabdomyolysis Caused by Commiphora mukul, a Natural Lipid-Lowering Agent by Antonio Bianchi, MD Director, Traditional Medicine Department, Centro di Orientamento Educativo, Barzio, Italy Paola Cantù, MD Head, Nephrology Department, Gallarate Hospital, Gallarate, Italy Fabio Firenzuoli, MD Phytotherapist, Director, Centre for Natural Medicine, S. Giuseppe Hospital, Empoli, Italy Gabriela Mazzanti, PhD Associate Professor, Faculty of Pharmacy, University “La Sapienza,” Rome, Italy.

OBJECTIVE: To report a case of rhabdomyolysis caused by Commiphora mukul, a natural

lipid-lowering agent.
CASE SUMMARY : 55-year-old man was taking an extract of C. mukul 300 mg 3 times daily

to lower his cholesterol level. He developed rhabdomyolysis with hemoglobinuria after 2 weeks of treatment. Laboratory tests showed creatine kinase 144 600 IU/L (reference range 24– 195), myoglobin >3000 ng/mL (28–72), lactate dehydrogenase 7157 IU/L (230–460), aspartate aminotransferase 1115 IU/L (10–35), and alanine aminotransferase 205 IU/L (10–35). Analysis of a urine sample was 2+ positive for hemoglobin. All parameters returned to normal after the herbal preparation was discontinued.
DISCUSSION: The Naranjo probability scale indicates C. mukul as the possible cause of

rhabdomyolysis in our patient. Drug-induced rhabdomyolysis is an established but rare adverse effect of high doses of cholesterol-lowering agents (statins) or interactions between drugs (eg, statins and fibrates). As of May 28, 2004, to our knowledge, this is the first reported case of rhabdomyolysis following C. mukul ingestion.
CONCLUSIONS: Our report describes a case of rhabdomyolysis possibly caused by C.

mukul and underlines the need for active surveillance of natural products.

5. Cardio protective effect of Commiphora mukul resin extract in isoproterenol induced myocardial infarction in rats by Dr.K.Kannabiran, Ms.R.Sangeetha.

The present study investigates cardio protective effect of Commiphora mukul (Guggul) resin extract on LDH, CPK, SGOT and Na+- K+ levels against isoproterenol induced myocardial infarction (MI) in rats. The male Wistar rats were randomized into four groups. Group-I rats were administered with normal saline (0.9% NaCl), Group-II rats were administered with isoprenaline (2 mg/kg body wt) whereas Group-III rats were administered with Verapamil (5µmole/kg body wt) as standard drug intravenously followed by isoproterenol after 10mins of administration. Group-IV rats were administered with Commiphora mukul (Guggul) resin extract followed by isoproterenol after 10mins of administration. After 48 hours of drug administration, it showed significant reduction in serum cardiac enzymes like LDH, CPK, SGOT and Na+- K+ levels compared with isoproterenol induced group rats which showed increase in cardiac enzymes with myofibrillar damage. Hence, the present study is the effective remedy for the myocardial infarction in rats using Commiphora mukul

PLANT PROFILE

PLANT PROFILE
Indian Name :Guggul Botanical Family Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Sapindales Family: Burseraceae Genus: Commiphora Popular Name(s): Commiphora, Mukul, Indian Bedellium Tree, Gugulipid, Mukul myrrh tree, Parts , Used: Whole guggul. Plant Name(s): Commiphora Mukul, Name: Burseraceae

Habitat: Rocky tracks of Western India and Eastern Himalayas.

INTRODUCTION
Commiphora mukul is one of the most respectable herbs known in Indian Herbal System (Ayurveda). Commiphora mukul gum resin is used in treatment of rheumatism, neurological disorders, obesity and related disorders, hypothyroidism, syphilis, skin and urinary disorders. Commiphora mukul resin oil possesses a powerful stimulant, anti-inflammatory, antioxidant, astringent.

DESCRIPTION
Guggul is a yellowish resin, which is secreted by a small, thorny mukul myrrh tree called Commiphora mukul. The shrub reaches a maximum height of 4 to 6 feet and bears thorns on its branches. The leaves are small similar to those of neem. The flowers are red and the fruit is oval in shape and pulpy in nature. The gum resin excreted by the bark of the plant is called guggul. The plant is grown throughout the north India. The herb has been playing a major role in the traditional medicine of India. It is also known as guggul gum, guggul, gugglesterone.

ORIGIN
The mukul myrrh (Commiphora mukul) tree is a small, thorny plant distributed throughout India. Guggul and gum guggul are the names given to a yellowish resin produced by the stem of the plant.

CHEMICAL COMPOSITION / KEY ACTIVE CONSTITUENTS
Ketone fraction that is extracted from the resin contains the most potent cholesterol lowering components. This is composed of C21 or C27 steroids, with the major components being Z and E-guggulsterone. Guggul contains resin, volatile oils, and gum. The extract isolates ketonic steroid compounds known as Guggulsterones. These compounds have been shown to provide the lipid-lowering actions noted for guggul.

Guggul significantly lowers serum triglycerides and cholesterol as well as LDL and VLDL cholesterols (the "bad" cholesterols). At the same time, it raises levels of HDL cholesterol (the "good" cholesterol), inhibits platelet aggregation, and may increase thermogenesis through stimulation of the thyroid, potentially resulting in weight loss. In Ayurvedic medicine, it is used to remove "Ama", or deposits of waste or toxic material in the body, including mucus and mineral deposits in the joints, Thus reducing a possible cause of sluggishness, inflamed joints, and many other conditions. A direct anti-inflammatory effect has been observed for guggulsterones. Guggul has also been shown to reduce the stickiness of plateletsanother effect that lowers the risk of coronary artery disease. REMEDIES FOR :  High cholesterol levels,
 High triglyceride levels & Atherosclerosis,  Obesity. Guglipid offers considerable benefit in preventing and treating

atherosclerotic vascular disease (Heart disease).  It is most effective in lowering LDL cholesterol and triglycerides.  It also raises the level of good cholesterol (HDL).

ADVERSE EFFECTS Studies with the crude oleoresin reported numerous side
effects, including diarrhea, anorexia, abdominal pain, and skin rash. Hence, Ayurveda has not only cautioned the use of Guggul but also given many combined formulary with other herbs which when used as medicine causes no side effects

USES & BENEFITS OF GUGGUL

Guggul possesses strong disinfecting properties and is a weight loss and fat burning It lowers elevated serum cholesterol and triglycerides, while maintaining or improving The herb increases white blood cells count and reduces the risk of coronary artery Guggul was historically used for a condition called medoroga, a disease similar to the Studies report that it increases the production of thyroid hormone. Since this hormone

agent.

the HDL to LDL ratio.

disease.

modern atherosclerosis.

breaks down cells of protein, fat and carbohydrates, this herb is believed to be promoting weight loss.

The herb is used as an anti-inflammatory and pain-relieving herb by many Indian Guggul is useful in arthritic pains and helps in reversing the degenerative changes that The herb is widely used in diseases like rheumatoid arthritis, gout, osteoarthritis, Since it acts as a blood purifier, it is widely used in skin diseases. Guggul helps in promoting the production of red blood corpuscles (RBC) and

herbal doctors.

occur in joints and bones.

sciatica, paralysis, hemiplegia, lymphadenopathy, etc.
• •

improving the action of white blood corpuscles (WBC).

It helps in strengthening the digestive system, easy secretion of digestive juices, works The herb is beneficial in hemorrhoids and colitis and relieves from hyperacidity and Regular use of guggul helps in improving sexual ability, sperm count and sperm Guggul helps in treating leprosy and eczema. Its extract also helps in fighting tumors. The herb is useful in menstrual disturbances and painful menses.

as an appetizer and avoids indigestion and constipation.

belching.

quality.
• •

CAUTION

Guggul extract can generate side effects like headache, nausea, diarrhea, anorexia, Thyroid patients should consult a doctor before using guggul extract.

abdominal pain and skin irritation in some individuals.

AIMS AND OBJECTIVES

AIMS AND OBJECTIVES
AIM:
Anti inflammatory prospective study of herbal extract of Commiphora mukkul (guggul) bark by adopting carrageenan induced paw oedama in wistar albino rats.

OBJECTIVES:

• • • • •

To evaluate the inflammation caused by carrageen, formalin, acetic acid. To evaluate the paw oedema by plethysmometer. To evaluate the efficacy of the Commiphora mukul. To study the anti-inflammatory activity of alcoholic extract of acetone extract of Commiphora mukul on carrageen induced hind paw oedema in rats. To study the (control) anti-inflammatory activity of the extracts with the standard marketing drugs.

MATERIALS & METHODS

MATERIALS AND METHODS

MATERIALS:

1)

Soxhlet extractor

2) Collection of plant materials 3) Experimental animals

4) Inflammation inducers 5)
Lab materials(Plethysmometer)

Soxhlet extractor :

1: Stirrer bar/anti-bumping granules

2: Still pot (extraction pot) 3: Distillation path 4: Soxhlet Thimble 5: Extraction solid (residue solid) 6: Syphon arm inlet 7: Syphon arm outlet 8: Reduction adapter 9: Condenser 10: Cooling water in 11: Cooling water out

Collection of plant materials : • The stem of commiphora mukkul were collected during the months of March and April from the hobby hub located at nacharam (Hyderabad). • • •

The samples were washed with distilled water and dried under shade. mechanically pounded to get coarse powder and passed through 40 number sieve meshes. The sample powders were processed in such a way that they are suitable for both powder studies and phytochemical analysis. All the stem were Shade dried at room temperature until they were free from moisture pulverized in electric grinder. The powder was obtained and extracted separately by continuous hot extraction process using soxhlet extractor.

Experimental animals:
 Healthy albino rats of either sex (Wistar strain) weighing 150‐160 g

belonging to Wistar strain were used for present study.
 The animals

had free

access to

food and water and were

maintained under controlled temperature (27±20c) and 12 h: 12 h light and da rk cycle.

Initial body weight of each animal was recorded.

Inflammation inducers:  Carrageenan  Histamine  Formalin  Acetic acid

lab materials(plethysmo meter):

 The instrument designed to measure of small volume changes. For precise and rapid screening of small rodents for inflammation or edema of the paw. Model Measurement Range Resolution Display Output Power Requirements (Rat&Mouse) 0-80 ml 0.01ml LCD Printer and RS232C 100V 50/60Hz

METHODS:
Preparation of extracts

Extraction process Carrageenan-induced inflammation in Rats Test for anti-inflammatory activity

Preparation of extracts : The coarse powder 150 grams of the given samples were extracted with 600 ml of ethanol (95%) by continuous hot percolation using Soxhlet apparatus until the completion of extraction procedure. The successive extractions were done separately for each solvent namely Chloroform, Ethanol, Ether, Benzene, Hexane and water. The powder solvent ratio employed for the present study was 1:4. On completion, the extracts were filtered and the solvents were removed by distillation and dried under reduced pressure and controlled temperature 50°-60°. They were refrigerated until use. Both stem and bark powder samples were subjected to various analyses such as organoleptic characters ,fluorescence studies physico-chemical properties ,preliminary phytochemical screening.

Extraction process: Ethanol extract: Dried stem of commiphora mukkul were reduced to a fine powder with a mechanical grinder. The powder plant material (200 g) was soaked in 3 l of 80% ethanol and stand for 3 days. The extract was concentrated to dryness and stored at a temperature of – 4oC until use. Diagrammatic representation of a soxhlet extractor: A Soxhlet extractor is a piece of laboratory apparatus invented in 1879 by Franz von Soxhlet. It was originally designed for the extraction of a lipid from a solid material. However, a Soxhlet extractor is not limited to the extraction of lipids. Typically, a Soxhlet extraction is only required where the desired compound has a limited solubility in a solvent, and the impurity is insoluble in that solvent. If the desired compound has a significant solubility in a solvent then a simple filtration can be used to separate the compound from the insoluble substance.

In order to remove the contaminants from the blubber samples, we used an extraction protocol which put them, and the lipid material, into solution. First we weighed each sample for later reference, and cut the blubber samples into small pieces. These pieces were then homogenized with a mortar and pestle in sodium sulphate, to remove any water. The homogenate was transferred to a cellulose extraction thimble, and covered with glass wool. We ran these samples in a Soxhlet extractor for 4 hours; reflux events occurred every 7-10 minutes. The sample, which has been homogenized in sodium sulphate, is covered with glass wool and contained in a porous cellulose thimble. The thimble is placed in the extraction tube, which itself sits on a flask containing an organic solvent (like hexane).

The solvent is boiled, and its vapor travels upward through the extraction tube into the condenser tube. The cool water flowing around the outside of the condenser tube condenses the vapor, which then drips into the thimble, containing the sample. Because the contaminants and lipid are soluble in organic solvents, they move into the condensed solvent as it accumulated in the thimble. The solution, now containing the contaminants and dissolved lipid, build up in the thimble. Once the liquid reaches the level of the bypass arm, it is siphoned back into the flask. This continuous condensation, buildup, and siphoning is known as the reflux event.

The advantage of the soxhlet is that once the contaminants and lipid material are brought into solution, and siphoned back into the flask, they stay in the flask--so that the sample in the extraction thimble is continuously re-exposed to fresh, heated solvent--thus greatly increasing the extraction rate. After extraction, we reduced the volume of solvent in our samples by rapid rotary evaporation down to about 2mL, using a Rotovap. After extraction the solvent is removed, typically by means of a rotary evaporator, yielding the extracted compound. The non-soluble portion of the extracted solid remains in the thimble, and is usually discarded. Liebermann – burchard test : The Liebermann–Burchard or acetic anhydride test is used for the detection of cholesterol. The formation of a green or green-blue colour after a few minutes is positive Method : Dissolve few drops of Commiphora mukul extrac in dry chloroform in a dry test tube. Add several drops of acetic anhydride and then 2 drops of conc.H2SO4 and mix carefully. After the reaction finished, the concentration of cholesterol can be measured using spectrophotometry. Carrageenan-induced inflammation in Rats : • Carrageenan is a sulphated polysaccharide obtained from sea weed (Rhodophyta). It causes inflammation by releasing histamine, 5HT, bradykinins and prostaglandins that produce inflammation and Oedema • Injection of carrageenan 1% (50 μl) in the rat paw causes a biphasic response: an early inflammatory response that lasts 6 h and a second late response that peaks at 72 h, declining at 96 h. • • Western blot analysis showed that cyclooxygenase 2 (COX-2) at 24 h point. In conclusion, rat paw oedema is biphasic and age-weight dependent. The present results are the first report on the differential expressions of COX-1 and COX-2 in response to carrageenan injection in the two phases of the rat paw oedema.

Carrageenan solution injected in the albino rat:

Test for Anti inflammatory Activity:  These extracts were tested for anti‐inflammatory activity by carrageenan (inflamagens) induced rat paw edema method.
 Different groups of animals were taken for experiment is as follows:

 Group I served as control (5 ml/kg Normal Saline)
 Group II received ethanol extract of Commiphora mukkul (100 mg/kg body

weight)  Group III served as reference standard drug (10 mg/kg b.w).

The mean paw oedema volume of the extract treated groups was compared both with control group and the standard drug Indomethacin treated groups. Thus, oedema volume in control (Vc) and extract treated groups (Vt) was computed. The percentage inhibition was calculated using the formula as shown below.

where, Vc-oedema volume of control group Vt-oedema volume of test group The results are tabulated and analysed using student's 't' test to know the level of statistical significance. Rats paw oedema: Data are expressed as mean ± s.e.m. The level of statistical significance was determined by one-way analysis of variance (ANOVA) followed by Bonferroni's t-test for multiple comparisons, using the Graph Pad Prism software. Each group of animals received subplantar administration of 50 μl of saline or 50 μl of carrageenan 1% (w /v) in saline . The paw was marked in order to immerge it always at the same extent in the measurement chamber. The volume was measured by using a hydropletismometer specially modified for small volumes immediately before subplantar injection, and 2, 4, 6, 24, 48, 72 and 96 h thereafter. The assessment of paw volume was performed always in double blind and by the same operator. The increase in paw volume was calculated by subtracting the initial paw volume (basal) to the paw volume measured at each time point. After Carrageenan induced in left Paw of albino rat:

RESULTS

EVALUATION OF ANTI-INFLAMMATORY ACTIVITY
Anti-inflammatory activity of the commiphora mukkul was assessed by using Plethysmograph. It is a glass tube of 20 mm internal diameter and one end fabricated to a glass tube with0.5 mm bore. This tube is fused to a flexible tube and a pump (glass - syringe) and fixed to other end of the tube. This pump is used to adjust the level of mercury in both the flexible tube and graduated glass tube up to zero level. For the evaluation of anti inflammatory activity of commiphora.carrageenan induced paw model rats were selected and divided into four groups of 6 in each group. The paw oedema was produced by sub-plantar administration of 0.1 ml of a 1% freshly prepared solution of carrageenan into the right hind paw of rats of each group. The paw volume was recorded before (0h) and 1 h after histamine injection. The percent inhibition of the inflammation is calculated using the formula and compared with control group. % Inhibition = Vc - Vt /Vc x 100 Vt - edema volume in the drug treated groups Vc- edema volume in the drug control groups

TABLE: 1 Effect of alcoholic extract of commiphora mukul on inflammation produced in the paw of rats using carrageenan

DRUG

DOSE (mg/kg)

%oedem a omin 25±1.2 (24.9)

15min

30min

45min

60min

75min

90min

120mi n

Control

-----

29±1.9 (28.0)

32.68±2. 5 (30.0)

35.89±4. 2 (31.5) 22.4±4.2 (48.98)

39.8 ± 5.6
(32.7)

44.9±5.8 (28.08) 34.8±4.2 (36.87)

52.22±6.8 (27.56) 44.79±5.8 (32.84)

40±6. 8 (25.9) 44 ± 2.5 (28.5) 33.5 ± 6.8 (48.0) 32.4 ±2.8 (46.0)

GUGGU L GUGGU L GUGGU L

200mg/k g 400mg/k g 600mg/k g

25±5.4 (65.2) 25±5.4 (64.2) 25±5.4 (65.2)

21.0±5. 4 (65.2) 22.0±6. 2 (60.0) 22.0±6. 2 (60.0)

22.0±6. 2 (60.0)
21.56±4. 4 (64.65)

25.0±チ2.5 (38.6) 21.0±5.4 (65.2) 22.2±6.2 (60.0)

21.0±5. 4 (65.2)
22.87±5. 4 (59.56)

21.0±5. 4 (65.2)
26±5.4 (54.0)

25.0±チ2.5 (38.6)
29±1.9 (42.0)

22.0±2. 2 (58.0)

The increase in paw volume was highest at 1hr after carrageenan injection. The significant difference was seen in rats treated with Ethanolic extract of Commiphora mukul. The difference is seen at 1:15hr and 1:45hr which reflex the decrease of release of inflammatory mediators in the rats which were treated with Ethanolic extract of Commiphora The antiinflammatory activity at 0:30min, 11/2h, 2hrs respectively.

DISCUSSION:

DISCUSSION:
The traditional and conventional allopathic treatments suffers from severe effects of ulcers and perforations, already the anti ulcer activity of plant was reported previously. Thus the

extract of this plant helps in reinforcing the anti-inflammatory activity without causing any side effects via ulceration and perforation. Thus the use of ethanolic extract of commiphora mukkul shows the superior effect in treating inflammation without side effect. The antiinflammatory activity of extract is mainly due to active principle like sterols, alkaloids, flavonoids tannins etc. Already are reported to inhibit the COX-2 enzyme and inhibit PGE synthesis so these presence may be the cause in curing the inflammation. Hence ethanolic extract of commiphora mukkul can be of great potential in treating inflammation without causing ulceration hence promoting the health of people. According to vineagar et al (1987), the development of paw edema is derived from the release of cytoplasmic enzymes and serotonin from mast cells and the increase of Postaglandins(PGE) in the inflammatory area. The macrophage in carrageenan inserted dermal tissue release interleukin-1 causing accumulation of polymorphic nuclear cells (PMNs) in the inflammatory area and then releases lysosomal enzymes and active oxygen which induces paw swelling. The Ethanolic extract of commiphora mukul were evaluated in wistar rats. Using carragenan Induced hind paw Oedema test after oral administration. The results show the promising anti inflammatory activity both against acute and chronic inflammation.

CONCLUSION:

CONCLUSION:
The results of the present study shows that commiphora mukul formulation possesses significant anti inflammatory activity in the wistar rat such as Carragenan Induced hind paw edema . Synthetic products are rich source of free radicals which are carcinogenic 6 but plant extracts are rich source of antioxidants, which can prevent and even help in curing the cancer and also possesses anti-ulcer activity. But anti-inflammatory activity may not yet proved or documented therefore we have selected these herbals to investigate its anti-inflammatory activity.

BIBLIOGRAPHY

BIBLIOGRAPHY

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df
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