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PHYTOCHEMICAL SCREENING AND ANTIOXIDANT ACTIVITY OF CAESALPINIA SAPPAN AN INVITRO ANALYSIS

DISSERTATION SUBMITTED TO THIRUVALLUVAR UNIVERSITY, IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF PHILOSOPHY IN BIOCHEMISTRY

By Under the guidance of Associate professor, Department of Biochemistry , D.K.M. College for women, (Autonomous) Sainathapuram, Vellore-632 001

DHANABAGIYAM KRISHNASWAMY MUDALIAR COLLEGE FOR WOMEN, (Autonomous) Sainathapuram, Vellore-632 001. June - 2011

M.Phil.,Scholar, Department of Biochemistry, D.K.M.College for women (Autonomous), Sainathapuram, Vellore

DECLARATION
The thesis entitled PHYTOCHEMICAL SCREENING AND ANTIOXIDANT Submitted for the

ACTIVITY OF CAESALPINIA SAPPAN AN INVITRO ANALYSIS

M.Phil., degree of the Thiruvalluvar university is a record of research work done by me during 2010-2011 and it has not formed the basis for the award of any other degree, diploma, associate ship, fellowship (or) any other similar title. . PLACE :VELLORE DATE :

ACKNOWLEDGEMENT

First and Foremost, I sincerely thank the God Almighty for blessing me to complete the project successfully.
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My heartful thanks to Mr. T.Sivakumar M.A., the president of D.K.M. College for Women for giving a congenial atmosphere in the college. I am thankful to Mr. Thiru. D. Maninathan, B.E., Secretary, of D.K.M. College for Women for giving a congenial atmosphere in the college. I would like to extend my gratitude to Mrs. Dr. K. Revathi, M.A., M.Phil, B.Ed., P.G.T.S., Ph.D., the Principal and Selvi. R. Manimegalai, M.Sc., M.Phil., Administrative officer of D.K.M College for Women, (Autonomous) Vellore. I especially thanks to my guide and Head of the Department of Biochemistry Mrs.

K. Sarumathy, M.sc M.Phil., for her valuable guidance and continous encouragement through the course of this study. The research work was the result of her found of inspiration . It was a great privilege for me to be a research student under her gudiance.We are also thhankful to the Dr.Seethalakshmi and other lab collegues from Life Teck Research institute, Vadapalani,Chennai for offering their help to perform this project. I sincerely thank all faculty members for their encouragement and support during the course of my study. My whole hearted thank goes to my parents for their care, affection and encouragements, which made me to come up to this level .

CERTIFICATE

This is to certify that this Dissertation entitled, PHYTOCHEMICAL SCREENING AND ANTIOXIDANT ACTIVITY OF CAESALPINIA SAPPAN AN INVITRO ANALYSIS submitted to the Thiruvalluvar university, vellore in partial fulfillment for the
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Degree of Master of Philosophy in Biochemistry is a record of original research work done by Miss. during her study in this Department under my supervision and the Dissertation has not formed the basis for any award of any Degree/ Diploma/ Associateship/ Fellowship or other similar titles to any candidate of any university.

Certified By Mrs.K.Sarumathy, M.Sc., M.Phil., Associate Professor, The Head of the Department, D.K.M. College for women, Sainathapuram, Vellore-632 001.

Submitted to university examination in Biochemistry Examiners Date : 1. 2.

Station:

LIST OF ABBREVIATIONS

MTT - (3,4,5- dimethyl thiazol -2yl)-2, 5-diphenyl tetrazolium bromide) DPPH - 1,1 diphenyl 2 picryl hydrazyl MEM - minimal essential medium FCS - foetal calf serum

DMSO - dimethyl sulphoxide TPVG - trypsin, phosphate buffer saline versene, glucose EDTA - ethylene diamine tetra-acetic acid CO2 BTH GM H Kg Mg Min g Ml l % Eg - carbon dioxide - butylated hydroxyl tolvene - gram - hour - kilogram - milligram - minutes - microgram - milliliter - micro litre - percentage - example

HEP2 - human laryngeal tumor cells IU Nm - international units - nano metre

CONTENTS

S.NO 1 2 3 4 5 6 INTRODUCTION

TITLE

PAGE NO 1 41 53 54 71 81

REVIEW OF LITERATURE AIM AND OBJECTIVE MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSION

SUMMARY

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BIBLIOGRAPHY

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HERBAL THERAPY Herbalism is a traditional medicinal or folk medicine practice based on the use of plants and plant extracts. Herbalism is also known as botanical medicine, medical herbalism, herbal medicine, herbology, and phytotherapy. The scope of herbal medicine is sometimes extended to include fungal and bee products, as well as minerals, shells and certain animal parts.( Acharya, Deepak and Shrivastava Anshu et al., 2008): Pharmacognosy is the study of medicines derived from natural sources. Traditional use of medicines is recognized as a way to learn about potential future medicines. In 2001, researchers identified 122 compounds used in mainstream medicine which were derived from "ethno medical" plant sources; 80% of these compounds were used in the same or related manner as the traditional ethno medical use. (Fabricant DS,Farnsworth NR et al., 2001). Plants have evolved the ability to synthesize chemical compounds that help them defend against attack from a wide variety of predators such as insects, fungi and herbivorous mammals. By chance some of these compounds whilst being toxic to plant predators turn out to have beneficial effects when used to treat human diseases. Such secondary metabolites are highly varied in structure , Many are aromatic substances, most of which are phenols or their oxygen-substituted derivatives. At least 12,000 have been isolated so far; a number estimated to be less than 10% of the total. Chemical compounds in plants mediate their effects on the human body by binding to receptor molecules present in the body; such processes are identical to those already well understood for conventional drugs and as such herbal medicines do not differ greatly from conventional drugs in terms of how they work. This enables herbal medicines to be in principle just as effective as conventional medicines but also gives them the same potential to cause harmful side effects. Many of the herbs and spices used by humans to season food yield useful medicinal compounds.( Lai PK, Roy J et al.,2004). Antimicrobial and chemo preventive properties of herbs and spices. ( Tap sell LC, Hemphill I, Cobiac L, et al. 2006). Similarly to prescription drugs, a number of herbs are thought to be likely to cause adverse effects. (Talalay, P; Talalay, P et al.,2001). Furthermore, adulteration, inappropriate formulation, or lack of understanding of plant and drug interactions have led to adverse reactions that are sometimes life threatening or lethal. (Elvin-Lewis M.et al., 2001).

Herbal therapy is a broad term used to refer to any type of healthcare treatment using fresh or dried herbs. The use of herbs may take the form of supplements, fusions or teas, tinctures, topical creams and poultices. Herb therapy may also include the creation of a healing steam that is scented with various combinations of herbs. One of the strengths of herbal therapies in general is that the use of herbs helps to provide the body with essential nutrients that may be lacking in the daily diet. When the body does not receive sufficient nutrition, various types of aches, pains, and emotional disorders begin to appear. By using herbs to restore a nutritional balance, the body is equipped with what it needs to overcome illness and regain health. Herbal aromatherapy is a specialized type of herbal therapy that utilizes herbs to restore the balance between the body and the mind. Often, the process involves simmering fresh or dried herbs in water to release the scent. For example, simmering lavender is said to help calm the nerves after a trying day and may also be effective in easing the after effects of an anxiety attack. Herbs are natural botanical substances that have effects on the body. Many herbs have long been used in detoxification. Kudzu has the potential for moderating alcohol abuse. Kava and valerian can be used to treat the insomnia that accompanies withdrawal. Milk thistle has been shown to improve liver function. The use of herbs in the recovery process may be most effective when used in conjunction with other strategies that support the whole person including nutrition, bodywork, acupuncture, relaxation and exercise. ALTERNATIVE MEDICINE FOUNDATION (AMF) This site, highly recommended by herbal therapists and physicians who use herbs, features Herb Med, an evidence-based resource on medicinal herbs providing a variety of detailed information on approximately 125 herbs. The site also links to clinical and scientific publications.

CAESALPINIA SAPPAN

SCIENTIFIC CLASSIFICATION Kingdom Division class Order Family Genus Species Plantae Magnoliophyta magnoliopsida fabales fabeaceae Caesalpinia sappan

ACTION AND USES IN AYURUVEDA According to ayurveda it contains Gunna (properties) ruksh (dry) Rasa (taste) kashaya (astringent), tickta (bitter) and madhur (sweet) Virya (potency) sheet (cold) PLANT DISTRIBUTION Caesalpinia sappan is a small to medium-sized, shrubby tree, 4-8(-10) m tall; A small thorny tree, 6-9 mm in height and 15-25 cm in diameter with a few prickly branches; leaves compound, with 8-12 pairs of oblong leaflets and small prickles, leaflets vary, in-equilateral and rounded at the apex; flowers yellow in terminal and auxiliary panicles; stamens waxy-white, filaments densely woolly at the base, fruits woody pods, sub-compressed with a hard recurred short beak, seeds 3-4. The heartwood which is used in medicine is light yellow when freshly cut, but it quickly changes to red. The color diffuses out easily in hot water. In about 7-10 hours the extract becomes deep orange in color. PLANT PART USED Leaf FUNCTIONAL USES The wood is used for firewood and its energy value is about 25 000 kJ/kg. Timber: The tree is the source of the commercial redwood or Brazil wood. Sapwood is white, heartwood makes up to 90 % of the total volume, is yellow or deep orange when fresh turning to dark red. The wood is straight grained with a fine to moderately fine texture, fairly heavy (600-780 kg/m), hard and lustrous. It is difficult to dry and susceptible to warping and collapse, but moderately easy to work; it takes high finish and is tough and resistant to termite attack. It is used for inlaying work, cabinet making, violin bows and for walking sticks. Gum or resin: The stem produces a gum. Tannin or dyestuff: The heartwood yields a valuable red crystalline dye, brazilin, used on cotton, silk and wool fabrics. Bakam gives bright red and violet shades, and with garcine produces a chocolate tint. Bark and pods yield similar dyes, pods contain ca. 40% tannin used for production of light leather goods. Roots give a yellow dye. Essential oil: Leaves contain a pleasant smelling volatile oil. Medicine: A decoction of the wood is a powerful emmenagogue and, because of its
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tannic and gallic acids, is an astringent used in mild cases of dysentery and diarrhoea. It is also given internally for certain skin ailments. The sappan is given as a tonic to women after confinement and to relieve vomiting of blood. It is one of the ingredients in a mixture prescribed for malaria. The dried heartwood is widely used in oriental medicine, particularly against inflammation. Seeds serve as a sedative. Other products: Seeds are reported to contain trypsin and chymotrypsin inhibitors. Protosappanin A isolated from Caesalpinia sappan heartwood has a mild sedative effect. Six 3-benzylchroman derivatives (isoflavonoids) were isolated from Sappan Lignum, the dried heartwood of Caesalpinia sappan. Screening showed that the methanolic extract had significant anti-hypercholesteraemic activity. Brazilin (7,11b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10(6H)-tetrol), the principle component of C.sappan has been found to exhibit hypoglycaemic properties and to increase glucose metabolism in diabetic rats.

USES

The heartwood is bitter, astringent, sweet, acrid, refrigerant, vulnerary, depurative, constipating, sedative and haemostatic. It is useful in vitiated conditions of pitta, burning sensation, wounds, ulcers, leprosy, skin diseases, diarrhea, dysentery, epilepsy, convulsions, menorrhagia, leucorrhoea, diabetes, haemoptysis, hemorrhages, stomatopathy and odontopathy.

DPPH DPPH is a common abbreviation for an organic chemical compound 2,2-diphenyl-1picrylhydrazyl. It is a dark-colored crystalline powder composed of stable free-radical molecules. DPPH has two major applications, both in laboratory research: one is a monitor of chemical

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reactions involving radicals and another is a standard of the position and intensity of electron paramagnetic resonance signals.

IUPAC NAME di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium OTHER NAMES 2,2-diphenyl-1-picrylhydrazyl 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl Diphenylpicrylhydrazyl MODE OF ACTION OF DPPH (DRUGS) DPPH is efficient radical trap, it is also a strong inhibitor of radical-mediated polymerization. (Cowie, J. M. G.; Arrighi, Valeria et al.,2008).

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Whereas DPPH is normally a paramagnetic solid, it transforms into an antiferromagnetic state upon cooling to very low temperatures of the order 0.3 K. ( A. M. Prokhorov and V.B. Fedorov et al., (1963),Teruaki Fujito et al., (1981), Stig Lundqvist (1998), Aleksandr M. Prokhorov et al.,(1964). PROPERTIES AND APPLICATIONS DPPH has several crystalline forms which differ by the lattice symmetry and melting point (m.p.). The commercial powder is a mixture of phases which melts at ~130 C. DPPH-I (m.p. 106 C) is orthorhombic, DPPH-II (m.p. 137 C) is amorphous and DPPH-III (m.p. 128 129 C) is triclinic. (Kiers, C. T.; De Boer, J. L.; Olthof, R.; Spek, A. L. et al.,1976). DPPH is a well-known radical and a trap ("scavenger") for other radicals. Therefore, rate reduction of a chemical reaction upon addition of DPPH is used as an indicator of the radical nature of that reaction. Because of a strong absorption band centered at about 520 nm, the DPPH radical has a deep violet color in solution, and it becomes colorless or pale yellow when neutralized. This property allows visual monitoring of the reaction, and the number of initial radicals can be counted from the change in the optical absorption at 520 nm or in the EPR signal of the DPPH. (Mark S. M. Alger et al., 1997). EFFECTS OF DPPH Although the material is not thought to be an irritant, direct contact with the eye may cause transient discomfort characterized by tearing or conjunctival redness (as with windburn). Slight abrasive damage may also result. The material may produce foreign body irritation in certain individuals. The material is not thought to produce either adverse health effects or irritation of the respiratory tract following inhalation (as classified using animal models). Nevertheless, adverse effects have been produced following exposure of animals by at least one other route and good hygiene practice requires that exposure be kept to a minimum and that suitable control measures be used in an occupational setting. Accidental ingestion of the material may be damaging to the health of the individual. The substance and/or its metabolites may bind to hemoglobin inhibiting normal uptake of oxygen.

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Symptoms include cyanosis (a bluish discoloration skin and mucous membranes) and breathing difficulties. IN VITRO STUDY In vitro (Latin: within glass) refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. In contrast, the term in vivo refers to work that is conducted with living organisms in their normal, intact state, while ex vivo refers to studies on functional organs that have been removed from the intact organism. Common examples of in vitro experiments include (a) cells derived from multicellular organisms (cell culture or tissue culture), (b) subcellular components (e.g. mitochondria or ribosomes), (c) Cellular or subcellular extracts (e.g. wheat germ or reticulocyte extracts), or (d) purified molecules in the test tube (often proteins, DNA, or RNA, either individually or in combination).

ADVANTAGES OF IN VITRO STUDIES Living organisms are extremely complex functional systems that are made up of, at a minimum, many tens of thousands of genes, protein molecules, RNA molecules, small organic compounds, inorganic ions and complexes in an environment that is spatially organized by membranes and, in the case of multicellular organisms, organ systems.( Alberts, Bruce et al.,2008). For a biological organism to survive, these myriad components must interact with each other and with their environment in a way that processes food, removes waste, moves components to the correct location, and is responsive to signalling molecules, other organisms, light, sound, temperature and many other factors. This extraordinary complexity of living organisms is a great barrier to the identification of individual components and the exploration of their basic biological functions. The primary advantage of in vitro work is that it permits an enormous level of simplification of the system under study, so that the investigator can focus on a small number of components.( Vignais, Paulette M.; Pierre Vignaiset al., 2010). (Jacqueline Nairn; Price, Nicholas C. et al.,2009) For example, the identity of proteins of the immune system (e.g. antibodies), and the mechanism by
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which they recognize and bind to foreign antigens would remain very obscure if not for the extensive use of in vitro work to isolate the proteins, identify the cells and genes that produce them, study the physical properties of their interaction with antigens, and identify how those interactions lead to cellular signals that activate other components of the immune system. ( Sunshine, Geoffrey; Coico, Richard et al.,2009). EFFECTS OF IN VITRO STUDIES The primary disadvantage of in vitro experimental studies is that it can sometimes be very challenging to extrapolate from the results of in vitro work back to the biology of the intact organism. Investigators doing in vitro work must be careful to avoid over-interpretation of their results, can sometimes lead to erroneous conclusions about organismal and systems biology. (Rothman, S. S.et al., (2002) For example, scientists developing a new viral drug to treat an infection with a pathogenic virus (e.g. HIV-1) may find that a candidate drug functions to prevent viral replication in an in vitro setting (typically cell culture). However, before this drug is used in the clinic, it must progress through a series of in vivo trials to determine if it is safe and effective in intact organisms (typically small animals, primates and humans in succession). Typically, many candidate drugs that are effective in vitro prove to be ineffective in vivo because of issues associated with delivery of the drug to the affected tissues, or toxicity towards essential parts of the organism that were not represented in the initial in vitro studies. (De Clercq E (October 2005).

EXAMPLES OF IN VITRO WORK Polymerase chain reaction is a method for selective replication of specific DNA and RNA sequences in the test tube. Protein purification involves the isolation of a specific protein of interest from a complex mixture of proteins, often obtained from homogenized cells or tissues. In vitro fertilization is used to allow spermatozoa to fertilize eggs in a culture dish before implanting the resulting embryo or embryos into the uterus of the prospective mother.

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In vitro diagnostics refers to a wide range of medical and veterinary laboratory tests that are used to diagnose diseases and monitor the clinical status of patients using samples of blood, cells or other tissues obtained from a patient. IN VITRO TEST METHODS For a typical skin-penetration study, test formulations are applied to excised skin mounted on either the Franz static or Bronaugh flow-through diffusion cell systems. Skin should be exposed to the drug a maximum of 24 h because of deterioration of skin integrity with time. Receptorfluid samples are collected at regular intervals over the exposure period for each cell. Several methods can be used to remove the residual formulation from the skin surface following the designated exposure period: washing, wiping, tape-stripping, and combinations thereof. After the removal of the residual dose from the skin surface, the epidermis is separated from the dermis by physical methods. Diffusion-cell washes, skin surface material removal samples,epidermis-, dermis-, and receptor-fluid samples are then analyzed for drug content. A greater number of formulations can be characterized in a study using the Bronaugh flow-through diffusion cell system because of the automated receptorfluid collection and generally smaller amount of skin used per cell compared with the static diffusion cell systems. In vitro drug-release studies are simplified skin-penetration studies. After the application of the test formulations to the membrane mounted on the Franz static diffusion cell system, receptor-fluid samples are collected at defined intervals of drug exposure and are subsequently analyzedfor drug content. CELL LINE A cell line is a product of immortal cells that are used for biological research. Cells used for cell lines are immortal, that happens if a cell is cancerous. The cells can perpetuate division indefinitely which is unlike regular cells which can only divide approximately 50 times. These cells are 'useful' for experimentation in labs as they are always available to researchers as a product and do not require what is known as 'harvesting' (the acquiring of tissue from a host) every time cells are needed in the lab. (cell culture) A cell line is a permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space.
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Lines differ from cell strains in that they have escaped the Hayflick limit and become immortalised. Some species, particularly rodents, give rise to lines relatively easily, whereas other species do not. No cell lines have been produced from avian tissues and the establishment of cell lines from human tissue is difficult. Many cell biologists would consider that a cell line is by definition already abnormal and that it is on the way towards becoming the culture equivalent of a neoplastic cell. Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture. Animal cell culture became a routine laboratory technique in the 1950s, but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century. HISTORY The 19th-century English physiologist Sydney Ringer developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside of the body. In 1885 Wilhelm Roux removed a portion of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the principle of tissue culture. Ross Granville Harrison, working at Johns Hopkins Medical School and then at Yale University, published results of his experiments from 1907-1910, establishing the methodology of tissue culture. (Schiff, Judith Ann et al., 2002) Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology. Growing viruses in cell cultures allowed preparation of purified viruses for the manufacture of vaccines. ISOLATION OF CELLS Cells can be isolated from tissues for ex vivo culture in several ways. Cells can be easily purified from blood, however only the white cells are capable of growth in culture. Mononuclear cells can be released from soft tissues by enzymatic digestion with enzymes such as collagenase, trypsin, or pronase, which break down the extracellular matrix. Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are available for culture. This method is
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known as explant culture. Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived from tumours, most primary cell cultures have limited lifespan. After a certain number of population doublings cells undergo the process of senescence and stop dividing, while generally retaining viability. An established or immortalised cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene. There are numerous well established cell lines representative of particular cell types. MAINTAINING CELLS IN CULTURE Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37C, 5% CO2) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed. Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrient components. The growth factors used to supplement media are often derived from animal blood, such as calf serum. These blood-derived ingredients pose the potential for contamination of derived pharmaceutical products with viruses or prions. Current practice is to minimize or eliminate the use of these ingredients where possible. Some cells naturally live without attaching to a surface, such as cells that exist in the bloodstream. Others require a surface, such as most cells derived from solid tissues. Cells grown unattached to a surface are referred to as suspension cultures. Other adherent cultures cells can be grown on tissue culture plastic, which may be coated with extracellular matrix components to increase its adhesion properties and provide other signals needed for growth. ESTABLISHED HUMAN CELL LINES One of the earliest human cell lines, descended from Henrietta Lacks, who died of the cancer that those cells originated from, the cultured HeLa cells shown here have been stained with Hoechst turning their nuclei blue. Cell lines that originate with humans have been somewhat controversial in bioethics, as they may outlive their parent organism and later be used in the discovery of lucrative medical treatments. In the pioneering decision in this area, the Supreme Court of California held in 1990 that human patients have no property rights in cell lines derived from organs removed with their consent. It
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is estimated that about 20% of human cell lines are not the kind of cells they were generally assumed to be. The reason for this is that some cell lines exhibit vigorous growth and thus can cross-contaminate cultures of other cell lines, in time overgrowing and displacing the original cells. The most common contaminant is the HeLa cell line. While this may not be of significance when general properties such as cell metabolism are researched, it is highly relevant e.g. in medical research focusing on a specific type of cell. Results of such research will be at least flawed, if not outright wrong in their conclusion, with possible consequences if therapeutic approaches are developed based on it. (Drexler, HG; Dirks, WG; Macleod, RA et al.,Oct 1999). APPLICATIONS OF CELL CULTURE Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and many products of biotechnology. Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes, hormones, immunobiologicals (monoclonal antibodies, interleukins, lymphokines), and anticancer agents. Although many simpler proteins can be produced using rDNA in bacterial cultures, more complex proteins that are glycosylated (carbohydrate-modified), currently must be made in animal cells. An important example of such a complex protein is the hormone erythropoietin. The cost of growing mammalian cell cultures is high, so research is underway to produce such complex proteins in insect cells or in higher plants. HEP2 CELL LINE The HEp-2 cell line was established in 1952 by A. E. Moore, L. Sabachewsky, and H. W. Toolan (Cancer Res. 15: 598, 1955) from tumors that had been produced in irradiated-cortisonized weanling rats after injection with epidermoid carcinoma tissue from the larynx of a 56-year-old male (H. Toolan, Cancer Res. 14: 660, 1954). A hardy cell line, HEp-2 resists temperature, nutritional, and environmental changes without a loss of viability. It has supported growth of 10 of 14 arboviruses (Texas Rep. Biol. Med. 15: 588, 1957) and measles virus (Proc. Soc. Exp. Biol. Med. 93: 107, 1956), and it has been used for experimental studies of tumor production in rats, hamsters, mice, embryonated eggs and volunteer terminal cancer patients (Ann. N.Y. Acad. Et al., 1958). HepG2 cells are a suitable in vitro model system for the study of polarized human hepatocytes. (Another well-characterized polarized hepatocyte cell lines includes the rat hepatoma-derived
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hybrid cell line WIF-B (Ihrke et al., 1993)). With the proper culture conditions, HepG2 cells display robust morphological and functional differentiation with a controllable formation of apical and basolateral cell surface domains (van IJzendoorn et al., 1997; 2000) that resemble the bile canalicular (BC) and sinusoidal domains, respectively, in vivo. Because of their high degree of morphological and functional differentiation in vitro, HepG2 cells are a suitable model to study the intracellular trafficking and dynamics of bile canalicular and sinusoidal membrane proteins and lipids in human hepatocytes in vitro. This can be important for the study of human liver diseases that are caused by an incorrect subcellular distribution of cell surface proteins, e.g. hepatocanalicular transport defects such as DubinJohnson Syndrome and progressive familial intrahepatic cholestasis (PFIC), and familial hypercholesterolemia. HepG2 cells and its derivatives are also used as a model system for studies of liver metabolism and toxicity of xenobiotics, the detection of cytoprotective, anti (environmental and dietary) genotoxic and cogenotoxic agents, understanding hepatocarcinogenesis, and for drug targeting studies. HepG2 cells are also employed in trials with bio-artificial liver devices. CELL MORPHOLOGY This slide is an illustration of a HEp-2 or HEp-2000 cell with several nuclear and cytoplasmic structures indicated. Antibodies to DNA, histones, centromere, nuclear RNP, cytoplasmic RNP, mitochondria, ribosomes, lysosomes, golgi apparatus, as well as a variety of cytoskeletal proteins such as microfilaments, intermediate filaments, and microtubules, can be detected using the HEp-2 or HEp-2000 cell lines. It is important to note that RNA constitutes the bulk of the nucleic acid in the cells, being 5-10 times more abundant than DNA. Generally we consider 2 compartments in the cell when detecting autoantibodies in systemic rheumatic disease: 1) nucleus, and 2) cytoplasm. Staining of the nucleus includes staining inside the nucleoli. LIVER The liver is the largest organ in the body. It weighs about 3 lb (1.36 kg). It is reddish brown in color and is divided into four lobes of unequal size and shape. The liver lies on the right side of the abdominal cavity beneath the diaphragm. Blood is carried to the liver via two large vessels called the hepatic artery and the portal vein. The hepatic artery carries oxygen-rich blood from the aorta (a major vessel in the heart). The portal vein carries blood containing digested food from the small intestine. These blood vessels subdivide in the liver repeatedly, terminating in
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very small capillaries. Each capillary leads to a lobule. Liver tissue is composed of thousands of lobules, and each lobule is made up of hepatic cells, the basic metabolic cells of the liver. It is essential in keeping the body functioning properly. It removes poisons from the blood, produces agents to control infection, and removes germs and bacteria from the blood. It makes proteins that regulate blood clotting and produces bile to help absorb fats and fat-soluble vitamins. One consequence of this complexity is that hepatic disease has widespread effects on virtually all other organ systems. At the risk of losing sight of the forest by focusing on the trees, we will focus on three fundamental roles of the liver:
1. Vascular functions, including formation of lymph and the hepatic phagocytic system. 2. Metabolic achievements in control of synthesis and utilization of carbohydrates,

lipids and proteins.


3. Secretory and excretory functions, particularly with respect to the synthesis of

secretion of bile. The latter is the only one of the three that directly affects digestion - the liver, through its bilary tract, secretes bile acids into the small intestine where they assume a critical role in the digestion and absorption of dietary lipids. However, understanding the vascular and metabolic functions of the liver is critical to appreciating the gland as a whole. The liver is one of the largest, most important, and least appreciated organs in the body. The bulk of the liver consists of hepatocytes, which are epithelial cells with a unique conFiguration. The liver is essentially an exocrine gland, secreting bile into the intestine. and significantly so The liver has a diversity of functions not typically associated with glands. The liver is a metabolic factory, synthesizing and breaking down a variety of substances. It's functions include all of the following: Formation and secretion of bile. Storage of glycogen, buffer for blood glucose. Synthesis of urea. Metabolism of cholesterol and fat.
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Synthesis and endocrine secretion of many plasma proteins, including clotting factors. Detoxification of many drugs and other poisons. Cleansing of bacteria from blood. Processing of several steroid hormones and vitamin D. Volume reservoir for blood. Catabolism of hemoglobin from worn-out red blood cells.

CORE CONCEPTS IN HEPATIC PHYSIOLOGY ARE PRESENTED AS THE FOLLOWING TOPICS: Architecture of the liver and biliary tract Physiology of the hepatic vascular system Secretion of bile and the role of bile acids in digestion Biliary excretion of waste products: elimination of bilirubin Metabolic functions of the liver ADVANCED AND SUPPLEMENTAL TOPICS RELATED TO PHYSIOLOGY OF THE LIVER: Regeneration of the Liver The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemical necessary for digestion. The liver is necessary for survival; there is currently no way to compensate for the absence of liver function long term, although liver dialysis can be used short term. This organ plays a major role in metabolism and has a number of functions in the body, including glycogen storage, decomposition of red blood cells, plasma protein synthesis, hormone production, and detoxification. It lies below the diaphragm in the abdominal-pelvic region of the
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abdomen. It produces bile, an alkaline compound which aids in digestion via the emulsification of lipids. The liver's highly specialized tissues regulate a wide variety of high-volume biochemical reactions, including the synthesis and breakdown of small and complex molecules, many of which are necessary for normal vital functions.( Maton, Anthea; Jean Hopkins, Charles William McLaughlin, Susan Johnson, Maryanna Quon Warner, David LaHart, Jill D. Wright et al., 1993).

ANATOMY The liver is a reddish brown organ with four lobes of unequal size and shape. A human liver normally weighs 1.41.6 kg (3.13.5 lb),( Cotran, Ramzi S.; Kumar, Vinay; Fausto, Nelson; Nelso Fausto; Robbins, Stanley L.; Abbas, Abul K.et al., 2005). and is a soft, pinkish-brown, triangular organ. It is both the largest internal organ (the skinbeing the largest organ overall) and the largest gland in the human body.

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STRUCTURAL ORGANISATION OF LIVER

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It is located in the right upper quadrant of the abdominal cavity, resting just below the diaphragm. The liver lies to the right of the stomach and overlies the gallbladder. It is connected to two large blood vessels, one called the hepatic artery and one called the portal vein. The hepatic artery carries blood from the aorta, whereas the portal vein carries blood containing digested nutrients from the entire gastrointestinal tract and also from the spleen and pancreas. These blood vessels subdivide into capillaries, which then lead to a lobule. Each lobule is made up of millions of hepatic cells which are the basic metabolic cells. BLOOD FLOW The liver receives a dual blood supply from the hepatic portal vein and hepatic arteries. Supplying approximately 75% of the liver's blood supply, the hepatic portal vein carries venous blood drained from the spleen, gastrointestinal tract, and its associated organs. The hepatic arteries supply arterial blood to the liver, accounting for the remainder of its blood flow. Oxygen is provided from both sources; approximately half of the liver's oxygen demand is met by the hepatic portal vein, and half is met by the hepatic arteries. (Benjamin L. Shneider; Sherman, Philip M.et al., 2008). THE BILIARY TREE The term biliary tree is derived from the arboreal branches of the bile ducts. The bile produced in the liver is collected in bile canaliculi, which merge to formbile ducts. Within the liver, these ducts are called intrahepatic (within the liver) bile ducts, and once they exit the liver they are considered extrahepatic (outside the liver). The intrahepatic ducts eventually drain into the right and left hepatic ducts, which merge to form the common hapatic duct. The cystic duct from the gallbladder joins with the common hepatic duct to form the common bile duct. Bile can either drain directly into the duodenum via the common bile duct, or be temporarily stored in the

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gallblader via the cystic duct. The common bile duct and the pancreatic duct enter the second part of the duodenum together at the ampulla of vater. SURFACE ANATOMY Apart from a patch where it connects to the diaphragm (the so-called "bare area"), the liver is covered entirely by visceral peritoneum, a thin, double-layered membrane that reduces friction against other organs. The peritoneum folds back on itself to form the falciform ligament and the right and left triangular ligaments. These lits are in no way related to the true anatomic ligaments in joints, and have essentially no functional importance, but they are easily recognizable surface landmarks. An exception to this is the falciform ligament, which attaches the liver to the posterior portion of the anterior body wall. LOBES Traditional gross anatomy divided the liver into four lobes based on surface features. The falciform ligament is visible on the front (anterior side) of the liver. This divides the liver into a left anatomical lobe, and a right anatomical lobe. If the liver is flipped over, to look at it from behind (the visceral surface), there are two additional lobes between the right and left. These are the caudate lobe (the more superior) and the quadrate lobe (the more inferior). From behind, the lobes are divided up by the ligamentum venosum and ligamentum teres (anything left of these is the left lobe), the transverse fissure (or porta hepatis) divides the caudate from the quadrate lobe, and the right sagittal fossa, which the inferior vena cava runs over, separates these two lobes from the right lobe. Each of the lobes is made up of lobules; a vein goes from the centre, which then joins to the hepatic vein to carry blood out from the liver. On the surface of the lobules, there are ducts, veins and arteries that carry fluids to and from them.

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The central area where the common bile duct, hepatic portal vein, and hepatic artery proper enter is the hilum or "porta hepatis". The duct, vein, and artery divide into left and right branches, and the portions of the liver supplied by these branches constitute the functional left and right lobes. The functional lobes are separated by an imaginary plane joining the gallbladder fossa to the inferior vena cava. The plane separates the liver into the true right and left lobes. The middle hepatic vein also demarcates the true right and left lobes. The right lobe is further divided into an anterior and posterior segment by the right hepatic vein. The left lobe is divided into the medial and lateral segments by the left hepatic vein. The fissure for the ligamentum teres also separates the medial and lateral segments. The medial segment is also called the quadrate lobe. In the widely used Couinaud (or "French") system, the functional lobes are further divided into a total of eight subsegments based on a transverse plane through the bifurcation of the main portal vein. The caudate lobe is a separate structure which receives blood flow from both the right- and leftsided vascular branches. (Holger Strunk et al., 2009) PHYSIOLOGY The various functions of the liver are carried out by the liver cells or hepatocytes. Currently, there is no artificial organ or device capable of emulating all the functions of the liver. Some functions can be emulated by liver dialysis, an experimental treatment for liver failure. FUNCTIOS OF LIVER The liver is separated into a right and left lobe, separated by the falciform ligament. The right lobe is much larger than the left lobe. The working cells of the liver are known as Hepatocytes. Hepatocytes have a unique capacity to reproduce in response to liver injury. Liver regeneration can occur after surgical removal of a portion of the liver or after injuries that destroy parts of the liver. Although the liver's ability to react to damage and repair itself is remarkable, repetitive insults can produce liver failure and death. The liver is a metabolically active organ responsible for many vital life functions. The primary functions of the liver are: Bile production and excretion Excretion of bilirubin, cholesterol, hormones, and drugs Metabolism of fats, proteins, and carbohydrates
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Enzyme activation Storage of glycogen, vitamins, and minerals Synthesis of plasma proteins, such as albumin, and clotting factors Blood detoxification and purification Due to these important activities, the liver is exposed to a number of insults and is one of the body's organs most subject to injury. The biliary system consists of the gallbladder, and the hepatic, cystic, and common bile ducts. The gallbladder is located beneath the right lobe of the liver. The primary function of the gallbladder is to store and concentrate bile. Bile is comprised of bile salts, bilirubin, phospholipids, cholesterol, bicarbonate and water. Bile salts mix with ingested fats to promote absorption of fats from the gastrointestinal tract. Bilirubin, cholesterol, and phospholipids are end products of metabolism. Bicarbonate and water are needed in the gastrointestinal tract to help neutralize stomach acid, as digestion and absorption require an alkaline environment. After its formation in the liver, bile flows into the hepatic duct. The hepatic duct joins with the cystic duct of the gallbladder to form the common bile duct, which then enters the duodenum at the ampulla of Vater. The sphincter of Oddi surrounds the ampulla of Vater. When food in the gastrointestinal canal enters the duodenum, cholecystokinin is released from the duodenal mucosa to stimulate contraction of the gallbladder and common bile duct, and relaxation of the sphincter of Oddi so that bile can enter the small intestine. Lipogenesis, the production of triglycerides (fats). The liver produces coagulation factors I (fibrinogen), II (prothrombin), V, VII, IX, X and XI, as well as protein C, protein S and antithrombin. In the first trimester fetus, the liver is the main site of red blood cell production. By the 32nd week of gestation, the bone marrow has almost completely taken over that task. The liver produces and excretes bile (a yellowish liquid) required for emulsifying fats. Some of the bile drains directly into the duodenum, and some is stored in the gallbladder. The liver also produces insulin-like growth factor 1 (IGF-1), a polypeptide protein hormone that plays an important role in childhood growth and continues to have anabolic effects in adults.

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The liver is a major site of thrombopoietin production. Thrombopoietin is a glycoprotein hormone that regulates the production of platelets by the bone marrow. The liver converts ammonia to urea. The liver has many functions. Some of the functions are: to produce substances that break down fats, convert glucose to glycogen, produce urea (the main substance of urine), make certain amino acids (the building blocks of proteins), filter harmful substances from the blood (such as alcohol), storage of vitamins and minerals (vitamins A, D, K and B12) and maintain a proper level or glucose in the blood. The liver is also responsible for producing cholesterol. It produces about 80% of the cholesterol in your body. OTHER FUNCTIONS The liver stores a multitude of substances, including glucose (in the form of glycogen), vitamin A (12 years' supply), vitamin D (14 months' supply), vitamin B12 (1-3 years' supply), iron, and copper. The liver is responsible for immunological effects- the reticuloendothelial system of the liver contains many immunologically active cells, acting as a 'sieve' for antigens carried to it via the portal system. The liver produces albumin, the major osmolar component of blood serum. The liver stores a multitude of substances, including glucose (in the form of glycogen), vitamin A (12 years' supply), vitamin D (14 months' supply), vitamin B12 (1-3 years' supply), iron, and copper. The liver synthesizes angiotensinogen, a hormone that is responsible for raising the blood pressure when activated by renin, an enzyme that is released when the kidney senses low blood pressure.

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LIVER FUNCTION TESTS (LFTs or LFs), which include liver enzymes, are groups of clinical biochemistry laboratory blood assays designed to give information about the state of a patient's liver. Most liver diseases cause only mild symptoms initially, but it is vital that these diseases be detected early. Hepatic (liver) involvement in some diseases can be of crucial importance. This testing is performed by a medical technologist on a patient's serum or plasma sample obtained by phlebotomy. Some tests are associated with functionality (e.g., albumin); some with cellular integrity (e.g., transaminase) and some with conditions linked to the biliary tract (gamma-glutamyl transferase and alkaline phosphatase). Several biochemical tests are useful in the evaluation and management of patients with hepatic dysfunction. These tests can be used to (1) detect the presence of liver disease, (2) distinguish among different types of liver disorders, (3) gauge the extent of known liver damage, and (4) follow the response to treatment. Some or all of these measurements are also carried out (usually about twice a year for routine cases) on those individuals taking certain medicationsanticonvulsants are a notable example- in order to ensure that the medications are not damaging the person's liver.

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As the liver performs its various functions it makes chemicals that pass into the bloodstream and bile. Various liver disorders alter the blood level of these chemicals. Some of these chemicals can be measured in a blood sample. Some tests that are commonly done on a blood sample are called liver function tests (LFTs). These usually measure the following: ALANINE TRANSAMINASE (ALT) Normal value 9 to 40 IU/L Alanine transaminase (ALT), also called Serum Glutamic Pyruvate Transaminase (SGPT) or Alanine aminotransferase (ALAT) is an enzyme present in hepatocytes (liver cells). When a cell is damaged, it leaks this enzyme into the blood, where it is measured. ALT rises dramatically in acute liver damage, such as viral hepatitis or paracetamol (acetaminophen) overdose. Elevations are often measured in multiples of the upper limit of normal (ULN). This is an enzyme that helps to process proteins. (An enzyme is a protein that helps to speed up chemical reactions. Various enzymes occur in the cells in the body.) Large amounts of ALT occur in liver cells. When the liver is injured or inflamed (as in hepatitis), the blood level of ALT usually rises. ASPARTATE TRANSAMINASE (AST) Normal value 10 to 35 IU/L This is another enzyme usually found inside liver cells. When a blood test detects high levels of this enzyme in the blood it usually means the liver is injured in some way. However AST can also be released if heart or skeletal muscle is damaged. For this reason ALT is usually considered to be more specifically related to liver problems. Aspartate transaminase (AST) also called Serum Glutamic Oxaloacetic Transaminase (SGOT) or aspartate aminotransferase (ASAT) is similar to ALT in that it is another enzyme associated with liver parenchymal cells. It is raised in acute liver damage, but is also present in red blood cells, and cardiac and skeletal muscle and is therefore not specific to the liver. The ratio of AST to ALT is sometimes useful in differentiating between causes of liver damage. Elevated AST levels are not specific for liver damage, and AST has also been used as a cardiac marker. (Nyblom H, Berggren U, Balldin J, Olsson R (2004). et.al.,) (Nyblom H, Bjornsson E, Simren M, Aldenborg F, Almer S, Olsson R (September 2006).et.al.,)
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ALKALINE PHOSPHATASE (ALP) Normal value 30 to 120 IU/L This enzyme occurs mainly in liver cells next to bile ducts, and in bone. The blood level is raised in some types of liver and bone disease. Alkaline phosphatase (ALP) is an enzyme in the cells lining the biliary ducts of the liver. ALP levels in plasma will rise with large bile duct obstruction, intrahepatic cholestasis or infiltrative diseases of the liver. ALP is also present in bone and placental tissue, so it is higher in growing children (as their bones are being remodelled) and elderly patients with Paget's disease. GAMMA GLUTAMYL TRANSPEPTIDASE (GGT) Normal value 0 to 42 IU/L Although reasonably specific to the liver and a more sensitive marker for cholestatic damage than ALP, Gamma glutamyl transpeptidase (GGT) may be elevated with even minor, sub-clinical levels of liver dysfunction. It can also be helpful in identifying the cause of an isolated elevation in ALP. (GGT is raised in chronic alcohol toxicity). LACTATE DEHYDROGENASE (LDH) Lactate dehydrogenase is an enzyme found in many body tissues, including the liver. Elevated levels of LDH may indicate liver damage

ALBUMIN (ALB) Normal value 3.5 to5.0g/dL

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This is the main protein made by the liver, and it circulates in the bloodstream. The ability to make albumin (and other proteins) is affected in some types of liver disorder. A low level of blood albumin occurs in some liver disorders. It is the main constituent of total protein; the remaining fraction is called globulin (including the immunoglobulins). Albumin levels are decreased in chronic liver disease, such as cirrhosis. It is also decreased in nephrotic syndrome, where it is lost through the urine. Poor nutrition or states of protein catabolism may also lead to hypoalbuminaemia. The half-life of albumin is approximately 20 days. Albumin is not considered to be an especially useful marker of liver synthetic function; coagulation factors are much more sensitive BILIRUBIN. The liver has many functions. One of the liver's functions is to produce and secrete bile into the intestines to help digest dietary fat. Another is to remove toxic chemicals or waste products from the blood, and bilirubin is a waste product. The liver removes bilirubin from the blood. After the bilirubin has entered the liver cells, the cells conjugate (attaching other chemicals, primarily glucuronic acid) to the bilirubin, and then secrete the bilirubin/glucuronic acid complex into bile. The complex that is secreted in bile is called conjugated bilirubin. The conjugated bilirubin is eliminated in the feces. (Bilirubin is what gives feces its brown color.) Conjugated bilirubin is distinguished from the bilirubin that is released from the red blood cells and not yet removed from the blood which is termed unconjugated bilirubin. Jaundice occurs when there is 1) too much bilirubin being produced for the liver to remove from the blood. (For example, patients with hemolytic anemia have an abnormally rapid rate of destruction of their red blood cells that releases large amounts of bilirubin into the blood), 2) a defect in the liver that prevents bilirubin from being removed from the blood, converted to bilirubin/glucuronic acid (conjugated) or secreted in bile, or 3) blockage of the bile ducts that decreases the flow of bile and bilirubin from the liver into the intestines. (For example, the bile ducts can be blocked by cancers, gallstones, or inflammation of the bile ducts). The decreased conjugation, secretion, or flow of bile that can result in jaundice is referred to as cholestasis: however, cholestasis does not always result in jaundice

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A raised level of 'unconjugated' bilirubin occurs when there is excessive breakdown of red blood cells. For example, in haemolytic anaemia. TOTAL BILIRUBIN (TBIL) Normal value 0.21.2 mg/dL Bilirubin is a breakdown product of heme (a part of hemoglobin in red blood cells). The liver is responsible for clearing the blood of bilirubin. It does this by the following mechanism: bilirubin is taken up into hepatocytes, conjugated (modified to make it water-soluble), and secreted into the bile, which is excreted into the intestine. Increased total bilirubin causes jaundice, and can signal a number of problems: 1. Prehepatic: Increased bilirubin production. This can be due to a number of causes, including hemolytic anemias and internal hemorrhage. 2. Hepatic: Problems with the liver, which are reflected as deficiencies in bilirubin metabolism (e.g. reduced hepatocyte uptake, impaired conjugation of bilirubin, and reduced hepatocyte secretion of bilirubin). Some examples would be cirrhosis and viral hepatitis. 3. Posthepatic: Obstruction of the bile ducts, reflected as deficiencies in bilirubin excretion. (Obstruction can be located either within the liver or in the bile duct. DIRECT BILIRUBIN (CONJUGATED BILIRUBIN) Normal value 02.3 mg/dL The diagnosis is narrowed down further by looking at the levels of direct bilirubin. If direct (i.e. conjugated) bilirubin is normal, then the problem is an excess of unconjugated bilirubin, and the location of the problem is upstream of bilirubin excretion. Hemolysis, viral hepatitis, or cirrhosis can be suspected. If direct bilirubin is elevated, then the liver is conjugating bilirubin normally, but is not able to excrete it. Bile duct obstruction by gallstones or cancer should be suspected. TOTAL PROTEIN. This measures albumin and all other proteins in blood.
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OTHER TESTS COMMONLY REQUESTED ALONGSIDE LFTS 5' nucleotidase (5'NTD) 5' nucleotidase is another test specific for cholestasis or damage to the intra or extrahepatic biliary system, and in some laboratories, is used as a substitute for GGT for ascertaining whether an elevated ALP is of biliary or extra-biliary origin. Coagulation test (e.g. INR) The liver is responsible for the production of coagulation factors. The international normalized ratio (INR) measures the speed of a particular pathway of coagulation, comparing it to normal. If the INR is increased, it means it is taking longer than usual for blood to clot. The INR will only be increased if the liver is so damaged that synthesis of vitamin K-dependent coagulation factors has been impaired: it is not a sensitive measure of liver function. It is very important to normalize the INR before operating on people with liver problems (usually by transfusion with blood plasma containing the deficient factors) as they could bleed excessively. SERUM GLUCOSE (BG, GLU) The liver's ability to produce glucose (gluconeogenesis) is usually the last function to be lost in the setting of fulminant liver failure. LIVER FUNCTION TEST USES: To help diagnose liver disorders if you have suggestive symptoms (such as jaundice). The pattern of the blood results may help to say which disorder is causing the problem. For example, depending on which enzyme is highest it may point to a particular disorder.To monitor the activity and severity of liver disorders.As a routine precaution after starting certain medicines to check that they are not causing liver damage as a side-effect. OTHER TESTS OF THE LIVER

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LFTs are useful, and are often the first marker of disease in the liver. However, other tests of the liver may also be done to confirm the diagnosis of a particular disorder, and/or to monitor the activity of the disorder and response to treatment. BLOOD CLOTTING TESTS. The liver makes many of the proteins needed to make blood clot. In certain liver disorders the liver cannot make enough of these proteins and so blood does not clot so well. Therefore, blood clotting tests may be used as a marker of the severity of certain liver disorders. See separate leaflet called 'Blood Test - Clotting Tests'. Gamma-glutamyl transferase (GGT or 'gamma GT'). This is another enzyme that occurs in liver cells. A high level of this enzyme is particularly associated with heavy alcohol drinking. (The liver breaks down and clears alcohol from the body and this enzyme is involved in the process.) BLOOD TESTS: Viruses and antibodies to viruses. Various virus infections can cause hepatitis (inflammation of the liver). For example, hepatitis A virus, hepatitis B virus, etc. Auto-antibodies. These are antibodies which attack a part of your own body and occur in autoimmune disorders. The most common autoimmune disorders of the liver are: Primary biliary cirrhosis (associated with anti-mitochondrial antibodies). Autoimmune hepatitis (associated with smooth muscle antibodies). Primary sclerosing cholangitis (associated with antinuclear cytoplasmic antibodies). Other types of protein in the blood can point to specific liver diseases. For example: Ceruloplasmin is reduced in Wilson's disease. Lack of 1-antitrypsin is an uncommon cause of cirrhosis. A high level of ferritin is a marker of haemochromatosis. MEDICATIONS THAT NEGATIVELY EFFECT THE LIVER

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Medications have side effects that may harm your liver. Some of the medications that can damage your liver are: serzone, anti-cancer drugs (tagfur, MTX, and cytoxan), and medications used to treat diabetes. Serzone is a prescription drug manufactured by Bristol-Myers Squibb for the treatment of depression. The possible side effects of Serzone are: agitation, dizziness, clumsiness or unsteadiness, difficulty concentrating, memory problems, confusion, severe nausea, gastroenteritis, abdominal pain, unusually dark urine, difficult or frequent urination, fainting, skin rash or hives yellowing of the skin or whites of the eyes (jaundice) or a prolonged loss of weight or loss of appetite. If you or a family member have suffered serious side effects or a fatal injury after taking Serzone, you or the family member may be eligible to file a claim against the manufacturer. You should contact an attorney that specializes in class action lawsuits immediately. To help prevent liver damage, let your doctor know about your liver condition when being treated for other conditions. Medications come in many forms and it is best to find out what is in them and what it can do to your liver. LIVER DISEASE Liver disease (also called hepatic disease) is a broad term describing any single number of diseases affecting the liver. Many are accompanied by jaundice caused by increased levels of bilirubin in the system. The bilirubin results from the breakup of the hemoglobin of dead red blood cells; normally, the liver removes bilirubin from the blood and excretes it through bile.

DRUG-INDUCED LIVER DISEASE Drug-induced liver diseases are diseases of the liver that are caused by physician-prescribed medications, over-the-counter medications, vitamins, hormones, herbs, illicit ("recreational") drugs, and environmental toxins.

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DISEASES OF THE LIVER Hepatitis, inflammation of the liver, caused mainly by various viruses but also by some poisons (e.g. alcohol), autoimmunity (autoimmune hepatitis) or hereditary conditions. Diagnosis is done by checking levels of Alanine transaminase. Non-alcoholic fatty liver disease, a spectrum in disease, associated with obesity and characterized as an abundance of fat in the liver; may lead to a hepatitis, i.e. steatohepatitis and/or cirrhosis. Cirrhosis is the formation of fibrous tissue in the liver from replacing dead liver cells. The death of the liver cells can be caused by viral hepatitis, alcoholism or contact with other liver-toxic chemicals. Diagnosis is done by checking levels of Alanine transaminase and Asparatine transaminase (SGOT). Chronic liver diseases such as hepatitis, fatty liver, or cholestasis can lead to the necrosis (death) of liver cells. Scar tissue forms as part of the healing process that is associated with the dying liver cells, and severe scarring of the liver can lead to cirrhosis. The most common example of drug-induced cirrhosis is alcoholic cirrhosis . Examples of drugs that can cause chronic liver diseases and cirrhosis include methotrexate (Rheumatrex), amiodarone (Cordarone), and methyldopa (Aldomet). Haemochromatosis, a hereditary disease causing the accumulation of iron in the body, eventually leading to liver damage. Cancer of the liver (primary hepatocellular carcinoma or cholangiocarcinoma and metastatic cancers, usually from other parts of the gastrointestinal tract).

Wilson's disease, a hereditary disease which causes the body to retain copper. Primary sclerosing cholangitis, an inflammatory disease of the bile duct, likely autoimmune in nature. Primary biliary cirrhosis, autoimmune disease of small bile ducts.
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Budd-Chiari syndrome, obstruction of the hepatic vein. Gilbert's syndrome, a genetic disorder of bilirubin metabolism, found in about 5% of the population. Glycogen storage disease type II, the build-up of glycogen causes progressive muscle weakness (myopathy) throughout the body and affects various body tissues, particularly in the heart, skeletal muscles, liver and nervous system. There are also many pediatric liver disease, including biliary atresia, alpha-1 antitrypsin deficiency, alagille syndrome, and progressive familial intrahepatic cholestasis, to name but a few. The liver supports almost every organ in the body and is vital for survival. Because of its strategic location and multidimensional functions, the liver is also prone to many diseases. (Cirrhosis Overview National Digestive Diseases Information Clearinghouse. The most common include: Infections such as hepatitis A, B, C, E, alcohol damage, fatty liver, cirrhosis, cancer, drug damage (especially acetaminophen (also known as paracetamol) and cancer drugs) Many diseases of the liver are accompanied by jaundice caused by increased levels of bilirubin in the system. The bilirubin results from the breakup of the Hemoglobin of dead red blood cells; normally, the liver removes bilirubin from the blood and excretes it through bile. There are also many pediatric liver diseases including biliary atresia, alpha-1 antitrypsin deficiency, alagille syndrome, progressive familial intrahepatic cholestasis, and Langerhans cell histiocytosis, to name but a few. Diseases that interfere with liver function will lead to derangement of these processes. However, the liver has a great capacity to regenerate and has a large reserve capacity. In most cases, the liver only produces symptoms after extensive damage. SYMPTOMS OF A DISEASED LIVER

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The external signs include a coated tongue, itchy skin, excessive sweating, offensive body odor, dark circles under the eyes, red swollen and itchy eyes, acne rosacea, brownish spots and blemishes on the skin, flushed facial appearance or excessive facial blood vessels. Other symptoms include jaundice, dark urine, pale stool, bone loss, easy bleeding, itching, small, spider-like blood vessels visible in the skin, enlarged spleen, fluid in the abdominal cavity, chills, pain from the biliary tract or pancreas, an enlarged gallbladder, and in rare cases, tremors in the left hand. The symptoms related to liver dysfunction include both physical signs and a variety of symptoms related to digestive problems, blood sugar problems, immune disorders, abnormal absorption of fats, and metabolism problems. Patients with mild liver disease may have few or no symptoms or signs. Patients with more serious disease develop symptoms and signs that may be nonspecific or specific. Nonspecific symptoms, that is, symptoms that don't suggest that the liver is their cause, include: Fatigue, Weakness, Vague abdominal pain, and Loss of appetite. Symptoms and signs that are specific for liver disease include: Yellowing of the skin (jaundice) due to the accumulation of bilirubin in the blood, Itching, and Easy bruising due to decreased production of blood clotting factors by the diseased liver. Severe, advanced liver disease with cirrhosis can produce symptoms and signs related to cirrhosis; these symptoms include: Fluid accumulation in the legs (edema) and abdomen (ascites), Mental confusion or coma, Kidney failure,
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Vulnerability to bacterial infections, and Gastrointestinal bleeding. THE CLASSIC SYMPTOMS OF LIVER DAMAGE INCLUDE THE FOLLOWING: Pale stools occur when stercobilin, a brown pigment, is absent from the stool. Stercobilin is derived from bilirubin metabolites produced in the liver. Dark urine occurs when bilirubin mixes with urine Jaundice (yellow skin and/or whites of the eyes) This is where bilirubin deposits in skin, causing an intense itch. Itching is the most common complaint by people who have liver failure. Often this itch cannot be relieved by drugs. Swelling of the abdomen, ankles and feet occurs because the liver fails to make albumin. Excessive fatigue occurs from a generalized loss of nutrients, minerals and vitamins. Bruising and easy bleeding are other features of liver disease. The liver makes substances which help prevent bleeding. When liver damage occurs, these substances are no longer present and severe bleeding can occur. ( Crohn's & Colitis et al., 2010) DIAGNOSIS The diagnosis of liver function is made by blood tests. Liver function tests can readily pinpoint the extent of liver damage. If infection is suspected, then other serological tests are done. Sometimes, one may require an ultrasound or a CT scan to produce an image of the liver. Physical examination of the liver is not accurate in determining the extent of liver damage. It can only reveal presence of tenderness or the size of liver, but in all cases, some type of radiological study is required to examine it. BIOPSY The ideal way to determine damage to the liver is with a biopsy. A biopsy is not required in all cases, but may be necessary when the cause is unknown. A needle is inserted into the skin just below the rib cage and a biopsy is obtained. The tissue is sent to the laboratory, where it is

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analyzed under a microscope. Sometimes, a radiologist may assist the physician performing a liver biopsy by providing ultrasound guidance. REGENERATION The liver is the only internal human organ capable of natural regeneration of lost tissue; as little as 25% of a liver can regenerate into a whole liver. This is predominantly due to the hepatocytes re-entering the cell cycle. That is, the hepatocytes go from the quiescent G0 to the G1 phase and undergo mitosis. This process is activated by the p75 receptors (Suzuki K, Tanaka M, Watanabe N, Saito S, Nonaka H, Miyajima A et al., 2008) Scientific and medical works about liver regeneration often refer to the Greek Titan Prometheus who was chained to a rock in the Caucasus where, each day, his liver was devoured by an eagle, only to grow back each night. Some think the myth indicates the ancient Greeks knew about the livers remarkable capacity for self-repair, though this claim has been challenged. (Chen P et al., 1994, Power C and Rasko J et al., 2008). LIVER TRANSPLANTATION Human liver transplants were first performed by Thomas Starzl in the United States and Roy Calne in Cambridge, England in 1963 and 1965, respectively. Liver transplantation is the only option for those with irreversible liver failure. Most transplants are done for chronic liver diseases leading to cirrhosis, such as chronic hepatitis C, alcoholism, autoimmune hepatitis, and many others. Less commonly, liver transplantation is done for fulminant hepatic failure, in which liver failure occurs over days to weeks. Liver allografts for transplant usually come from donors who have died from fatal brain injury. Living donor liver transplantation is a technique in which a portion of a living person's liver is removed and used to replace the entire liver of the recipient. This was first performed in 1989 for pediatric liver transplantation. Only 20% of an adult's liver (Couinaud segments 2 and 3) is needed to serve as a liver allograft for an infant or small child. More recently, adult-to-adult liver transplantation has been done using the donor's right hepatic lobe, which amounts to 60% of the liver. Due to the ability of the liver to regenerate, both the donor and recipient end up with normal liver function if all goes well. This procedure is more
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controversial, as it entails performing a much larger operation on the donor, and indeed there have been at least two donor deaths out of the first several hundred cases. A recent publication has addressed the problem of donor mortality, and at least 14 cases have been found. (Bramstedt K et al., 2006). The risk of postoperative complications (and death) is far greater in right-sided operations than that in left-sided operations. DEVELOPMENT FETAL BLOOD SUPPLY In the growing fetus, a major source of blood to the liver is the umbilical vein which supplies nutrients to the growing fetus. The umbilical vein enters the abdomen at the umbilicus, and passes upward along the free margin of the falciform ligament of the liver to the inferior surface of the liver. There it joins with the left branch of the portal vein. The ductus venosus carries blood from the left portal vein to the left hepatic vein and then to the inferior vena cava, allowing placental blood to bypass the liver. In the fetus, the liver develops throughout normal gestation, and does not perform the normal filtration of the infant liver. The liver does not perform digestive processes because the fetus does not consume meals directly, but receives nourishment from the mother via the placenta. The fetal liver releases some blood stem cells that migrate to the fetal thymus, so initially the lymphocytes, called T-cells, are created from fetal liver stem cells. Once the fetus is delivered, the formation of blood stem cells in infants shifts to the red bone marrow. After birth, the umbilical vein and ductus venosus are completely obliterated in two to five days; the former becomes the ligamentum teres and the latter becomes the ligamentum venosum. In the disease state of cirrhosis and portal hypertension, the umbilical vein can open up again.

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A literature review is a body of text that aims to review the critical points of current knowledge including substantive findings as well as theoretical and methodological contributions to a particular topic. Literature reviews are secondary sources, and as such, do not report any new or original experimental work. Most often associated with academic-oriented literature, such as a thesis, a literature review usually precedes a research proposal and results section. Its ultimate goal is to bring the reader up to date with current literature on a topic and forms the basis for another goal, such as future research that may be needed in the area. A well-structured literature review is characterized by a logical flow of ideas; current and relevant references with consistent, appropriate referencing style; proper use of terminology; and an unbiased and comprehensive view of the previous research on the topic.
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In spite of many synthetic compounds, the most efficient drugs available have their roots directly or indirectly related with the plant kingdom. Many of the extracts have proven to possess pharmacological action. Caesalpinia sappan, a plant widely used in the traditional medicinal systems of India has been reported to possess antibacte-rial, anti-inflammatory, antioxidant, anticancer and immunosuppressive activities. This review highlights some of the phytochemical and pharmacological aspects. (Chaitali R. Pawaret al., 2009) Antioxidant activities of the 95% ethanol extract from Caesalpinia sappan heartwood (ECS), protosappanin A, protosappanin B, and brazilein were studied in vitro. The inhibition of the formation of malondialdehyde (MDA) and the scavenging of superoxide anions, hydrogen peroxide, and hydroxyl radicals were assayed. The experimental results show that all four substances had antioxidant activity in vitro but their capabilities differed for the different indicators. ECS, protosappanin A, and protosappanin B show more inhibition of MDA and scavenging of hydrogen peroxide, while brazilein shows more scavenging of hydroxyl radicals. All the samples show little scavenging of superoxide anions. (Jun Hu,Xiaoling Yan, Wei Wang, Hao Wu , Lei Hua and Lijun Duet al.,2008) The Argentinean legume Caesalpinia paraguariensis Burk. (Fabaceae) was selected forfurther fractionation work based on the strong antimicrobial activity of its CH2Cl2-MeOH(1:1 v/v) extract against a host of clinically significant microorganisms, including antibioticresistant strains. 1D and 2D NMR enabled the identification of the novel benzoxecin derivative caesalpinol along with the known compounds bilobetin, stigma-5-en-3-O -6 stearoylglucopyranoside, stigma-5-en-3-_-6_-palmitoylglucopyranoside, stigma-5-en-3-_-

glucopyranoside, oleanolic acid, 3-O-(E)-hydroxycinnamoyl oleanolic acid, betulinic acid, 3-O(E)-hydroxycinnamoyl betulinic acid, and lupeol from the active fractions. Oleanolic acid was found active against Bacillus subtilis and both methicillin-sensitive and -resistant Staphylococcus aureus with MICs of 8 (17.5 m), 8 and 64 (140 m) g/ml, respectively. The rest of the compounds, however, did not show activity. (Girma M. Woldemichaela, Maya P. Singhb, William M. Maieseb, and Barbara N. Timmermanna 2003)

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References and further reading may be available for this article. To view references and further reading you must purchase this article. The antioxidant activity (AA) of ethyl acetate extracts of Caesalpinia sappan, Lithospermum erythrorhizon, Anemarrhena asphodeloides, Paris polyphylla and Illicium verum were tested in refined peanut oil at 60 0.5 C. The concentrations of the extracts added were 0.20% (w/w). The rate of oxidation was assessed by the measurement of peroxide value (PV) and calculation of such characteristics as induction period (IP), when PV reaches 20 meq kg1, protection factor (PF), which is the ratio of IP of the sample with additive' and IP of the sample without additive', and AA (the ratio of IP increase of the sample with extract' and IP increase of the sample with butylated hydroxytoluene'). All of C. sappan, L. erythrorhizon extracts and their combinations were found to be high effective in peanut oil. But the extracts of A. asphodeloides, P. polyphylla and I. verum slightly decrease the formation of peroxides in peanut oil as compared with pure oil. (Pan Yingming, Liang Ying, Wang Hengshan and Liang Min et al.,2004) In this work we have applied Fourier-transform Raman spectroscopy to the analysis of several archival samples of brazilwoods from different geographical origins and of different ages. Samples of Caesalpinia echinata Lam. (from Brazil, South America), Caesalpinia sappan L. (East Indies, Asia), Haematoxylum brasiletto Karsten (Central America) and Haematoxylum campechianum L. (North America) were analysed in order to isolate key Raman biomarker bands which could provide the basis for an identification protocol. Previously recorded Raman spectra of brazilin and brazilein pigments extracted from genuine brazilwood of Brazilian origin provided a foundation for the nondestructive spectral discrimination between a sample of false brazilwood, which consisted of an inferior wood substratum stained with genuine pigment, and the true specimens. The provision of well-documented specimens of determinable age from the archival collection facilitated the evaluation of the effects of temporal degradation on the observed spectra, which could be used to further test the experimental protocols for nondestructive verification of samples in the archive with questionable assignment or provenance.(Howell G. M. Edwards, Luiz F. C. de Oliveira and Mark Nesbitt 2003). Trichloroethylene (TCE) induces liver cancer in mice but not in rats. Three metabolites of TCE may contribute--chloral hydrate (CH), dichloroacetate (DCA), and trichloroacetate (TCA). CH
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and TCA appear capable of only inducing liver tumors in mice, but DCA is active in rats as well. The concentrations of TCA in blood required to induce liver cancer approach the mM range. Concentrations of DCA in blood associated with carcinogenesis are in the sub-microM range. The carcinogenic activity of CH is largely dependent on its conversion to TCA and/or DCA. TCA is a peroxisome proliferator in the same dose range that induces liver cancer. Mice with targeted disruptions of the peroxisome proliferator-activated receptor alpha (PPAR-alpha) are insensitive to the liver cancer-inducing properties of other peroxisome proliferators. Human cells do not display the responses associated with PPAR-alpha that are observed in rodents. This may be attributed to lower levels of expressed PPAR-alpha in human liver. DCA treatment produces liver tumors with a different phenotype than TCA. Its tumorigenic effects are closely associated with differential effects on cell replication rates in tumors, normal hepatocytes, and suppression of apoptosis. Growth of DCA-induced tumors has been shown to arrest after cessation of treatment. The DCA and TCA adequately account for the hepatocarcinogenic responses to TCE. Low-level exposure to TCE is not likely to induce liver cancer in humans. Higher exposures to TCE could affect sensitive populations. Sensitivity could be based on different metabolic capacities for TCE or its metabolites or result from certain chronic diseases that have a genetic basis. (R J Bull et al.,2009). High doses of pyrethrins have been shown to produce liver tumors in female rats. Pyrethrins are not genotoxic agents. Pyrethrins produce liver tumors in rats by a mode of action (MOA) involving induction of hepatic xenobiotic metabolising enzymes, hypertrophy, increased cell proliferation, and the development of altered hepatic foci. The relevance of pyrethrins-induced rat liver tumors to human health was assessed by using the 2006 International Programme on Chemical Safety Human Relevance Framework. The postulated rodent tumor MOA was tested against the Bradford Hill criteria and was found to satisfy the conditions of dose and temporal concordance, biological plausibility, coherence, strength, consistency, and specificity that fit with an established mode of action for rodent liver tumor formation by phenobarbital and related compounds, which are activators of the constitutive androstane receptor. Other possible MOAs including mutagenicity, cytotoxicity, hepatic peroxisome proliferation, porphyria, and hormonal pertubation were excluded. The proposed MOA is considered not to be plausible in humans because pyrethrins, like phenobarbital, do not induce cell proliferation in human hepatocytes. Moreover, epidemiological studies with phenobarbital demonstrate that such compounds do not

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increase the risk of liver tumors in humans. It is concluded that pyrethrins do not pose a hepatocarcinogenic hazard for humans. (Osimitz TG, Lake BG et al.,2007) The demonstrated modified spectrophotometric method makes use of the 2,2-diphenyl-1picrylhydrazyl (DPPH) radical and its specific absorbance properties. The absorbance decreases when the radical is reduced by antioxidants. In contrast to other investigations, the absorbance was measured at a wavelength of 550 nm. This wavelength enabled the measurements of the stable free DPPH radical without interference from microalgal pigments. This approach was applied to methanolic microalgae extracts for two different DPPH concentrations. The changes in absorbance measured vs. the concentration of the methanolic extract resulted in curves with a linear decrease ending in a saturation region. Linear regression analysis of the linear part of DPPH reduction versus extract concentration enabled the determination of the microalgaes methanolic extracts antioxidative potentials which was independent to the employed DPPH concentrations. The resulting slopes showed significant differences (6 - 34 mol DPPH g-1 extract concentration) between the single different species of microalgae (Anabaena sp., Isochrysis galbana, Phaeodactylum tricornutum, Porphyridium purpureum, Synechocystis sp. PCC6803) in their ability to reduce the DPPH radical. (Kai Marxen 1, Klaus Heinrich Vanselow , Sebastian Lippemeier , Ralf Hintze , Andreas Ruser and Ulf-Peter Hansen et al.,2007) Extracts of 4 medicinal and aromatic plants were investigated for their antioxidant potency employing six various established in vitro system: H. officinalis L. var. angustifolius aerial parts, C. speciosum flowers, V. odorata and B. hyrcana leaves.With regard to IC50 values (g/ ml), the order in DPPH radical-scavenging were CS (585.6) > HO (311) > VO (245.1) > and BH (113.1). Effectiveness in reducing powers were high and in a descending order of HO > CS > BH > VO (at the concentrations of 25-800 g/ ml). IC50 for Fe2+ chelating ability were 188, 750 and 980 g/ ml for VO, CS and HO, respectively. BH extract has shown only 38% inhibition at 800 g/ ml. The extracts showed weak nitric oxide-scavenging activity. All extracts exhibited very low and moderate concentration-dependent antioxidant activity in FTC methods. IC50 for scavenging of H2O2 were 169 for BH, 175 for CS, 640 for VO and 663 g/ ml for HO. The content of total phenolic compounds and flavonoids were measured in plant extracts. The data obtained in the in vitro models clearly establish the antioxidant potency of all extracts.( Mohammad ali

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ebrahimzadeha, Seyed mohammad nabavia,b, Seyed fazel nabavia, Fatemeh bahramianc and Ahmad reza bekhradnia et al.,2008) The active principle was extracted from Trygon pastinaca (the fish from Black Sea) liver. Byusing the DPPH and DMPD methods the photoprotective action was demonstrated.The results demonstrate a protective effect of anisole for the tasted oil when the dose level was 0.15mg for both methods. Comparing with the vitamin E, the tested principle presents a lower activity, but this activity is enough to have a photoprotective action (protective factor of 26.04% and 28.6 % when DPPH and DMPD methods were used). (1Florica busuricu, Lantoanela popescu, 2Doina paula balaban, 1Ticua prjol negreanu, 3Stela zamfirescu et al.,2008) Cajanus indicus is a herb with medicinal properties and is traditionally used to treat various forms of liver disorders. Present study aimed to evaluate the effect of a 43 kD proteinisolated from the leaves of this herb against chloroform induced hepatotoxicity. Male albino mice were intraperitoneally treated with 2mg/kg body weight of the protein for 5 daysfollowed by oral application of chloroform (0.75ml/kg body weight) for 2 days. Different biochemical parameters related to physiology and pathophysiology of liver, such as, serumglutamate pyruvate transaminase and alkaline phosphatase were determined in the murine sera under various experimental conditions. Direct antioxidant role of the protein was also determined from its reaction with Diphenyl picryl hydraxyl radical, superoxide radical and hydrogen peroxide. To find out the mode of action of this protein against chloroform induced liver damage, levels of antioxidant enzymes catalase, superoxide dismutase and glutathione-S-transferase were measured from liver homogenates. Peroxidation of membrane lipids both in vivo and in vitro were also measured as malonaldialdehyde. Finally, histopathological analyses were done from liver sections of control, toxin treated and protein pre- an posttreated (along with the toxin) mice. Levels of serum glutamate pyruvate transaminase and alkaline phosphatase, which showed an elevation in chloroform induced hepatic damage, were brought down near to the normal levels with the protein pretreatment. On the contrary, the levels of antioxidant enzymes such as catalase, superoxide dismutase and glutathione-S-transferase that had gone down in mice orally fed with chloroform were significantly elevated in protein pretreated ones. Besides, chloroform induced lipid peroxidation was effectively reduced by protein treatment both in vivo and in vitro. In cell free system the protein effectively quenched diphenyl picryl hydrazyl radical and superoxide radical, though it could not catalyse the breakdown of hydrogen peroxide. Post
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treatment with the protein for 3 days after 2 days of chloroform administration showed similar results. Histopathological studies indicated that chloroform induced extensive tissue damage was less severe in the mice livers treated with the 43 kD protein prior and post to the toxin administration. Results from all these data suggest that the protein possesses both preventive and curative role against chloroform induced hepatotoxicity and probably acts by an anti-oxidative defense mechanism. ( Ayantika Ghosh, Kasturi Sarkar and Parames C. Sil et al.,2006) Natural products have provided a variety of lead structures, which serve as templates for the development of new drugs. The water kept in Caesalpinia sappan Linn. (Sappan lignum) heartwood is being used in Kerala as herbal drinking water for its antithirst, blood purifying, antidiabetic, improvement of complexion and several other properties. The plant is also being used worldwide for a large number of traditional medicinal purposes. Modern day research confirms its cytotoxic, antitumor, antimicrobial, antiviral, immunostimulant and several other activities. Several triterpenoids, flavonoids, oxygen heterocycles, etc. were isolated. Brazilin is found to be the main constituent of the plant responsible for several of its biological activities. The use of heartwood as a colouring agent for wine, meat, fabric, etc. is well established. It has the potential to hit the market as a safe natural colouring agent with good medicinal value for food products, beverages and pharmaceuticals. There is also a scope for further research to establish its medicinal properties and to identify lead compounds for drug development. (Shrishailappa Badami , Sudheer Moorkoth and Suresh B et al.,2004) Antioxidant activity of Caesalpinia sappan heartwood was studied both by in vitro and in vivo models. The ethyl acetate, methanol and water extracts exhibited strong antioxidant activity as evidenced by the low IC50 values in both 1,1-diphenyl-2-picryl hydrazyl (DPPH) and nitric oxide methods. The values were found to be less or comparable to those of ascorbic acid and rutin, the standards used. Administration of the successive methanol and water extracts at 50 and 100 mg/kg body weight given for four days prior to carbon tetrachloride (CCl4) treatment caused a significant increase in the level of superoxide dismutase (SOD) and catalase and a significant decrease in the level of thiobarbituric acid reactive substances (TBARS), when compared to CCl4 treated control in both liver and kidney. These changes observed at 100 mg/kg body weight treatment were comparable to those observed for standard vitamin E at 50 mg/kg treatment. The results support significant antioxidant nature of Caesalpinia sappan heartwood extracts. (Badami S, Moorkoth S, Rai SR, Kannan E, Bhojraj S.et al.,2006)
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Antiradical activities of some commercial cognacs were evaluated by the DPPH test. Different mathematical models for the evaluation of the antiradical efficiency of the cognac samples were proposed and discussed. Nonflavonoid phenols were found to be the main substances responsible of the radical scavenging activity of cognacs. In particular the strongest correlations between antiradical activity measurements and cognac chemical characteristics was found for ellagitannins, high molecular weight polyphenols, which are extracted from the wood and solubilized in the spirit mainly during first year aging. (Carla Da Porto, Sonia Calligaris, Emilio Celotti, and Maria Cristina Nicoli et al.,2000)Full-size image (8K) The reaction mechanisms of three antioxidants are proposed in order to explain experimental results obtained from a kinetic study using the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, previously adapted in our laboratory. In its radical form, DPPH shows an absorbance maximum at 515 nm which disappears upon reduction by an antiradical compound. BHT, a synthetic antioxidant, slowly reacts with DPPH reaching a steady state within 5 h. This 2.8-stoichiometric complete reaction follows a 1.5-order with respect to DPPH and 0.5 to BHT. The kinetic rate constant, k, is estimated to be 5.0 L/(mols) at 20 C and the energy of activation, Ea, is equal to 35 kJ/mol in methanol. Eugenol reacts with DPPH reaching a steady state within 2 h. This 1.9-stoichiometric reaction follows a 2-order with respect to both DPPH and eugenol, k and Ea are estimated to be 5.4 3 1010 L3/(mol3s) at 20 C and 30 kJ/mol, respectively. The eugenol mechanism may involve a dimerization between two phenoxyl radicals. The reaction with isoeugenol is rapid and reversible, with a stoichiometry of 1.1. It is first order with respect to isoeugenol with k (direct reaction) equal to 8.9 3 102 s1 at 10 C. This reaction is consistent with a pseudo-monomolecular mechanism. .( V. Bondet, W. BrandWilliams and C. Berset et al.,1997) 5,7,3V,4V-hydroxy-substituted flavonoids are considered as very efficient radical scavengers. This study examines the effect of the structural elements of the C-ring, on the radical scavenging activity of these compounds. The impact of di-hydroxy substitution of A- or B-ring on the activity of fully substituted C-ring flavonoids was also studied. Quercetin, luteolin, taxifolin, eriodictyol, rutin, (+)-catechin, (_)-epicatechin, fisetin and kaempferol were studied during the reaction with the DPPH radical; they revealed two distinctive steps of reaction, a first rapid and a second slower. DPPH scavenging followed second order kinetics during the rapid step; stoichiometric factors and rate constants were determined.
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Quercetin and fisetin scavenged four radicals by each molecule, while the other flavonoids scavenged two radicals with a consequent production of B-ring diquinone. Also the rate constants were affected by C-ring structure. The kinetics of the slow step was much more complicated and the contribution of C-ring was based on stoichiometry. (Dimitrios I. Tsimogiannis, Vassiliki Oreopoulou et al.,2005) (Hydromethanol extracts of 15 Bangladeshi medicinal plants, traditionally used in different ailments, were evaluated for antioxidant potential using DPPH (1,1-diphenyl-2-picryl hydrazyl) radical scavenging assay. Among the extracts Cocos nucifera, Caesalpinia pulcherrima, Punica granatum and Syzygium cumini were found displaying strong (90% or more) DPPH radical scavenging action. Syzygium cumini exhibited the highest radical scavenging, with an IC50 value of 4.25 _g/ml compared to the IC50 value of 5.15 _g/ml as shown by the reference antioxidant ascorbic acid, in a dose dependent fashion. (S. M. Raquibul Hasan1, Md. Mokarram Hossain, Raushanara Akter, Mariam Jamila3, Md. Ehsanul Hoque Mazumder and Shafiqur Rahman et al., 2009) The ethanolic extract of Ipomoea batatas was evaluated for its in vitro cytotoxic and antioxidant activities. The extract showed potent cytotoxic activity in trypan blue dye exclusion method using DLA cell lines with EC50 value of 305g/ml and exhibited a dose dependent decrease in cell count for all the concentrations tested. The antioxidant activity was evaluated by DPPH free radical method. The extract exhibited potent antioxidant activity with an EC50 of 36.5g/ml. (Prasanth nv, Dilip C, Sanal dev kt, LIS Augustine, Saraswathi r. et al.,2010) Mesalamine (5-aminosalicylic acid, 5-ASA) is used because of its local effects in the treatment of inflammatory bowel disease. Therefore, the aims of this work were to compare and validate three analytical methods for the quality control of commercial coated tablets containing 5-ASA: high performance liquid chromatography (HPLC), 1,1- diphenyl-2-picrylhydrazyl radicals (DPPH) and nitrosation. The parameters linearity, precision and accuracy were studied in this work. HPLC with ultraviolet detection at 254 nm was carried out with a C18 column and a mobile phase constituted of 30 mmol/L monobasic phosphate buffer (pH 7.0) and methanol (70:30; v/v), with 25% tetrabutylammonium hydrogen sulphate. The DPPH method was performed at 517 nm and using 100 mmol/L acetate buffer, pH 5.5, ethanol and 250 mol/L ethanolic solution of DPPH. The nitrosation method was accomplished by using a platinum electrode and standard mol/L sodium nitrite as titrant solution. Repeatability (intra-day) and
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intermediate precision (inter-day) expressed as RSD, were lower than 3%. The experimental recoveries were between 72.5 and 99.9%. Statistical analysis by one-way ANOVA, followed by the multiple comparison test of Bonferroni showed no significant difference among the three methods. All proposed methods can be used for the reliable quantitation of 5-ASA in pharmaceutical dosage forms. (Janice Aparecida Rafael, Jos Roberto Jabor, Rbia Casagrande, Sandra Regina Georgetti, Maria de Ftima Borin, Maria Jos Vieira Fonseca et al.,2007) Antioxidation properties and mechanism of action of dihydromyricetin from Ampelopsis grossedentata. To assess the antioxidative properties and the mechanism of action of dihydromyricetin (DMY) from Ampelopsis grossedentata.( Zhang YS, Ning ZX, Yang SZ, Wu H. Yao Xue Xue Bao et al.m2003)

References and further reading may be available for this article. To view references and further reading you must purchase this article. Measuring the AC susceptibility and registering the ESR spectra of the stable 1,1-diphenyl-2picryl-hydrazyl (DPPH) radical at 77.4125 K, we have studied the effect of the radical on the current properties of the intergranular contacts in the superconducting ceramics Bi1.8Pb0.3Sr1.9Ca2.8Cu3.4Oy. Critical current in the ceramics is decreased by the magnetic field generated by the radical spin. The effect of the field on the critical current corresponds to the location of the adsorbed radical at a distance of 56 from the superconducting phase.( L. L. Makarshin and V. N. Parmon Et al.,2000) The Flavonoids extracted from tartary buckwheat seeding was tested by the DPPH elimination method to study its antioxidant activity.The results showed that the Flavonoids extracted from tarrtary buckwheat seedling was of a strong anti-oxidizing action. The DPPH elimination rate was up to 70.07% when the Flavonoids concentration was 46.674 g /mL and its elimination rate was significantly higher than that of Vc and Ve with the same concentration. The Flavonoids extracted from tartary buckwheat seedling is a natural oxidizer with the development.(Fang YuMei; Tan Ping; Wang YiHong; Zhang ChunSheng et al.,2007)

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The factors contributing to the loss of phenolic content and DPPH radical scavenging activity of Taiwanese yam, Dioscorea alata Tainung No. 1 (TNG1), in the 20% TNG1-containing pollock surimi gel were investigated. Heating at 90C for 30 min decreased both the total phenolic content and DPPH radical scavenging activity in 50% ethanolic extract from TNG1, but no significant effect was found for adding 2% NaCl. Blending 20% TNG1 with pollock surimi without heating or adding 2% NaCl could reduce the DPPH radical scavenging activity. For 20% TNG1-containing surimi samples, heating at 90C for 30 min decreased the total phenolic content, while the combination of the heat treatment and 2% NaCl resulted in further decrease. It was suggested that heat treatment might also cause an interaction between the denatured fish proteins and phenolic compounds in TNG1, thus decreasing the extractability of the phenolic compounds. (Jing-Chung chen1, Jan-ying yeh1, et al.,2010) Free radicals are implicated for more than eighty diseases including diabetes mellitus, arthritis, cancer, ageing, etc. in treatment of these diseases; antioxidant therapy has gained an utmost importance. Current research is now directed towards finding naturally occurring antioxidant of herbal drugs. Antioxidant activity of methanol extract of Chitrakadi Vati was evaluated by using Phosphomolybdenum assay, DPPH radical scavenging assay, superoxide radical scavenging assay and ABTS assay. The total phenolic, total tannins and total flavonoids content were determined. Antibacterial activity was also studied against Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Streptococcus pyogenes by using cup-plate method. Erythromycin was used as standard antibacterial agent. The methanol extract was diluted into different concentration (1, 2, 4, 6, 8, 10 mg/l00 l) with DMSO. The results of the study revealed that, the Chitrakadi Vati exhibited significant antibacterial activity. (Bagepalli Srinivas Ashok Kumar, Vontoor Byrappa Narayana Swamy, Peresandra Avalakondarayappa Arun Kumar 3, and SaleemullaKhan et al.,2010). An ultrasonic technique was employed to extract polysaccharides fromInonotus obliquus(UPS). The effects of ultrasonic conditions on the recovery and 1, 1-diphenyl -2-picrylhydrazyl (DPPH) radical scavenging activity of UPS were evaluated. The physicochemical properties of UPS were analyzed by differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and UV-visible spectrophotometry. Molecular weights of UPS were determined by gel permeation chromatography (GPC). The optimal ultrasonic conditions to obtain the highest
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recovery of polysaccharides were 80 min, 75 C, and 100 w. While the optimal ultrasonic conditions to obtain the strongest DPPH scavenging activity of polysaccharide were 80 min, 95 C, and 160 w. The differences were found by DSC, SEM, GPC analysis and no apparent differences were found from the UV-vis spectra of the polysaccharides after the ultrasonic treatment, which indicated that ultrasonic treatment might cause the physicochemical changes in UPS conformation and degradation of UPS chain but not the main groups. Ultrasonic technique was an effective method to extract bioactive polysaccharides fromI. obliquus(UPSPractical Application: Polysaccharides fromInonotus obliquusexhibited many biological activities including antitumor, antioxidant, hypoglycemic, immune-stimulating effects, and so on. Ultrasonic treatment is now an usual proceeding technique for the extraction of some bioactive constituents. But there was no information about the effects of ultrasonic treatment on the physicochemical properties and DPPH radical scavenging activity of polysaccharides fromI. obliquusuntil now. Ultrasonic technique was an effective method to extract bioactive polysaccharides fromI. obliquus(UPS) according to this study. ( Fu, L., Chen, H., Dong, P., Zhang, X. and Zhang, M.et al.,2010), Inhibition of polyphenol oxidase (PPO)-mediated browning in apple juice using different concentrations of floral honey was evaluated by performing the kinetics of PPO inhibition, effect of honey concentrations on activity of PPO and rate of quinone formation (browning) in apple juice. The antioxidant status of honey was demonstrated by 2, 2-diphenyl-1-picryl hydrazine (DPPH) radical-scavenging assay. Agar diffusion assay was carried out to demonstrate the antimicrobial activity of honey. Kinetic experiment suggests that honey is a noncompetitive inhibitor of PPO having Km = 3.33 mM. There exist a negative correlation ( = 0.65) between different concentrations (50100%, v/v) of honey and PPO activity with significant retardation in rate of browning reaction in apple juice. The DPPH radical-scavenging activity showed that the antioxidant potential of honey strongly depends on its concentration with a positive correlation ( = +0.96). Assessment of the antimicrobial activity of honey using agar diffusion assay demonstrated the significant inhibition of Candida albicans (Microbial Technology & Culture Collection [MTCC-3018]), Escherichia coli (MTCC-1588), Pseudomonas aeruginosa (MTCC2488) and Staphylococcus aureus (MTCC-96) as compared to standard antibiotics.( Gacche, R., Shinde, B., Dhole, N., Pund, M. and Jadhav, A. 2009).

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The scavenging behavior of a series of catechol and guaiacol acid derivatives toward DPPH was examined having as a starting point the order of activity derived on the basis of theoretically calculated BDE values. The studied compounds were protocatechuic, homoprotocatechuic, dihydrocaffeic, and caffeic acids and also vanillic, homovanillic, dihydroferulic, and ferulic acids. Catechol and guaiacol were used as reference compounds. Observations from the parallel study were made with regard to structural features (number and position of hydroxyl groups and the side-carbon chain characteristics) that regulated the behavior of the compounds experimentally. The exceptional DPPH scavenging behavior observed for homoprotocatechuic acid in ethanol and for caffeic acid in acetonitrile could not be supported by the respective BDE values. Ferulic was the most active among guaiacolic acids, whereas dihydroferulic exhibited the highest stoichiometry. Ionizable carboxylic groups seem to affect considerably the relative order of activity as was also evidenced using the ORAC assay. Questions raised about the validity of widely accepted views on criteria for SARs are discussed with regard to literature findings. (Stella A. Ordoudi, Maria Z. Tsimidou,Anastasios P. Vafiadis, and Evangelos G. Bakalbassis 2006).

Liver damage can be a very serious condition. Treatment is to discontinue the substance or substances that have caused the damage and work toward managing the symptoms. Herbalism is a traditional medicinal or folk medicine practice based on the use of plants and plant extracts. Sometimes the scope of herbal medicine is extended to include fungi and bee products, as well as minerals, shells and certain animal parts. The use of herbs to treat disease is almost universal among non-industrialized societies. A number of traditions came to dominate the practice of herbal medicine at the end of the twentieth century. The aim of our study was to investigate functional, therapeutic implications and applications of free radical antioxidant and anti-cancer activities of the medicinal plants.

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PLANT USED Caesalpinia sappan COLLECTION OF THE PLANT The Aerial part of caesalpinia sappan were collected freshly from the Thirunelveli District, Tamil Nadu, India . During the month of March 2010 and indentified authenticated by V.Chelladurai (Research officer),Department of Botany (C.C.R.A.S),Government of India . PREPARATION OF LEAF POWDER The leaves were shade dried for about 10-15 days to remove the moisture content and to obtain fully dried leaves. The sunlight drying of leaves are avoided. As it may bring about concentration changes in the constituents of the leaves due to photosynthesis.The dried leaves are then weighed and grinded. The fine powder is obtained by sieving. It is weighed and stored in sterile containers.
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AQUEOUS EXTRACT PREPARATION The fine powder was subjected to Soxhlet and the aqueous pure extract of the leaf was obtained. Soxhlet extractor A schematic representation of a Soxhlet extractor Stirrer bar Still pot Distillation path Thimble Solid Siphon top Siphon exit Expansion adapter Condenser Cooling water in Cooling water out A Soxhlet extractor is a piece of laboratory apparatus invented in 1879 by Franz von Soxhlet. It was originally designed for the extraction of lipids 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 high solubility in a solvent then a simple filtration can be used to separate the compound from the insoluble substance. The sample is placed in the thimble. Normally a solid material containing some of the desired compound is placed inside a thimble made from thick filter paper, which is loaded into the main chamber of the Soxhlet
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extractor. The Soxhlet extractor is placed onto a flask containing the extraction solvent. TheSoxhlet is then equipped with a condenser. The solvent is heated to reflux. The solvent vapour travels up a distillation arm, and floods into the chamber housing the thimble of solid. The condenser ensures that any solvent vapour cools, and drips back down into the chamber housing the solid material. The chamber containing the solid material slowly fills with warm solvent. Some of the desired compound will then dissolve in the warm solvent. When the Soxhlet chamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask. This cycle may be allowed to repeat many times. Over hours or days.

SOXHLET EXTRACTOR

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During each cycle, a portion of the non-volatile compound dissolves in the solvent. After many cycles the desired compound is concentrated in the distillation flask. The advantage of this system is that instead of many portions o f warms solvent being passed through the sample, just one batch of solvent is recycled.
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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. The aqueous leaf extract obtained using Soxhlet apparatus was dried and the fine powder was used for the experiment. Extraction by using different solvents Chloroform Methanol Petroleum ether Soxhelet extractions 5g of sample Dissolved in 250 ml of the corresponding solvent Three solvents completed Chloroform Methanol Petroleum ether Extracts obtained Caesalpinia sappan (chloroform extracts) Empty weight Of the beaker : 105.44 Beaker containing extracts : 105.99

(105.99 -105.44) =0.55mg extracts Dissolved in 5500l of DMSO to make 100mg/ml concentration Caesalpinia sappan (Petroleum Ether extracts) Empty weight Of the beaker : 105.44
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Beaker containing extracts

: 105.63

(105.63 -105.44) =0.19mg extracts Dissolved in 1900l of DMSO to make 100mg/ml concentration Caesalpinia sappan (methanol extracts) Empty weight Of the beaker : 105.44 Beaker containing extracts : 105. 78

(105.78 -105.44) =0.34mg extracts Dissolved in 3400l of DMSO to make 100Mg/ml concentration CHEMICALS USED DMSO was purchased from Merck India Ltd, Mumbai.MTT were Sigma Chemical co .(MO,USA), Media HI MEDIA, Chloroform CSRL Chemicals, Mumbai, FCS(Fetal calf serum) Axiva sichem pvt .Ltd . Delhi, DPPH ,TPVG ,DET,MET, and antibiotics were from Life Technologies in chennai .All other chemical and reagent were of pure analytical grade .

BIOCHEMICAL TESTS PHYTOCHEMICAL ANALYSIS TANNINS: 1 ml of sample was taken, to that few drops of 0.1% ferric chloride was added and observed for blue colourization/brownish green.

63

SAPONINS: 1 ml of sample was taken,to that 2 ml of H 2O (shaken vigorously) was added and observed for foaming appearance. FLAVONOIDS: 1 ml of sample was taken, to that concentrated HCL and magnesium chloride was added and observed for pink tomoto red colour. ALKALOIDS: 1 ml of sample was taken, to that few drops of dragandoff reagent was added and observed for orange red colour. PROTEINS: 1 ml of sample was taken, to that few drops of Bradford reagent aws added and observed for blue colour development. STEROIDS: 1 ml of sample was taken, to that 10% concentrated H 2SO4 was added and observed for green colour. ANTHRAQUINONES: 1 ml of sample was taken, to that aqueous ammonia (shaking) was added and observed for change in colour of aqueous layer (pink, red or violet). PRELIMINARY PHYTOCHEMICALS SCREENING OF VARIOUS EXTRACTS OF CAESALPINIA SAPPAN AERIAL PART.

S.No

PHYTOCHEMICAL TEST

EXTRACTS

64

PETROLEUM ETHER 1 2 3 4 4 5 6 TEST FOR TANNINS TEST FOR SAPPONINS TEST FOR FLAVONOIDS TEST FOR ALKALOIDS TEST FOR PROTEINS TEST FOR STEROIDS TEST FOR + + + + +

CHLOROFORM

METHANOL

+ + + + +

+ + + + + +

ANTHRA + (-)

QUINONES

CS caesalpinia sappan (+) (-) indicates presence of constituents. indicates absence of constituents.

INVITRO ANALYSIS ANTIOXIDANT ASSAY Anti oxidant study: Anti oxidant is a molecule capable of inhbiting the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions that damage cells.
65

Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols. The antioxidant activities of the above plants extracts, such as super oxide dismutase (SOD)- like and scavenging of diphenyl picryphydrazyl (DPPH) radicals were abserved. All the plant extracts of this study had an antioxidant activity, but the petroleum other extracts of clitoria ternatea showed the highest antioxidant activities. Assessment of invitro antioxidant activity 1, 1Diphenyl2picrylhydrazyl radical scavenging activity The ability of the extract to scavenge DPPH radicals were determined by the method of (Gyamfi et al.,1997). with minor modifications. A 20l of aliquot of test extract at different concentrations in methanol was mixed with 0.5 ml of 100 mM methanolic solution of DPPH. After 30 min incubation in darkness and at ambient temperature, the resultant absorbance was recorded at 517 nm. The percentage inhibition was calculated using the following formula. Percentage inhibition = (Abs control Abs sample) X 100/ Abs control

CYTOTOXICITY Anti-cancer studies on plants: Epidemicological studies have revealed that a diet rich in plant-derived foods has a protective effect on human health. Identifying bioactive dietary constituents is an active atea of scientific investigation that may lead to new drug discovery. More than 50% of todays anticancer drugs are natural products or derived from a natural origin. To discover new entities with potential to treat prostate cancer at androgen-refractory stages, 3b structurally diverse natural products were screened using functional based assays.

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The tested compounds were selected broadly from major secondary metabolites of plants, marine invertebrates, and fungi. These diverse entities were prescreemed for their anti-invarive ability against prostate cancer cells, Pc-3M, using spheroid disaggregation assay. Active representative including three selected structural classes, a macrolide, a - carboline alkaloid, and a phenyl methylene hydantoin (PMH), were then tested for their ability to stabilize junctional complexes and enhance cell-cell adhesion of androgen independent prostate cancer cells. Transepithelial resistance (TER) and paracellular permeability assays were used to elicit the a for mentioned properties. These studies led to the emergence of PMHs as a small molecules class from the marine sponge hemimycle Arabica with a unique potential to attenuate CTstimulated prostate cancer growth, metasis, paracellular permeability, and enhance TER and cellcell adhesion of prostate cancer cells. The unique activities of PMHs were validated using several invitro assays followed by in vivo testing in two mice models. A 3D QSAR was established using Sybyl 8.1 comparative molecular field analysis (Comfa) model. This chapter includes the methodology for evaluation of structural and biological properties of new antiinvasive molecules with an exceptional potential to stabilize junctional complexes from diverse natural product sources. MATERIALS AND METHOD: MATERIALS REQUIRED IN MEM: i) Monolayer culture bottle of Hep2 cell lines. ii) 5ml,10ml serological pipette iii) Minimal essential media (MEM) with 10%,2% foetal calf serum iv) TPVG (Trypsin PBS versene glucose) v) Discarding jar, inverted microscope, desiccators vi) Gloves, spirit, cotton, label pad, marker pen MATERIALS REQUIRED IN CYTOTOXICITY ASSAY: 1. Monolayer culture in log phase 2. Drug extracts (different concentrations) 3. MEM without FCS
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4. 0.45 filter 5. 5ml sterile storage vial 6. Tissue paper, spirit, cotton, marker pen and gloves 7. Micropipette and tips MATERIALS REQUIRED IN MTT ASSAY 1. MTT (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide)stock solution 5mg/ml 2. DMSO-dimethyl sulfoxide 3. Micropipette and 200l of sterile tips 4. Spectrophotometer with 1ml cuvette holder.

MINIMAL ESSENTIAL MEDIA PREPARATION: Media is defined as a complex source of nutritional supplementation vital for the growth proliferation and maintenance of cells in vitro The MEM dissolved in the pre sterilized Millipore distilled water and mixed well, closed and sterilized at 15lbs 121c for 15mins. Allow ingredients in the quantity, depending on the concentration of foetal calf serum (2% or 10%) mix well by shaking. Take care avoid spills pass CO2 using sterile pipette, Shake the bottle, check Ph and adjust to 7.2 to 7.4. The MEM bottles are kept for 2 days at 37c and checked for sterility, PH drop and floating particles they are then transferred to the refrigerator. MEDIA PREPARATION: INGREDIENTS 10% GROWTH MEDIA 2%GROWTH MEDIA MAINTANCE MEDIA WITHOUT FCS MEM 857ml
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937ml

957ml

Penicillin and streptomycin Phenol red Amphotericin B 3% L-glutamine Foetal calf serum 7.5%NaHCo3 Total volume

1ml 1ml 1ml 10ml 100ml 30ml 1000ml

1ml 1ml 1ml 10ml 20ml 30ml 1000ml

1ml 1ml 1ml 10ml nil 30ml 1000ml

PREPARATION OF INGREDIENT: 1. penicillin and streptomycin: (concentration 100IU of penicillin and 100 g 0f streptomycin) Dissolve both antibiotics in sterile Millipore distilled water, so as to aliquots. Store at -20 C Check sterility. 2. Fungi zone (amphotericin B): (conc.: 20g/ml) Dissolve in sterile Millipore distilled water so as to give a final concentration of 20g/ml and distribute in 1ml aliquots in vials. Store at -20c. Check sterility. 3. L.glutamine: 3% Weigh 3g of l-glutamine accurately and dissolve in 100ml sterile vials. Store at -20c. Check sterility. 4. 7.5% sodium-bi-carbonate
69

give a final

concentration 100 IU of penicillin and 100g of streptomycin/ml. Mix well and distribute in 1ml

Millipore distilled water

and mix well. Filter through Millipore membrane filter 0.22 and distribute in 5ml aliquots in

Weigh requisite quantity of sodium-bi-carbonate (to give 7.5% solution) accurately and dissolve in 100ml of sterile Millipore distilled water. Filter through what man filter paper No.1, distribute into bottles and at 121c, 15lbs, 15mins. Cool and store at +4c. 4. Foetal calf serum Bring FCS at room temperature. Inactivated at 56c in water bath for hour and cool at room temperature. If floating particles are seen filter through Seitz filter. Distribute in 100ml, 50ml, 20ml quantities in sterile bottles. Store at -20c. 5. Trypsin, PBS,versene, glucose solution: (TPVG) 2% trypsin: 100ml Weigh 2g of trypsin accurately; dissolve in 100 ml sterile Millipore distilled water with magnetic stirrer for hour. Filter through membrane filter. Store at -20c 0.2%EDTA (versene) Weigh 200mg of EDTA accurately. Dissolve in 100 ml of sterile Millipore distilled water. Autoclave at 121C 15lbs/15mins. 10%glucose -100ml Weigh 1g of glucose accurately. Dissolve in 100 ml of sterile Millipore distilled water and filter through what man filter paper and autoclave at 15lbs/15mins. TPVG-100ml PBS 2%trypsin 0.2%EDTA 10%glucose - 840ml -50ml -100ml -5ml

Penicillin & streptomycin -5ml Mix all ingredients and adjust the pH to 7.4 with 0.1 N HCl or 0.1 N NaOH. Distribute in 100 ml aliquots. Store at -20c.
70

METHOLDOLOGY: MAINTENANCE OF CELL LINE: Maintenance of cells involves the following operations: Dispersion and Sub culturing (seeding) of cells. Preservation of cells in repository. Revival of cells from repository.

SUBCULTURING AND MAINTENANCE OF CELL LINE: 1. Bring the medium and TPVG to room temperature for thawing. 2. Observe the tissue culture bottles for growth, cell degeneration, pH and turbidity by seeing in inverted microscope. 3. If the cells become 80% confluent it goes for sub culturing process 4. Wipe the mouth of the bottle with cotton soaked in spirit to remove the adhering particles. 5. Discard the growth medium in a discarding jar keep distance between the jar and the flask. 6. Then add 4 5 ml of MEM without FCS and gently rinsed with tilting. The dead cells and excess FCS are washed out and then discard the medium. 7. TPVG was added over the cells. And incubate at 37 C for 5 minutes for

disaggregation. The cells become individual and its present as suspension. 8. Add 5ml of 10% MEM with FCS by using serological pipette. 9. Gently give passaging by using serological pipette. If any clumbs is present then repeat the process. 10. After passaging split the cells into 1:2, 1:3 ratio for cytotoxicity studies for plating method
71

SEEDING OF CELLS: After homogenize take one ml of suspension and pour in to 24 well plates. In each well add 1ml of the suspension and kept in a desiccators in 5% CO2 atmosphere. After 2 days incubation observe the cells in inverted microscope. The cells were observed for confluence. CYTOTOXICITY ASSAY: In order to study the antitumor activity of a new drug, it is important to determine the cytotoxicity concentration of the drug. Cytotoxicity tests define the upper limit of the extract concentration, which is non-toxic to the cell line. The concentration nontoxic to the cells is chosen for antiviral assay. After the addition of the drug, cell death and cell viability was estimated. The result is confirmed by additional metabolic intervention experiment such as MTT assay STOCK DRUG CONCENTRATION 0.5ml of drug is dissolved in 4.5 ml of DMSO giving a working concentration of 1mg/ml. the working concentration is prepared fresh and filtered through 0.45 filter before each assay. 1. To prepare 5 ml of extract and giving conc. (1mg/ml). 2. 500l of MEM without FCS was taken in 9 eppendroff tubes./each samples 3. Then 500l of the working conc. was added to the first eppendroff tube and mixed well then 500l of this volume was transferred from first to last tube by serial dilution to obtain the desired concentration of the drug. 4. As a result the volume remains constant but there is a change in concentration. SAMPLING: 1. 48hr monolayer culture of Hep2cells at a concentration of one lakh /ml /well (10 cells / ml / well) seeded in 24 well titer plate. 2. The plates were microscopically examined for confluent monolayer, turbidity and toxicity if the cells become confluent. 3. The growth medium (MEM) was removed using micropipette. Care was taken so that the tip of the pipette did not touch the cell sheet. 80%

72

4. The monolayer of cells was washed twice with MEM without FCS to remove the dead cells and excess FCS. 5. To the washed cell sheet, add 1ml of medium (without FCS) containing defined concentration of the drug in respective wells. 6. Each dilution of the drug ranges from 1:1 to 1:64 and they were added to the respective wells of the 24 well titer plates. 7. To the cell control wells add 1ml MEM (w/o) FCS. 8. The plates were incubated at 37c in 5% CO2 environment and observed for cytotoxicity using inverted microscope. MTT ASSAY: MTT assay is called as (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide.MTT assay was first proposed by Mossman in 1982.

Formazan

PRINCIPLE: MTT is cleaved by mitochondrial dehydrogenase in viable cells, yielding a measurable purple product formazan. This formazan production is proportionate to the viable cell number and inversely proportional to the degree of Cytotoxicity

73

PROCEDURE: After incubation, remove the medium from the wells carefully for MTT assay. In each well wash with MEM (w/o) FCS for 2 3 times. And add 200l of MTT conc of (5mg/ml). And incubate for 6-7hrs in 5% CO2 incubator for Cytotoxicity. After incubation add 1ml of DMSO in each well and mix by pipette and leave for 45sec If any viable cells present formazan crystals after adding solublizing reagent (DMSO) it shows the purple color formation. The suspension is transferred in to the cuvette of spectrophotometer and an O.D values is read at 595nm by taking DMSO as a blank. Graph is plotted by taking concentration of the drug on X axis and relative cell viability on Y axis. Cell viability (%) = Mean OD/Control OD x 100

Disease are common and increasing day by day, which makes the man to search for new and high effective medicine as therapeutic agents. Caesalpinia sappan are used as a folk traditional medicine to treat various types of disease.the phytochemical analysis of Caesalpinia sappan extracts was followed by using various components have identified the different types ingredients. In this phytochemical analysis which is screened both possitive and negative results. In This study the plant Caesalpinia sappan was used to test the cytotoxicity again Hep2 cell line. The result were clear that the find contain it chemical which are basically toxicity in nature or not to cell. So that chemicals are comparable to many broad spectral antibiotics. The results have been clearly supported wider use of this as traditional medicine in all parts of the world,

74

particularly in the Indian system of medicine. Though this is a scientific proof for its anticancer activity, it would be better if the toxicity were clearly studied. The study as clearly indicated the plant can be as an anticancer agent and contains most of the phytochemicals. The following studies are comparable to the result of the presents study.further study are in progress in our laboratory in synthesis of novel derivatives and investigation of molecular mechanism responcible for the cytotoxicity activity and antioxidant of the plant. This study may contribute the improvement of scientific understanding of chemical constituens and functionally of the tested traditional medicinal plants. Phytochemical Analysis: Phytochemical screening of crude extracts The phytochemical screening using organic solvents like petroleum ether , chloroform and methanol extracts of Caesalpinia sappan were carried out .It reveals the presence of following phyto constituents such as tannin,saponins , flavanoids , protein steroids and anthrax quonone in petroleum ether and tannin, flavanoids , protein steroids antra quionones in chloroform extracts and tanins, sapponins, Flavonoids, proteins, steroids and anthra quinones in methanol (Harborne JB .,et al 1998).The results were shown in Fig 1 and 1.1,2 and 2.1,3 and 3.1.

OBSERVATIONS Fig 1 and Fig 1.1 shows the presence of tannin, flavonoid, protein steroid and anthra quonone. It also indicates the absence of sapponins and alkaloids. Fig 2 and Fig 2.1 shows the presence of tannin, sapponins,flavonoid, protein , steroid and anthra quonone. It also indicates the absence of alkaloids. Fig 3 and Fig 3.1 shows the presence of tannin, sappanin, flavonoid, protein, steroid and anthra quonone. It also indicates the absence of alkaloids.
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ANTIOXIDANT Scavenging activity for free radicals of DPPH has been widely used to evaluate the antioxidant activity of natural products from plant and microbial sources. Different organic solvent extracts of plant from the caesalpinia sappan plants listed in table -1 were prepared for investigation of their antioxidant activities. Free radical scavenging activity of crude extracts from the three different solvent extracts of caesalpinia sappan was quantitatively determined using a DPPH Assay .The dosage of solvent extract is expressed in g of dry weight of the extract (COMPOUND) per mL of the assay mixture .Ic 50 value represents the concentration of test extract or compound where the inhibition of test activity reached 50The different solvent extracts of caesalpinia sappan such as petroleum ether ,chloroform and methanol. Most of the plant extracts investigated in this

76

report exhibited good antioxidant DPPH activity is petroleum ether extracts of caesalpinia sappan. The aim of the present study is to be determine radical scavenging compounds in different solvent extracts of caesalpinia sappan based on in vitro reaction of the antioxidant with the DPPH radicals.

OBSERVATION DPPH is one of the free radicals widely used for testing preliminary radical scavenging activity of a compound or a plant extract. In the present study Petroleum ether extracts of caesalpinia sappan showed potential free- radical scavenging activity. The antioxidant activities of the individual compounds, present in the extracts may depended on structural factors, Such as the number of phenolics hydroxyl, keto group, free carboxylic groups and other structural features (Pat DE ., et al 1990).Petroleum ether of caesalpinia sappan extracts has shown good antioxidant activity as compared with other two solvent extracts which is clear from Table -1.From the Fig 4,it is found that the inhibition. In the previously given table 1 shows the optical densities and their respective percentage activities of the antioxidant present have. It was observed that three different organic solvent extracts of caesalpinia sappan have a certain percentage of antioxidant activity . The Methanol of caesalpinia sappan have the lowest antioxidant activity at 1.64 .The Petroleum ether
77

extracts of caesalpinia sappan

have the highly significant antioxidant activity standing at

60.43%. And then chloroform extracts of caesalpinia sappan showed 20.32% antioxidant activity. When compare to these three extracts petroleum ether extracts of caesalpinia sappan acts as a good antioxidant activity. These extracts are used to analyze the presence of anticancer agents. (Tabular column showing optical density and activity of standard and test samples are shown in Table-1)

CYTOTOXICITY The plant extracts caesalpinia sappan with petroleum ether showed effective cell viability. The percentage of cell viability increase with concentration .A cell viability of more than 50% was observed at a concentration lesser then 5mg/ml (sample 6 to sample 8) the sample no.1 had the lowest cell viability standing at 19.60%. The sample no.8 has the highest cell viability 94.11% the plant sample which were analyzed all posses a certain percentage of antioxidant properties. In this three samples out of an sample petroleum ether shows that good antioxidant properties. These sample tested for the presence of anticancer agent they yield positive result.

Various extracts of dried aerial parts exhibited strong cytotoxic properties when tested invitro on several cancerous cell line. The petroleum ether and methanol which may shows the good antioxidant property. The results were shown in table 3, table 4 and Fig 6 ,6.1 and7,7.1. Chloroform extracts shows the lowest cell viability. The results were shown in table 2 and Fig 5
78

and5.1. The comparison between these three extracts (petroleum ether, chloroform and methanol) the results were shown in Fig 8.

OBSERVATIONS The plant extracts caesalpinia sappan showed effective cell viability. The % of cell viability increased on increase of concentration. Table 2, fig 5, fig 5.1 indicates the lowest cell viability. Table 3, table 4 and fig 6, 6.1 and 7, 7.1 indicates the highest cell viability. Among these three extracts petroleum ether which may shows the good antioxidant activity.

79

OBSERVATIONS The comparison between these three extracts (petroleum ether, chloroform and methanol) the results were shown in Fig 5. The plant extracts of methanol shows the highest cell viability standing at 98.03% The plant extracts of chloroform and petroleum ether shows the cell viability standing at 92.15%, and 94.11%

80

OBSERVATIONS Fig a,b,c and d was observed that cell viability increases with increase in concentration of the plant extract. Fig a) and b) shows the lowest cell viability standing at 5.88 and 43.13% Fig c) and d) shows the highest cell viability standing at 68.62 and 92.15%

81

OBSERVATIONS Fig a, b, c and d was observed that cell viability increases with increase in concentration of the plant extract. Fig a) and b) shows the lowest cell viability standing at 13.72 and 37.25% Fig c) and d) shows the highest cell viability standing at 62.74 and 98.03%

82

OBSERVATIONS Fig a, b, c and d was observed that cell viability increases with increase in concentration of the plant extract. Fig a) and b) shows the lowest cell viability standing at 19.60 and 49.01% Fig c) and d) shows the highest cell viability standing at 58.94 and 94.11%

83

Hep G2 is a perpetual cell line which was derived from the liver tissue of a 15 year old Caucasian American male with a well differentiated hepatocellular carcinoma. These cells are epithelial in morphology, have a model chromosome number of 55 and are not tumorigenic in nude mice. The cells secrete a variety of major plasma proteins; e.g., albumin, transferrin and the acute phase proteins fibrinogen, alpha 2-macroglobulin, alpha 1-antitrypsin, transferrin and plasminogen. They have been grown successfully in large scale cultivation systems. Hepatitis B virus surface antigens have not been detected. Hep G2 cells have been shown to be G418 resistant (400 g/mL). The cells will respond to stimulation with human growth hormone. The plant extracts caesalpinia sappan was tested on HEP2 cell lines to check whether they induce apoptosis on the cells. The percentage of cell viability was calculated. Caesalpinia sappan had good antioxidant properties and hence they formed the basis for the performance of our project study. From the above mentioned data was clear that the specific plant caesalpinia sappan shows a significant role of antioxidant and anticancer activity. They were tested using three different extracts. The samples which had good antioxidant activities were studied for the presence of anticancer properties. Caesalpinia sappan extracts were then compared to the standard antioxidant Butyrate hydroxyl toluene (BHT) and their respective antioxidant activities were calculated.
84

Disease are common in the world and increases in day to day life. Which makes the man to search for new and high effective medicine as therapeutic agents. Caesalpinia sappan are used as folk traditional medicines to treat various types of diseases. Cancer is the main types of diseases. Which is largely treated using different types of herbal medicines. The resistant mechanism of the caesalpinia sappan is used as medicine for humans and animals. The phytochemical analysis of Caesalpinia sappan extracts was followed using various components to identify the different types of compounds in the Caesalpinia sappan. The results have been clearly supported is wider use of this a folk traditional medicine, in all parts of world and particularly in the Indian system of medicine. Though this is a scientific proof for its anticancer activity, it would be better if the toxicity were clearly studies. The study has clearly indicated the plant can be as an anticancer and antioxidant properties contains most of them phytochemicals. In summary, we observed that some of our selected plant extracts could dose-dependently and significantly inhibit free radical and superoxide anion. In addition, DNA damage by hydroxyl radicals could also be effectively prevented in the presence of specific organic solvent extracts. Our results in the present report suggest that the 3 different extracts of caesalpinia sappan have a certain percentage of antioxidant activity.

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TABLE 1 TABULATION SHOWING THE ANTIOXIDANT ASSAY OF CAESALPINIA SAPPAN BY USING THREE DIFFERENT SOLVENT EXTRACTS
93

S.no

Plants

Extracts

Blank

Absorbance at 517 nm

%DPPH Scavenging 1.64 20.32 60.43

Caesalpinia sappan 1

Methanol Chloroform Petroleum ether

0.182 0.182 0.182

0.179 0.145 0.072

BHT

100 % activity

ANTIOXIDANT ACTIVITY OF DIFFERENT MEDICINAL PLANTS

TABLE 2 TABULATION SHOWING THE ABSORBANCE AND % OF CELL VIABILITY FOR CAESALPINIA SAPPAN+ CHLOROFORM
94

S.no 1 2 3 4 5 6 7 8

Concentration (mg/ml) 5 2.5 1.25 0.625 0.3125 0.156 0.078 Cell control

Dilutions Neat 1:1 1:2 1:4 1:8 1:16 1:32 -

Absorbance 0.03 0.16 0.22 0.29 0.35 0.39 0.47 0.51

Cell viability 5.88 31.37 43.13 56.86 68.62 76.47 92.15 100

95

TABLE 3 TABULATION SHOWING THE ABSORBANCE AND % OF CELL VIABILITY FOR CAESALPINIA SAPPAN+ METHANOL

S.no 1 2 3 4 5 6 7 8

Concentration (mg/ml) 5 2.5 1.25 0.625 0.3125 0.156 0.078 Cell control

Dilutions Neat 1:1 1:2 1:4 1:8 1:16 1:32 -

Absorbance 0.07 0.11 0.19 0.23 0.32 0.46 0.50 0.51

Cell viability 13.72 21.56 37.25 45.09 62.74 90.19 98.03 100

96

S.no 1 2 3 4 5 6 7 8

Concentration (mg/ml) 5 2.5 1.25 0.625 0.3125 0.156 0.078 Cell control

Dilutions Neat 1:1 1:2 1:4 1:8 1:16 1:32 -

Absorbance 0.10 0.18 0.25 0.27 0.30 0.35 0.48 0.51

Cell viability 19.60 35.29 49.01 52.94 58.82 68.62 94.11 100

TABLE 4 TABULATION SHOWING THE ABSORBANCE AND % OF CELL VIABILITY FOR CAESALPINIA SAPPAN+ PETROLEUM ETHER

97

98