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, professor, Department 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
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M.Phil.,Scholar, M.Phil.,Scholar, Department of Biochemistry, D.K.M.College D.K.M.College for women (Autonomous), (Autonomous), Sainathapuram, Vellore DECLARATION
The The
thesi hesiss
ent entitled led “PHYTOC PHYTOCHEM HEMICAL ICAL SCREEN SCREENING ING AND ANTIOX ANTIOXIDA IDANT NT
ACTIVITY OF CAESALPINIA SAPPAN AN INVITRO ANALYSIS ”
Subm Submiitted ted for for the
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. 2
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 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 p erform 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 affection and encouragements, which made me to come up to this level .
CERTIFICATE
This is to certify that this Dissertation entitled, “ PHYTOCHEMICAL SCREENING AND AND
ANTI ANTIOX OXID IDAN ANT T
ACTI ACTIVI VITY TY
OF
CAESAL CAE SALPIN PINIA IA
SAPPAN SAPPAN AN
INVI INVITR TRO O
ANALYSIS” submitted to the Thiruvalluvar university, vellore in partial fulfillment for the 3
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, Department, D.K.M. College for women, Sainathapuram, Vellore-632 001.
Submitted to university examination in Biochemistry Examiners Date
:
1.
Station:
2.
LIST OF ABBREVIATIONS
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MTT - (3,4,5(3,4,5- dimethy dimethyll thiazol thiazol -2yl)-2 -2yl)-2,, 5-diphenyl 5-diphenyl tetrazoliu tetrazolium m bromide) bromide) DPPH - 1,1 diphenyl diphenyl 2 picryl picryl hydrazyl hydrazyl MEM - minim minimal al essen essentia tiall medium medium FCS
- fo foetal ca calf ser seruum
DMSO- dimethyl sulphoxide TPVG - trypsin, trypsin, phosphate phosphate buffer saline saline versene, glucose glucose EDTA - ethylene ethylene diamine diamine tetra-aceti tetra-aceticc acid CO2
- carbon dioxide
BTH BTH - but butyl ylat ated ed hyd hydro roxy xyll tol tolven venee GM
- gram
H
- hour
Kg
- kilogram
Mg
- milligram
Min
- minutes
µg
- microgram
Ml
- milliliter
µl
- micro litre
%
- percentage
Eg
- example
HEP2 HEP2 - human human laryn laryngea geall tumor tumor cells cells IU
- international units
Nm
- nano metre
CONTENTS
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S.NO
TITLE
PAGE NO
1
INTRODUCTION
1
2
REVIEW OF LITERATURE
41
3
AIM AND OBJECTIVE
53
4
MATERIALS AND METHODS
54
5
RESULTS AND DISCUSSION
71
6
CONCLUSION
81
7
SUMMARY
82
8
BIBLIOGRAPHY
83
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HERBAL THERAPY
Herbalism is a traditional medicinal or folk medicine practice based on the use of plants plants and plant extracts extracts.. Herbal Herbalism ism is also also known known as botani botanical cal medici medicine, ne, medica medicall herbal herbalism ism,, herbal herbal medicine, herbology, and phytotherapy. and phytotherapy. The scope of herbal medicine is sometimes extended to include fungal and bee and bee products, products, as well as minerals, 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 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 harmful side effects. effects. Many of the herbs and spices used by humans to season food yield useful medici medicinal nal compou compounds nds.( .( Lai PK, Roy J et al.,200 al.,2004). 4). Antimi Antimicro crobia biall and chemo chemo preven preventiv tivee properties of herbs and spices. ( Tap sell LC, Hemphill I, Cobiac L, et al. 2006). Similarly to prescription drugs, 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).
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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. 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 other strate strategie giess that that suppor supportt the whole whole person person includ including ing nutrit nutrition ion,, bodywor bodywork, k, acupunc acupunctur ture, e, 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.
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CAESALPINIA SAPPAN
SCIENTIFIC CLASSIFICATION
Kingdom
Plantae
Division
Magnoliophyta
class
magnoliopsida
Order
fabales
Family
fabeaceae
Genus
Caesalpinia
Species
sappan
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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 terminal and auxiliary auxiliary panicles; stamens waxy-white, waxy-white, filaments 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/m³), 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 10
tannic tannic and gallic acids, is an astringent astringent used in mild cases of dysentery 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 Caesalpinia sappan. Screening showed that the methanolic extract had significant anti-hypercholesteraemic activity. Brazilin (7,11b-dihydrobenz[b]indeno[1,2-d]pyr [1,2-d]pyran-3,6a an-3,6a,9,10( ,9,10(6H)-t 6H)-tetrol etrol), ), the principle principle component component of C.sappan 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 ulcers,, lepros leprosy, y, skin skin diseas diseases, es, diarrh diarrhea, ea, dysent dysentery ery,, epilep epilepsy sy,, convuls convulsion ions, s, menorr menorrhagi hagia, a, leucorrhoea, diabetes, haemoptysis, hemorrhages, stomatopathy and odontopathy.
DPPH
DPPH DPPH is a comm common on abbr abbrev eviiati ation for for an orga organi nicc chemical chemical compoun compoundd 2,2-diphenyl-1 picrylhydrazyl. 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
11
reactions involving radicals and another is a standard of the position and intensity of electron 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 of radical-mediated polymerization. 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 DPPH has severa severall crysta crystall lline ine forms forms which which differ differ by the lattic latticee symmet symmetry ry 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, orthorhombic, DPPH-II DPPH-II (m.p. 137 °C) is amorphous and DPPH-III DPPH-III (m.p. 128– 128– 129 °C) is triclinic. 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: (Latin: within within glass) glass) refers refers to studies studies in experimental biology that are conducted conducted using components of an organism that have been isolated from their usual biological b iological 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 or tissue culture), culture), (b) subcellular components (e.g. mitochondria or ribosomes or ribosomes), ), (c) Cellul Cellular ar or subcel subcellul lular ar extrac extracts ts (e.g. (e.g. wheat wheat germ germ or reticulocyte extrac extracts) ts),, or (d) purifi purified ed molecules in the test tube (often proteins, DNA, or RNA 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 inorganic ions and complexes complexes in an environment environment that is spatially spatially organized organized by membranes and, in the case case of multic multicellul ellular ar organisms organisms,, organ syste systems.( ms.( Alberts Alberts,, Bruce et al.,2008). 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 Paulette M.; Pierre Vignaiset Vignaiset al., 2010). (Jacqueline (Jacqueline Nairn; Nairn; Price, Nicholas Nicholas C. et al.,2009) al.,2009) For example, the identity of proteins of the immune system (e.g. antibodies), and the mechanism by 14
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 primar primaryy disadv disadvant antage age of in vitro vitro experim experimenta entall studie studiess is that that it can someti sometimes mes be very very challenging challenging to extrapolate extrapolate from the results of in vitro vitro work back to the biology 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 that are effect effective ive in vitro vitro prove prove to be ineffe ineffecti ctive ve 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 system sys tems. s. In vit vitro ro dru drug-r g-rele elease ase stu studie diess are sim simpli plifi fied ed ski skin-p n-penet enetrat ration ion stu studie dies. s. Aft After er 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 lines are immor immortal tal,, that that happens happens if a cell cell is cancer cancerous ous.. The cells cells can perpet perpetuat uatee divisi division on 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) culture) A cell line is a permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space. space. 16
Lines differ from cell strains strains in that that they they have have esca escape pedd the the Hayf Hayfli lick ck limi limitt and becom becomee immortalised. Some species, species, particularly rodents, rodents, give rise to lines relatively easily, whereas other species do not. No not. No cell lines have been produced from avian tissues and the establishment of cell lines from human tissue is difficult. difficult. Many cell biologists 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 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. 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, Harrison, working at Johns Hopkins Medical School and then at Yale University, University, published results of his experiments from 1907-1910, establishing the methodology of tissue of tissue culture. culture. (Schiff, Judith Ann et al., 2002) Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology. virology. Growin Growingg viruse virusess in cell cell cultur cultures es allowe allowedd prepar preparati ation on of purifi purified ed viruse virusess for the manufacture of vaccines. of vaccines. ISOLATION OF CELLS
Cells can be isolated from tissues for ex 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, collagenase, trypsin, trypsin, or pronase, pronase, which break down the extracellular matrix. matrix. Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are available available for culture. This method is 17
known as explant culture. 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 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. 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, 37°C 37°C,, 5% CO2) 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, pH, glucose concentration, growth factors, factors, and the presence of other nutrient components. The growth factors used to supplement media are often derived from animal blood, blood, such as calf serum. calf serum. These blood-derived ingredients pose the potential for contamination of derived pharmaceutical products with viruses or prions. 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, 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 their nuclei blue. Cell lines that originate with humans have been somewhat controversial in bioethics 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 18
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 produc products ts of biotec biotechnol hnology ogy.. Biolog Biologica icall produc products ts produc produced ed by recomb recombina inant nt DNA (rDNA) technology in animal cell cultures include enzymes, hormones, immunobiologicals (monoclonal (monoclonal antibodies, interleukins, interleukins, lymphokines), lymphokines), and anticancer agents. 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. 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. 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 19
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 Because of their high degree of morphologic morphological al and functional functional different differentiatio iationn in vitro, HepG2 cells are a suitable model to study the intracellular trafficking and dynamics of bile canalicular and and sinu sinuso soid idal al memb membra rane ne prot protei eins ns and and lipi lipids ds in huma humann hepat hepatocy ocyte tess in vitr vitro. o. This 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 DubinJohnso Johnsonn Syndro Syndrome me and progre progressi ssive ve famili familial al intrah intrahepa epatic tic choles cholestas tasis is (PFIC) (PFIC),, and famili familial al 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 (envi enviro ronm nmen enta tall
and and
diet dietar aryy)
geno genoto toxi xicc
and and
coge cogeno nottoxi oxic
agen agentts,
unde unders rsta tand ndin ingg
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 acid in the the cell cells, s, bein beingg 5-10 5-10 time timess more more abund abundant ant than than DNA. DNA. Gene Genera rall llyy we cons consid ider er 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 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). heart). The portal vein carries blood containing digested food from the small intestine. These blood vessels subdivide in the liver repeatedly, terminating in 20
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 he patic phagocytic system.
2.
Metabolic achievements in control of synthesis synthesis and utilization utilization of carbohydrates, lipids and proteins.
3.
Secretory and excretory functions, particularly particularly with respect to the synthesis 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 impo import rtan ant, t, and and leas leastt appr apprec ecia iate tedd orga organs ns in the the body body.. The The bulk bulk of the the live liverr cons consis ists ts of hepatocytes, hepatocytes, which are epithelial cells with a unique conFiguration. conFiguration. The liver is essentia essentially lly an exocrine exocrine gland, secreting secreting bile into into the intestine. intestine. and significant significantly ly so The The liver liver has a dive divers rsit ityy of functi functions ons not typic typicall allyy associ associate atedd with with glands glands.. The liver is a metabolic factory, synthesizing synthesizing and breaking down a variety of substances. It's functions include 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. 21
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 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 This orga organn play playss a majo majorr role role in meta metabo boli lism sm and and has has a number number of func functi tion onss in the the body 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 22
abdomen. It produces bile, an alkaline compound which aids in digestion via the emulsification of lipids lipids.. The liver' liver'ss highly highly specia specializ lized ed tissue tissuess regula regulate te a wide wide variet varietyy of high-v high-volu olume me 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 1.4–1.6 kg (3.1–3.5 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 located in the right upper quadrant quadrant of the abdominal abdominal cavity, cavity, restin restingg just just below below the diaphragm. The liver lies to the right of the stomach and overlies the gallbladder. It is connected to two large blood vessels vessels,, one called called the hepatic hepatic artery artery and one called called the portal portal vein. 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 The live liverr rece receiv ives es a dual dual blood blood supp supply ly from from the the hepa hepati ticc port portal al vein vein and hepa hepati ticc arte arteri ries. es. Supplying approximately 75% of the liver's blood supply, the hepatic portal vein carries venous blood drained from the spleen, spleen, gastrointe gastrointestina stinall tract, tract, and its associated associated organs. The hepatic hepatic arteries supply arterial blood to the liver, accounting for the remainder of its blood flow. Oxygen is provided provided from both sources; approximat approximately ely 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 hapatic duct. The cystic duct from the gallbladder joins with the common hepatic duct to form the common bile duct. Bile can either drain drain direct directly ly into into the duodenum duodenum via the common common bile duct, duct, or be tempor temporari arily ly stored 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 covered entirely entirely by visceral visceral peritoneum, peritoneum, a thin, double-layer double-layered ed membrane 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
Tradit Tradition ional al gross gross anatom anatomyy divide dividedd the liver liver into into four four lobes lobes based based on surfac surfacee featur features. es. 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 caudate lobe (the more superior) and the quadrate lobe (the more inferior). From behind, the lobes are divided up by the ligamentum ligamentum venosum and ligamentum teres (anything left of these is the left lobe), the transverse fissure (or porta (or porta hepatis) 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, duct, hepatic portal vein, vein, and hepatic artery proper enter proper enter is the hilum or " porta hepatis". hepatis". The duct, vein, and artery divide into left and right branches, and the portions of the liver supplied by these branches b ranches 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 anterior and posterior posterior segment 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. 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. 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 for liver failure. 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. 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 27
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 is exposed to a number of insults 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 gall gallbl bladd adder er is to stor storee and and conce concent ntra rate te bile bile.. Bile Bile is compr compris ised ed of bile bile salt salts, s, bili biliru rubi bin, n, phospholipi phospholipids, ds, cholesterol cholesterol,, bicarbonate bicarbonate and water. water. Bile salts mix with ingested fats to promote 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 an d absorption require an alkaline environment. After its formation formation in the liver, liver, bile flows flows into the hepatic 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 gastro gastroint intest estina inall canal canal enters enters the duodenu duodenum, m, cholecy cholecysto stokin kinin in is releas released ed from from the duodena duodenall mucosa to stimulate stimulate contraction contraction of the gallbladder gallbladder and common bile duct, and relaxation relaxation of the sphincter of Oddi so that bile can enter the small intestine. Lipogenesis, Lipogenesis, the production of triglycerides of triglycerides (fats). The liver produces coagulation factors I (fibrinogen), II (prothrombin), V, VII, VII, IX, IX, X and XI, as well as protein as protein C, C, protein S and antithrombin. antithrombin. In the first first trimester trimester fetus, fetus, the liver is the main site of red blood cell production. By the 32nd week of gestation of gestation,, the bone 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, duodenum, and some is stored in the gallbladder . The liver also produces insulin-like growth factor 1 (IGF-1), a polypeptide a polypeptide protein 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. production. Thrombopoietin Thrombopoietin is a glycoprotein hormone that regulates the production of platelets of platelets by the bone the bone marrow. marrow. The liver converts ammonia to urea. 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), proteins), filter harmful substances from the blood (such as alcohol), storage of vitamins and minerals (vitam (vitamins ins A, D, K and B12) B12) and maintain a proper level or glucose in the blood. The liver is also responsible for producing cholesterol. 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 of glycogen), ), vitamin A (1–2 years' supply), vitamin D (1–4 months' supply), vitamin B12 (1-3 years' supply), iron, 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, albumin, the major osmolar component osmolar component of blood blood serum. serum. The liver stores a multitude of substances, including glucose (in the form of glycogen), glycogen), vitamin A (1–2 years' supply), vitamin D (1–4 months' supply), vitamin B12 (1-3 years' supply), iron, iron, and copper . The liver synthesizes angiotensinogen, angiotensinogen, a hormone that is responsible for raising the blood pressure when activated by renin, renin, an enzyme that is released released when the kidney senses low blood pressure. pressure.
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LIVER FUNCTION TESTS
(LFTs (LFTs or LFs), LFs), which which includ includee liver liver enzymes 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 plasma sample obtained by phlebotomy by phlebotomy.. Some tests are associated with functionality (e.g., albumin); some with cellular integrity (e.g., transaminase) transaminase) and some with conditions linked to the biliary tract (gamma-glutamyl ( gamma-glutamyl transferase and alkaline phosphatase). 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 paracetamol (acetaminophen) overdose. overdose. Elevations are often measured in multiples of the upper up per 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. d amaged. 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 parenchymal cells. 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, 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.,) 32
ALKALINE PHOSPHATASE (ALP) Normal value 30 to 120 IU/L
This enzyme occurs mainly in liver cells next to bile b ile 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 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. 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 prot protei einn cata catabo boli lism sm may may also also lead lead to hypo hypoal albu bumi minae naemi mia. a. The The half half-l -lif ifee of album albumin in 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 co lor.) 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 w ith 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, o r 3) blockage of the bile ducts d ucts 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: ch olestasis: 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 0.2–1.2 mg/dL mg/dL Bilirubin is a breakdown product of heme of heme (a part of hemoglobin 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, hepatocytes, conjugated conjugated (modified (modified to make it water-solub water-soluble), le), and secreted secreted into the bile the bile,, which is excreted into the intestine. Increased total bilirubin causes jaundice 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: Hepatic: Problems with the liver, which are reflected reflected as deficiencies deficiencies in bilirubin bilirubin metabolism 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 Posthepatic:: Obstructio Obstructionn of the bile ducts, reflected reflected as deficienci deficiencies es in bilirubin bilirubin excretion. excretion. (Obstruction can be located either within the liver or in the bile the bile duct. duct. DIRECT BILIRUBIN (CONJUGATED BILIRUBIN)
Normal value 0–2.3 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 bilirubin is elevated, then the liver is conjugating conjugating bilirubin bilirubin normally, 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. 35
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 b iliary or extra-biliary origin. Coagulation test (e.g. INR)
The liver is responsible for the production of coagulation 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 of vitamin K -dependent -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 (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 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, v irus, 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. diabetes. Serzone is a prescription drug manufactured by Bristol-Myers Squibb for the treatment of depression. The possib possible le side side effect effectss of Serzon Serzonee are: are: agitat agitation ion,, dizziness, dizziness, clumsi clumsines nesss or unstea unsteadin diness ess,, difficulty concentrating, memory problems, confusion, severe nausea, gastroenteritis, 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) jaundice) or a prolonged loss of weight we ight 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; 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 medications,, over-the-coun over-the-counter ter medication medications, s, vitamins, vitamins, hormones, hormones, herbs, herbs, illicit illicit ("recreati ("recreational") onal") 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), alcohol), autoimmunity (autoimmune (autoimmune hepatitis) hepatitis) or hereditary conditions. Diagnosis is done by checking levels of Alanine of Alanine transaminase. transaminase. Non-a Non-alco lcohol holic ic fatty fatty liver liver diseas disease, e, a spec spectr trum um in dise diseas ase, e, asso associ ciat ated ed with with obes obesit ityy and and characterized as an abundance of fat in the liver; may lead to a hepatitis, i.e. steatohepatitis and/or cirrhosis 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 chemic chemicals als.. Diagno Diagnosis sis is done by checki checking ng levels levels of Alanine Alanine transamina transaminase se and Asparatine Asparatine transaminase (SGOT (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 that can can caus causee chron chronic ic liver liver dise diseas ases es and and cirr cirrho hosi siss incl include ude meth methot otre rexa xate te (Rheu (Rheuma matr trex) ex),, amiodarone (Cordarone), and methyldopa (Aldomet). Haemochromatosis, Haemochromatosis, a hereditary disease causing the accumulation of iron 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). tract).
Wilson's disease, disease, a hereditary disease which causes the body to retain copper . Primary sclerosing cholangitis, cholangitis, an inflammatory disease of the bile duct, duct, likely autoimmune in nature. Primary biliary cirrhosis, cirrhosis, autoimmune disease of small bile ducts. 39
Budd-Chiari syndrome, obstruction of the hepatic vein. Gilbert's Gilbert's syndrome syndrome,, a genetic disorder of bilirubin metabolism, found in about 5% of the population. Glycogen storage disease type II, 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 There are also also many many pediatr pediatric ic liver liver diseas disease, e, includ including ing biliary biliary atresia, atresia, alpha-1 alpha-1 antitryps antitrypsin in deficiency, deficiency, alagille syndrome, syndrome, and progressive familial intrahepatic cholestasis, 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 Overview National Digestive Diseases Information Clearinghouse. The most common include: include: Infections Infections such as hepatitis A, A, B, C, E, alcohol damage, fatty liver , cirrhosis, cirrhosis, cancer , drug damage (especially acetaminophen (also known as paracetamol) and cancer drugs) Many diseases of the liver are accompanied by jaundice by jaundice caused by increased levels of bilirubin bilirubin in the system. The bilirubin results from the breakup of the Hemoglobin of dead red blood cells; cells; normally, the liver removes bilirubin from the blood and excretes e xcretes it through bile. There There are also also many many pediat pediatric ric liver liver diseas diseases es includ including ing biliary biliary atresia atresia,, alpha-1 alpha-1 antitrypsi antitrypsinn deficiency, deficiency, alagille syndrome, progressive syndrome, progressive familial intrahepatic cholestasis, 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
40
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, 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 non specific or specific. Nonspecific symptoms, that is, symptoms that don't suggest that the liver is their cause, include: Fatigue, Weakness, Vague abdominal pain, 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 (edema)) and abdomen (ascites (ascites), ), Mental confusion or coma or coma,, Kidney failure, 41
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 where bilirubin deposits in skin, causing an intense intense itch. 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 by blood tests. 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.. 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
42
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; 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. cycle. That is, the hepatocytes 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 Saito S, Nonaka H, Miyajima A et al., 2008) Scientific and medical works about liver regeneration often refer to the Greek Titan 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 liver’s 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, 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 leading to cirrhosis, cirrhosis, such as chronic hepatitis C, C, alcoholism, alcoholism, autoim autoimmun munee hepatit hepatitis, is, and many many others others.. Less Less common commonly, ly, liver liver transp transplan lantat tation ion is done for fulminant hepatic failure, failure, in which liver failure occurs over days to weeks. Liver allografts Liver allografts for transplant for transplant usually come from donors who have died from fatal brain fatal brain injury. 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, regenerate, both the donor and recipient end up with normal liver function if all goes well. This procedure is more 43
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 venosus carries blood from the left portal vein to the left hepatic vein and then to the inferior vena cava, 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 placenta. The fetal liver releases some blood stem cells that migrate to the fetal thymus, 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 red bone marrow. 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 of cirrhosis and portal hypertension, hypertension, the umbilical vein can open up again.
44
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, sources, and as such, do not report any new or original experimental work. Most often associated with academic-oriented literature, such as a thesis, 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; style; proper use of terminology of terminology;; and an unbiased and comprehensive view of the previous research on the topic. 45
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 Caesalpinia sappan, a plant widely used in the traditional medicinal pharmacological action. Caesalpinia
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 heartwood (ECS), (ECS), Caesalpinia sappan heartwood protosappan protosappanin in A, protosappani protosappaninn B, and brazilein brazilein were studied in vitro. vitro. The inhibition inhibition of the format formation ion of malondi malondiald aldehy ehyde de (MDA) (MDA) and the scaven scavengin gingg of superox superoxide ide anions anions,, hydrog hydrogen en peroxide, and hydroxyl radicals were assayed. The experimental results show that all four substa substance ncess had antiox antioxida idant nt activi activity ty in vitro vitro but their their capabil capabiliti ities es differ differed ed for the differ different ent 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 Argentinean legume legume Caesalpini Caesalpiniaa paraguarien paraguariensis sis Burk. (Fabaceae) was selected forfurther fractionation work based on the strong antimicrobial activity of its CH2Cl2-MeOH(1:1 v/v) extract extract against against a host of clinically clinically significant significant microorgani microorganisms, sms, including including antibioti antibioticresis cresistant tant stra strain ins. s. 1D and and 2D NMR NMR enab enable ledd the the ident identif ific icat atio ionn of the the novel novel benzo benzoxe xeci cinn deriv derivat ativ ivee caesalpinol
along
with
stearoylgl stearoylglucopyr ucopyranoside anoside,,
the
kno known
com compound oundss
bilobe obetin,
stigma-5-en-3-O
stigma-5-e stigma-5-en-3-_ n-3-_-6_-pa -6_-palmit lmitoylgl oylglucopyra ucopyranosid noside, e,
-6
stigma-5-e stigma-5-en-3-_n-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 μm), 8 and 64 (140 μm) μg/ml, respectively. The rest of the compou compounds nds,, howeve however, r, did not show show activi activity. ty. (Girma (Girma M. Woldem Woldemicha ichaela ela,, Maya Maya P. Singhb Singhb,, William M. Maieseb, and Barbara N. Timmermanna 2003) 2 003)
46
References and further reading may be available for this article. To view references and further reading you must purchase must purchase this article. The antioxidant activity (AA) of ethyl acetate extracts of Caesalpinia Lithospermum um Caesalpinia sappan, Lithosperm 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 kg−1, 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 sample with butylated butylated hydroxytol hydroxytoluene') uene').. All of C. sappan, sappan, L. erythrorhi erythrorhizon zon extracts extracts and their combinations were found to be high effective in peanut oil. But the extracts of A. asphodeloides, P. polyph polyphyl ylla la and I. verum verum slight slightly ly decrea decrease se the formati formation on of peroxi peroxides des in peanut 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 Sampl es of Caesal Caesalpinia pinia echinata Lam. (fro (from m Brazi Brazil, l, South America), America), Caesalpinia sappan L. (East Indies, Asia) Asia),, Haemat Haematoxylum oxylum brasiletto brasiletto Karst Karsten en (Cent (Central ral Ameri America) ca) and Haemat Haematoxylu oxylum m 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 provi ded a founda foundation tion for the nondestructive nondestructive spectral discriminat discrimination ion betwee betweenn a sampl samplee 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 archiv arc hival al col collec lectio tionn fac facil ilita itated ted the eva evalua luati tion on of the eff effect ectss of tem tempor poral al degr degrada adatio tionn on the obse ob serv rved ed sp spec ectr tra, a, whi which ch cou could ld be us used ed to fu furt rthe herr te test st th thee ex expe peri rime ment ntal al pr prot otoc ocol olss fo for r nonde no ndest stru ruct ctiv ivee ve veri rifi fica cati tion on of sa samp mple less in th thee ar archi chive ve wi with th que quest stio ionab nable le as assi sign gnme ment nt or provenance.(Howell provenance. (Howell G. M. Edwards, Luiz F. C. de Oliveira and Mark Nesbitt 2003). 2003). Trichloroethylene (TCE) induces liver cancer in mice but not in rats. Three metabolites of TCE may contribute contribute--chl --chloral oral hydrate hydrate (CH), dichloroacetate dichloroacetate (DCA), (DCA), and trichloro trichloroacetate acetate (TCA). CH 47
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 could affect affect sensit sensitive ive populat population ions. s. Sensiti Sensitivit vityy could could be based based on differ different ent metabol metabolic ic capacities capacities for TCE or its metabolite metabolitess or result from certain chronic diseases that have a genetic 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 prolifer proliferation, ation, and the development of altered altered hepatic hepatic foci. The relevance relevance of pyrethrins pyrethrins-induc -induced ed rat liver tumors to human health was assessed by using the 2006 International Programme on Chemical Chemical Safety Safety Human Relevance Framework. Framework. The postulated rodent tumor MOA was tested 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
48
increase the risk of liver tumors in humans. It is concluded that pyrethrins do not pose a hepatocarcinogenic hazard for humans. (Osimitz TG, TG, Lake BG et al.,2007) The demons demonstra trated ted modifi modified ed spectr spectropho ophotom tometr etric ic method method makes makes use of the 2,2-di 2,2-diphen phenylyl-11 picrylhydrazyl (DPPH) radical and its specific absorbance properties. The absorbance decreases when the radical radical is reduced reduced by antioxidants. antioxidants. In contrast contrast to other investigatio investigations, ns, the absorbance 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 microalgae’s methanolic extracts antioxidative potentials which was independent to the employed DPPH concentrations. The resulting slopes showed significant differences (6 - 34 μmol DPPH g-1 extrac extractt concent concentrat ration ion)) betwee betweenn the single single differ different ent specie speciess of microa microalga lgaee (Anabae (Anabaena na sp., sp., Isochrysi Isochrysiss galbana, galbana, Phaeodactyl Phaeodactylum um tricornutu tricornutum, m, Porphyridi Porphyridium um purpureum, purpureum, Synechocyst Synechocystis is sp. PCC6 PCC6803 803)) in thei theirr abil abilit ityy to reduc reducee the the DPPH DPPH radi radica cal. l. (Kai (Kai Marx Marxen en 1, Klau Klauss Heinr Heinric ichh Vansel Vanselow ow , Sebasti Sebastian an Lippemeie Lippemeierr , Ralf Ralf Hintze Hintze , Andreas Andreas Ruser Ruser and Ulf-Pet Ulf-Peter er 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 (μg/ 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 HO > CS > BH > VO (at the concentrations of 25-800 μg/ ml). IC50 for Fe2+ chelating ability were 188, 750 and 980 μg/ ml for VO, CS and HO, respectively. BH extract has shown only 38% inhibition at 800 μg/ 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 BH , 175 for CS , 640 for VO and 663 μg/ ml for HO HO. The content of total of H2O2 were 169 for BH
phenolic compounds and flavonoids were measured in plant extracts. The data obtained in the in vitro vitro models models clearl clearlyy establ establish ish the antiox antioxida idant nt potency potency of all extrac extracts. ts.(( Mohamm Mohammad ad ali
49
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 DPPH and DMPD DMPD methods were used). (1Florica busuricu, Lantoanela popescu, 2Doina paula balaban, 1Ticuţa pârjol 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 rela relate tedd to phys physio iolo logy gy and and patho pathophy physi siol olog ogyy of live liver, r, such such as, as, seru serumg mglu luta tama mate te pyru pyruva vate te transa transami minas nasee and alkali alkaline ne phospha phosphatas tasee were were determ determine inedd in the murine murine sera sera under under variou variouss 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 antiox antioxida idant nt enzyme enzymess catala catalase, se, supero superoxid xidee dismut dismutase ase and glutat glutathio hione-S ne-S-tr -trans ansfer ferase ase were 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 sections of control, control, toxin treated treated and protein pre- an posttreate posttreatedd (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 cell free free syste system m the protei proteinn effect effective ively ly quenche quenchedd dipheny diphenyll picryl picryl hydraz hydrazyl yl radica radicall and superoxide radical, though it could not catalyse the breakdown of hydrogen peroxide. Post 50
treatment treatment with the protein for 3 days after 2 days of chloroform chloroform administrat administration ion showed showed similar 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 curative role against chloroform chloroform induced hepatotoxicity hepatotoxicity and probably probably acts by an anti-oxidat anti-oxidative ive 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 Caesalpinia sappan Linn. (Sappan lignum) development of new drugs. The water kept in Caesalpinia
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 constituent of the plant responsible responsible for several of its biological biological activities. 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 establ establish ish its medici medicinal nal proper propertie tiess and to identi identify fy lead lead compoun compounds ds for drug drug developm development ent.. (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. models. The ethyl acetate, acetate, methanol methanol and water extracts extracts exhibited exhibited strong antioxidant antioxidant activity 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 (Badami S, Moorkoth S, S, Rai SR , Kannan E, E, Bhojraj S.et S.et al.,2006) 51
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 anti antira radic dical al acti activi vity ty meas measur urem ement entss and and cogna cognacc chem chemic ical al chara charact cter eris isti tics cs was was found found for 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 al.,2000)Full-size image (8K) The reaction mechanisms of three antioxidants are proposed in order to explain experimental result resultss obtain obtained ed from from a kineti kineticc study study using using the free free radica radicall 2,2-di 2,2-diphen phenylyl-1-p 1-picr icryl ylhyd hydraz razyl yl (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 slowly reacts with DPPH• reaching a steady 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/(mol·s) at 20 °C and the energy of activation, activation, Ea, is equal to 35 kJ/mol in methanol. methanol. Eugenol reacts with DPPH• reaching reaching a steady state within 2 h. This 1.9-stoichiometric reaction follows a 2-order with respect to both DPPH• and eugenol, eugenol, k and Ea are estimat estimated ed to be 5.4 3 1010 1010 L3/(mo L3/(mol3· l3·s) s) at 20 °C and 30 kJ/mol kJ/mol,, respec respectiv tively ely.. The eugenol eugenol mechan mechanism ism may involv involvee a dimeri dimerizat zation ion betwee betweenn two phenoxy phenoxyll radicals. radicals. The reaction reaction with isoeugenol isoeugenol is rapid and reversible reversible,, with a stoichiome stoichiometry try of 1.1. It is first first order with respect respect to isoeugenol isoeugenol with k (direct reaction) reaction) equal to 8.9 3 10–2 s–1 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 structural elements of the C-ring, 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 seco second nd slowe slower. r. DPPH DPPH scav scaven engi ging ng foll follow owed ed seco second nd order order kine kineti tics cs duri during ng the the rapi rapidd step; step; stoichiometric factors and rate constants were determined. 52
Quercetin and fisetin scavenged four radicals by each molecule, while the other flavonoids scaven scavenged ged two radica radicals ls with with a conseq consequent uent productio productionn of B-ring B-ring diquin diquinone. one. Also the rate rate constants were affected by C-ring structure. The kinetics of the slow step was much more comp compli lica cate tedd and and the the contr contrib ibut utio ionn of C-ri C-ring ng was was base basedd on stoi stoich chio iome metr try. y. (Dim (Dimit itri rios os 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 radical scavenging scavenging assay. assay. Among the extracts extracts Cocos nucifera, Caesalpinia Caesalpinia pulcherrima pulcherrima,, Punica granat granatum um and Syzygi Syzygium um cumini cumini were were found found displa displayin yingg strong strong (90% (90% or more) more) DPPH radica radicall scavenging scavenging action. Syzygium cumini exhibited the highest highest radical radical scavenging, scavenging, with an IC50 value value of 4.25 _g/ml _g/ml compared compared to the IC50 IC50 value value of 5.15 5.15 _g/ml _g/ml as shown shown by the refere reference nce 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 305μg/ml 305μg/ml and exhibited exhibited a dose dependent decrease 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 36.5μg/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 inflammatory bowel disease. Therefore, Therefore, the aims of this work were to compare and validate validate three analytical methods for the quality control of commercial coated tablets containing 5-ASA: high high perfor performan mance ce liquid liquid chroma chromatog tograp raphy hy (HPLC) (HPLC),, 1,11,1- diphen diphenylyl-2-p 2-picr icryl ylhyd hydraz razyl yl radica radicals ls (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 (70:30;; v/v), v/v), with with 25% tetrab tetrabuty utylam lammon monium ium hydrog hydrogen en sulphat sulphate. e. The DPPH DPPH method 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 53
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 meth method ods. s. All All propo propose sedd meth methods ods can can be used used for for the the reli reliab able le quant quantit itat atio ionn of 5-AS 5-ASA A in pharmaceutical dosage forms. (Janice Aparecida Rafael, José Roberto Jabor, Rúbia Casagrande, Sandra Regina Georgetti, Maria de Fátima Borin, Maria José Vieira Fonseca et al.,2007) Antiox Antioxida idati tion on proper propertie tiess and mechan mechanism ism of action action of dihydr dihydromy omyric riceti etinn from from Ampelo Ampelopsi psiss gros grosse sede dent ntat ata. a. To asse assess ss the the anti antiox oxid idat ativ ivee prop proper erti ties es and and the the mech mechani anism sm of acti action on of dihydromyricetin (DMY) from Ampelopsis grossedentata.( Zhang YS, YS, Ning ZX, ZX, Yang SZ, 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 must purchase this article. Measuring the AC susceptibility and registering the ESR spectra of the stable 1,1-diphenyl-2 picryl-hydrazyl (DPPH) radical at 77.4–125 K, we have studied the effect of the radical on the curr curren entt prop proper erti ties es of the the inte interg rgra ranu nula larr cont contac acts ts in the the supe superc rcon ondu duct ctin ingg cera cerami mics cs Bi1.8Pb0.3Sr 1.9 1.9Ca2.8Cu3.4Oy. 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 Å from the superconducting phase.( L. L. Makarshin and V. N. Parmon Et al.,2000) The Flavonoids extracted extracted from tartary tartary buckwheat buckwheat seeding seeding was tested by the DPPH eliminatio eliminationn 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 µg µg /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 development.(Fang YuMei;; Tan Ping; YuMei Ping; Wang YiHong; YiHong; Zhang ChunSheng et al.,2007)
54
The factors contributing to the loss of phenolic content and DPPH radical scavenging activity of Taiwanese Taiwanese yam, Dioscorea Dioscorea alata Tainung Tainung No. 1 (TNG1), (TNG1), in the 20% TNG1-contai TNG1-containing ning pollock surimi gel were investigated. Heating at 90°C 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 90°C 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. determined. Antibacterial Antibacterial activity activity was also studied studied against against Bacillus Bacillus subtilis, subtilis, Escherichia Escherichia coli, coli, Staphylococc Staphylococcus us aureus and Streptococcus Streptococcus pyogenes by using cup-plate method. Erythromy Erythromycin cin was used used as standa standard rd antiba antibacter cterial ial agent. agent. The methan methanol ol extrac extractt was dilute dilutedd into into differ different ent concentration (1, 2, 4, 6, 8, 10 mg/l00 μl) with DMSO. The results of the study revealed that, the Chitrakadi Vati exhibited significant antibacterial activity. (Bagepalli Srinivas Ashok Kumar, Vontoo Vontoorr Byrappa Byrappa Naraya Narayana na Swamy, Swamy, Peresa Peresandr ndraa Avalak Avalakond ondara arayap yappa pa Arun Arun Kumar Kumar 3, and SaleemullaKhan et al.,2010). An ultrasonic ultrasonic technique was employed to extract polysaccharides fromInonotus obliquus(UPS). The effects of ultrasonic condit condition ionss on the recover recoveryy and 1, 1-diph 1-dipheny enyll -2-pic -2-picryl rylhyd hydraz razyl yl (DPPH) (DPPH) radical radical scavenging scavenging activity of UPS were evaluated. The physicochemi physicochemical cal properties properties of UPS were analyzed by differential scanning calorimetry (DSC), scanning electron microscopy (SEM), (SEM), and UV-visible UV-visible spectrophotom spectrophotometry etry.. Molecular Molecular weights of UPS were determined determined by gel permeation chromatography (GPC). The optimal ultrasonic conditions to obtain the highest 55
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 effective method to extract extract bioactive bioactive polysacchari polysaccharides des fromI. obliquus( obliquus(UPS UPS Practical Applic Applicati ation: on: Polysa Polysacch cchari arides des fromIn fromInonot onotus us obliqu obliquuse usexhi xhibit bited ed many many biolog biologica icall activi activitie tiess including including antitumor, antitumor,
antiox antioxida idant, nt, hypogl hypoglyce ycemic mic,, immuneimmune-sti stimul mulati ating ng effect effects, s, 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 physicoche physicochemical mical properties properties and DPPH radical radical scavenging scavenging activity activity of polysacchar polysaccharides ides fromI. obliquusunti obliquusuntill now. Ultras Ultrasoni onicc techni technique que was an effect effective ive method method to extrac extractt bioact bioactive ive polysaccharides fromI. obliquus(UPS) according to this study. ( Fu, L., Chen, H., Dong, P., Zhang, X. and Zhang, M.et al.,2010), Inhibi Inhibiti tion on of polyph polyphenol enol oxidas oxidasee (PPO)(PPO)-med mediat iated ed browni browning ng in apple apple juice juice using using differ different ent 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 K m = 3.33 mM. mM. There exist a negative negative correlation correlation (γ = −0.65) between different concentrations (50–100%, 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 (γ = +0.96) +0.96).. Assess Assessmen mentt of the antimi antimicro crobia biall activi activity ty of honey honey using using agar agar diffus diffusion ion assay assay demonstrat demonstrated ed the significan significantt inhibition inhibition of Candida Candida albicans albicans (Microbial (Microbial Technology Technology & Culture Culture Collection Collection [MTCC-3018] [MTCC-3018]), ), Escherichia Escherichia coli (MTCC-1588) (MTCC-1588),, Pseudomonas Pseudomonas aeruginosa aeruginosa (MTCC(MTCC2488) and Staphylococcus aureus (MTCC-96) as compared to standard antibiotics.( Gacche, R., Shinde, B., Dhole, N., Pund, M. and Jadhav, A. 2009).
56
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 calcul calculate atedd BDE values values.. The studie studiedd compoun compounds ds were were protoc protocate atechui chuic, c, homopr homoproto otocat catech echuic uic,, 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 the side side-c -car arbo bonn chai chainn char charac acte teri rist stic ics) s) that that regu regula late tedd the the beha behavi vior or of the the comp compou ound ndss 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 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.
57
PLANT USED
Caesalpinia sappan COLLECTION OF THE PLANT
The Aerial part of caesalpinia sappan were collected freshly from the Thirunelveli District, Tamil Tamil Nadu, India India . During During the month month of March 2010 and
indent indentifi ified ed authent authentica icated ted 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 full fullyy drie driedd leav leaves es..
The The sunl sunlig ight ht drying drying of leave leavess are are avoi avoide ded. d. As it may may brin bringg about about
concentration changes in the constituents of the leaves due to photosynthesis.The dried leaves are then weighed weighed and grinded. The fine powder is obtained obtained by sieving. sieving. It is weighed and stored stored in sterile containers. 58
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 originally designed designed for the extraction extraction of lipids from a solid material. material. However, However, a Soxhlet extractor is not limited 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. solvent. If the desired compound compound has a high solubility solubility in a solvent then a simple filtrati filtration on 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 59
extrac extractor tor.. The Soxhlet Soxhlet extrac extractor tor is placed placed onto onto a flask flask contai containin ningg the extrac extractio tionn solven solvent. t. TheSoxhlet is then equipped with a condenser. The solvent is heated to reflux. The solvent vapour travels up a distillation arm, and floods floods into the chamber housing housing the thimble of solid. solid. The condenser condenser ensures that any solvent 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. flask. This cycle may be allowed allowed to repeat many times. Over hours or days.
SOXHLET EXTRACTOR
60
During each cycle, a portion of the non-volatile compound dissolves in the solvent. After many cycles cycles the desired compound compound is concentrated concentrated in the distilla distillation tion flask. flask. The advantage 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. 61
After extraction the solvent is removed, typically by means of a rotary evaporator, yielding the extracted compound. compound. The non-soluble portion of the extracted solid remains 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 Beak Beaker er cont contai aini ning ng ext extra ract ctss
: 105 105.9 .999
(105.99 -105.44) =0.55mg extracts Dissolved in 5500µl of DMSO to make 100mg/ml concentration Caesalpinia sappan (Petroleum Ether extracts)
Empty weight Of the beaker : 105.44 62
Beak Beaker er cont contai aini ning ng ext extra ract ctss
: 105 105.6 .633
(105.63 -105.44) =0.19mg extracts Dissolved in 1900µl of DMSO to make 100mg/ml concentration Caesalpinia sappan (methanol extracts)
Empty weight Of the beaker : 105.44 Beake Beakerr cont contai aini ning ng extr extrac acts ts : 105 105.. 78 78 (105.78 -105.44) =0.34mg extracts Dissolved in 3400µl of DMSO to make 100Mg/ml concentration CHEMICALS USED
DMSO was purchased from Merck India Ltd, Mumbai.MTT were Sigma Chemic Chemical al co .(MO,U .(MO,USA) SA),, Media Media – HI MEDIA, MEDIA, Chloro Chlorofor form m –CSRL –CSRL Chemic Chemicals als,, Mumbai Mumbai,, FCS( FCS(Fe Feta tall calf calf seru serum) m) – Axiv Axivaa sich sichem em pvt .Ltd .Ltd . Delh Delhi, i, DPPH DPPH ,TPV ,TPVG G ,DET ,DET,M ,MET ET,, and and antibiotics were from Life Technologies in chennai .All other chemical and reagent were 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 .N o
PHYTOCHEMICAL TEST
EXTRACTS
64
PETROLEUM
CHLOROFORM
METHANOL
ETHER 1
TEST FOR TANNINS
+
+
+
2
TEST FOR SAPPONINS
+
-
+
3
TEST FOR FLAVONOIDS
+
+
+
4
TEST FOR ALKALOIDS
-
-
-
4
TEST FOR PROTEINS
+
+
+
5
TEST FOR STEROIDS
+
+
+
6
TEST
+
+
FOR
ANTHRA + (-)
QUINONES
CS→ caesalpinia sappan (+)
indicates presence of constituents.
(-)
indi ndicate catess abse absenc ncee of cons consti tittuent uents. s.
INVITRO ANALYSIS ANTIOXIDANT ASSAY Anti oxidant study:
Anti Anti oxidan oxidantt is a molecu molecule le capabl capablee of inhbit inhbiting ing the oxidation oxidation of other other molecu molecules les.. 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 antioxidant activity 1, 1Diphenyl2picry 1Diphenyl2picrylhydraz lhydrazyl yl radical scavenging activity
The ability of the extract to scavenge DPPH radicals were determined by the method of (Gyamfi et al.,1 al.,1997 997). ). with with minor minor modi modifi ficat catio ions ns.. A 20µl 20µl of aliq aliquot uot of test test extr extrac actt at diff differ eren entt 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 scient scientif ific ic invest investiga igati tion on that that may lead to new drug drug discov discovery ery.. More More than than 50% of today’ today’ss 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.
66
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 abilit abilityy agains againstt prosta prostate te cancer cancer cells, cells, Pc-3M, Pc-3M, using using sphero spheroid id disagg disaggreg regati ation on assay. assay. Active Active representative including three selected structural classes, a macrolide, a β- carboline alkaloid, and a phenyl phenyl methylene methylene hydantoin hydantoin (PMH), (PMH), were then then tested tested for their abilit abilityy to stabil stabilize ize 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 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 67
4. 0.45µ 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 200µl 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 121ºc 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 37ºc and checked for sterility, PH drop and floating particles they are then transferred to the refrigerator. MEDIA PREPARATION: INGREDIENTS
10% GROWTH
2%GROWTH
MAINTANCE
MEDIA
MEDIA
MEDIA WITHOUT FCS
MEM
857ml
937ml 68
957ml
Penicillin and
1ml
1ml
1ml
streptomycin Phenol red
1ml
1ml
1ml
Amphotericin B
1ml
1ml
1ml
3% L-glutamine
10ml
10ml
10ml
Foetal calf serum
100ml
20ml
nil
7.5%NaHCo3
30ml
30ml
30ml
Total volume
1000ml
1000ml
1000ml
PREPARATION OF INGREDIENT: 1. penicillin penicillin and streptomycin: streptomycin: (concentr (concentration ation 100IU 100IU of of penicillin penicillin and and 100 µg 0f streptomycin)
Dissol Dissolve ve both antibi antibioti otics cs in sterile sterile Milli Millipor poree distille distilledd water, water, so as to
give give a final final
concentration 100 IU of penicillin and 100µg of streptomycin/ml. Mix well and distribute in 1ml aliquots. Store at -20º C Check sterility. 2.
Fungi Fungi zone zone (amphot (amphoteric ericin in B): (conc.: (conc.: 20µg/m 20µg/ml) l)
Dissolve in sterile Millipore distilled water so as to give a final concentration of 20µg/ml and distribute in 1ml aliquots in vials. Store at -20ºc. Check sterility. 3. L.gl L.glut utam amin ine: e: 3%
Weigh 3g of l-glutamine accurately and dissolve in 100ml sterile sterile
Millipore distilled water
and mix well. Filter through Millipore membrane filter 0.22µ and distribute in 5ml aliquots in vials. Store at -20ºc. Check sterility. 4. 7.5% sodium-bi-carbonate 69
Weigh requisite requisite quantity quantity of sodium-bisodium-bi-carbona carbonate te (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 121ºc, 15lbs, 15mins. Cool and store at +4ºc. 4. Foet Foetal al cal calff seru serum m
Bring FCS at room temperature. Inactivated at 56ºc in water bath for½ 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 -20ºc. 5. Trypsin, Trypsin, PBS,v PBS,verse ersene, ne, glucose glucose solut solution ion:: (TPVG) (TPVG) 2% trypsin: 100ml
Weigh 2g of trypsin accurately; dissolve in 100 ml sterile Millipore distilled water with magnetic stirrer for ½ hour. Filter through membrane filter. Store at -20ºc 0.2%EDTA (versene)
Weigh 200mg of EDTA accurately. Dissolve in 100 ml of sterile Millipore distilled water. Autoclave at 121ºC 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
- 840ml
2%trypsin
-50ml
0.2%EDTA
-100ml
10%glucose
-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 -20ºc. 70
METHOLDOLOGY: MAINTENANCE OF CELL LINE:
Maintenance of cells involves the following operations: op erations: 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 medium and TPVG TPVG to room room temperat temperature ure for thawing. 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% 8 0% 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. TPVG VG was was added added over over the the cell cells. s. And And incub incubat atee 7. TP
at 37º 37º C for for
5 minut minutes es for for
disaggregation. The cells become individual and it’s present as suspension. 8. Add 5ml of 10% MEM with FCS by using serological pipette. 9. Gently Gently give passagi passaging ng by using using serologica serologicall pipette. pipette. If any any clumbs clumbs is present 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 incub incubat atio ionn obse observ rvee the the cells cells in inve invert rted ed micr micros oscop cope. e. The cell cellss were were obse observ rved ed for for
80% 80%
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 an d filtered through 0.45 µfilter before each assay. 1. To prepare 5 ml of extract and giving conc. (1mg/ml). 2. 500µl of MEM without without FCS was taken in 9 eppendroff tubes./each samples 3. Then 500µl of the working working conc. was added to the first eppendroff tube and mixed well then 500µl of this this volume was was transferr transferred ed from first first to last last tube by serial serial dilution dilution to obtain 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 monolaye monolayerr culture culture of Hep2cells Hep2cells at a concentrat concentration ion of one lakh lakh /ml /well /well (10 cells 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.
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.
The plat plates es were were incu incuba bate tedd at 37ºc 37ºc in 5% CO2 CO2 envir environ onme ment nt and and obse observ rved ed for for 8. The cytotoxicity using inverted microscope. MTT ASSAY:
MTT assay assay is called called as (3-(4, (3-(4, 5-dime 5-dimethy thyll thiazo thiazol-2 l-2yl yl)-2 )-2,, 5-diph 5-dipheny enyll tetraz tetrazoli olium um 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 200µl 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 Caesalpinia sappan medicine to treat various types of disease.the phytochemical analysis of Caesalpinia
extr extrac acts ts was was foll follow owed ed by usin usingg vari variou ouss comp compone onent ntss have have ident identif ifie iedd the the diff differ eren entt type typess 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. medicine. Though this is a scientific 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 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 phytochemi phytochemical cal screening screening using organic solvents solvents like petroleum petroleum ether , chloroform chloroform and methanol extracts of Caesalpinia sappan were carried out .It reveals the presence of following phyto constituents such as tannin,saponins tannin,saponins , flavanoids flavanoids , protein , protein steroids and anthrax quonone in petroleum petroleum ether and tannin, flavanoids flavanoids , protein protein steroids steroids antra quionones quionones in chloroform chloroform extracts 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, tannin, sapponins,flavonoid, protein , steroid and anthra 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. 75
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 µg of dry weight of the extract (COMPOUND) per mL of the assay mixtur mixturee .Ic 50 value value repres represent entss the concent concentrat ration ion of test test extrac extractt or compoun compoundd where where the inhibition of test activity reached 50℅The 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 activities of the antioxidant antioxidant present have. It was observed that three different different organic organic solvent extracts of caesalpinia sappan have a certain certain percentage of antioxidant antioxidant activity activity . The Methanol Methanol of caesalpinia sappan have the lowest lowest antiox antioxida idant nt activity activity at 1.64℅ .The Petroleu Petroleum m ether ether 77
caesalpinia sappan have the highly significant antioxidant activity standing at extracts of caesalpinia caesalpinia sappan showed 20.32% antioxidant 60.43% 60.43%.. And then then chloro chlorofor form m extrac extracts ts of caesalpinia antioxidant
activity. activity. When compare compare to these three extracts extracts petroleum petroleum ether extracts extracts of caesalpinia 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 percentage of cell viability viability increase with concentration concentration .A cell viability viability of more than 50% was observed observed at a concentration concentration lesser then 5mg/ml (sample (sample 6 to sample 8) the sample no.1 had the lowest lowest cell viability viability standing at 19.60%. 19.60%. The sample no.8 has the highest highest cell viability viability 94.11% the plant sample sample which were analyzed analyzed all posses a certain certain percentage percentage of antioxidant antioxidant properties. 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. and5.1. 1. The The comp compar aris ison on betwe between en thes thesee thre threee extr extrac acts ts (pet (petro role leum um ethe ether, r, chlo chloro rofo form rm and 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% 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 observed that cell viability viability increases increases with increase increase in concentratio concentrationn of the plant extract. Fig a) and b) shows the lowest cell viability standing at 19.60 and 49.01% 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 number of 55 and are not tumorigenic in nude mice. The cells secrete a variety of major plasma proteins; e.g., albumin, albumin, transferrin and the acute phase proteins fibrinogen, fibrinogen, alpha 2-macroglobu 2-macroglobulin lin,, alpha 1-antitryp 1-antitrypsin sin,, 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 µg/mL). The cells will respond to stimulation with human growth hormone. hormone. The plant extracts extracts caesalpini caesalpiniaa sappan was tested tested on HEP2 cell lines to check whether 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 mentioned data was clear that the specific specific plant caesalpinia 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 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 caesalpinia sappan is used as medicine for herbal medicines. The resistant mechanism of the caesalpinia
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 The stud studyy has has clea clearl rlyy indi indicat cated ed the the plan plantt can can be as an anti antica cance ncerr and and anti antiox oxid idan antt properties contains most of them phytochemicals. In summary, we observed that some of our select selected ed plant plant extrac extracts ts could could dose-d dose-depen ependen dently tly and signif significa icantl ntlyy inhibi inhibitt free free radica radicall 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 caesalpinia sappan have sugg sugges estt that that the the 3 diff differ erent ent extra extract ctss of caesalpinia have a cert certai ainn perc percen enta tage ge of
antioxidant activity.
85
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TABLE 1 TABULATION SHOWING THE ANTIOXIDANT ASSAY OF CAESALPINIA SAPPAN BY USING THREE DIFFERENT SOLVENT EXTRACTS
93
S.no
Plants
Caesalpinia Caesalpinia sappan
Extracts
Blank
Absorbance at
%DPPH
517 nm
Scavenging
Methanol
0.182
0.179
1.64
Chloroform
0.182
0.145
20.32
Petroleum ether
0.182
0.072
60.43
1
2
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 S.no
Conc Conceentr ntrati ation
Dilutions
Absorbance
1
(mg/ml) 5
Neat
0.03
5.88
2
2 .5
1:1
0.16
31.37
3
1.25
1:2
0.22
43.13
4
0.625
1:4
0.29
56.86
5
0.3125
1:8
0.35
68.62
6 7
0.156 0.078
1:16 1:32
0.39 0.47
76.47 92.15
8
Cell control
-
0.51
100
95
Cell viability
TABLE 3 TABULATION SHOWING THE ABSORBANCE AND % OF CELL VIABILITY FOR CAESALPINIA SAPPAN+ METHANOL
S.no
Concentration
Dilutions
Absorbance
1
(mg/ml) 5
Neat
0.07
13.72
2
2.5
1:1
0.11
21.56
3
1.25
1:2
0.19
37.25
4
0.625
1:4
0.23
45.09
5
0.3125
1:8
0.32
62.74
6
0.156
1:16
0.46
90.19
7
0.078
1:32
0.50
98.03
8
Cell control
-
0.51
100
96
Cell viability
S.no
Concentration
Dilutions
Absorbance
Cell viability
1
(mg/ml) 5
Neat
0.10
19.60
2
2.5
1:1
0.18
35.29
3
1.25
1:2
0.25
49.01
4
0.625
1:4
0.27
52.94
5
0.3125
1:8
0.30
58.82
6 7 8
0.156 0.078 Cell control
1:16 1:32 -
0.35 0.48 0.51
68.62 94.11 100
TABLE 4 TABULATION SHOWING THE ABSORBANCE AND % OF CELL VIABILITY FOR CAESALPINIA SAPPAN+ PETROLEUM ETHER
97
98