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Lipotropic and anti cirrhotic effects of Disulphides in rats fed high fat diet or Ethanol, Sunanda M. Dept of Bio Chemistry,Dr.B.R.Ambedkar Medical College,Bangalore – 45, Karnataka, India.

Lipotropic and anti cirrhotic effects of Disulphides in rats fed high fat diet or Ethanol, Sunanda M. Dept of Bio Chemistry,Dr.B.R.Ambedkar Medical College,Bangalore – 45, Karnataka, India.


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  • 1. Lipotropic and anti cirrhotic effects of Disulphides in rats fed high fat diet or Ethanol Thesis Submitted to Rajiv Gandhi University of Health Sciences For the award of DOCTOR OF PHILOSOPHY In Biochemistry [Medical Faculty] By Sunanda M. Dept of Bio Chemistry, Dr.B.R.Ambedkar Medical College, Bangalore – 45, Karnataka, India.
  • 2. Declaration I here by declare that the matter embodied in thisthesis is the result of experiments carried out by me inthe Department of Biochemistry, Dr. B. R. Ambedkarmedical college, Bangalore under the guidance ofDr. R. T. Kashinath Prof. and Head, Department ofBiochemistry, Dr. B. R. Ambedkar medical college,Bangalore and has not been submitted for the award ofany degree, diploma, associate ship, fellowship etc ofany university or institute. Sunanda M.M.Sc. Dept of Bio Chemistry, Dr.B.R.Ambedkar Medical College, Bangalore – 45, Karnataka, India.
  • 3. Dr. B. R. Ambedkar Medical College, Kadugondanahalli, Bangalore-560045 Certificate This is to certify that the thesis entitled“Lipotropic and anti cirrhotic effects of DiSulphides in Rats fed high fat diet or Ethanol”submitted by Sunanda M. to Rajiv GandhiUniversity Of Health Sciences (Medical faculty)Bangalore for the award of the degree of Doctor ofPhilosophy in Biochemistry is based on the resultsof the studies carried out by her under the guidanceand supervision of Dr. R.T. Kashinath M.Sc. Ph.D. The thesisor any part there of has not been submittedelsewhere for any other degree. Dr. G. Mohan, M. S. Principal Dr.B.R.Ambedkar Medical College, Bangalore.
  • 4. Dr. B. R. Ambedkar Medical College, Kadugondanahalli, Bangalore-560045 Certificate This is to certify that the thesis entitled“Lipotropic and anti cirrhotic effects of DiSulphides in Rats fed high fat diet or Ethanol”submitted by Sunanda M. to Rajiv GandhiUniversity Of Health Sciences (Medical faculty)Bangalore for the award of the degree of Doctor ofPhilosophy in Biochemistry is based on the resultsof the studies carried out by her under my guidanceand supervision. The thesis or any part there of hasnot been submitted elsewhere for any other degree. Dr. R.T. Kashinath M.Sc. Ph.D (Medical faculty). Prof and Head, Dept of Bio Chemistry, Dr .B. R. Ambedkar Medical College, Bangalore - 45
  • 5. Acknowledgements I wish to acknowledge my sincere thanks and indebtedness toDr. R. T. Kashinath, M.Sc. Ph.D. Professor and Head, Department ofBiochemistry, Dr B. R. Ambedkar Medical college, Bangalore for hisvaluable guidance, encouragement and continued support throughout thecourse of work. His kind counsel and patient understanding wereinvaluable in the preparation of this thesis. I wish to express my gratitude to Shri. M. K. Kempasiddayya LL. M.Trustee and Shri. B. Gurappaji, the then Chairman, Dr B. R. AmbedkarMedical College, Bangalore for encouraging me and allowing me topursue the course. I am extremely thankful to Dr. G. Mohan, M. S. Principal, Dr B. R.Ambedkar Medical College, Bangalore, for the support and help duringmy course of study. My thanks to madam Dr. A. Premarathna, Dr. R. Hemalatha, Dr.B. Ravi, Dr. T. M .R. Usha, Dr. G. Rajeshwari, Dr. C. D. Dayananda, Dr.B. Sudeer, Mrs. Sheema Ahamed, Mrs. T. M. Rashmi, Dr. Girish Desai,
  • 6. Dr. B. Kala Suresh, Dr. Rama, Dr. Saroj Golia and all my other friends inthe college for their encouragement throughout my course. My sincere thanks to Dr. Y.A. Manjunath, Dr. D.S. VasudevaDepartment of pathology, Dr B. R. Ambedkar Medical College, Bangalorefor their valuable suggestions during my course.. My special thanks to the technical staff Mrs. Darly Abharam, Mr. S.Swamy, Mrs. Violet, Mr. S. Shivalingaiah, Mrs. Ayesha, Mr. Nagaraj,Mr. Manjunath, Mr. Shivamahadeva, Mr. Ramesh, Mr. Devaraj andMrs. Valli for their help and assistance in maintaining experimentalanimals and to conduct experiments. I would like to extend my thanks to Mrs. Jayashri Rao, M.Lib.Sci. ChiefLibrarian who helped me to do the reference work. I remain ever grateful to my sister Mrs. Shobha Chinnobaiah andfamily, my brothers Mr. Prakash Adnur, Mr. Chandshekar Adnur,Babu Adnur and Girish Ramangoudar for their encouragement andmoral support throughout my course. I am grateful to my husband Mr.SreenivasMurthy and specialthanks to my son Sandeep Murthy who helped me in typing this thesis. Sunanda. M.
  • 7. ContentsChapter - 1 IntroductionChapter - 2 Materials and Methods.Chapter - 3 Results Tables, Graphs and FiguresChapter - 4 Discussions.Chapter - 5 ConclusionsChapter - 6 Summary Bibliography.
  • 8. List of Abbreviations usedAbs AbsorbanceADH Alcohol dehydrogenaseAEG Aqueous Extract of GarlicALP Alkaline phosphotaseALT Alanine TransaminaseAMP Adenosine MonophosphateAST Aspartate TransaminaseAR Grade or Analar Analytical reagent gradeBSA Bovine Serum AlbuminCamp Cyclic AMPDADS Diallyl DisulphideDPDS Dipropyl DisulphideEFA Esterfied fatty acidFFA Free fatty acidg GramHDL-Cholesterol High Density Lipoprotein ChlosterolHEG Hexane Methanol ExtractHLD High Lipid DietIU International UnitsL LitreLDH Lactate DehydrogenaseLDL Low Density Lipoprotein
  • 9. MEOS Microsomal Ethanol Oxidising systemm mol milli moleµ mol micro molem eq / l milli equivalents / litreml milli litreµ gram micro gramNAD Nicotin amide Adenine DinucleiotideNADP Nicotin amide AdenineDinucleiotide Phosphate ( oxidized)NADH Reduced Nicotin amide Adenine DinucleiotideNADPH Reduced Nicotin amide Adenine Dinucleiotide Phosphate ( oxidized)NEFA Non Esterfied Fatty AcidOAA Oxalo AcetateOD Optical DensityODFR Oxygen Derived Free RadicalPPM Parts Per MillionSER Smooth Endoplasmic Reticulum-SH groups Sulphydryl groupsSI Units System international unitsTBARS Thio barbituric acid reactive substanceUEFA Unesterfied Fatty AcidVLDL Very Low Density Lipoprotein
  • 10. Chapter-1Introduction
  • 11. 1.1. Plants and their medicinal use Plants have been the major source of drugs in the Indiansystem of medicine. The earliest reference of the medicinal plantsis found in Rig Veda (3500-1800 B.C). The important works ofmedicine of later period (1000-400 B.C) namely Charaka samhitaand Susruta samhita also give extensive description of variousmedicinal herbs. Apart from the written records, some knowledgeon the subject of medicinal plants has descended through times.Many other Asian and African countries have also reported themedicinal values of the plants and their extracts. Many medicinal plants, when subjected to scientificexperiments have yielded useful drugs, which could be taken up bythe modern system of medicine. One such plant is Ammi visanga.Decoction of the dried seeds of this plant is used as a diuretic andas an antispasmodic in renal colic in Mediterranean countries andAmerica. Investigation on this plant showed the active constituentto be ‘Khellin’ which was found to be effective vasodilator with aselective action on coronary arteries (Anrep G. V. etal 1946). Thescattered information on the medicinal plants in India has beensystematically organized by Kirtikar and Basu in 1933 (Kirtikar K.H. etal 1933) and later by Chopra in 1956 (Chopra R. N. etal1956). These works cover an extensive list of medicinal plantsavailable in various parts of the country with their reportedmedicinal values. Scientific studies and clinical trials haveconfirmed the medicinal properties of many of these plants. TheCentral Drug Research Institute, Lucknow, India has screenedaround three thousand plant materials for a wide variety of
  • 12. chemotherapeutic and pharmacological activities of which manyhave been clinically confirmed (Dhar M. L. etal 1973, Bhakuni D.S. etal 1969, Dhawan B. N. 1977, Aswal B. S. 1984).1.2 Hypolipidemic effects of plants and their extracts Among the various biological activities of the medicinalplants, the hypolipidemic activity has been the most commonlystudied one. Extracts of various plants have been shown to producehypolipidemia in normal experimental animals. Some of thecommonly studied plants are Allium sativum, Allium cepa,Momordica charantia, Ficus bengalensis, Coccinia indica etc.Active principles have been isolated from some of these plants. The plants belonging to the genus Allium group have beenextensively used as hypolipidemic agents in medicine forcenturies. Studies of products and essential oils of garlic and onionfor their physiological and therapeutic effects have been conductedsince early part of this century probably even before. But thesestudies were necessarily limited by the lack of knowledge aboutthe nature and inter relationship of the chemical components in thefresh tissue processed products or essential oils. The studies ofCavallito and Coworker (Cavallito etal 1985) and Stoll andSeeback (Stoll and Seeback 1951) may thus be seen as crucial inthe development of chemical and biochemical basis of therapeuticstudies.Table 1.2 gives the detailed list of plants having hypolipidemicaction.
  • 13. Table-1.2 List of some plants having hypolipidemic activity Name of the plant Fractions found to have References hypolipidemic or hypoglycemic action1.Acacia catechu, Singh etal 1976 Acacia sumo and Seeds Albizza odoratissima2.Allium cepa Linn Petroleium ether and Bramachari and Augusti (onion) ethanolic extracts and 1961, Jain and vyas allicin 1974, Augusti 19733.Allium sativum Linn Petroleium ether, Diethyl Bramachari and Augusti (garlic) ether and ethanolic 1962, Jain and vyas extracts and allicin 1975, Mathew, Augusti 19734.Bamusa Aqueous extract of the Bapat etal 1969 dendrocalamus leaves5.Bougainvillea Ethanolic extracts of the Narayana etal 1984. Soectaoilis roots and aqueous extract of the leaves6.Coccinia indica Ethanolic extracts Bramachari and Augusti 1963, Mukherjee etal 1972, Khan etal 19807.Eugenia Jambolana Ethanolic extracts of Bramachari and Augusti, seeds Shroti etal 1963, Bansal etal 19818.Ficus bengalensis Ethanolic and aqueous Joglekar etal 1963,
  • 14. extracts of the bark Augusti 1975, Babu and bengalenoside Murthy1984.9.Ficus glomerrata Ethanolic extracts of Shrotri and Aiman 1960, bark Gupta 1964.10.Ficus religiosa Water extracts of bark Bramachari and Augusti 1962.11.Gymnema Aqueous extract Gupta 1963. svelvestre12.Melia uzadirachta Oil from the seeds and Pillai Shanthakumari nimbdin 1981.13.Momordica Juice of the fresh fruit Sharma etal 1960, Charantia benzene and ethanolic Chatterjee extract of the dried fruit 1963,Pugazhenthi and and charantin Murthy 1979, Leatherdale etal 1981.14.Pterocarpus Aqueous and ethanolic Gupta 1963 Trivedi marsupium extract of the wood and 1963. pterostilbena15.Trigonella foenum Trigonella and coumarin Shani etal 1974. graceum16.Vinca rosea Aqueous extracts of the Shrotri etal 1963. leaves17.Xanthium A sulphur containing Kupiecki etal 1974. Strumarium hypoglycemic agent
  • 15. 1.3. Garlic1.3.1. Chemistry and uses Garlic is used widely in food and pharmaceuticalpreparations in India. It is a member of allium species and itsbotanical name is Allium sativum Lin. The botanical word allium isderived from the celtic word, all means pungent and it betrays thepresence of a host of remarkable flavorants and odorants all ofthem having in common, one element, sulphur. Garlic has been used for its therapeutic effects such asantidiabetic, antioxidant, antiatherogenic, anticancer as well asfibrinolytic action for centuries. Biological actions of alliumproducts are ascribed to organo sulphur compounds having allylgroup (CH2=CH-CH2) or its isomer propenyl group (CH3-CH=CH-) (Itokayway etal 1973, J. L. Brewster etal 1990). Presence of theseorgano sulphur compounds is the characteristics of this genus. Following the innumerable claims about the miraculousmedicinal properties of garlic, scientific basis for these weresought much later. Thus, the important ingredients of garlic are sulphurcompounds, mainly sulphoxide and disulphide and are principallyresponsible for its medicinal properties. Various chemical contents present in garlic are given intable 1.3.1.
  • 16. Table-1.3.1Table showing chemical constituents present in garlic Contents Chemical contents (g / 100)Moisture (%) 61.3-86.3 Carbohydrate 9.5-27.4Proteins 2.2-6.2 Ash 0.6-1.5Energy (cal) 39-140 Fat 0.2-0.3Bulk elements (mg / 100 g wet wt)Ca 50-90 Mg 43-77Fe 2.8-3.9 Al 0.5-3.9P 390-460 Ba 0.2-1.0K 100-120 Na 10-20Sulphur, Chlorine and trace elements (mg / 100 mg, wet wt)S 65.0 Zn 1.8-3.1Cl 43.0 Mn 0.2-1.0B 0.3-0.6 Cr 0.3-0.5Cu 0.02-0.03 Vitamins (mg/100mg wet wt)Thiamine 0.25 Ascorbic acid 5.0Pyridoxine Traces Retinol 15.0Riboflavin 0.08 Nicotinic acid 0.5
  • 17. 1.3.1.1. Carbohydrates of garlic Ananthakrishna and Ventakataraman (Ananthakrishna etal 1940.) determined various forms of carbohydrates of garlic. Apart from starch, reserve polysaccharides were chiefly made up of mannose, fructose and a non-reducing sugar. Srinivasn etal qualitatively estimated the water soluble carbohydrates in the bulbs of garlic. (Srinivasan etal 1953). 1.3.1.2. Lipids of garlic The garlic contains about 0.5% lipids on dry weight basis. Fractionation showed that garlic lipids comprised of neutral lipids, glycolipids and phospholipids, cholesterol, campesterol. The fatty acid compositions of total and component lipids have been determined.The presence of β-sitosterol and sigma sterol have also been reported. The ethyl acetate extract of condensed garlic residue also yielded more than two saponins (Danata etal 1976).1.3.1.3. Amino acids and peptides of garlic Ananthakrishan and Venkataraman (Ananthakrishan etal 1940) reported the presence of appreciable amounts of lysine and histidine and 30.5% of total phosphorus as phytin in garlic. The alcoholic extract of garlic on paper chromatography showed the presence of major amino acids such as Alanine, Arginine, Aspartic acid. Aspargine, Histidine, Leucine, Methionine, Phenyl alanine, Proline, Serine, Threonine, Trytophan and Valine. These are present as per the WHO specifications. The occurrence of six new gamma-glutamyl peptides from root, bulb and aerial parts of garlic
  • 18. were reported by Suzuki etal (Suzuki T. etal 1961) of which fourwere identified. Virtanen etal (Virtanen A. I. 1987) isolated at leastnine gamma-glutamyl peptides from garlic with gamma glutamyls-allyl cysteine and gamma-glutamyl s-propyl cysteine beingcharacteristics of garlic only. Anthocyanins, pectins, quercetin, flavonides andsulphoxides are glycosides from garlic. Amino acid composition ofgarlic protein, which showed hypolipidemic action, has beenreported earlier (Biju C. Mathew 1996). Flavonoids and sulfoxidesalso act as antioxidants. The active principles involved inantioxidant activity were considered to be s-alkenyl cysteinesulfoxides (Natto. S.1981) (alliins) and quercetin and its flavoneaglycone analogues (Pratt D. E. etal 1964).1.3.1.4. Other constituents of garlic Denjelak (Denielak Roman etal 1973) revealed theapplication of TLC in the determination of glucosinolates andsulphides of garlic. Rakhimber and coworker (Rakhimber I. R. etal1981) made use of TLC and four specific biological tests toidentify and confirm gibberlins A1 and A3 from germinating garlicbulbs.1.3.1.5. Allithiamine Perhaps the most exquisite property of garlic is its reactionwith thiamine to form allithiamine. Since the discovery ofallithiamine, many properties have been characterized (Watanabe
  • 19. H. etal 1953). One of the striking properties of allithiamine is therapid permeability across the intestinal walls as evidenced byurinary excretion of thiamine after an oral administration ofallithiamine compared to thiamine hydrochloride in human subject. Since, the discovery of allithiamine various other derivativesof thiamine (thio and thiol) have been reported. The disulphidederivative of thiamine also exhibits growth inhibiting effects onsome cells and tissues (Hamaji M. etal 1966, Kazuo H. etal 1958).1.3.1.6. Volatiles of garlic Steam distillation of garlic cloves yields essential oil knownas garlic oil (0.1-0.2 %). Various chemical compounds present inthe oil are as follows.Composition of essential oil of garlic is as followsDiallyl disulphide 60.0 %Diallyl trisulphide 29.0 %Diallyl tetrasulphide 10.5 %Propyl disulphide 6.0 %1.3.1.7. Allicin Raw garlic contains 0.4 % by weight of “alliin” which is s-allyl cysteine sulphoxide. On crushing the garlic an enzyme“allinase” convert alliin to allicin, which is responsible for garlic
  • 20. smell. Allinase is also known as “Alliin lyase” or “Alliin alkylsulphonate lyase”[EC: 4,4,4,4.]. Cavallito and Baily (Cavallito etal1950) showed the production of allicin by enzyme acting on alliinfor the first time. Later, it was Stool and Seeback (Stoll A. etal 1951) whoisolated and determined the optimal conditions with regard to time,temperature, pΗ and substrate specificity and also elucidated thebiosynthetic pathway for allicin. Allinase catalyses the conversionof allyl cysteine sulphoxide (alliin) to allyl-sulphinic acid whichspontaneously changes to diallyl thiosulphinate which on warming/ heating becomes diallyl disulphide (DADS). Goryachenkovademonstrated the stimulation of the enzyme ‘allinase’activity bypyridoxal phosphate. Subsequently, many workers reported thepresence of this enzyme in other allium species. Later it wasstudied in detail by Kupiecki and Virtanen (Kupiecki F. P. etal1913) and Schwimmer and Mazelis (Schwimmer S. etal 1963) inAllium cepa. Mazelis (Mazelis M. etal 1963) and Jacobson(Jacobson J. etal 1965) reported the presence of Allinase inBrassica species and Tulbaghia violanceae. Based on pH optima, Jacobson (Jacobson J. etal, 1965)classified Allinase into two categories with optimum pH 5.6 to 6.5(Allium satium) and pH 8.5 (Allium cepa). In 1968, Mazelies andCrew (Mazelies etal 1968) purified garlic allinase seven fold fromthat of homogenate and determined a number of characteristics.Allicin formed by the action of allinase on alliin was extractedfrom crushed garlic bulbs with water (Stoll A. and Seebeck 1951).
  • 21. Biosynthetic path way for Allicin CH2=CH-CH2-S-CH2-CH-COOH | O NH2 Allyl-L-Cysteinyl Sulphoxide (Alliin) CH3COCOOH (Pyruvic acid) Allinase NH3 CH2=CH-CH2-SH O Allyl sulphinic acid (2molecules) 2H2O CH2=CH-CH2-S-S-CH2-CH=CH2 ODiallyl thio sulphinate (allicin) Warming / Distillation CH2 CH2 || || CH CH | | CH2 – S – S – CH2 Diallyl Disulphide (DADS)
  • 22. Raghunandan Rao etal (Raghunandan Rao etal 1946) reported animprovised procedure for allicin extraction, its comparativestability in presence of blood and artificial gastric juice, itsactivation by artificial pancreatic juice and its enzyme inhibitingeffects on milk clotting activity of papain and amylolytic activityof alpha amylase. Srenivas Murthy etal reported (Srenivas Murthyetal 1960) the instability of allicin in aqueous extracts of garlicwhen stored at different temperatures. Allicin when heated orupon steam distillation looses oxygen and becomes a disulphidei.e. Diallyl disulphide (DADS). Allicin inhibits nearly allsulphydryl enzymes but very few non-sulphydryl enzymes whichwere associated with the presence of (-S-O-S-) group and not (-SO-), (-S-S-) or-S- groups. Such an enzyme inhibition by allicinwas prevented by reducing agents like cysteine or glutathione(Wills E. D. 1956).Therapeutic uses of garlic and its products Garlic as a therapeutic agent has been found to cure variousdiseases of diverse etiology. The therapeutically active garlicpreparation with high content of alkyl sulphides and allicin wasmade by the enzyme decomposition of glucoside with otherchemicals, which stabilized the alkyl sulphides and allicin in theirnascent state, after allowing for 5 to 8 days for completefermentation. This preparation could also be stabilized by vacuumdrying (Spinka etal 1956). The enzymes inhibited by garlic and its fractions areenlisted in table 1.3.1.7.
  • 23. Table 1.3.1.7 Enzyme inhibiting activity of Garlic and its fractions. Enzyme inhibited Garlic References Fractions1 Alcohol dehydrogenase Allicin Wills 19562 Alkaline phosphatase Allicin Wills 19563 Alpha amylase Garlic Suh. M. Yuna-Jah 19764 Beta amylase Allicin Raghunandan Rao etal 19465 Choline estrase Allicin Wills 19566 Fatty acid oxidase Garlic oil Vanderhock etal 19807 Glyoxylase Allicin Wills 19568 Hexokinase Allicin Wills 19569 Lactate dehydrogenase Allicin Wills 195610 Papain Allicin Raghunandan Rao etal 194611 Protease Garlic extract Mamoru and Yoshika 197712 Succinate Dehydogenase Allicin Wills 195613 Triose phosphate isomerase Allicin Wills 195614 Trypsin Garlic extract Sumathi and Pattabhiraman15 Tyrosinase Allicin Wills 1956, Agarwala etal 195216 Urease Allicin Wills 195617 Xanthine oxidase Allicin Wills 1956
  • 24. 1.3.2.1 Hypolipidemic effects of garlic Augusti and Mathew (Augusti etal 1973) first reported thatan aqueous extract of garlic has hypolipidemic action on normalrats. Later it was showed that allicin also reduces serum and tissuecholesterol and triglycerides (Augusti etal 1974). Mirhadi and others (Mirhadi etal 1991) reported thatsupplementation of garlic to rabbits, which were fed cholesterolrich diet suppressed the increased levels of cholesterol in plasma,aorta and liver. Total lipids, phospholipids and free fatty acids inaorta and liver. Gebhardt (Gebhardt etal 1991) found that cultureof rat hepatocytes on incubation with water-soluble extracts ofgarlic powder diminished cholesterol biosynthesis and its export into the medium. Pure alliin alone or after incubation with alliinase(that produces allicin) in concentration corresponding to itscontent in the extracts does not exert any inhibition. HMG COAreductase activity is significantly inhibited by garlic extracts(Diallyl Disulphide (DADS), a component of garlic extract). Fattyacid synthetase is the only enzyme, inhibited by alliin even athigher concentrations. Thus alliin does not seem to be of majorsignificance in the cholesterol biosynthesis (Kumar etal 1991). Bordia and Verma (Bordia etal 1978,1980) reported thatsupplementation of garlic to rabbits fed with a hypercholesteremicdiet showed significant decrease in LDL and VLDL andsignificant increase in HDL levels. In another report by Lau etal(Lau etal 1983) an increased HDL level was demonstrated in rats
  • 25. fed freeze-dried garlic powder making up 2% of an atherogenicdiet. Jain (Jain 1978) compared the effects of garlic and onion inrabbits fed with a diet containing cholesterol at 0.5 % g / day. Thegroup supplemented with garlic juice equivalent to 0.25 g of garlic⁄ day showed approximately 20 % of the cholesterol level of thecontrol group after 16 weeks. Using garlic oil, Jain and Komar (Jain etal 1978) showedthat the cholesterol lowering effect was dose tested. Rabbits werefed with a diet of 2g ⁄ day of cholesterol for 16 weeks.Administration of garlic oil at the dose of 0.25, 0.5 and 1g ⁄ dayresulted in 6.2 %, 21 % and 30 % reduction in serum cholesterolrespectively. According to Benjamin Lau (Lau B. H. S. etal 1987) kyolic,an odour modified garlic product when given to patients havingelevated cholesterol level (220-440 mg⁄dl) increased thecholesterol level and triglycerides level for the first two months,from the third month onwards a significant drop in serum lipidsbegan, by six months, normal levels of lipids reached in 65 % ofthe subjects. The initial rise in serum lipids may be due to the shiftof the lipid deposits from the tissues in to the blood stream (Changetal 1980, Bordia A. 1981, Chi M. S. 1982, Jain, R. C. 1975,Nakumura H. etal 1971, Krichevsky D. etal 1980).
  • 26. On continued garlic consumption, the excess lipids werebroken down and finally excreted from the body. Similarly aninitial rise occurred with LDL ⁄ VLDL before significant reductionfollowed while HDL steadily rose after the first month. Accordingto Krichevsky (Kritohevsky etal 1991) garlic can lower serumlipid levels in rats and significantly reduce the severity ofcholesterol induced atherosclerosis in rabbits. Qureshi etal(Qureshi A. A. etal 1983) reported that the odorless water solublecomponent of garlic was equally effective in lowering bloodcholesterol and triglycerides levels. Shoetan etal (Shoetan A. etal 1984) reported that whenalcohol mixed with garlic oil was fed to rats on a high fat diet noincrease of tissue lipids was observed. Several animal studies have demonstrated thatcomponents of garlic inhibit synthesis by liver cells (KritchevskyD. etal 1980, 1991, Qureshi A. A. etal 1983). On feeding garlic torats decreased the activity of several important enzymes involvedin the synthesis of lipids not only in the liver but also in adiposetissues such as fat pads (Chang etal 1980).1.3.2.2. Hypoglycemic effects of garlic Laland and Havre old (Laland P. etal, 1933) first reported onthe hypoglycemic effect of garlic. They extracted an ether soluble,steam volatile, alkaloid substance which when mixed with thedisulphides found in garlic and injected in to dogs and rabbitsshowed a hypoglycemic action. A hypoglycemic effect of garlic
  • 27. was later confirmed by others (Brahmachari H. D etal 1962, Jainetal 1973, Chang M. L. W. etal 1980, Farva D. etal 1988). Another study by Wakunga Pharmaceutical Investigatorsshowed that liquid kyolic extract, (kyolic is an odorless garlicproduct from Japan) prevented the rise of blood sugar after oralloading of glucose in a standard glucose tolerance test (Nagai K.etal 1975). Zaman et al (Zaman Q. A. M. etal 1981) and Mahantaetal (Mahanta R. K. etal 1980) administered garlic oil and rawgarlic to human volunteers and found considerable blood sugarreductions. Blood sugar lowering effect of garlic was ascribed to allicinand its various sulphur containing compounds. The -SH groupcompounds are antagonistic to the action of insulin. Allicin andrelated disulphides remove these thiols from the system and thusspare some insulin from inactivation by thiols (Farva D. etal 1988,Mathow P. T. etal 1973).R1-S-S-R1 + 2R2 SH R1-S-S-R2 + R1-S-S-R2 + H2OAllicin Thiols Mixed disulphidesR1-S-S-R2 + R3 SH R1- S-S-R3 + R2 SH (Disulphide) R1 = C3H5 and R2 & R3 = aliphatic chains or protein in enzymeswith -SH group.
  • 28. Rashiah (Rasiah S.V. etal 1985) demonstrated thehypoglycemic effect of garlic oil in experimental diabetes studies.All these findings illustrate that garlic is beneficial to diabetics.1.3.2.3. Anti tumor effects of garlic Garlic and its components significantly inhibited tumorformation (Mazelis M. 1968). Aqueous extract of garlic bulbsmarkedly suppressed the mutaganesis in both E.coil WP2 tryp-andE coli WP2 tryp. (Zhang etal 1989). Garlic and onion essential oilsand related compounds inhibited soybean lipoxygenase (Belmanetal 1989). According to Belman (Belman S. etal 1983) onion andgarlic oil inhibit tumor promotion. In cancer inhibition related studies comparison of effect ofgarlic with that of standard cancer drug, it was found that garlicshowed more positive results than BCG (Bacillus Calmette-Guerin), a live vaccine used to treat bladder cancer (Lau B. H. S.etal 1985). After five treatments of garlic extract, injected directlyinto the tumors of mice, no cancer cells were seen. In animal models, garlic compounds have been shown toinhibit chemical carcinogens and thus prevents various types ofcancer (Belman S. 1983, Wattenburg L. W.1983, Wargovich M. J.1987) Wattenburg L. W. has reviewed the anticarcinogenic activityof garlic and its principles. The allyl derivatives present in garliceffectively block the carcinogenic effects of many carcinogens.Western Reserve University reported that garlic extracts preventedtumor growth by inactivating –SH compounds of tumor cells
  • 29. (Weisberger A. S. etal 1957). All these studies show that garlichelps to inhibit tumor growth and can also enhance body’s ownimmune system.1.3.2.4. Antimicrobial action of garlic Garlic has been reported to posses a broad spectrum ofantimicrobial properties. Crude extract of garlic was found to beeffective against gram +ve and gram –ve bacteria. It inhibited thegrowth of some bacterial cultures that were resistant to commonlyused antibiotics (Sharma A.D. etal 1977, Kumar A. Sharma etal1982). Garlic can be used as a prophylactic agent againstenterotoxigenic E.Coil induced diarrhoea (Sharma V. D. etal 1977,Kumar A. etal 1982). Reports showed that garlic extract exhibitedpromising antibacterial activity against several clinical strains ofStaphylococous, Escherichia and Pseudomonas Adetumbi etal(Adetumbi etal 1986) demonstrated that garlic extracts stop thegrowth of a yeast organism (Candia Albicans) by preventingformation of lipids in the membrane of these germs andobstructing the intake of oxygen. Garlic possesses an antimicrobialactivity against Coccidioides immitis, an opportunistic fungus thatmay be involved with AIDS (Adetumbi etal 1986). Tsai etal (Tsai Y. etal 1985) reported that garlic has antiviralactivity against influenza virus and Herpes simplex virus.Benjamin Lau found that human immunodeficiency virus (HIV) orAIDS did not grow well in the presence of garlic in tissue culture
  • 30. (Lau B. H. S. 1988). Garlic was also used for treating leprosy(Chaudary D. S. etal 1962) and diarrhoea caused by Entamoebahistolytica (Varon S. 1987).1.3.2.5. Anti platelet aggregation effects of garlic Platelet aggregation is a normal response to bleeding orvascular injury. Excessive platelet aggregation is undesirable inindividuals with thrombosis tendencies. Bordia (Bordia A. 1978)showed that platelet aggregation induced by ADP epinephrine orcollagen could be inhibited by an in vitro addition of garlic oil in adose related manner. Makheja and Baily (Makheja A. N. etal 1990) reported thatfrom garlic and onion three anti platelet constituents wereidentified namely adenosine, allicin and paraffinic polysulphides.The antiplatelet effects of garlic and onion ingested are attributablemore to adenosine than to allicin and paraffinic polysulphides.Inhibition of platelet aggregation in vitro has been reported bymany investigators using garlic oil (Bordia A. 1975, Boulin D. J.1981 Makheja A. N. etal 1979, Vanderhock J.Y. 1980) and anaqueous extract of garlic (Srivastava K. C. 1984, Mohammed S. F.etal 1986, Srivastava K.C.1986). A compound with potent antithromobotic activity wasisolated by Apitz Castro etal (Apitz Castro etal 1986) in 1983 andlater identified and synthesized by Block etal (Block etal 1984). Acompound with the empirical formula C9H14S3O was named,“Ajoene”after the Spanish word ‘ajo’ for garlic, which is a
  • 31. decomposed and rearranged form of allicin that is present in agedgarlic. Commercially “Ajoene” a product of garlic is available(Apitz Castro etal 1986, Block E. 1984).. 3C3H5-S-S-C3 H5 2C3 H5-S-CH2-CH=CH-S-S-C3H5 O -H2O O (Allicin) (Ajoene)1.3.2.6. Effects of garlic on blood coagulation parameters Thrombosis is believed to be one of the precipitating factortriggering myocardial infarction and stroke. A high percentage ofpatients with acute myocardial infarction had either low ordefective fibrinolytic activity (Sassa H. etal 1975) and increasedlevels of plasma fibrinogen and shortened blood coagulation time. Bordia and Bansal (Bordia etal 1973) reported in 1973 thatfresh garlic juice or equivalent essential oil extract prevented thefat induced increase in plasma fibrinogen and decrease incoagulation time and fibrinolytic activity. Bordia etal (Bordia A.K. etal 1997) compared the effects of garlic oil (extract from 1ggarlic ⁄ kg body weight) on serum fibrinolytic activity in patientswith myocardial infarction. Patients with previous myocardialinfarction showed 83 % increase in fibrinolytic activity overcontrol patients. Patients with acute myocardial infarction fed withgarlic oil showed an increase in fibrinolytic activity of 63 % to95.5 %.
  • 32. Chutani and Bordia (Chutani S. K. etal 1981) found thatwhen raw or fried garlic (30g ⁄ day) was administered to 20patients with ischaemic heart disease for four weeks, theirfibrinolytic activity increased significantly. Bordia etal (Bordia etal 1985) compared essential oils ofonion and garlic with the antilipidemic agent clofibrate. Essentialoil of both onion and garlic reduced the elevated serum cholesteroland blood coagulability in cholesterol fed rabbits (0.2g ⁄ kg ⁄day).Garlic and onion proved more effective than clofibrate and garlicappeared superior to onion. These findings definitely enlist garlicas a source of therapeutic agents. Hasenberg etal (Hasenberg J. etal 1988) found that intake ofdried garlic by hyperlipoproteinemic patient decreased fibrinogenand fibrino peptide (Adoga etal 1991). It is also reported that garlicoil when added in the diet of Streptozocin (STZ) induced diabeticrats, reduced the hypercoagulable state of their blood, there byminimizing the risk of intravascular clotting abnormalities seen indiabetes mellitus.1.3.2.7. Effects of garlic on blood pressure Hypotensive effects of garlic extract were described as earlyas 1921 by Leoper and Debrayl (Leoper M. etal 1921). Accordingto Bolton etal (Bolton etal 1985) garlic has been used for treatinghypertension in China and Japan for centuries and is recognizedofficially for this purpose by the Japanese Food and Drug
  • 33. Administration. Malik etal (Malik etal, 1981) also reportedhypotensive effects of garlic in dogs. The hypotensive effect of garlic has been postulated to bedue to a prostaglandin formed by the action of unsaturatedpolysulphides (viz.ajoene) formed from allicin. Bulgarianresearcher Pettov (Petkov V. etal 1953, 1981) conducted extensivestudies involving both animals and humans in an attempt todemonstrate the effects of garlic on high blood pressure. Hereported that the garlic stored for seven to twelve months, theblood pressure lowering activity was significantly increased.Petkov summarized that storage enabled certain enzyme processesto release the active components of garlic. A recent study by the Chinese Co-operative Group(Zheziang 1986) on 70 hypertensive patients who were given theequivalent of 50 g of raw garlic a day. As per the study, 33 of thesubjects showed a marked lowering of blood pressure, 14 showedmoderate reductions in blood pressure, with an overall success rateof 61.7 %. Another study using essential oil of garlic conducted at thePeoples Experimental Academy of Health of Zheziang provincedemonstrated similar results in lowering of blood pressure. (BordiaA. K. 1991)
  • 34. 1.3.2.8. Other therapeutic effects of garlic Benjamin Lau (Lau B. H. S. 1991) conducted experimentsto find out the effect of garlic on stress. In his experiments twogroups of mice were employed, one group on a regular laboratorydiet and the other on the regular diet plus 25mg / day / mouse of aspecial garlic powder (Wakunaga Pharmaceutical Preparation,Japan). At the end of one week the levels of blood corticoid the“stress hormone”, secreted by the body under stress weremeasured. The mice on a regular diet had an average bloodcorticoid level of 500 ng / ml, while those who took supplementshad a dramatically lower level of 100 ng / ml. 1.3.4. Toxic effects of garlic The overuse or misuse of either oils or aqueous extracts ofgarlic often produces certain undesirable effects. As compared tothe studies conducted on the benefits of garlic there are relativelyfew studies on the toxicity of the garlic. Further Osamu Imada etal (Osamu Imada etal 1990) showedthat raw garlic causes extensive edema, bleeding and ulceration offore-stomach mucosa, reduction of red blood cells count andhematocrit and increase of reticulocytes and anemia (Joseph P. K.etal 1989). On ingestion of garlic to nursing mothers their milk getsodour of garlic. Some people have allergic reaction like dermatitisor asthma to fresh garlic or onion.
  • 35. Benjamin Lau and co-workers (Lau B. H. S. 1991)incubated human lymphocytes in kyolic or fresh garlic extract. Forcomparisons they also set up tests using L-cysteine, a compoundused to protect against radioactivity. They found that L-cysteineenjoyed significant protection. It was also found that the freshgarlic extract was quite toxic for lymphocytes in their test system.This may be due to the cytotoxicity of allicin in fresh garlic. Allthe cells were dead after incubating with fresh garlic for 24 hours.Studies have been conducted by others showing that too muchfresh garlic can have harmful side effects. The administration of Diallyl disulphide and S-allyl-L-cysteine at sub acute levels caused hematological disorders,increased serum transaminases and alkaline phsophotase and bloodurea levels. It was observed when 100 mg / Kg body weight garlicoil fed to 24 hours fasted rats, died of pulmonary edema (Joseph P.K. etal 1989). The negative as well as positive effects of alliummay be due to the action of their principles on thiol group systems.Excessive reactions may retard many enzymes and there by retardprotein synthesis. Further studies conducted by Kashinath (Kashinath R. T.1993 established that the toxic effects of garlic may be due to theproduction of acrolein, a metabolite of DADS which is a toxicproduct (Reynold E. F. 1993). Therefore only customary amounts of these species may beused as part of diet, salads and pickles (Augusti P.K. 1996).
  • 36. The studies of Dr.Osamu Imade (Osamu Imade etal 1990)indicated that the orla LD (50) values (mg/kg body weight) inmices for various garlic components were as follows. LD (50) values for various garlic componentsGarlic components LD (50) Values (mg/Kg Body) weight) Males FemalesAllicin 309 363Diallyl disulphide 145 130S- ally mercapto cysteine 600 922Diallyl sulphide 2029 1089S-allyl 8890 93901.3.3. Other garlic preparations There are a number of preparations of garlic other than thetherapeutic ones. As garlic is widely used as a flavoring agent,essentially powdered and stable garlic preparations are preferredfor storage. The current trend is to produce deodorized garlicpreparations for cosmetics, metal detoxications etc. Table 1.3.3 summarizes the various garlic preparations withtheir possible uses.
  • 37. Table1.3.3 Non-therapeutic garlic preparations and their usesPreparation Application References1.Stable garlic powder As a spice for flavour Pruthi etal 19592.Deodorised garlic -do- Yamamato 19603. -do- -do- Chrobe, 19724. -do- -do- Hauro, 19735. -do- -do- Askazu, 19786.Stable garlic -do- Spinka and Preparation Stampler 19527.Sake Deodorised garlic wine Iuemon 19708.Garlic wine Garlic odour free wine Kemichi Goku 19779.Hair dressing Stop of hair loss, Ameroetta 1967 re growth and revitalization of hair.
  • 38. 10.Garlic preparations To keep the nails Madeleine and for Strengthening flexible from hardening Mavala 1968 of nails. with the contact of foreign material.11.Metal stabilizer For decomposition of Tolok etal 1972 (Garlic waste) Nickel and Cobalt product12.Cooking oil To prepare flavour Suzuki, 1973 enhanced fried vegetables.13.Cosmetic pack Prevents wrinkling Masao 1977 dis-colourlisation of skin.14.Transparent aqueous Cosmetics, flavour Toshio etal 1977 garlic solutions and therapeutics.15.Garlic To prepare off flavoured Carl etal 1969 condiments free condiments for salad dressing.16.A formulation To mask the strong Joii O. etal 1979 mutton odour.
  • 39. 1.4. Ethanol metabolism1.4.1. Ethanol and its metabolic effects Ethanol or ethyl alcohol (C2H5OH) generally known asalcohol is commonly used organic solvent in the laboratory.Although a trace amount of ethanol can be synthesizedendogenously (McManus etal 1966, Iwata K.1972) ethanol isprimarily exogenous compound, which can be readily absorbedfrom gastro intestinal tract [GIT]. Only 2-10 % of the absorbeddose can be eliminated through the kidneys and lungs, the restmust be oxidized in the body and most of this occurs in the liver.The liver is the only organ capable of metabolizing significantamount of ethanol and magnitude of gastrointestinal ethanolmetabolism has been reported to be negligible. Since liver is themajor organ of ethanol induced toxicity many of the derangementsof hepatic functions have been attributed to the products of ethanoloxidation rather than to ethanol perse. (Liber C. S. 1984, Orrego H.1981, Sorrel M. E. etal 1979). Although ethanol perse has been shown to alter membranefluidity and in this case impaired function in a variety of organs,the susceptibility of the liver to the toxic action of ethanolindicates that metabolism of ethanol in this organ likely plays akey role in alcoholic liver injury (Rubin E. etal 1982). Despite the vast number of studies reported concerning theeffects of ethanol on liver the mechanism by which ethanolexhibits its hepato toxic effects is still an enigma. The following
  • 40. mechanisms are proposed to explain the pathogenesis of alcoholicliver injury. Most of the absorbed ethanol is degraded by oxidativeprocess primarily in the hepatocytes first to acetaldehyde and thento acetate.CH3CH2OH CH3CHO CH3COOH(Ethanol) (Acetaldehyde) (Acetic acid) The acetaldehyde formed, covalently binds tohepatocellular macromolecules especially to proteins and there byalters hepatocellular structure and function ultimately resulting inthe liver injury (Liber C. S. 1984, Orrego H. 1981). The hepatocytes contain three main pathways for themetabolism of ethanol, each located in a different sub cellularcompartment. They are,1.Alcohol dehydrogenase pathway of the cytosol or soluble fraction of the cell.2.Microsomal ethanol oxidizing system (MEOS) located in the endoplasmic reticulum, and3.Catalase degradative route located in peroxisomes. Major amount of ethanol is oxidized to acetaldehyde in theliver by alcohol dehydrogenase (ADH). Catalase and mixedfunction dehydrogenase - microsomal ethanol oxidizing system,
  • 41. MEOS, which accounts for about 25% of ethanol oxidation[Racker etal 1949].1.4.1.1. Alcohol dehydrogenase - [ADH] [EC: 1.1.1.1.] ADH is a major and rate limiting step for alcoholmetabolism, although alternate pathway exists. ADH is an NAD+dependent enzyme of the cell sap (cytosol) with an optimum pH of10-11 and catalyses the conversion of ethanol to acetaldehyde. InADH mediated oxidation of ethanol, hydrogen is transformed fromsubstrate to cofactor Nicotinamide Adenine Dinucleotide [NAD+]resulting in the conversion of its reduced form NADH. AlcoholCH3CH2OH + NAD+ CH3CHO +NADH +H+ dehydrogenase This results in the generation of reducing equivalent in thecytosol in the form of NADH and H+ with concomitant depletionof NAD+. This depletion of NAD+ can have profound effects onintermediary metabolism and is responsible for some of themetabolic consequences of alcohol intoxication (Jatlow P. 1980).During metabolism of alcohol, NADH is accumulated and NAD+is depleted resulting in lactate not being oxidized to pyruvate andlatter accumulates (Jatlow P. 1980). ADH is a heterodimer with multiple forms which arise fromthe association of different types of subunits, namely α, β and γ.The β chain is mutated in some individuals [β1 / β2] and mutationrate is more in Orientals. In Orientals 85 % are αβ2, so ethanol israpidly converted to acetaldehyde. In Whites this is minor i.e. 5-10
  • 42. %, and it has optimum pH of 8.5. The racial difference in degree ofsusceptibility to alcohol intoxication is attributed to theisoenzymes of Alcohol dehydrogenase and Aldehydedehydrogenase, which catalyses further oxidation of acetaldehydeto acetate. Aldehyde dehydrogenase CH3CHO CH3COOH(Acealdehyde) NAD+ NADH + H+ (Acetic acid) Oxidation of ethanol mainly by ADH in vivo was supportedby pyrazole, a potent inhibitor of ADH, which reduces ethanoloxidation in vivo (Lester D. etal 1968). ADH normally accountsfor the bulk of ethanol oxidation at low blood ethanol levels, butnot necessarily at high ethanol levels or during long-term use ofalcohol. ADH level is reduced in chronic alcoholics.ADH has broad specificity as it can oxidize methanol, retinol,dehydrogenation of steroid, and omega oxidation of fatty acids(Lieber C. S. 1982). It is generally recognized that the liver is the main site ofethanol metabolism although gastrointestinal metabolism isreported which is negligible (Lin G. W. etal 1980). However whenalcohol is ingested in moderate amounts, a notable fraction (about20%) (Caballeria J. etal 1987) does not enter the systemiccirculation and is oxidized mainly in the stomach. (Julkunen R. J.etal 1985, DiPadova C. etal 1987). Thus gastric ethanolmetabolism appears to decrease the bioavailability of ethanol andmay represent a barrier to the penetration of ethanol, therebymodulating its systemic effects and potential toxicity. After long
  • 43. term ethanol consumption, much of that barrier is lost, an effectthat may be due in part to diminished ADH activity (DiPadova C.etal 1987). This gastric barrier is low in women, therebycontributing to their increased susceptibility to ethanol.1.4.1.2. Microsomal ethanol oxidizing system [MEOS] This microsomal enzyme is in smooth endoplasmicreticulam [SER], proved by morphological observation in rats.Ethanol feeding resulted in proliferation of smooth endoplasmicreticulum (SER), which is a part of microsomal fraction (Iseri O.A. 1964, Lieber etal 1966).Ethanol +O2+NADPH + H+ Acetaldehyde +2H2O + NADP+ This proves that in addition to its oxidation by ADH in thecytosol, ethanol may also be metabolized by microsomes. TheMEOS requires NADPH or NADPH generating system andoxygen. Its availability is optimum at physiological pH i.e. 7.4. Therate of ethanol oxidation by this is ten times higher. MEOS systemrequires higher conc. of ethanol for half maximum activity (LieberC. S. etal 1972). MEOS is an inducible enzyme, associated withchronic alcohol, drug metabolism such as cyto-p450 reductase,cyto-p450. Ethanol increases the activity of variety of hepaticmicrosomal enzymes, which metabolizes other drugs. Theconverse also occurs, administration of variety of drugs results inincreased MEOS activity (Lieber C. S. 1972). These changes
  • 44. render the alcoholic more resistant to the effects of many commonsedative like barbiturates, resulting in the necessarily for largerthan normal dose when sedation is required. Competition betweenalcohol and for oxidation by cyto-p450 causes depression of drugmetabolism. However, ethanol and barbiturates are consumedsimultaneously competitive inhibition for those enzymes results ina reduction in clearance (Lieber C. S. 1974, Kater etal 1969) andabnormally high blood levels. MEOS utilizes cyto-p450 as anelectron carrier. This system becomes prominent at higher alcoholconcentration. Microsomal induction affects total body energy metabolism.Alcoholics given ethanol in addition to normal diet did not gainweight (Lieber C.S. etal 1965, Piroda R.C. etal 1972). Onepostulated mechanism for this apparent energy wastage isoxidation without phosphorylation by MEOS. Indeed, ethanoloxidation to acetaldehyde by ADH is associated with thegeneration of NADH, a high energy compound. But when ethanolis oxidized by MEOS, a high energy compound NADPH is usedand no high energy compound is formed, the reaction onlygenerates heat, to the extent that calorigenesis exceeds the needsfor thermoregulation which is a energy wastage.1.4.1.3. Catalase [EC – 1.11.1.6] The enzyme catalase is capable of oxidizing ethanol in vitroin presence of H2O2, a generating system shown by Keilin etal(Keilin 1945).
  • 45. CatalaseEthanol + H2O2 2H2O + Acetaldehyde It plays minor role in ethanol metabolism in vivo, becauseits activity depends on H2O2 production, which has been shown tobe low under normal circumstances in liver. Hepatic microsomescontain an enzyme, NADPH oxidase, which in presence ofNADPH and O2 generates H2O2 addition of catalase to this systemallows it to oxidize ethanol (Gillette 1957). Catalase resides primarily in the micro bodies, which areseparated with mitochondrial fraction. Other organelles howeverincluding the microsomes contain traces of catalase. Thus, thecombination of H2O2 generation from NADPH oxidase andcatalase could account for microsomal oxidation (Roach etal 1969,Tephly etal 1969). It appears that slow rate with which H2O2 canbe generated from NADPH oxidase or xanthine oxidase preventscatalase from contributing to more than 2 % of the in vivo ethanoloxidation. The NADPH oxidase activity increases upon ethanolfeeding. As a result catalase activity remained unchanged (Lieberand DeCarli 1970). MEOS +CH3CH2OH+NADPH+H +O CH3CHO +NADP+2H2O NADPH Oxidase + NADP + O2 NADP+ + H2O2 + Catalase CH3CH2OH +H2O2 CH3CHO + 2H2O
  • 46. Chronic ethanol consumption results in an enhanced rate ofethanol disappearance from blood. This is not due to the increasein ADH or catalase activity but it is due to significant change inMEOS. Increased rate of alcohol metabolism following ethanolconsumption contributes in addition to the central nervous systemadaptation to the increased tolerance to ethanol which alcoholicsare known to develop. The product of these varied oxidation pathway isacetaldehyde. It appears that either ethanol or acetaldehyde but notnecessarily both, induce various organ disorders and biochemicalalterations. For example, the fetal alcohol syndrome appears to bean ethanol effect independent of acetaldehyde (Mathions P. R etal1982) where as liver fibrosis and collagen formation is moreclosely associated with acetaldehyde than ethanol (Mendenhall C.L. 1981). The increase in NADH with concomitant decrease inNAD+ associated with ADH activity may also produce sequentialmetabolic changes with clinical consequences.1.4.2. Biochemical and metabolic effects of ethanol The oxidation of ethanol results in the transfer of hydrogento NAD+, which is reduced to NADH. This excess reducingequivalent of NADH reflects shift in redox potenicial of thecytosol and change in ratio of some metabolites such as pyruvateand lactate, as measured by changes in the lactate and pyruvateratio.
  • 47. The altered redox state, in turn is responsible for a variety ofmetabolic abnormalities. The redox changes associated withoxidation of ethanol results in increased lactate level resultingfrom either decreased hepatic utilization of lactate derived fromextra hepatic tissues or depending on the metabolic state of liverincreased hepatic lactate production. As a consequence lactatelevel rises in blood resulting in hyperlacticidemia and lacticacidosis (Daughaday W. H. etal 1962). In addition to acidosis the hyper lactiacdemia also hasclinically significant effects on uric acid metabolism. The rise inblood lactate decreases urinary uric acid out put, which leads to anincrease in uric acid level, i.e. hyperuricemia (Lieber etal 1962).Various hepatotoxic agents result in an increased breakdown ofliver nucleoproteins and enhance the release of uric acid into theblood. Both hyper uricemia and hyper lactacidemia play a role inthe aggravation or precipitation of gouty attacks traditionallyassociated with alcoholism. Enzyme activities related to ethanol oxidation and drugmetabolism are frequently altered these changes may be producedeither directly by enzyme induction or suppression or indirectly bythe shift in reducing equivalents NAD+ / NADH.1.4.2.1. Effects of ethanol on carbohydrate metabolism In vitro ethanol has been shown to inhibit the activetransport of D-glucose (Chang T. etal 1967) into intestinal cells.Ethanol impairs galactose utilization by inhibiting its conversion to
  • 48. glucose by UDP galactose 4-epimerase (Isselbacher K. J. 1961) asthis reaction is NAD+ dependent and is impaired by NADHgenerated from ethanol. Gluconeogenesis is similarly impaired byethanol by variety of mechanisms. The principle amino acidglutamate, entering gluconeogenetic pathway after deamination isconverted to α-ketoglutarate due to Glutamate dehydrogenase[GDH] reaction or other compounds of citric acid cycle (Friden C.1959). GlutamateGlutamate + NAD+ Ketoglutarate + NADH + H+ dehydrogenase This reaction is opposed by the oxidation of ethanol. Thusdecrease in the citric acid cycle activity from these precursors.Glutamate dehydrogenase is NAD+ dependent enzyme, theavailability of NAD+ is reduced due to ethanol oxidation by ADHreaction (Madison L. L. etal 1967). Increased NADH producesdissociation of glutamate dehydrogenase. The excess of NADH byethanol also favors lactate formation and retards its conversion topyruvate for gluconeogenetic pathway. Lactate Pyruvate +NADH + H+ Lactate + NAD+ dehydrogenase Ethanol promotes glycerol-lipid formation and impairsamino acid transport there by availability of the amino acid isdecreased, and hence reduced gluconegenesis. The NADH
  • 49. generated by ethanol oxidation are shuttled into the mitochondria,supplant, the citric acid cycle as a source of Hydrogen, there bycitric acid cycle activity is affected. Increased NADH / NAD+slows the reactions of the citric acid cycle, which require NAD+.Moreover, the redox change associated with ethanol oxidationdecreases hepatic oxaloacetate (Williamson etal 1969) theavailability of which controls the activity of citrate synthase.Under these conditions, mitochondria will utilize the hydrogenequivalents from the ethanol oxidation rather than oxidize twocarbon fragments derived from fatty acids, which normallyrepresent the main mitochondrial fuel (Fritz 1961). By combination of these and possibly other metabolicblocks both in extra and intra mitochondrial compartments ofhepatocytes, ethanol contributes to decreased gluconeogenesis,which in turn cause alcoholic hypoglycemia in individuals whoseglycogen stores are already depleted or who have pre existentabnormal carbohydrate metabolism (Arky R. A. etal 1966). In addition to hypoglycemia, hyperglycemia has also beendescribed. Pancreatites that occurs in alcoholic could play a role.The increase in circulating catecholamines observed after ethanolabuse could also result in hyperglycemia. The mechanism ofhyperglycemia is still obscure (Lieber C. S. 1972). Typically the storage of glycogen in the liver is alsodiminished. This results from poor dietary intake and liver diseaseso frequently associated with chronic alcoholism.
  • 50. 1.4.2.2. Effects of ethanol on protein metabolism The abnormal redox state affects protein metabolism andprotein function. Inhibition of protein synthesis has been observedafter addition of various preparations invitro (Jeejeebhoy K. etal1975). Ethanol administration increases glutamate level in the liverdue to the decreased activity of glutamate dehydrogenase becauseof low level of NAD+ in the tissue (Madones J. 1963). Further, increased NADH produces dissociation ofglutamate dehydrogenase into inactive subunits (Frieden 1959) anddecreases the availability of alpha ketoglutarate necessary fortransamination before its conversion into glucose. Thus synthesisof other amino acid in the liver is affected. The availability ofamino acid for protein synthesis is altered because ethanoldecreases Na+, K+, and Mg++ ATPase activity necessary for theactive transport with concomitant suppression of neutral aminoacid (Israel Y. etal 1963, 1968). Not all the proteins are necessarily affected, synthesis ofconstituent protein of fibrous tissue, mainly collagen may infact beincreased. This may be due to increased synthesis or decreaseddegradation or both. Thus, the process of formation or degradation of collagen inliver is complex. Increased collagen synthesis may be observed byincreased activity of hepatic peptidyl proline hydroxylase leadingto increase incorporation of proline into hepatic collagen in ratliver slices (Feinman L. 1972).
  • 51. However during early stage of alcoholic liver injury (MakerA. B. etal 1970) increased activity of collagenase is observed,subsequently collagenase activity may decrease contributing to theaccumulation of collagen (Maker A. B. etal 1968) on the otherhand, ethanol consumption may increase, the tissue lactateresulting in increased peptidyl proline hydroxylase activity both invivo (Green H. etal 1964) and invitro (Lindy S. etal 1971).1.4.2.3. Effects of ethanol on lipid metabolism Due to ethanol metabolism there is increased NADH /NAD+ ratio which results in accumulation of lipids in most tissuesin which ethanol is metabolized. The mechanism is multifactorialresulting from both increased accumulation and decreased removalof lipid. The ingestion of ethanol with a fatty meal greatlyincreases uptake of fat into chyle (Mendenhall C. L. etal 1974)with an accompanying increase in both hepatic and intestinallymph flow (Baraona E. etal 1975). Lipid synthesis is alsoaccelerated. The increase in NADH/NAD+ ratio results inenhanced fatty acid synthesis (Lieber C. S. etal 1959) possiblyinvolving fatty acid elongation system of outer mitochondria. Inliver, the increased NADH / NAD+ ratio raises the concentration ofα-glycerol phosphate (Nikkila E. A. etal 1963) which favorsaccumulation of hepatic triglyceride by trapping fatty acids(Johnson D. 1974). Ethanol consumption enhances the activity ofhepatic microsomal α-glycerophosphate acyltranferase (Joly etal1971) and phosphotidate phospho hydrolase, which is a rate-limiting enzyme in hepatic triglyceride synthesis (Savolainen M. J.1978). It is reported that increased quantity of phospholipid
  • 52. content of the liver is due to increased activity of enzymes namelycholine phospho transferase and phosphotidyl ethanolaminemethyl transferase. Moreover, if reduction of oxaloacetate tomalate is coupled with oxidation of ethanol, according to themetabolic scheme, enhanced NADH may result in increasedNADPH. Theoretically, to the extent that trans hydrogenation fromNADH to NADPH occurs, mixed function oxidase activity ofmicrosomes, which utilizes NADPH, will contribute to thedisposal of excess hydrogen generated by ethanol oxidation incytosol. Thus, in addition to promoting fatty acid elongation, ethanolmetabolism may result in production of two building blocksneeded for fatty acid synthesis, namely NADPH and acetylcoenzyme-A (Co-A) which is a means of disposing the excess ofHydrogen produced during ethanol oxidation in the liver.Hydrogen equivalents are transformed from cytosol intomitochondria by various shuttle mechanisms, namely malateshuttle and fatty acid elongation cycle. Normally, fatty acids are oxidized by beta oxidation andcitric acid cycle of the mitochondria, which serve as hydrogendonors for the mitochondrial electron transport chain. Thehydrogen equivalents generated by ethanol oxidation are shuttledinto the mitochondria; supplant the citric acid cycle as a source ofhydrogen there by depressing the citric acid cycle. Citric acid cycleactivity is affected by slowing down of the reactions that requireNAD+ and by decreasing hepatic oxaloacetate (OAA) (Williamsonetal 1969), the availability of which controls the activity of citrate
  • 53. syntheses. Under these conditions mitochondria will utilizehydrogen equivalents from ethanol oxidation rather than from fattyacids oxidation there by decreasing fatty acid oxidation (Fritz1961). Administration of ethanol with cholesterol rich dietenhances hepatic cholesterol accumulation (Lefevre etal 1969).This is due to decreased cholesterol catabolism due to reduction inbile acid production by ethanol. Ethanol administration induces mild hyperlipemia (Baronaetal 1970) from enhanced lipoproteins production by esterificationof fatty acids. The hyperlipemia is probably potentiated byunderlying defective lipid metabolism, like diabetics, a lowlipoprotein lipase activity (Losowsky M. S. etal 1963) orassociated pancreatitis (Dimagno E. P. etal 1973) as risk ofdeveloping pancreatitis doubles with low intake. Increasedavailability of fatty acids in liver may also contribute to the hyperketonemia and ketonuria, which is seen in alcoholics. The over all effects of ethanol in all three main sub cellularsites of hepatocytes contribute to alterations in the lipidmetabolism. The increased availability of NADH also results in alterationof the hepatic steroid metabolism in favor of the reducedcompound (Cronholm etal 1970). Ethanol oxidation also producesmarked changes in the lipid membrane composition and affects the
  • 54. fluidity, but it has not been clearly demonstrated (Uthus E. etal1976).1.4.2.4.Miscellaneous alterations associated with ethanol metabolism a. Vitamin abnormalities Excessive alcohol use commonly leads to vitamin deficiency(Leevy C. M. etal 1970). Liver not only converts vitamins intometabolically useful form (Cherrick G. R. etal 1965, Fennely J.etal 1967) but also a storage depot for vitamins, so injury to theliver alters vitamin metabolism. Vitamin can be released fromnecrotic liver, lost from the body and not adequately replaced(Frank O. etal 1964). Need for vitamins are increased, as there isliver regeneration (Leevy C. M. 1963, 1964) and increased nucleicacid synthesis. Fat soluble vitamins may not be adequatelyabsorbed by intestine because of increased fat loss in the stool,malnutrition associated with alcohol abuse, and inhibition ofabsorption of some vitamin (Hoyump A. M. etal 1975). The poordietary intake of alcoholics can also lead to vitamin deficiency. Inalcoholics low serum vitamin values are observed due to poordietary intake by alcoholics (Leevy C. M. 1965). Vitamin-A (retinal), which is absorbed by intestine and isconverted to retinal by alcohol dehydrogenase, is competitivelyinhibited by ethanol in liver (Thomson A. D. 1981).
  • 55. Vitamin-D, deficiency is not a major problem amongalcoholics (Leevy C. M. etal 1965). However ethanol may alter tosome extent vitamin-D and related compounds. Vitamin-E deficiency is observed because of low dietaryintake and general malnutrition in alcoholics (Thomson A. D. etal1981). Vitamin-K stores are small, billary obstruction or severeparenchymal liver disease can produce bleeding abnormalities(Melntyre N. etal 1979). Among water-soluble vitamins deficiency of Thiamine,Nicotinic acid, Folic acid, Pyridoxine, Cyanacobalamine andAscorbic acid are encountered (Leevy C. M. etal 1970). Active transport of thiamine across the intestine is inhibitedby alcohol (Hoyumpa A. M. 1975). Moreover the carbohydraterequirement is more in alcoholics, so also thiamine (Melntyre N.etal 1979) requirement. Thiamine deficiency can produceneuropsychiatry disorder called “Wernicke Korasakoff syndrome”in small number of alcoholics this can be genetically determined(Blass J. P. etal 1977). There is also increasing evidence thatthiamine deficiency may contribute to other forms of brain injury(Thomson A. D. etal 1981). The nicotinic acid is converted to coenzymes like NAD+ andNADP+. The deficiency symptoms pellagra of nicotinic acid found
  • 56. in the alcoholics may be probably due to impaired absorption ofnicotinic acid. Folic acid deficiency is very common in alcoholics. This isdue to the malnutrition, which is associated with decreased folicacid absorption associated with alcoholics (Halsted C. H. etal1971). In addition, alcohol may directly block folate metabolism(Sullivan L. W. 1964). Pyridoxine deficiency affects the transformation of aminoacids as a result of which there is increase in both aminotransferases AST and ALT especially, greater increase in AST(Ludwig S. etal 1979). Cyanacobalamine absorption at ileum is inhibited by alcohol(Lindenbaum F. etal 1973, Powell L. W. 1982) but as suchCyanacobalamine deficiency is rarely a problem (Leevy C. M.1970). Because Cyanacobalamine is stored in liver, acute liver cellnecrosis of alcoholic hepatitis may actually produce a noticeableincrease in serum levels that parallels the severity of the liverinjury. Vit-C deficiency is not common in alcoholics but low levelshave been attributed (Beattle A. D. 1976) in alcoholics.b. Mineral abnormalities It is observed that there is mild to moderate ironaccumulation in the liver of alcoholics which leads to cellular
  • 57. damage resulting in cirrhosis, heart failure etc. Inhaemochromatosis, there is accumulation of iron the mechanism ofwhich is unclear and controversial. This may be either due to theiron content of ethanol especially in red wine, or due to theinfluence of folate on iron absorption. Deficiency of zinc in alcoholics is due to reduced intake andincreased urine loss (Flink E. B. 1971) resulting in manysymptoms associated with zinc deficiency. Lead toxicity is due tothe high content of lead in some wines (Thomson A. D. 1981),which interferes with incorporation of iron into hemoglobin. Deltaaminolevulinic acid is increased and excreted in the urine (Flink E.B. 1971). Magnesium deficiency in alcoholics is quite common, asmagnesium activates many of the enzymes there by enhancingthiamine deficiency (Zieve L. 1969). Calcium level in alcoholic liver disease is very low as it isexcreted in the urine (Kalbfleisch J. M. etal 1963) and also due tolower Calcium absorption because of decreased liverhydroxylation of vit-D. Phosphorus level is decreased in 50% of hospitalizedalcoholics [Jung R. T. etal 1978) resulting in hypophosphotaemia,which is due to cellular uptake and formation of phosphate esters(Krane S. M. etal 1980) poor food intake, diarrhea, vomiting andmagnesium deficiency.
  • 58. Ethanol also affects the microsomal metabolism ofexogenous and endogenous steroid (Lieber C.S. 1968). The effectsinclude decreased blood testosterol levels (Gorden G. G. etal 1976)due in part to enhanced testosterone degradation and conversion toestrogens, as well as to decreased testicular synthesis of steroid.1.4.2.5. Alcohol induced lipid peroxidation Lipid peroxidation is a complex process where bypolyunsaturated fatty acids (PUFA) in phospholipids of cellularmembranes under go reaction with oxygen to yield hydroperoxides (LOOH). The reaction occurs through a free radicalchain mechanism initiated by the abstraction of H+ atom fromPUFA by a reactive free radical, followed by a complex sequenceof propagative reactions. The LOOH and conjugated dienes thatare formed can decompose to form numerous other productsincluding alkanals, hydroxy alkanals, malonaldehydes and volatilehydrocarbons (Halliwell B. etal 1989). These diffuse from originalsite of attack and spread the damage to other parts of the cell. O2 LHLH + R* RH + L* LOO* L* + LOOH LO*, LOO*, aldehydes (These are more radical species)LH is target PUFA LOO *is fatty acid peroxyl radicalL* is fatty acid radical R* is initiating oxidizing radicalLOOH is Lipid hydroperoxides.
  • 59. TBARS assay is the most popular and easiest method usedas an indicator of lipid peroxidation and free radical activity inbiological samples. The liver damage due to the reactive free radicals is througha variety of mechanisms, Ex. Lipid peroxidation, covalent binding,depletion of glutathione and protein thiols, derangements ofintracellular free calcium, homeostasis, DNA fragmentation withdifferent relevance in various condition (Freemann B. A. 1982). The liver injury due to acute or chronic abuse in ethanol intake (steatosis, plus necrosis, inflammation and fibrosis in lattercases) has been proved to be dependent to its oxidative metabolismat the cytosolic or microsomal (Lieber C. S. 1988). But despiteextensive investigation the molecular mechanism leading to thedamage is still need to be classified. Recent studies on the subjectsucceeded in directly demonstrating the involvement of freeradical species in ethanol metabolism and through their possiblerole in the pathogenesis of tissue changes. By using electron spin resonance, Albano etal were able todetect in rat liver microsomes incubated with ethanol, the hydroxyethyl free radical (CH3C*HOH) (Albano E. etal 1986, 1988).Further, these authors demonstrate that the formation of ethanolderived radical was mostly due to the activity of cyto-P450,dependant mono oxygenase system and minor amount of radicalcan be attributed to ethanol reaction with hydroxyl radicaloriginating (OH*) from iron catalyzed degradation of H2O2.Theformation of hydroxy ethyl radical is now proved to occur also in
  • 60. vivo by its detection in the bile of deer mice which do not expressalcohol dehydrogenase (Knecht K. T. etal 1990) in the liver(Reinke L. A. etal 1987). The role of cyto-P-450 in catalyzing the free radicalactivation of ethanol was more stressed by Albano etal (Albano E.etal 1991) who showed that reconstituted membrane vesiclescontaining cyto-P450 reductase in presence of NADPH, spin trapand ethanol do not give rise to hydroxy ethyl radicals unless cyto-P450 is incorporated. An ethanol inducible form of cyto-P450 has beencharacterized in the liver and shown mainly responsible for theformation of ethanol free radical (Johansson I. 1988). There isevidence of presence of an analogous form of cyto-P 450 also inhuman liver (Ekstrom G. etal 1989). The hydroxyethyl radicaltogether with the reactive oxygen species whose endogenousproduction in the endoplasmic reticulum is strongly enhanced bythe induction of ethanol related cyto-P450 isoenymes can inprinciple trigger oxidative damage in chronic ethanol intoxication.As regards acute intoxication, it is likely that the excess ofacetaldehyde in the liver cytosol is oxidized by alternativepathways such as xanthine oxidase, aldehyde oxidase, with theproduction of super oxide radical (O2*) (Shaw S. 1987). The most investigated mechanism of free radical inducedliver injury during metabolism is lipid peroxidation and is linked toacetaldehyde oxidation. (Comporti M. etal 1967). Acetaldehydereacts quite readily with mercaptons and cysteine, and could
  • 61. complex with acetaldehyde to form a hemiacetal. Binding ofacetaldehyde with cysteine or cysteine containing GSH or bothmay contribute to a depression of liver GSH (Shaw S. etal 1987)there by reducing the scavenging of toxic free radicals by thistripeptide. The depression of glutathione is predominant in themitochondrial compartment and may contribute to the strikingfunctional and structural damage produced by long term alcoholconsumption in that organ. The increased activity of microsomalNADPH oxidase after ethanol consumption may result in enhancedsuperoxide and hydrogen peroxide production there bytheoretically favoring lipid peroxidation (Lieber C. S. etal1970).Following is the possible biochemical steps linking theobserved effects of ethanol on free radical production and on lipidperoxidation. Free radical mediated reactions potentially involved in ethanol induced liver damage Ethanol Alcohol dehydrogenase Acetaldehyde Cyto-P450, 11E1 Xanthine oxidase Aldehyde oxidase O2*, H2O2, OH*, CH3C*HOH Decreased GSH Lipid peroxidation Protein covalent binding
  • 62. Further, it has been shown that long term ethanolconsumption is accompanied by increased formation of hydroxyradicals. Thus, ethanol provokes a significant, if not dramaticdecrease of intracellular GSH through series of mechanisms.(Albano E. etal 1991). Severe glutathione reduction favors lipidperoxidation and peroxidation may be prevented by theadministration of methionine, (Fernandez Cheeca J. C. 1987) aprecursor of cysteine and glutathione. So, hepatic ethanol over load is followed both by an increaseof reactive oxidant species, mainly free radicals, or by a decline ofantioxidant defense. The ethanol intoxication induces oxidativestress, that is an increased ratio between prooxidant and oxidantreaction.1.5. Metabolism of acetaldehyde Ethanol metabolism results in the production ofacetaldehyde, which is converted to acetate by aldehydedehydrogenase (Racker E. etal 1949) and this occurspredominantly in the liver mitochondria. The acetate formed ishighly reactive compound and exerts some toxic effects of its own. Acetaldehyde dehydrogenase Acetaldehyde Acetate Although acetaldehyde is rapidly metabolized significantamount of this reactive aldehyde has been shown to accumulate inthe liver and blood during ethanol oxidation (Braggins J. 1963,
  • 63. Lindros K. O. 1982, Nuutinen H. etal 1983). This is associatedwith generation of reducing equivalents producing a rise incytosolic and mitochondrial NADH / NAD+ ratio (Lieber C. S.1984, Orrego H. etal and Sorrel M. F. etal 1979). Numerousstudies have implicated the role of either acetaldehyde or alteredredox state in many ethanol-induced alteration of hepatic functionsand structure. (Lieber C. S. etal 1984). The polymorphism of theseenzymes is interesting. Persons mainly Asians who harbor aninactive aldehyde dehydrogenase variant (Harada S. etal 1980,Yoshida A. etal 1984) have high blood acetaldehyde levels whenthey drink, with striking consequences in terms of ethanoltolerance and flushing. The beneficial effects include a relativeaversion to alcohol, with low consumption and related morbidity. Acetaldehyde has a carbonyl carbon, which is electrophilicin nature. This makes it susceptible to the attack by a variety ofnucleiophilic compounds (O. Donnel J. P. 1982). Since manynucleophilic groups are present in peptides, the likely targetmacromolecules of acetaldehyde in the liver would be proteins. Acetaldeyde can covalently bind to a variety of proteinssuch as albumin (Mohammed A. etal 1949) plasma proteins(Lumeng L. etal 1982) hepatic microsomal proteins andhaemoglobin under physiological pH of 7.4 and at 37°c (Gaines U.C etal 1979, Nomura F. etal 1981). It can also form adducts withlipids and nucleic acid, but the reaction products are unstable,reversible and formed to lesser extent (Stevens V. J. etal 1981,Kenny W. C. 1982, Ristow H. etal 1978) to form mainly unstableschiffs base and latter stable compound (Medina V. A. etal 1985)
  • 64. hence altering protein functions. This alteration in protein causesdisplacement of pyridoxal phosphate from its binding site, henceinterfering with the activity of some protein, inhibition of hepaticprotein secretion and induced liver function. Acetaldehyde reacts with protein to form unstable schiff ’sbase. The reaction is between carbonyl carbon of acetaldehydewith amino group of lysine of protein, which is later reduced byNADH to form stable secondary amines. This is supposed to be thechief causes of alcoholic cirrhosis. Any condition that wouldincrease acetaldehyde levels in the liver during ethanol oxidationwould also enhance the formation of acetaldehyde protein adducts. Chronic ethanol consumption elevates acetaldehyde levelsby both increasing its formation as well as decreasing its oxidation(Nuutinen H. etal 1983). Presence of excessive reducingequivalents formed from ethanol oxidation favor the reduction ofschiff’s base to form stable adducts. Conditions like fasting, intakeof certain drugs, particularly acute and chronic ethanolconsumption promote the formation of acetaldehyde proteinadducts (Pessayre D. etal 1979, Gillette J. R. etal 1981,Macclonald C. M. etal 1977, Fernaudez V. etal 1981). Evenacetaldehyde protein adduct formation is effectively reduced byreducing agent like vit-C. Biological nucleiophiles like cysteine, glutathione and lysinedecrease the binding of acetaldehyde to proteins presumably byvirtue of their ability to form addition products with acetaldehyde(Medina V. A. etal 1985).
  • 65. H R-NH2 HCH3CH2OH CH3C=O CH3C=NR CH3CH2-NR NAD+ NADH + H+ Schiff’s base Stable adduct H-carrier (reducing agent)1.5.1. Effect of acetaldehyde on lipid metabolism One of the most conspicuous lesions induced by acute orchronic ingestion of ethanol in animals and man is, theaccumulation of triglycerides and cholesterol in the liver. Acetaldehyde, which is formed from ethanol oxidation,causes accumulation of triglycerides and cholesterol in the liver byforming schiff’s base a stable adduct (as shown in) with protein inthe liver (Giudicelli Y. etal 1972) thus inhibiting the transport oftriglycerides and cholesterol out of liver. Thus, aldehyde stimulatesalcohol in many biochemical effects (Trult E. etal 1971) perhaps inmore toxic manner (Ttoltzman S. G. etal 1974).1.5.2. Effect of acetaldehyde on protein metabolism There is diminution in both liver protein content and in theincorporation of leucine in to the liver protein showing that proteinsynthesis is affected in acetaldehyde treated in animals (PrasannaC. V. 1980).
  • 66. 1.5.3. Effect of acetaldehyde on carbohydrate metabolism With respect to carbohydrate metabolism, there is a decreasein TCA cycle as shown by diminished incorporation of glucose into respired CO2. It was also found that both hexokinase andpyruvate kinase activities of the liver and brain were diminished(Prassana C. V. etal 1980). Glycogen synthesis of the liver was increased, as seen bydecrease in the up take of glucose into the liver glycogen. Therewas diminished level of liver glycogen due to increasedglycogenolysis as cyclic AMP phosphosphodiesteras activity ofliver was found to be diminished.1.6. Hyperlipidemia By definition hyperlipidemia is a condition with elevatedlevels of lipids in the blood. The hyperlipidemia may be due to anyof the following reasons.1. An over intake of dietary fat.2. An abnormal lipid metabolism in the body.3. Acute or prolonged alcohol ingestion.4. Exposure to hepato toxins.
  • 67. 5. Protein malnutrition and malabsorption. As liver plays an important and extremely active role in thelipid metabolism along with adipose tissue and epithelial cell, theamount of lipid in the liver at any given time is the resultant ofseveral influences, some acting in conjunction with, some inopposition to others. Normal levels of lipid in the liver are theresult of maintenance of the proper balance between the factorscausing deposition of fat in the liver verses factor causing removalof fat from the liver. Any alterations in these processes as well asvulnerability of liver results in the accumulation of abnormalquantity of lipid in the liver resulting in so called fatty liver. Ifprolonged may lead to enlargement of liver and fibrotic changesultimately leading to cirrhosis of liver. The fat that accumulates inthe liver is derived mainly from following three sources,1. Dietary lipids, which reach blood stream as chylomicrons.2. Adipose tissue lipids, which are transported to liver as FFA.3. Lipids synthesized in the liver itself. The fatty acids from various sources are converted by liverinto low-density lipoproteins that may be returned to plasma, oraccumulate in liver because of a large number of disturbancesresults in hyperlipemia or hyperlipidemia. The heavy influx of fatty acids results in greatly increasedsynthesis of low-density lipoproteins by liver. Their liberation into
  • 68. the plasma, in the face of decreased uptake by adipose tissuecontributes to the resultant hyperlipemia. The fat content of whichdecreases accordingly. (Owing to the non-utilization ofcarbohydrates, adipose tissue is unable to take up low-densitylipoproteins, thus, aggravating the hyperlipemia.)1.6.1. Fatty liver Fatty liver is an excessive accumulation of fat (lipid) in theliver parenchyma cells, as a result there is slight to moderateenlargement of liver. The fatty liver may result from number ofstresses or abnormality in lipid metabolism. The fat thataccumulates normally includes Triacyl glycerol, Cholesterol andPhospholipids, majority being neutral fat, Triacyl glycerol. Fattyliver can be separated into two categories based on whether the fatdroplets in hepatocytes are macro vesicular (large fat droplets) ormicro vesicular (small fat droplets). Macro vesicular fatty liver ismost common type and most frequently seen in the cases ofalcoholism, obesity and Diabetes Mellitus. In general the fat in the liver is not damaging perse, and willdisappear with improvement or elimination of predisposingcondition. Micro vesicular fatty liver is less common and isassociated with jaundice and hepatic failure. Fatty livers can bedivided into several types depending on the cause.The factors that tend to increase fat content are,a. Increased synthesis of FA in that organ by carbohydrates or
  • 69. proteins.b. Increased influx of dietary lipid.c. Increased mobilization of fat from depots to liver.d. Decreased synthesis of VLDL.e. Deficiency of lipotropic factors.f. Exposure to hepato toxins.Thus, fatty liver may be caused due to,1. High lipid diet (HLD), or over feeding Due to intake of high lipid diet (HLD), fat appears in theplasma as chylomicrons, major fraction of this has to be clearedfrom liver, minor fraction by lipoprotein lipase of plasma andsome taken up by adipose and other tissues. In response to thisincrease, the liver synthesizes larger quantity of low density lipoproteins (LDL), which it liberates into plasma for transport intoadipose tissue, increasing the stores of fat in those organs with noor with increase in Triacyl glycerol and low density lipo proteins. Over feeding results in over synthesis of fatty acids, Triacylglycerol and low-density lipo proteins from carbohydrates.
  • 70. 2. Deficiency of phospholipids Phospholipids are lipotropic factors and help in the normalthe mobilization of the fat from liver. Their deficiency anddecreased synthesis may lead to under mobilization of fat from theliver hence may result in the fatty liver.3. Decreased synthesis of lipoproteins Practically all the lipids of plasma are present as lipoproteincomplexes, which are synthesized in the liver and intestinalmucosa. They are involved in the transport of lipid from the liverto adipose tissue and vice versa. Thus possibly the decreasedproduction of lipoprotein may affect the fat transport, hence fatmay accumulate in the liver leading to fatty liver.4. Deficiency of Vitamins, flavoproteins and essential Fatty acids These help in the turn over of phospholipids. The Vitaminsspecially Pyridoxine (B6), Cyanacobalamine (B-12) as well asflavoproteins and essential fatty acids have been reported toproduce fatty liver in experimental animals by affecting their turnover. Their deficiency may lead to decreased production ofphsopholipid, hence may result in the accumulation of fat in theliver leading to fatty liver.Table –1.6.1 gives the list of different types of fatty liver.
  • 71. Table-1.6.1 Classification of Fatty liver Effect on Effect Immediate CurativeType Blood lipids Effect on depot cause agents (Postabsorptive) on lipids (lipotropic liver factors) lipids1. None or increase Increase Increase Excessive fat in Choline orOver diet precursors orfeeding substitutes2. None or increase Increase Increase ExcessiveOver carbohydrates, Choline orsynthesis cysteine, B- precursors or Vitamines in diet substitutes Deficiency of Threonine3.Over Increase, normal Increase, Decrease Carbohydratemobilization pattern. Increase Normal deprivation in unesterified pattern (dietary fatty acids hormonal)4. Decrease, Increase Deficiency ofUnder especially in fat and Decrease essential fatty Inositol, cholinemobilization in phsopholipid cholestero acids,Pyridoxine, and precursors and cholesterol l, Panthothenicacid Or substitutes, decrease Choline(direct or Lipoaeic acid in lecithin indirect, excess of cholesterol5.Under Increase in Increase Increase, Deficiency of Choline andUtilization phsopholipid and Decrease panthothenic precursors chlosterol acid, Hepato Decrease later if toxic agents. severe
  • 72. 1.6.2. Cirrhosis Cirrhosis is a major cause of death in young and middleaged individuals in many West European Countries and USA(WHO). Cirrhosis is the destruction of normal tissue that leaves non-functioning scar tissue surrounding areas of functioning livertissue. Pathologically it is a defined entity that is generallypreceded by fatty liver, and is associated with spectrum ofcharacteristic clinical manifestations. Cirrhosis is derived fromGreek word “Kirrhos” meaning ‘tawny’ as the projecting nodulesare of fawn or yellowish russet bordering on the greenish. Duringinitial stage, fat molecules infiltrate the cytoplasm of the cell,which later merge together so that most of the cytoplasm becomesladen with fat. By this time nucleus is pushed to the side of thecell, nucleus gets disintegrated and ultimately hepatic cell is lysed.As a healing process fibrous tissue is laid down causing fibrosis ofliver i.e. cirrhosis. Cirrhosis is classified as under, based onetiology or morphological features (Fauci Braunwald etal 1998).1.Alcoholic cirrhosis2.Cryptogenic and post viral or post necrotic cirrhosis.3.Biliary cirrhosis.4.Cardiac cirrhosis.5.Metabolic, inherited or drug mediated cirrhosis and,6. Miscellaneous type of cirrhosis.
  • 73. 1.6.2.1. Alcoholic cirrhosis Majority of the cases, cirrhosis occurs in chronic alcoholicsand is usually preceded by a stage of fatty liver (Lieber C.S.1972).Alcoholic cirrhosis, which is also known as alcoholic hepatitis, isprinciple consequence resulting from chronic alcohol ingestion.Alcohol may cause three types of liver damage,1.Fat accumulation (fatty liver).2.Inflammation of liver (alcoholic hepatitis), and3.Scarring of liver (cirrhosis). Alcoholic cirrhosis is historically referred as Laennec,scirrhosis, which is the most common type in developed countries.It is an inflammatory lesion characterized by infiltration of liverwith leucocytes, liver cell necrosis and alcoholic hyaline is thoughtto be major precursor of cirrhosis. Deposition of collagen inperivenular spaces may be the earliest manifestations of theprocess that ultimately leads to cirrhosis. Loss of functioning ofhepatocellular mass may lead to jaundice, edema, coagulopathyand variety of metabolic abnormalities. Biochemical blood tests are usually normal in patients withalcoholic fatty liver except modest elevation of amino transferases(AST and ALT), occasional increase in ALP and bilirubin. Thedisproportional elevation in AST leading to an AST/ALT ratiogreater than 2 is associated with alcoholic hepatitis. This mayresult from proportionally greater inhibition of ALT synthesis byethanol, which may be partially inhibited by Vitamin B6. Serum
  • 74. albumin level is depressed while serum globulin level is increased.This hypoalbuminemia indicates impairment of proteinbiosynthesis while hyperglobulinemia is thought to result fromnon-specific stimulation of reticuloendothelium system.1.7. Lipotropsim and lipotropic factors Various processes help in the removal fat from liver. Theseinclude, 1.Mobilisation of fat from liver to depots. 2.Passage of cholesterol and phospholipids into blood. 3.Degradation of fatty acid in the liver. Agents, which have the apparent effect of facilitating theremoval of fat from the liver, are said to be “Lipotropic”, thephenomenon being called “Lipotropism. Their effect is due totheir,1.Possible role in the synthesis of apolipoprotein, which helps in the transport of fat and fatty acid.2.They are directly or indirectly concerned with transmethylation reaction, thus involved in the synthesis of choline and choline related phsopholipid.3.They help in the passage of choline and phsopholipid into the blood. 4.They are involved in the degradation of fatty acid in the liver.
  • 75. Hence, choline, betaine, ethanol amine, methionine, casein,phospholipid, Vitamins like pyridoxine (B6), cyanacobalamine(B12), folic acid, folinic acid groups, panthothenic acid, carnitineand unsaturated fatty acids, etc are considered to be lipotropicfactors. Human organism is unable to synthesize methyl (-CH3)group and consequently must be obtained from preformed foods.Methionine is proved to be essential in the diet as its methyl groupcan be transformed to accepter molecules by transmethylation. Asa result many methylated derivatives like choline, carnitinebetaine, etc can be formed. Thus, the source of choline forphsopholipid synthesis is mainly of dietary origin. In liverphospholipid, mainly phosphotidyl choline in part may be formedfrom phsophotidyl ethalonamine by three successive methylationusing S-adenosyl methionine as a methyl donor by a minorpathway. -CH, -CH3,-CH3.Phsophotidyl ethanolamine Phsophotidyl choline This is of less significance as it drains off methionine, whichis an essential amino acid. Infact, choline can serve as theprecursor of the methyl groups for the synthesis of methionineprovided it is present in adequate amount in the diet. As choline isrequired for the synthesis of phospholipids, which is needed for theformation of lipoproteins specially LDL and HDL which arenecessary for the transport of lipids, hence choline along withmethionine, ethanolamine, betaine, function as lipotropic factors.
  • 76. Choline Betaine aldehyde Betaine Homocysteine Methionine Dimethyl glycene FH4 Favoproteins 5,10 Methylene FH4 Sarcosine FH4 5,10 Methylene FH4 Glycine Pyridoxine helps in the synthesis of phospholipids ingeneral, which are necessary for the transport of lipids hencepyridoxine acts as lipotropic factor. Coenzyme-A, the coenzyme form of panthothenic acid aswell as carnitine are involved in fatty acid oxidation, hence bothpanthothenic acid and carnitine act as lipotropic factor. Certain substances or agents, which act as antagonists tothese lipotropic substances are referred to as antilipotropic”factors. These include cholesterol, nicotinic acid or nicotinamide,saturated fat etc.
  • 77. Cholesterol which may compete with phospholipid for polyunsaturated fatty acids hence bring down the formation ofphsopholipid there by act as antilipotropic factor. Nicotinic acid, nicotinamide and guanido acetic acid aremethylated in the body to form N′methyl derivative (N′methylnicotinamide, N′methyl nicotinic acid and creatine) there bydepleting the supply of methyl groups for the synthesis of choline,hence acts as antilipotropic factor. Usually efforts to relieve fatty liver in alcoholics failbecause of continued alcohol abuse. Alcoholic fatty liver could notbe prevented by high protein diet, lipotropic factors or withclofibrate (Lieber C.S. 1972, 1965, Decarli etal 1967).
  • 78. 1.8. Aims and objectives of the study Garlic, a member of Allium species, is used in food andpharmaceuticals in India as well as in other parts of the world. It isclaimed that garlic has antidiabetic, antioxidant, andantiatherogenic, anticancer and fibrinolytic effects. These beneficial effects of garlic may be due to itsorganosulphur compounds, which consist of either allyl orpropenyl grouping. The predominant sulphur compound in garlicis Diallyl Disulphide (DADS). Consumption of garlic and its extracts, as stated in earlierpart of this thesis, have certain biochemical toxic effects likeincrease in transaminases, increase in urea, creatinine as well asincrease in tissue thiobarbutaric acid reactive substances (TBARS). Hence it is necessary to have much care while using orconsuming garlic and its products. In accordance with the information given so far in thepresent thesis regarding the effects of garlic and its components,the present work is under taken to study the effects of garlicextracts as well as synthetic disulphide specifically pertaining to,
  • 79. 1. Anti cirrhotic effects of garlic extracts (both aqueous i.e. AEG and Hexane methanol extract i.e. HEG) in chronic ethanol fed rats.2.Anti hyperlipidemic effects of garlic extracts (both aqueous i.e.AEG and Hexane methanol extract i.e. HEG) in high lipid diet (HLD) fed rats.3.Anti cirrhotic effects of Diallyl disulphide (DADS) and Dipropyl disulphide (DPDS) in chronic ethanol fed rats.4.Anti hyperlipidemic effects of Diallyl disulphide (DADS) and Dipropyl disulphide (DPDS) in high lipid diet (HLD) fed rats.
  • 80. Chapter-2 Materials And Methods
  • 81. 2.1. Chemicals The various general chemicals employed for differentextraction procedures were of analytical reagent grade(AR/Analar) supplied by various companies like British DrugHouse (BDH) Glaxo Laboratories Chemical Division. India:E.Merck. London, Sarabhai Chemicals: Loba Chemie: FinerChemicals: Qualigens etc. The Amino acids and keto acids usedfor transaminases were of chromatographic quality. DADS andDPDS employed were procured from Sigma Aldrich ForeignHolding Company.2. 2. Preparation of garlic extracts2.2.1. Aqueous extract of garlic (AEG) One-part of fresh garlic bulbs were crushed with three partsof water (w/w) in a waring blender. It is filtered through a gaugecloth one ml of this filtrate was considered equivalent to anaqueous extract of 250 mg garlic. This was prepared fresh eachtime.2.2.2. Hexane methanol extract of garlic (HEG) One part of fresh garlic bulbs was homogenized in enough(w/w) methanol. It was filtered through gauge cloth. The filtratewas collected separately. The garlic pulp was soaked in enoughmethanol for a day. Again it was filtered and filtrates collected in
  • 82. the same container. This extraction was repeated for three days andthe filtrates were collected. All the filtrate was combined andmethanol was distilled off from the extract. Methanol free extractwas treated with diethyl ether and ether soluble fraction wasseparated in a separating funnel. This extraction was repeated threetimes. The entire ether fraction was combined and ether wasdistilled off. The left over oil was treated with aqueous methanoland was stored at 25°C for four days. It was treated with hexaneand hexane layer was separated from aqueous layer. This was alsorepeated for 3 times. This was reduced to 1/3 by evaporatinghexane at 45-60°C and the left over oil was stored in refrigeratorfor feeding. This contains Diallyl disulphide, Diallyl trisulphide,Diallyl tetra sulphide, Allicin, Dithiens. The main component isDiallyl disulphide.2.3. Experimental animals Healthy adult male albino rats of 3 to 4 months old andweighing 100-150 g were selected from the stock of colonies ofthe animal house of Biochemistry Department, Dr. B. R. AMC,Bangalore were used for experiments. They were divided into 30groups of six rats each. They were given normal stock lab dietadilibitum until and unless specified.2.3.1. To establish the effective optimum hypolipidemic dose of AEG as well as HEG.Group-1 Normal rats fed stock lab diet and were given intragastrically using stomach
  • 83. tube, 30ml normal saline per kg body weight every day for 60 days. Water was given adlibitum.Group-2 Normal rats fed stock lab diet and were given intragastrically using stomach tube 30ml 30 % alcohol per kg body weight every day for 60 days. And water was replaced by 10 % alcohol and was given adlibitum.Group -3 Normal rats were given high lipid diet (HLD) adlibitum for 60 days. Water was given adlibitum.Group-4 to Group-7 Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with different doses of AEG - 1g, 3g, 5g, 10g per kg body weight respectively everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.Group-8 to Group-10 Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with different doses of HEG - 0.1g, 0.2g, 0.3 g in 30 ml warm
  • 84. water per kg body weight respectively everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.2.3.2.To study the hypolipidemic and other relevant biochemical effects including toxic effects of optimum dose of AEG as well as HEG in chronic alcohol fed rats.Group-1, Group-2 rats served as normal group and control group.Group-11 Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight along with 5g of AEG per kg body weight everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.Group-12 Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with 0.2g of HEG in 30 ml warm water per kg body weight everyday for 60 days and water was replaced by 10 % alcohol and was given adlibitum.
  • 85. 2.3.3.To study the hypolipidemic and other relevant biochemical effects including toxic effects of optimum dose of AEG and HEG in high lipid diet (HLD) fed rats. Group-1 and Group-3 rats served as normal and control group.Group-13 Normal rats were given high lipid diet (HLD) adlibitum and were given intragastrically using stomach tube 3g of AEG per kg body weight, everyday for 60 days and water was given adlibitum.Group-14 Normal rats were given high lipid diet (HLD) adlibitum and were given intragastrilcally using stomach tube 0.2g of HEG in 30 ml warm water per kg body weight everyday for 60 days. Water was given adlibitum.2.3.4.To establish the effective optimum hypolipidemic dose of Diallyl disulphide (DADS) as well as Dipropyl disulphide (DPDS).Group-1 and Group-2 rats served as normal and control group.Group-15 to Group-18 Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with different doses of
  • 86. DADS-50mg, 100mg, 150 mg and 200 mg in 30 ml normal saline per kg body weight everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.Group-19 to Group 22 Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with different doses of DPDS 50mg, 100mg, 150mg and 200mg in 30 ml normal saline per kg body weight everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.2.3.5.To study the hypolipidemic and other relevant biochemical effects including toxic effects of optimum dose of DADS and DPDS in chronic alcohol fed rats.Group-1 and Group-2 rats served as normal and control group.Group-23 Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with 100mg DADS in 30
  • 87. ml normal saline everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum. Group-24 Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with 100mg DPDS in 30 ml normal saline everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum. 2.3.6.To study the hypolipidemic and other relevant biochemical effects including toxic effects of optimum dose of DADS and DPDS in HLD fed rats. Group-1 and Group-3 rats served as normal and control group. Group-25 Normal rats were given high lipid diet (HLD) adlibitum and were given, intragastrically using stomach tube 100mg DADS in 30 ml normal saline per kg body weight, everyday for 60 days and water was given adlibitum.Group-26 Normal rats were given high lipid diet (HLD) adlibitum and were given intragastrilcally using stomach tube 100mg DPDS in 30 ml normal saline
  • 88. per kg body weight every day for days and water was given adlibitum.2.3.7.To study the biochemical effects of feeding garlic extracts (AEG and HEG), DADS and DPDS alone to normal rats. Group-1 rats served as normal.Group-27: Normal rats fed stock lab diet and were given intragastrically using stomach tube, 3g of AEG per kg body weight everday for 60 days. Water was given adlibitum.Group-28: Normal rats fed stock lab diet and were given intragastrically using stomach tube 0.2g HEG in 30 ml normal saline per kg body weight every day for 60 days. Water was given adlibitum.Group-29: Normal rats fed stock lab diet and were given intragastrically using stomach tube 100mg DADS in 30ml normal saline per kg body weight every day for 60 days. Water was given adlibitum.
  • 89. Group-30: Normal rats fed stock lab diet and were given intragastrically using stomach tube 100mg DPDS in 30ml normal saline per kg body weight every day for 60 days. Water was given adlibitum.2.4. Collection of blood and tissues and their processing. After the feeding period of 60 days the rats of Group-1 toGroup-30 were anesthetized and sacrificed. Blood was collected inheparinised beakers. The blood samples were centrifuged at 1500gfor 8-10 minutes and clear plasma was separated. The plasmasamples were used for estimation of Total lipid (TC), Triglyceride(TAG), Phospholipid (PL), Free fatty acids (FFA), Estresified fatty(EFA), Total protein (TP), Albumin (Alb) Uric acid (UA), Vit. Cas well as Aspartate transaminase (AST), Alanine transaminase(ALT), Lipo protein lipase (LPL) and alkaline phosphatase (ALP). The liver tissue was separated and put into pre weighedclean sterile beakers. A part of liver tissue was homogenized with chloroformmethanol (1:1 v/v) and extracts were used for TC, TL, TAG andPL. Another part of the liver was homogenized with 10% TCAand extracts were used for thiobarbutaric acid reactive substance(TBARS) analysis.
  • 90. Third part of the liver was homogenized with phosphatebuffer (pH 7.4) and extracts were used to estimate total -SH groups. Fourth part of the liver was immediately fixed in bufferedformalin and preserved for histomorphological studies.2.5. Histomorphological study of tissue Histomorphological technique to observe the tissue requiresfixing of the tissue, which is best done by taking the fresh tissue inbuffered formalin (Culling 1974). The fixed tissue was embeddedin paraffin and thin sections were taken using a microtome. Thesesections were then stained using hematoxylin eosin stainingprocedure. The procedure employed can be summarized asfollows,1.Sections were dewaxed in xylol and hydrated through graded alcohol to water.2. Stained with alum-hematoxylin stain.3. Washed well in running tap water until sections were blue (3-5 minutes).4.Differentiated in 1% acid alcohol (1% HCl in 70% alcohol for 3-5 seconds).5. Washed again in tap water until the sections were blue (3- 5 minutes).6. Stained using eosin Y (1%) for 10 minutes.7. Washed in running tap water for 5 minutes.8. Dehydrated through graded alcohols.9. Cleared in xylol. Mounted in DPX and observed.
  • 91. 2.6. Methods2.6.1. Estimation of total lipids The lipids in serum, plasma or tissue homogenates wereestimated by sulpho-vanillin reaction of Chabrol and Charronat, asgiven by Choudary (Chaudhary K.1989).Principle: Lipids on heating with the concentrated sulphuric acid areoxidized into ketones. The later forms a pink color withphosphoric acid and vanillin. The intensity of pink color was readusing green filter.Reagents1. Phosphoric acid: Reagent Grade.2. Conc. Sulphuric acid: A.R.Grade.3.Vanillin 0.6 %: 600 mg vanillin was dissolved in 100 ml of 1% benzoic acid solution. The reagent is stable at room temperature for several years.4. Standard Lipid solution: 1g of pure olive oil was dissolved in absolute ethanol and made up to 100 ml with absolute ethanol.
  • 92. Procedure1. 0.1 ml serum was mixed with 1ml conc. sulphuric acid in a test tube and was placed in boiling water bath for 10 minutes, after 10 minutes removed and cooled.2. 1ml vanillin (0.6%) was mixed with 4ml phosphoric acid in another test tube and 0.1 ml sulphated serum from step 1 was added to it.3. The tubes were vigoursly shaken and incubated at 37°C for 15 minutes. Later these tubes were removed and cooled to room temperature.4. For standard 0.1ml standard lipid solution and for blank 0.1ml distilled water was used in place of serum in the procedure.5. The absorbance was read against blank with green filter.Procedure for tissue homogenate 0.1 ml chloroform - methanol [1:1 v/v] extract was taken ina clean dry test tube, this was evaporated to dryness using waterbath maintained at 70°C. 1ml conc. sulphuric acid was added andpreceded as above from step 2.Calculations T- B 1. mg total lipids /100ml serum = -------- X 100 S-B
  • 93. T-B 1 2. mg Total lipids / g = -------- X ------- S–B 0.005 The values are reported in g / g or g / Kg of triolein2.6.2. Estimation of triacylglycerol Serum triacylglycerols [TAG] in serum, plasma or tissue homogenate were estimated colorimetrically using the Hantzsch Condensation Reaction as described in “In Practical clinical Biochemistry” by Varely etal (Varley H. etal 1980). Principle Triacylglycerol are extracted into isopropanol and saponified to glycerol by potassium hydroxide. Glycerol is oxidized to formaldehyde by metaperiodate. Formaldehyde reacts with acetylacetone and ammonia to give a yellow colored compound [Hantzsch reaction], whose absorbance is measured at 405 nm. Alumina is the absorbent for phospholipid. Reagents 1. Isopropanol : Reagent grade 2. Alumina: Washed with water and dried over night in an oven.
  • 94. 3. Saponifying agent: 50 g potassium hydroxide was dissolved in 600 ml of water to which 40 ml isopropanol was added.4.Sodium metaperiodate 77g anhydrous ammonium acetate was reagent: dissolved in about 700 ml water. 60ml glacial acetic acid and 650 g Sodium periodate was added to this and the volume was made to one liter with water.5.Actylacetone reagent: 7.5 ml of acetylacetone was added to 200 ml isopropanol and mixed, to this 800 ml water was added and mixed well.6.Standard triolein: A stock standard 200 mg of trolein was dissolved in 100 ml isopropanol, this was tightly sealed and kept at 4°C.Procedure1. 0.1ml serum, standard and water were taken in separate 13x100 mm screw capped tubes.2. 4 ml isopropanol was added to each tube and mixed well.3. To this 400 mg washed alumina was added and placed in a mechanical rotator for 15 minutes.4. It was then centrifuged and 2 ml supernatant was transferred into 13x100mm tubes.5. 0.6 ml potassium hydroxide was added to each tube, mixed,
  • 95. stoppered and incubated at 60°C - 70°C for 15 minutes.6.After cooling to room temperature, 1ml sodium metaperiodate reagent was added and mixed.7. 0.5ml acetylacetone reagent was then added to this and remixed similarly, stoppered and incubated at 50°C for 30 minutes.8. After cooling to room temperature, the color developed was read at 405 nm against the reagent blank .Procedure for tissue homogenate 0.05 ml chloroform - methanol (1:1 v/v) extract was taken inclean dry test tube, and proceeded as described above for serum.CalculationsSerum triacylglycerols [mg/100ml] = Reading of unknown concentration of standard X X 100 Reading of standard effective volume of the test T-Tc mg of triacylglycerol /g tissue = --------- X 100 S-Sc2.6.3. Estimation of cholesterol Cholesterol in the serum or plasma was estimated by themethod of Wybenga (Henry etal 1974) as described in “Clinical
  • 96. chemistry- Principles and practice” by Henry etal using ferricperchlorate (Henry etal 1974).Principle Cholesterol both free and esterified reacts with a combinedreagent composed of ethyl acetate, sulphuric acid andferricperchlorate. The resultant purple color was measuredphotometrically at 610 nm.Reagents1. Conc.H2SO4: A.R. Grade2. Ethyl acetate: “Spectra quality” solvent used without redistillation.3.Ferric perchlorate: Non-yellow, containing excess HClO4 (perchloric acid) containing usually 59- 75% ferric perchlorate. Kept desiccated after being opened.4. Cholesterol reagent: 520 mg ferric perchlorate was dissolved in 600 ml ethyl acetate contained in a two liter Erlenmeyer flask. The flask was kept in an ice bath and the contents were cooled to 4°C. To this 400 ml cold concentrated H2SO4 was added slowly in small
  • 97. portions. The contents were mixed after each addition the temperature was not allowed to exceed 45°C through out the preparation of the reagent. Then stored in amber colored bottle at 25°C. Reagent is stable for at least one year and for two years in a refrigerator.5.Cholesterol standard: 200 mg cholesterol in 100 ml glacial aceticacid. Solution is stable for one year and for two years in a refrigerator.Procedure1. 5.0 ml of cholesterol reagent was taken in 3 glass-stoppered test tubes marked blank, standard and test.2. 0.01 ml glass distilled water, 0.01 ml cholesterol standard and 0.01 ml serum were added to tubes marked blank, standard and test respectively. The contents of the tubes were thoroughly mixed.3. The tubes were kept in a vigorously boiling water bath for exactly 90 seconds.4. The tubes were removed and cooled in running water. The exterior of the tubes were wiped dry. The tubes were stoppered tightly and its contents were mixed by inversion.5. The absorbance of both the standard and the test were read against the blank at 610 nm.
  • 98. Procedure for tissue homogenates 0.2 ml chloroform - methanol extracts was taken in a cleandry test tube. The contents were evaporated to dryness on a waterbath maintained at 70ºC. To this tube 2 ml of n-hexane was addedand mixed well. This was decanted into tube marked ‘ T ’. Again 1ml aliquot of n-hexane added to the tube ‘T’ and mixed well anddecanted into tube ‘T’. This was repeated with another 1ml aliquotof n-hexane. Now the combined n-hexane extracts of tube ‘T’ wasevaporated to dryness on a water bath maintained at 60ºC. Thecholesterol present in tube ’T’ was estimated by adding 5mlcholesterol reagent and mixing for 2 minutes on vortex mixer. Thetube was kept in boiling water bath for exactly 90 seconds andcontinued as described above.Calculations Abs.T 1. mg cholesterol/100ml serum = X 200 Abs.S Abs.T 1 2. mg cholesterol / g Tissue = X X 200 Abs.S 0.012.6.4. Estimation of phospholipids Phospholipids in serum, plasma or tissue homogenates wereestimated by the modified method of Youngbera and Youngbera asdescribed in “Practice of Biochemistry in clinical Medicine” byR.L.Nath 1990.
  • 99. Principle Phospholipids were extracted with an ether-alcohol (1:3 v/v)mixture. An aliquot of the extract was dried and digested withsulphuric acid and nitric acid towards the end to liberatephosphoric acid. The liberated phosphoric acid was estimated.Reagents1. Ether-alcohol mixture: 1:3 v/v2. 10 N H2SO4: 10 ml concentrated H2SO4 was diluted to 36 ml with glass distilled water.3. Concentrated HNO3: A.R. grade.4. Ascorbic acid 1%: 1 g Ascorbic acid was dissolved in glass-distilled water and made up to 100 ml with the same.5. NH4 molybdate 1% 1 gm of pure NH4 molybdate dissolved in 0.5N H2SO4: in 50 ml glass distilled water with heating if necessary. This was transferred to 100 ml volumetric flask. 10 ml of 0.5N H2SO4 was added and diluted up to the mark with glass distilled water.6.Standard Phosphate Solution: a) Stock Standard: 0.439 g of properly dried KH2PO4
  • 100. 10 mg phosphorus (potassium dihydrogen phosphate) was per 100 ml) dissolved in glass distilled water and made up to 1000 ml with the same.b). Working Standard: 2 ml of stock phosphate standard was (0.2 mg phosphorus %) taken in a 100 ml volumetric flask. To this 2.5ml of 10 N H2SO4 was added and diluted up to the mark with glass distilled water. This contained 0.01 mg P / 5ml.Procedure for serum1. 0.2 ml serum and 15 ml ether-alcohol (1:3 v/v) mixture were taken in a glass-stoppered test tube and mixed well on a vortex mixer for 3 minutes.2. 10 ml supernatant was transferred in to another test tube and evaporated to dryness on a water bath maintained at 70° C.3. The residue was heated with 0.5 ml 10 N sulphuric acid slowly on a micro burner in a fume cupboard. Keeping the test tube in a slanting position.3. When white fumes start coming from the dark digest, it was cooled and one drop of concentrated nitric acid was added and heated again till the solution was clear and colorless (if any brown tinge was persisted the heating was repeated with another drop of nitric acid). The digest was made up to 10 ml with glass distilled water.5. 5.0 ml of this solution was transferred into another test tube and mixed with 0.5 ml ascorbic acid solution. 0.5 ml ammonium
  • 101. molybdate solution was added with the starting of the stopwatch and mixed well.6. The absorbance was read against water blank at 5th, 10th and 15th minutes at 640 nm. The readings were plotted against time and the zero minutes. Absorbance was found out by extrapolation. This absorbance was unknown as ‘U’.7. In another similar test tube 5 ml working phosphate standard solution was taken and preceded through the steps 5 and 6. The zero minute absorbance obtained by extrapolation was standard ‘S’.Procedure for tissue homogenate1. 1.0 ml Chloroform-methanol (1:1 v/v) extract was taken in a clean dry glass stoppered test tube and was evaporated to dryness on a water bath maintained at 70° C.2. The residue was extracted with 15 ml ether alcohol (1:3 v/v) mixture and mixed well.3. 10 ml of this ether- alcohol extract was taken in another test tube and preceded as described above for serum.Calculation U1.mg Lipid Phosphorus / 100ml serum = X 15 S U 12. mg Lipid Phosphorous / g tissue = X S 0.033
  • 102. 2.6.5. Estimation of free fatty acids (FFA) The determination of plasma or serum Free Fatty Acids[FFA](also know as Non-esterified or un-esterified fatty acids-NEFA or UEFA) is a measure of that portion of the total fatty acidpool that circulates in immediate readiness for metabolic needs.This is usually composed of C16 and C18 fatty acids. The FFA isestimated with fresh serum sample by the method of Dole andMeintertz as described by Nath.R.L (Nath R.L.1960).Principle The FFA is extracted from a sample of serum by a mixtureof heptane and isopropanol, which was then washed with dilutesulphuric acid to remove non-EFA contaminants. The purifiedFFA extract was titrated without any delay with alkali usingthymol blue as an indicator.Reagents1. n-Heptane : Redistilled .2. Extraction mixture: Prepared by mixing 30ml isoprpanol 30ml Heptane and 3 ml 1N H2SO4.3. Indicator: 0.1% solution of thymol blue was prepared in water and diluted to 10 times with re distilled ethanol.
  • 103. 4. Standard solution: 0.02N NaOH was prepared by diluting carbonate free saturated NaOH (1.2ml saturated NaOH to 1000 ml in carbon dioxide free glass distilled water). This was standardized and the normality was adjusted to 0.02 by adequate dilution.5. Standard solution 0.0512 g of re crystallized palmitic acid 0.2 m Eeq / l) was dissolved in n-heptane and made up to 1000 ml with the same solvent.ProcedureTest: 2ml of fresh serum was mixed with 10 ml extraction mixturein a glass stopperd tube. The contents were vigorously shaken andallowed to stand at room temperature for 5 to 7 minutes. Then 4 mlglass distilled water and 6 ml heptane was added and shaken again.3 ml aliquot of the upper phase in duplicate was taken in 15 mlcentrifuge tube. 1ml indicator was added and titrated against 0.02NNaOH solution taken in a micro burette till green yellow end pointwas obtained. The mean of the two titer readings were taken.Blank: The titration in duplicate, were carried on with 2 ml ofglass-distilled water instead of serum. The mean of the two-titerreadings were taken.
  • 104. Standard: The titration in duplicate, were carried on with 2 ml of glassdistilled water instead of serum and 6 ml standard solution ofpalmitic acid instead of 6 ml heptane. The mean of the titerreadings were taken.Calculation T-B meq FEA /L = -------- X 0.06 S-BWhere as,T = mean titre reading of testS = mean titre reading of standardB = mean titre of blank.2.6.6. Estimation of esterfied fatty acid (EFA) Esterfied fatty acids were estimated in plasma by the methodof Stern and Shabiro as described R.L.Nath 1990Principle: Hydroxylamine in alkaline solution reacts with esters offatty acids to give hydroxamic acids, which produces a red toviolet color with ferric chloride. The optical density of this color ismeasured at 540 nm.
  • 105. Reagents1. Ether –alcohol mixture: 1:3 v/v2.Hydroxylamine hydro: 1.4 g of hydroxylamine hydrochloride hydrochloride 14% was dissolved in 10 ml glass distilled water. Prepared fresh every time.3. Ferric chloride 10%: 10 g of FeCl3. 6H2O was distilled in 0.1N HCl made to upto 100 ml with the same.4. NaOH 14%: 14 g of NaOH pellets were dissolved in glass distilled water and made upto 100 ml with the same.5. HCl 33%: 1volume of conc. HCl [SG 1.18 was diluted with 2 volumes glass distilled water.6.Std solution of Triolein: 1.18 g of triolein was dissolved in ether: a. Stock std alcohol (1:3 v/v) mixture and made up to 100 ml with same mixture. b. Working std: 5ml of the stock std was diluted to 100 ml with Ether alcohol (1:3v/v) mixture 1ml of this contained 0.54 mg of fatty acid.
  • 106. Procedure 0.3 ml of serum was mixed with 6 ml ether alcohol mixturein a test tube, mixed and warmed on a water bath. It was cooledand filtered without pouring the precipitate through a fat free dryfilter paper into a 15ml test tube. The extraction was repeated inthe same way using 5ml and 4ml of the solvent successively. Thevolume of the combined filtrate was made up to 15 ml and wasmixed.Three stoppered cylinders were arranged as follows: Blank Standard Test1.Ether-alcohol mixture ml 5.0 5.0 -2.Working std ml - 1.0 -3.Filtrate of test ml - - 5.04.Hydroxylamine hydrochloride ml 5.0 5.0 5.0 Shaken and added5. NaOH ml 0.5 0.5 0.5 Shaken, stoppered and kept at 25°C for 20 minutes, then added.6. HCl ml 0.6 0.6 0.67.Ferric chloride ml 0.5 0.5 0.5The contents of each tube are mixed and absorbance of std (S) andtest (T) were taken against blank (B) at 540nm.Calculationmg Esterified fatty acid /100ml serum = T x 540 The values were converted into mEq /L and reported.
  • 107. 2.6.7. Estimation of Ascorbic acid Ascorbic acid is determined photo metrically with 2,4dinitrophenyl hydrazine to form the red bis hydrazone or with 2,4dichlorophenol indophenol which is reduced to a colorless form(Tietz 1994).Principle Ascorbic acid in plasma is oxidized by Cu++to form di hydroascorbic acid which reacts with acidic 2,4 dinitro phenyl hydrazineto form a red bis hydrazone which is measured at 520 nm.Reagents 1. Metaphosphoric acid: 6.0 g % Prepare fresh. Dissolve 30g HPO3 in distilled water, make up to 500 ml. 2. H2SO4 (4.5 mol/L): 250 ml conc. H2SO4 (AR grade) added to 500 ml of cold water in 1 liter flask and fill water up to the mark. Keep the flask in ice bath.3.H2SO4 (12 mol/L): Dilute 650 ml of conc. H2SO4 with 300 ml cold water, make up the volume to 1 liter.4. 2.4 Diniro phenyl: Dissolve 10 g of DNPH in 4.5mol /L
  • 108. hydrazine (DNPH) in H2SO4 and dilute to 500 ml. Let it 2.0 g/dl in 4.5mol /L stand fridge over night and then filter. H2SO4 :5. Thio urea (5.0g /L): Dissolve 5.0 g thio urea in glass distilled water and dilute to 100ml. Stable for 1 month.6. CuSO4. 0.6g/L: Dissolve 0.6g of anhydrous CuSO4 in a glass distilled water and dilute to100 ml.7. DTCS reagent: 5 ml Thio urea + 5 ml CuSO4 (100 ml 2.4 DNPH store in a bottle at 4°C for maximum of 1 week.(DTCS is Dinitro phenyl hydrazine Thio urea CuSO4 reagent)8. Calibrater: Prepare everyday Freshly. a. Stock 50 mg %: 50 mg Vit-C in 100 ml of HPO3. b. Intermediate 5.0mg%: 10 ml stock (a) into 100 ml of HPO3. c. Working: 0.5, 2.0, 4.0, 10.0 and 20.0 ml make up to 25 ml with 0.6g / 100 ml HPO3. (Conc. is 0.10, 0.4mg, 0.8, 1.2, 2.0, 3.0, 4.0 mg / 100ml).
  • 109. Procedure1.Add 0.5 ml heparinised plasma to 2.0 ml of freshly prepared meta phosphoric acid in a 13×10 mm test tube and mix well in a vortex mixture. Centrifuge the plasma meta phosphoric acid mixture for 10 minutes at 2500 × g. Pipet 1.2 ml of clear supernatant into a 13×100 mm Teflon lined screw cap test tube.2.Add 1.2 ml of each concentration of working calibrater in to a 13 × 100 mm screw cap test tube. Prepare calibrator in duplicate. Add 1.2 ml meta phosphoric acid to 2 tubes for use as blank.3.Add 0.4 ml of DTCS reagent to all tubes and cap all the tubes, mix the contents and incubate the tubes in a water bath at 37°C for 3 hours.4. Remove the tubes from the water bath and chill for 10 minutes in ice bath. While mixing slowly add 2.0 ml cold sulphuric acid (12 mol / L) to all tubes, mix in vortex mixer. (The temperature should not exceed room temperature).5. Adjust the spectrophotometer with blank to read zero and read the calibrators and unknown at 520 nm. Plot the concentration of each working calibrator versesabsorbance values.Calculation Value from calibration curve is multiplied by 5 to correct fordilution of the plasma by metaphosphoric acid to give conc/dl ofAscorbic acid in plasma.
  • 110. 2.6.8. Estimation of total protein Proteins in serum are estimated by Reinhold’s modificationof kingley Biuret method as described by Teitz 1986.Principle All proteins contain large number of peptide bonds. When asolution of protein is treated with Cu++ ions in moderate alkalinemedium a colored chelate complex of unknown composition isformed between the Cu++ions and carbonyl (-C=O) group and theamide (-NH) group of the peptide bonds. The intensity of the colorproduced is proportional to the number peptide bonds under goingreaction. Thus the biuret reaction used as basis for the simple andrapid colorimetric method for determining proteins. The intensityof the colored complex was measured at 550 nm or using greenfilter.Reagents1.Biuret diluent: A liter of NaOH was prepared by 0.5% KI in diluting 2.5 N (10% w/v) with CO2 free 0.25 N NaOH distilled water. To this 5 g KI was added. The solution was stored in stoppered polythene bottles.2.Stock Biuret Reagent: 15g of finely pulverized copper sulphate (Weichslebaun formula) (CuSo4.5H2O) were dissolved in about 80 ml of glass-
  • 111. distilled water. A solution of 45g of Rochelle salt (Potassium sodium tartrate tetra hydrate) was prepared in about 700ml biuret diluent and was added slowly to the copper sulphate solution with continuous stirring. Both solutions should be at room temperature when mixed to prevent reduction of Cu++ by tartrate. The volume was made up to 1000 ml with biuret diulent. The reagent was filtered to remove any deposit of cuprous oxide. Stored in polythene bottles away from direct sunlight.3.Working Biuret: The stock biuret reagent was diluted in 1:5 proportions with biuret diulent. Stored in the same way as the stock biuret reagent.4. Alkaline tartrate: 0.9 % w/v Rochelle salt in biuret diulent. The solution except Cu++ is omitted. This was used to correct for serum pigment error.5. Saline: NaCl 0.85% w/v in glass distilled water.6. Standard protein: Bovine serum album in solution of 6g /100ml.
  • 112. 7. Control Serum: This was prepared by pooling the normal non-lipemic and non–icteric sera. It was mixed and recentrifuged to remove the fine clots. This was preserved in small tubes and stored frozen. This was standardized by assaying against another serum of known value. Procedure 1. Test: 2ml saline was taken in a clean test tube and 0.1 ml of sera was added to it. Standard: 2ml saline was taken in a clean test tube and 0.1 ml of standard protein solution or assayed pooled sera was added. Blank:ml saline was taken in a test tube. 2. To all these tubes 8 ml of working biuret reagent was added. The content of the tubes were mixed thoroughly. 3. The tubes were kept for 30 minutes at room temperature for color development. 4. The absorbance of standard and test were measured against blank at 550 nm.
  • 113. Calculation Abs. Test g of protein / 100ml serum = X conc. (g/100ml) Abs.Std.The values are reported in g / L.2.6.9. Estimation of Albumin Albumin content of serum or plasma was estimated by dyebinding method using BCG (Bromo Cresol Green) as described byChaudhary 1989.Principle Bromocresol green solution has two forms monovalentyellow forms with phenol group undissociated and the divalentblue ion. Slightly below pK value (4.7) only a minor a part of thedye is in blue form and the solution has yellow colour. Albuminbinds with monovalent yellow form upsetting the balance and theblue color produced is proportional to the albumin concentration inthe sample.Reagents1. BCG reagent: 280 mg bromocresol green was dissolved in 125 ml 4% NaOH solution. To this 500 ml water, 28 mg
  • 114. succinic acid and 500 mg sodium azide were added. This as shaken to dissolve and diluted up to 100 ml with glass distilled water.2.Standard: Pooled human assayed serum.Procedure1.4 ml glass distilled water was mixed with 1 ml BCG Concentrate in a test tube. The absorbance was measured (Blank) against water adjusted to zero with orange red filter.2.To this was added 0.1 ml diluted serum (1vol serum +3vol normal saline). Mixed and absorbance was read immediately.3.For standard 0.1 ml assayed serum (1:3 diluted with normal saline) was used.Calculations Abs. T - Abs. Blankg Albumin/100 ml serum = X Conc. of std. Abs. Std - Abs. BlankThe values are reported as g / L2.6.10.Estimation of Lipoprotein Lipase (LPL) The name lipoprotein lipase [EC 3.1.1.3 lipase type XIII] ismeant to denote a specific lipase, which preferentially hydrolyses
  • 115. triacylglycerols when they are in the form of lipoproteins. As withall lipases, glycerol and unesterified fatty acids are produced. Theenzyme was first detected in the plasma of animals injected withheparin and was referred to as “clearing factor”. Considerable evidence show that this enzyme got a role inthe transport and metabolism of triglycerols of exogenous origin,the exact metabolic function of this enzyme remains uncertain.Lipoprotein lipase in the plasma has been estimated by methodexplained by Edward D.Korn.Principle The glycerol produced by the action of the enzyme onactivated triacylglycerol emulsion was oxidized by periodate toformaldehyde. The formaldehyde produced was then determinedcolourimetrically using the Hantzch reaction [Varley H.etal 1980].Reagents1. 0.5M (NH4)2SO4): 33.0 g of (NH4)2SO4 was dissolved in 500 ml glass distilled water.2. 20% Albumin: 2g of bovine serum albumin was dissolved in little glass distilled water, adjusted the pH to 8.5 with NaOH and made up the volume to 10 ml with glass distilled water.
  • 116. 3.Substrate: Activated triacylglycerol emulsion at a conc.of 2% with respect to TAG were prepared by incubating commercial TAG. (Activated TAG was prepared by incubating a commercial TAG emulsion (2%) with an equal volume of serum at 37°C for 30 minutes. The mixture was used as such).4. 0.05 M Sodium: 11.4g of HIO4.2H2O was dissolved in meta periodate 900 ml of glass distilled neutralised with 1N NaOH and was diluted to 1000 ml with glass distilled water.5. 1N sulphuric acid: A.R.grade5. Acetyl acetone solution. Procedure 0.4 ml of albumin was taken in a clean dry test tube, 0.1mlof Ammonium sulphate solution, 0.1 ml substrate, 0.1ml serumand 0.3 ml of glass distilled water were added. The contents weremixed and the tube was incubated at 37°C for 60 minutes. Analiquot of 0.1 ml was taken out at zero minute and transferred intoanother tube marked ‘control’ containing 0.1ml of 1N N H2SO4. Atthe end of the incubation period another 0.1 ml of aliquottransferred into a tube, marked ‘test’ containing 0.1ml of 1NH2SO4. The amount of glycerol in the ‘ control’ and ‘test’ tubes
  • 117. were determined by the procedure given earlier, starting fromaddition of metaperiodate. [refer TAG estimation procedure].CalculationsAmount of glycerol Glycerol GlycerolProduced in 60 minutes = present present X 10By 1ml serum at 37°C in test in control Enzyme activity: Activity is expressed in terms ofmicromole of Glycerol produced / ml serum / hour.2.6.11. Estimation of uric acid Uric acid in serum or plasma was estimated by the methodof caraways as described in “Practical clinical Biochemistrymethods and interpretations” by Ranjan chawla (Ranjan chawla,1995)Principle Phosphotungstic acid in alkaline medium oxidizes uric acidto allantoin and itself gets reduced to tungsten blue, a bluecoloured complex, which is estimated colorimetrically at 700nm.
  • 118. Reagents1.Sodium tungstate 10%:2. 2/3 N H2SO4:3. Tungstic acid: Add 50ml of 10% sodium tungstate 40 ml of 2/3 N H2SO4 and a drop of phosphoric acid with mixing to 800ml of distilled water.4.Phosphotungstic acid: Dissolve 50g sodium tungstate in 400 ml of distilled water slowly with constant shaking. Add 40 ml of 85 % phosphoric acid. Boil gently under reflux condenser for 2 hours. Cool and make volume to 500ml. Store in a brown bottle. Dilute 1 to 10 for use. The reagent is stable at room temperature in amber colored bottle for about 2 years. Arsenomolybdate can be used instead of phosphtungstate.5.10 % Na2Co3:6.Standard uric acid: For 100 mg % stock weigh 100mg of uric in a beaker. Weigh 60 mg LiCO3 and dissolve in 15-20 ml distilled water in a test tube. Heat the solution to 60ºC and pour to the uric acid. Stir to dissolve. When dissolved transfer to 100 ml flask. Add 2 ml of 40%
  • 119. formalin slowly with shaking. Add 1ml of 50% v/v acetic acid. Make up to mark with distilled water. Keep it away from light.7.Working uric acid: 1 mg %Procedure In a centrifuge tube pipette out 0.6 ml of serum and 5.4 mlof tungstic acid while shaking. Centrifuge and proceed as follows. Test Std BlankSupernatent 3.0mlStandard 3.0mlDistilled water 3.0mlSodium carbonate solution 0.6ml 0.6ml 0.6mlPhosphotungstate reagent 0.6ml 0.6ml 0.6mlCalcucation O.D of Test – O.D of BlankSerum uric acid in mg % = X 10 O.D of Test – O.D of Blank
  • 120. 2.6.12. Estimation alkaline phosphatase The alkaline phosphatase levels [Orthophophoric mono esterhydrolase: EC 3.1.3.1] were estimated by the modification of themethod of King and Armstrong [King E.J.etal] using disodiumphenyl phosphate substrate as given by IDP Wooten WootenI.D.P].Principle Phenol released by enzymatic hydrolysis of phenylphosphate under defined conditions of time temperature and pHwas estimated colorimetrically.Reagents1.Buffer pH 10.0: 6.3 g of anhydrous sodium carbonate and 3.36 g of NaHCO3 were dissolved in water and the volume was made up to a liter with glass distilled water. It was preserved in a refrigerator at or below 4°C.2.Substrate 0.01M: 2.18 g of disodium phenyl phosphate was disodium phenyl dissolved in a glass of distilled water and phosphate volume was made up to a liter with the same. The solution was brought quickly to boil to kill any
  • 121. organisms present. It was cooled immediately and preserved with little chloroform (4ml/L) at 4°C.3: Stock phenol: 1.0g pure crystalline phenol was dissolvedstandard (mg/ml) in 0.1N HCl and made upto a liter with the same solvent. It was kept in a fridge at 4°C in brown bottle.4.Working phenol: 1ml stock phenol standard was diluted to 100 ml with glass (1mg / 100ml) distilled water and was preserved at 4°C in a brown bottle with a few drops of chloroform.5. Sodium hydroxide: 0.5 N6. Sodium bicarbonate 0.5 N7.4-amino antipyrine: 0.6 g of 4-amino antipyrine dissolved in (4-amino phenazone) glass distilled water and volume was made up to a liter with the same solvent. Stored in brown bottles.8.Potassium ferricyanide: 24 g Potassium ferricyanide dissolved in glass-distilled water and was made upto a liter with the same. It was stored in brown bottle.
  • 122. ProcedureTest: 1ml buffer and 1ml phenyl phosphate substrate solutionwere mixed in a test tube and incubated at 37°C for 3 minutes. 0.1ml serum was added and mixed and incubated at this temperaturefor 15 minutes. The reaction was stopped by the addition of 0.8 ml0.5 N sodium hydroxide.Control 1ml buffer 1ml phenyl phosphate substrate and 0.8ml 0.5Nsodium hydroxide were mixed in a test tube. 0.1ml serum wasadded later and it was incubated for 15 minutes at 37°C.Standard 1.1ml buffer and 1ml working phenol standard solutionand 0.8 ml 0.5 N sodium hydroxide were mixed in a testtube.Blank 1.1ml buffer.1ml glass distilled water and 0.8 ml 0.5 Nsodium hydroxide were mixed in a test tube. To all these test tubes 1.2ml of 0.5 N sodium carbonate wasadded followed by 1ml of 4-amino antipyrine and 1ml ofpotassium ferricyanide, mixing thoroughly after each addition.
  • 123. The reddish brown color obtained immediately is read at510 nm avoiding exposure to strong sunlight.Calculations The amount of phenol present in the standard tube is 10μ g.Thus the phenol produced in 15 minutes in the test is, T-C ____ = -------- X μ gm S-B T-CHence 100 ml serum would liberate = X 10 mg phenol ___ S-B_ Since King-Armstrong unit is the production of 1mg ofphenol in 15 minutes, under the conditions of the test. T_- CSerum Alkaline Phosphotase (KA units/100ml = X 10 S-B The values are expressed in SI units [μ kat / L]2.6.13. Estimation serum transaminases Glutamic oxaloacetic transaminase GOT (Aspartate aminotransferase. AST) (EC: 2,6,1,1) and glutamic pyruvictransaminase, GPT Alanine amino transferase. ALT) (EC: 2,6,1,2)levels in serum were estimated by the colourimetric method ofReitman and Frankel as given by Teitz (Norbert W 1970).
  • 124. Principle AST: L-aspartic acid and α Ketoglutaric acid wereincubated with the sample and the rate of formation of oxaloaceticacid was determined directly as the dinitro phenyl hydrazone. Theabsorbance of the dinitro phenyl hydrazone derivatives wasmeasured using a green filter after adding alkali. ALT: L-Alanine and α-Ketoglutaric acid were incubatedwith the sample and the rate of formation of pyruvate wasdetermined directly as the dinitro phenyl hydrazones. Theabsorbance of the dinitro phenyl hydrazone derivatives wasmeasured using a green filter after adding alkali.Reagents:1.Phosphate buffer 23.86 g of Na2HPO4 (disodium 0.1M pH 7.4: hydrogen phosphate) and 4.36 g of KH2PO4 (potassium dihydrogen phosphate) were is dissolved in a glass-distilled water and made upto 2000 ml with the same.2. Pyruvate 2.0m mol/L: 0.110 g of sodium pyruvate was dissolved in 500 ml phosphate buffer. This was used for standard curve and was prepared fresh as needed.
  • 125. 3. AST Substrate: 0.146 g of α-Ketoglutaric acid and 13.3 g DL aspartic acid was taken in a beaker. Small quantities of 1N NaOH were added with stirring until the solution was complete. The pH of this solution was adjusted to 7.4 with 1N NaOH. This was made up to 500 ml with phosphate buffer. This is stable when refrigerated.4. ALT Substrate: 0.149 g of α- Ketoglutaric acid and 8.9 g of DL alanine was taken in a beaker. Small quantities of 0.1N sodium hydroxide solution were added with stirring until the solution was complete. The pH of this was adjusted to 7.4 with 1N NaOH. This was made upto 500 ml with phosphate buffer. This is stable when refrigerated.5. 2.4 Dinitro 0.198 g of 1,4 phenyl hydrazine was Phenyl hydrazine dissolved in a litre of 1N HCl and 1 M mol / L stored in a dark bottle in a fridge.6. Sodium Hydroxide: 0.4N
  • 126. Procedure:1. 1 ml AST / ALT substrate was put into clean dry test tubes and placed in a water bath maintained at 37°C for 10 minutes.2. 0.2 ml serum was added to both AST and ALT tubes and was mixed [Phosphate buffer].3. The tube was incubated at 37°C for 60 minutes for AST and 30 minutes for ALT.4. At the end of this incubation period the tubes were removed from water bath and 1ml 2,4 dinitro phenyl hydrazine reagent was added. The contents of these tubes were mixed well.5.The tubes were kept at room temperature for 20 minutes.6.1 ml of 0.4N sodium hydroxide solution was added to each tube and mixed well by inversion. The tubes were allowed to stand at room temperature for 10 minutes.7.The absorbance was measured against water blank using green filter.8.The units of activity were obtained from respective calibration curves.
  • 127. Calibration (Standard curve)1. Working pyruvate standard solution, AST or ALT substrate and water was mixed in test tubes as follows: - Test Pyruvate Substrate Water AST ALT Tube (ml) AST/ALT (ml) Units Units NO. (ml) 1 0.0 1.0 0.2 0.0 0.0 2 0.10 0.90 0.2 24.0 28.0 3 0.20 0.80 0.2 61.0 57.0 4 0.30 0.70 0.2 114.0 97.0 5 0.40 0.60 0.2 190.0 150.02. 1.0 ml di nitro phenyl hydrazine was added to each tube mixed and allowed to stand at room temperature for 20 minutes. Then 10 ml of 0.4N sodium hydroxide solution was added and mixed by inversion and let to stand for 10 minutes. The absorbance of each tube was measured with green filter against distilled water blank.3. The reading of each plotted against their corresponding AST/ALT units. The points were connected by smooth curves. The AST/ALT values are expressed in SI units (n Kat/L).
  • 128. 2.6.14. Estimation of total sulphydryl groups Total sulphydryl groups of tissues were estimated by thenitroprusside reaction as explained by Colowick and Kaplan(Colowick and Kaplan1957) using cysteine as standard sulphydrylcompound.Reagents1. Hydrochloric acid: 0.033 N2. Sodium hydroxide: 0.033 N3. Sodium chloride: Saturated4. Sodium carbonate: 1.5 M5. Sodium cyanide: 0.067 M6. Sodium nitroprusside: 0.067 M6. Sodium sulphydryl: 10 mg cysteine mono hydrochloride reagent in 100 ml glass distilled water. This contains 0.57µ-mol -SH group per ml.Procedure:1.1:10 homogenate of tissue (liver) was prepared in phosphate buffer of pH 7.4 was employed. 0.2 ml of this homogenate was used for -SH group determination. 0.2 ml aliquot of this in a test tube was mixed with 0.1 ml of 0.033 N hydrochloric acid, 1.2 ml saturated sodium chloride and 0.2 g solid sodium chloride. The tube was allowed to stand for 10 minutes at room temperature.
  • 129. Then 0.1 ml 0.033 N sodium hydroxide was added. The flocculated proteins were removed by centrifugation.2.0.8 ml of clear fluid was taken in a test tube. To this 0.1 ml 0.067M sodium nitroprusside solution and 0.1 ml each of and 0.1 ml each of 1.5 M sodium carbonate and 0.067 M sodium cyanide were added.3.The contents of the tubes mixed well and the Absorbance was taken exactly after 30 seconds using green filter.4.0.2 ml water for blank and 0.2 ml water containing 0.05 ml standard cysteine solution for standard were employed.Calculation Abs. Testµ mol -SH group/0.2g = X 0.056 Abs. StandardHence Abs. Test µ mol -SH group/g = X 2.8 Abs .Std2.6.15. Estimation of Thiobarbituric acid reactive (TBARS) substance Thiobarbituric acid reactive substances (TBARS) wereestimated in tissues by the procedure explained by Nadiger et al(1986).
  • 130. Principle TBARS in trichloroacetic acid homogenates was determinedby reacting them with thiobarbituric acid. The colour developedmeasured at 535 nm against distilled water.Reagents1.TCA 40%: 400 g triachloroacetic acid (40%)was dissolved in glass distilled water and diluted to 1000 ml.2. TCA 5%: 50 g triachloroacetic acid ( 5%) was dissolved in glass distilled water and diluted to 1000 ml. This was preserved in refrigerator.3. TCA 0.67%: 6.7 g triachloroacetic acid (0.67%) was dissolved in glass distilled water and was made up to 1000 ml .Procedure1. 1 g tissue was homogenized with 9 ml 5 % cold trichloroacetic acid for 10 minutes using Potter-Elvehiam type homogeniser fitted with Teflon piston.
  • 131. 2. 1 ml homogenate was taken in a long test tube and was mixed with 1 ml 40 % triachloroacetic acid followed by 2 ml of 0.67% triachloroacetic acid.3. The tube was kept in boiling water bath for 10 minutes.4. 4. After cooling it was centrifuged and the absorbance of the supernatant was measured at 535 nm against glass-distilled water.Calculation Using the extinction coefficient of mol MDA that is equal to1.56 X 10 –5 µ mol MDA was calculated. Abs. Test µ mol MDA = X 40 0.156High lipid high cholesterol diet (HLD)High lipid high cholesterol diet was prepared by mixing whole milkpowder, dalda, and cholesterol in the proportion of 1.0 : 0.5 : 0.1(w/w) with necessary vitamin supplements ; 1.2 mg thiamine, 2mgriboflavin and 3mg nicacin per 100 gm HLD. This was prepared fresheveryday
  • 132. Chapter-3Results [Tables Graphs Figures]
  • 133. 3.1. Optimum effective hypolipidemic dose of garlic extracts (both AEG and HEG). It is evident from the results given in Table-3.1a that 5g / kgbody weight has significant hypolipdemic effect in chronic alcoholfed rats. Similarly 0.2g / kg body weight HEG showed a significanthypolipdemic effect in chronic alcohol fed rats (Ref Table-3.1b). These optimum effective hypolipidemic dosages wereemployed in further studies with either chronic alcohol fed rats orHLD fed rats.3.2. Optimum effective hypolipidemic dose of synthetic disulphide (both DADS and DPDS). As seen from the Table - 3.2a significant hypolipidemiceffects were observed when 100 mg / kg body weight of DADS inchronic alcohol fed rats. Similarly an equivalent dose of DPDS (100 mg / kg bodyweight) showed a significant hypolipidemic effect in chronicalcohol fed rats (Ref Table - 3.2b). These effective hypolipidemic doses of both DADS andDPDS were employed in further studies with either chronicalcohol fed rats or HLD fed rats.
  • 134. 3.3. Hypolipidemic and other relevant biochemical effects in rats fed alcohol chronically along with AEG. The hypolipidemic effect of effective optimum dose of AEGin chronic alcohol fed rats is given in Table 3.3a. It is evident fromthe table that lipid parameters specially TL, TC, TAG, PL, FFA,and EFA are significantly elevated in chronic alcohol fed rats,(control group Group-2) as compared to normal group (Group-1)where as the same parameters are significantly decreased in AEGprotective group (Group-11), as compared to control group(Group-2) It is also seen from Table - 3.3a that plasma uric acid level iselevated where as plasma total proteins, plasma albumin, plasmaglobulin levels and plasma LPL activity are significantly decreasedin Group-2 as compared to Group-1. But plasma total protein,plasma globulin, plasma LPL activity is significantly raised inGroup-11 as compared to Group-2 rats. Where as plasma uric acidlevel is significantly decreased in Group-11 as compared to Group-2. There is no significant change observed in Vit-C levels. As given in Table-3.3b feeding alcohol for 60 days causes asignificant increase in the liver tissue levels of TL, TC, TAG andPL in control group (Group-2) as compared to normal group(Group-1) where as these parameters are significantly lowered inprotective group (Group -11) as compared to control group(Group-2).
  • 135. A significant decrease in total –SH groups is seen in Group-2 as compared to Group-1 (Ref table - 3.3b) where as a significantincrease is observed in Group-11 as compared to Group-2.3.4. Hypolipidemic and other relevant biochemical effects in rats fed alcohol chronically along with HEG. The hypolipidemic effects of optimum dose of HEG inchronic alcohol fed rats is given in Table - 3.4a. It is evident fromthe table that lipid parameters specially TL, TC, TAG, PL, EFA,and FFA are significantly elevated in chronic alcohol fed ratscontrol group (Group-2) as compared to normal group (Group-1)where as the same parameters are significantly decreased in HEGprotective group (Group-12). It is also seen from Table - 3.4a that plasma uric acid level iselevated where as plasma total proteins, plasma albumin, plasmaglobulin levels and plasma LPL activity are significantly decreasedin Group-2 as compared to Group-1. The plasma total protein,plasma globulin, plasma LPL activities are significantly raised inGroup-12 as compared to Group-2. Where as plasma uric acidlevel is significantly decreased in Group-12 as compared to Group-2.There is no significant change observed in Vit-C levels. As given in Table - 3.4b feeding alcohol for 60 days causesa significant increase in the liver tissue levels of TL, TC, TAG andPL in control group (Group-2) as compared to normal group(Group-1) where as these parameters are significantly lowered in
  • 136. protective group (Group-12) as compared to control group (Group-2). A significant decrease in total –SH groups is seen in Group-2 as compared to Group-1 (Ref table - 4.3b) where as a significantincrease is observed in –SH groups in Group-12 as compared toGroup-2.3.5. Hypolipidemic and other relevant biochemical effects in rats fed alcohol chronically along with DADS ( Diallyl Disulphide). The hypolipidemic effect of optimum dose of DADS inchronic alcohol fed rats is given in Table - 3.5a. It is evident fromthe table that lipid parameters specially TL, TC, TAG, PL, EFA,and FFA are significantly elevated in chronic alcohol fed ratscontrol group, (Group-2) as compared to normal group (Group-1)where as the same parameters are significantly decreased in DADSprotective group (Group-23). It is also seen from Table - 3.5a that plasma uric acid level iselevated where as plasma total proteins, plasma albumin, plasmaglobulin levels and plasma LPL activity are significantly decreasedin Group-2 as compared to Group-1. The plasma total protein,plasma globulin, plasma LPL activities are significantly raised inGroup-23 as compared to Group-2. Where as plasma uric acidlevel is significantly decreased in Group-23 as compared to Group-2.There is no significant change observed in Vit-C levels.
  • 137. As given in Table - 3.5b feeding alcohol for 60 days causesa significant increase in the liver tissue levels of TL, TC, TAG andPL in control group (Group-2) as compared to normal group(Group-1) where as these parameters are significantly lowered inprotective group (Group-23) as compared to control group (Group-2). A significant decrease in total -SH groups is seen in Group-2 as compared to Group-1 (Ref table-3.5b) where as a significantincrease is observed in Group-23 as compared to Group-2.3.6. Hypolipidemic and other relevant biochemical effects in rats fed ethanol chronically along with (DPDS) Dipropyl Disulphide The hypolipidemic effects of optimum dose of DPDS inchronic alcohol fed rats is given in Table - 3.6a. It is evident fromthe table that lipid parameters specially TL, TC, TAG, PL, EFA,and FFA are significantly elevated in chronic alcohol fed ratscontrol group, (Group-2) as compared to normal group (Group-1)where as the same parameters are significantly decreased in DPDSprotective group (Group-24). It is also seen from table - 3.6a that plasma uric acid level iselevated where as plasma total proteins, plasma albumin, plasmaglobulin levels and plasma LPL activity are significantly decreasedin Group-2 as compared to Group-1.The plasma total protein,plasma globulin, plasma LPL activities are significantly raised inGroup-24 as compared to Group-2. Where as plasma uric acid
  • 138. level is significantly decreased in Group-24 as compared to Group-2.There is no significant change observed in Vit-C levels. As given in Table - 3.6b feeding alcohol for 60 days causesa significant increase in the liver tissue levels of TL, TC, TAG andPL in control group (Group-2) as compared to normal group(Group-1) where as these parameters are significantly lowered inprotective group (Group-24) as compared to control group (Group-2). A significant decrease in total -SH groups is seen in Group-2 as compared to Group-1 (Ref table - 3.6b) where as a significantincrease is observed in Group-24 as compared to Group-2.3.7. Hypolipidemic and other relevant biochemical effects in rats fed HLD along with AEG. The hypolipidemic effect of optimum dose of AEG in HLDfed rats is given in Table - 3.7a. It is evident from the table thatlipid parameters specially TL, TC, TAG, PL and EFA aresignificantly elevated in HLD fed rats control group (Group-3) ascompared to normal group (Group-1) where as the sameparameters are significantly decreased in AEG protective group(Group-13). While FFA level is decreased in control group(Group-3) as compared to normal group (Group-1) and it isincreased in AEG protective group (Group-13).
  • 139. As seen from Table - 3.7a, no significant change was seen in serum uric acid level, plasma total proteins, plasma albumin, plasma globulin, Vit-C levels and plasma LPL activities. As given in Table – 3.7b feeding HLD for 60 days causes a significant increase in the liver tissue levels of TL, TC, TAG and PL in control group (Group-3) as compared to normal group (Group-1) where as these parameters are significantly lowered in protective group (Group-13) as compared to control group (Group- 3). A significant decrease in total -SH groups is seen in Group- 3 as compared to Group-1 (Ref table – 3.7b) where as a significant increase is observed in Group-13 as compared to Group-3.Hypolipidemic and other relevant biochemical effects in rats fed HLD along with HEG The hypolipidemic effect of optimum dose of HEG in HLD fed rats is given in Table - 3.8a. It is evident from the table that lipid parameters specially TL, TC, TAG, PL and EFA are significantly elevated in HLD fed rats control group (Group-3) as compared to normal group (Group-1) where as the same parameters are significantly decreased in HEG protective group (Group-14). While FFA level is decreased in control group (Group-3) as compared to normal group (Group-1) and it is increased in HEG protective group (Group-14).
  • 140. It is also seen from Table - 3.8a that plasma uric acid level,plasma total proteins, plasma albumin, plasma globulin, Vit-Clevels and plasma LPL activities no significant change is observedin Group-3 as compared to Group-1. Same observation is seen inGroup-14 as compared to Group-3. As given in Table 3.8b feeding HLD for 60 days causes asignificant increase in the liver tissue levels of TL, TC, TAG andPL in control group (Group-3) as compared to normal group(Group-1) where as these parameters are significantly lowered inprotective group (Group -14) as compared to control group(Group-3). A significant decrease in total -SH groups is seen in Group-3 as compared to Group-1 (Ref table - 3.8b) where as a significantincrease is observed in Group-14 as compared to Group-3.3.9. Hypolipidemic and other relevant biochemical effects in rats fed HLD along with Diallyl Disulphide (DADS). The hypolipidemic effect of optimum dose of DADS inHLD fed rats is given in Table - 3.9a. It is evident from the tablethat lipid parameters specially TL, TC, TAG, PL and EFA aresignificantly elevated in HLD fed rats, control group (Group-3) ascompared to normal group (Group-1). While FFA level isdecreased in control group (Group-3) as compared to normal group(Group-1) and it is increased in DADS protective group (Group-25).
  • 141. It is also seen from Table - 3.9a that plasma uric acid,plasma Total proteins, plasma albumin, plasma globulin, Vit-Clevels and plasma LPL activities no significant change is observedin Group-3 as compared to Group-1. Similar results areobservation is seen in Group-25 as compared to Group-3. As given in Table - 3.9b feeding HLD for 60 days causes asignificant increase in the liver tissue levels of TL, TC, TAG andPL in control group (Group-3) as compared to normal group(Group-1) where as these parameters are significantly lowered inprotective group (Group -25) as compared to control group(Group-3). A significant decrease in total -SH groups is seen in Group-3 as compared to Group-1 (Ref table - 3.9b) where as a significantincrease is observed in Group-25 as compared to Group-3.3.10. Hypolipidemic and other relevant biochemical effects in rats fed HLD Dipropyl Disulphide (DPDS). The hypolipidemic effect of optimum dose of DPDS in HLDfed rats is given in Table - 3.10a. It is evident from the table thatlipid parameters specially TL, TC, TAG, PL, and EFA aresignificantly elevated in HLD fed rats (control group, Group-3) ascompared to normal group (Group-1) where as the sameparameters are significantly decreased in DPDS protective group(Group-26) while FFA level is decreased in control group (Group-3) and same is increased in protective group (Group-26).
  • 142. It is also seen from Table - 3.10a that plasma uric acid,plasma total proteins, plasma albumin, plasma globulin, Vit-Clevels and plasma LPL activities no significant change is observedin Group-3 as compared to Group-1. Similar results are seen inGroup-26 as compared to Group-3. As given in Table – 3.10b feeding HLD for 60 days causes asignificant increase in the liver tissue levels of TL, TC, TAG andPL in control group (Group-3) as compared to normal group(Group-1) where as these parameters are significantly lowered inprotective group (Group-26) as compared to control group (Group-3). A significant decrease in total -SH groups is seen in Group-3 as compared to Group-1 (Ref table – 3.10b) where as asignificant increase is observed in Group-26 as compared toGroup-3.3.11. Comparative biochemical toxic effects of garlic as well as DADS and DPDS in chronic alcohol fed rats. The effect of feeding optimum dose of AEG, HEG, DADS,and DPDS on plasma AST, ALT, ALP levels as well as liver tissueTBARS levels is given in Graphs 3.11a, 3.11b, 3.11c and 3.11d. As seen from the graphs, there is a significant raise in thelevels of AST, ALT, and ALP in chronic alcohol fed rats (Group-2) compared to normal rats (Group-1). Feeding effective optimumdose of AEG, HEG or DADS causes a slight modification in these
  • 143. levels as compared to Group-2 showing that they themselves maybe toxic, in spite of being hypolipidemic. The raise with respect toplasma ALT, AST, and ALP are maximum with DADS (Group-23) as compared to both control group (Group-2) and garlic extract(AEG and HEG) fed groups (Group-11, Group-12). This is furtherevidenced by raise in liver TBARS levels (Ref graph-3.11d) andTBARS levels generally indicate the free radical levels. Hence itsuggests that the garlic extracts may be toxic in spite of beinghypolipidemic.3.12. Comparative biochemical toxic effects of garlic extracts, DADS and DPDS in HLD fed rats. It is evident from the graphs (graphs-3.12a, 3.12b, 3.12c and3.12d) that there is a significant raise in plasma AST (Graph-3.12a) in HLD fed rats control group (Group-3) as compared tonormal group (Group-1). There is no variation seen in plasma ALTand ALP levels in HLD fed rats control group (Group-3) ascompared to normal group (Group-1) (Graph-3.12b and 3.12c).There is slight variation seen in plasma AST as well as ALT andALP levels in rats given HLD along with AEG, HEG or DADS,but a little more increase was observed in case of plasma ALP (Refgraphs - 3.12c). This probably may be due to toxic nature of theseextracts as well as DADS. This is further evidenced by steep raisein liver TBARS levels. The raise in liver TBARS level in HLD fedDADS given rats is maximum (Graph-3.12d). A parallel decrease with respect to liver total -SH grouplevel in both chronic alcohol fed rats (control group, Group-2,) as
  • 144. well as HLD fed rats (control group Group-3) is seen whencompared to normal group (Group-1)(Graph-3.11e and3.12e). Thismay be in part due to a raise in liver TBARS levels. (Graphs–3.11dand 3.12d). The total –SH group is increased in protective groups(Group-11, Group-12, Group-13, Group-14, Group-23, Group-24,Group-25 and Group-26) as compared to control groups (Group-2and Group-3) with concomitant decrease in TBARS levels (Graph3.11e and 3.12e).3.13. The biochemical effect of feeding garlic extracts (AEG and HEG),DADS and DPDS alone to normal rats. In order to establish whether the garlic extracts (AEG andHEG), DADS and DPDS alone are toxic, certain experiments wereconducted by feeding effective optimum doses of AEG, HEG,DADS and DPDS to normal rats. The results obtained with respectto plasma AST, ALT, ALP as well as liver tissue TBARS levelsand total –SH groups are given in Table-3.13. As per the Table-3.13 it is evident that, there is increase inAST, ALT, ALP and TBARS levels in normal rats fed garlicextracts (AEG and HEG) and DADS (Group-27, Group-28 andGroup-29) as compared to normal group (Group-1). This rise inAST, ALT, ALP and TBARS levels may be due to the presence ofunsaturated disulphides in garlic extracts as well as DADS. Thesame is not true with DPDS, which is saturated disulphide (Group-30).
  • 145. 3.14. Histomorphological studies. The histomorphological studies of liver of rats given 30 ml30 % (v/v) ethanol daily for 60 days (Group -2) and of rats givenAEG, HEG, DADS and DPDS along with 30 ml 30 % (v/v)ethanol daily for 60 days (Group- 7, 8, 9 and 10 respectively) aregiven in figure (3.14a to 3.14f). The figures (3.15a to 3.15f) givesimilar studies conducted with rats given HLD alone daily for 60days (Group -3) and of rats given AEG, HEG, DADS and DPDSalong with HLD daily for 60 days (Group- 13, 15, 17 and 19respectively). The results indicate micro vescicular fattyinfiltration in liver of rats given alcohol or HLD alone. Where as amild changes observed in rats fed AEG, HEG and DADS alongwith alcohol or HLD but no such changes are seen in the liver ofrats given DPDS along with HLD.
  • 146. Chapter-4Discussion
  • 147. 4.1.Hyperlipidemia and fatty liver in rats fed ethanol for long period. The results given in the Table – 3.3a and 3.3b indicate thatfeeding alcohol to rats for a long period (60days) promotes lipidaccumulation in the serum and liver. It seems that the lipidsaccumulated may be of endogenous origin (Lieber C.S.1973). Asalcohol feeding is known to increase endogenous synthesis oflipids. Alcohol also increases triacylglycerol synthesis probably byincreasing the α-glycerol phosphate concentration through the α-glycerol phosphate dehydrogenase (Lieber C.S.1968). Further thealcohol feeding is known to increase not only the endogenoussynthesis of fatty acid and cholesterol but decreases their oxidation(Lieber C.S.1966). The decreased lipoprotein lipase activity in rats fed, ethanolfor 60days (Table–3.3a) could also contribute to thehyperlipidemia.4.2. Alcohol and liver disease Ethanol and / or its metabolites are hepatotoxins and play amajor role in the pathogenesis of alcoholic liver injury (LieberC.S.1981). The ethanol is metabolized in the liver to acetaldehydeproducing NADH. The acetaldehyde is further oxidized to aceticacid (Lieber C.S.1984). Hence oxidation of ethanol in liver produces large amountof NADH. This decreases the cellular and mitochondrial NAD+
  • 148. and upsets the NAD+/NADH ratio (Lieber C.S.1961). This inhibitsthe activity of NAD+ dependent reactions but accelerates theactivity of NADH dependent reactions. This results in increasedsynthesis of fatty acids and cholesterol as their synthesis demandsNADPH. NADPH level is also likely to increase during alcoholmetabolism.CH3CH2OH CH3CHO CH3COOH NAD + NADH+H + NAD+ NADH+H+ Acetaldehyde which is formed from ethanol oxidationcauses accumulation of triglycerides and cholesterol in the liver byforming schiff’s base with protein in the liver [Giudicelli,Y etal1972]. These schiff’s bases are reduced by NADH to form a stableadducts. These stable adducts in the liver alter the functions of theproteins and probably play a major part in the formation of fattyliver and cirrhosis (Tuma etal 1984). H R-NH2 HCH3CH2OH CH3C=O CH3C=NR CH3CH2-NR NAD+ NADH + H+ Schiff’s base (Stabilised adduct) H-carrier (reducing agent)
  • 149. Ethanol metabolism also reduces the oxidation of both fattyacids and cholesterol (Lieber C.S.1961). Alcoholic liver injury is also related to lipid peroxidaton.The acetaldehyde, which is the metabolite of ethanol, is known tobind glutathione and cysteine and reduce their bioavailability(Lieber C.S.1984). In present series of experiments in the groupfed ethanol for 60 days the liver titrable –SH groups are reducedprobably due to the binding of acetaldehyde. The depression ofliver glutathione is predominant in the mitochondrial compartment(Lieber C.S.1984) and may contribute to the striking functionaland structural damage produced by long-term alcohol consumptionin that organelle (Lieber C.S.etal 1968). Severe glutathionereduction favors lipid peroxidation. Thus the depletion of tissuethiols by ethanol may promote peroxidation especially in humans(Videla A. 1980). The increased TBARS levels in the liver of rats fed ethanolfor 60 days, as seen from the tables (3.3b) may be due to theoxygen derived free radical injury to the liver. The oxygen-derivedfree radicals are known to produce changes in membrane fluidityand membrane permeability that ultimately lead to cell necrosis(Slater T. F. 1984). This action of free radicals is due to oxidationof polyunsaturated fatty acids of cell and plasma membranesleading to the formation of lipid peroxides (Gupta V. K. 1992). Similarly a significant rise in serum AST, ALT and ALPlevel is seen in rats fed ethanol for 60 days (Ref graph 3.11a, 3.11band 3.11c). This is probably due to the free radical injury to the
  • 150. liver inducing the change in fluidity and membrane permeabilitythat could lead to a leakage of these enzymes. Chronic ethanol consumption is known to inhibit proteinbiosynthesis (Prof. Shadaksharaswamy 1982) as ethanol oxidationforms acetaldehyde which reacts with free amino groups ofproteins leading to the extensive formation of schiff’ base. Thisalteration include displacement of pyridoxal from its binding siteon proteins and interference with activity of some proteinsespecially those forming schiff’s base enzyme complexes asintermediate in their catalytic activity. (Lumeng L. 1978 and Grazietal 1963). Thus the accumulation of lipid in the liver, covalent bindingof acetaldehyde to the hepatic proteins and the increase in theoxygen-derived free radicals could be the contributing factors tothe alcoholic liver injury.4.3. Hyperlipidemia and fatty liver in HLD fed rat. Rats fed a high lipid diet (HLD) exhibit markedhyperlipidemia and fatty liver. (Table-3.7a and 3.7b) This is inagreement with earlier reports (Harrision, Ahrens M.A. etal andConner W. E. etal 1980). However the HLD fed rats havesignificantly higher TBARS levels and significantly lower titrable–SH groups in liver. The rise in oxygen-derived free radicals asevidenced by the TBARS level could also partly be responsible forthe fatty liver in HLD fed rats (Lefevre A. etal and Ahrens M.A.etal 1970). The increase in serum AST levels in HLD fed rats
  • 151. demonstrates decreased liver function. The free radical injury tothe liver and / or the reduction in titrable –SH groups could beresponsible for the leakage of this enzyme into the blood. Howeverserum lipoprotein lipase activity was not altered significantly inHLD fed rats. There was no significant change in proteins byfeeding HLD to rats (Ref Table - 3.7a).4.4. Optimum dose of garlic extract and synthetic disulphide. Hypolipidemic effects are reported by feeding garlic extracts(both AEG and HEG) in rats fed ethanol chronically or in rats fedHigh lipid diet (HLD) for a long period. The present series of experiments by feeding garlic extract(both AEG and HEG) and synthetic disulphides (DADS andDPDS) produced significant hypolipidemic effects in rats fedethanol chronically or in rats fed High lipid diet (HLD) for a longperiod. The synthetic disulphide, Diallyl disulphide (DADS) is aaliphatic unsaturated disulphide and Dipropyl disulphide (DPDS)is a aliphatic saturated disulphide. CH2 CH2 CH3 CH3 || || | | CH CH CH2 CH2 | | | | CH2 - S - S - CH2 CH2 - S - S - CH2Diallyl Disulphide (DADS) Dipropyl Disulphide (DPDS)
  • 152. The optimum dose for producing the hypolipidemic effect is5g / kg body weight and 0.2g / kg body weight for aqueous extract(AEG) and hexane ethanol extract (HEG) respectively (Table -3.1a and 3.1b). Similarly the optimum dose for producing hypolipidemic effectfor both DADS and DPDS is 100 mg / kg body weight. (Table -3.2a and 3.2b) Very large doses above 150 mg / Kg body weight DADS andDPDS when fed along with ethanol seems to be too toxic probablydue to synergestic action.4.5. Hypolipidemic effects of AEG and HEG as well as synthetic disulphides like DADS and DPDS. The results shown in the tables 3.3a, 3.3b, 3.4a and 3.4bclearly indicate that, the extracts of garlic, both aqueous andhexane ethanol confirm beneficial effects of garlic (Allium sativumLinn). (Sargeev D.M., Leonou I.D.1958, Chaudray D. M.). Thehypolipidemic effect of garlic is much studied and welldocumented (Augusti K.T. Itaokaw 1973, Sumiyosh H. 1997,Joseph P.K. and Augusti K.T.1982). It is further confirmed that sulphur compounds i.e.sulphoxides and disulphides of garlic are responsible for thishypolipidemic effect. (Augusti K.T.1996).
  • 153. Normally aqueous extract consist of sulphoxides (SheelaC.G. Augusti K.T. 1992), where as ether extracts or oil are rich indisulphides (Mathew P. T. Augusti K. T. 1973). These disulphidescould under go exchange reactions with various –SH groupcompounds in the body.R1-S-S-R1 + 2 R-SH R1-S-S-R2 + R1-S-S-R2 + H2OAllicin Thiols Mixed disulphideR1-S-S-R2 + R3 SH R1- S-S-R3 + R2 SH (Disulphide) R1=C3H5, R2 & R3 = aliphatic chains The –SH group of fatty acid synthetase and HMG CO-Areductase could also under go such exchange reactions with thealiphatic S-S compounds in these extracts resulting in diminishedsynthesis of these lipids.C3H5-S-S-C3H5 + R-SH C3H5-S-S-R + C3H5SH The results shown in the graphs (Ref graphs 3.12a, 3.12b,3.12c and 3.12d) clearly indicate that the undesirable side effectslike increase in serum AST, ALT and ALP levels and increase inthe levels of thiobarbutaric acid reactive substances in liver ismaximum in rats fed garlic extracts (both AEG and HEG) andDADS. The garlic extracts contain allyl disulphides. Thesedisulphide are known to be reduced by NADPH (Black S. 1962).Thus diallyl disulphide could have been metabolized as follows,
  • 154. Thus the metabolism of DADS could produce allylmercaptan. Further, probably by oxidation and desulphurationreaction could produce acrolein from allyl mercaptan. Metabolic pathway of DADS CH2 CH2 || || CH CH | | (Diallyl Disulphide) (DADS) CH2 – S – S – CH2 NADPH + H + NADP+ CH2 = CH – CH2 – SH ( Allyl mercaptan) CH2 = CH – CHO (Acrolein) Thus it can be concluded that hypolipidemic effects of garlicextracts (both AEG and HEG) and DADS may be due to theunsaturated nature of aliphatic disulphide. The toxic effects are dueto the production of acrolein produced from the allyl side chains ofthis aliphatic disulphide.4.6. Protective role of DPDS in ethanol fed rats. DPDS when fed along with alcohol for 60 days seems tocause an over all protective effect. The tissue and serum lipidsshow significant decrease in both the group of rats. (Ref Table -
  • 155. 3.6 and 3.6b). This action of DPDS might be due to its reactionwith NADPH like any other Disulphide (Black S. 1962).R-S-S-R + NADPH +H+ 2RSH Which could be similar to the metabolism of garlic extractand DADS. The metabolism of DPDS could have reduced thehepatic NADPH levels, which would reduce the fatty acid andcholesterol synthesis. Also DPDS could under go exchangereactions with endogenous enzymes and proteins similar to theaction of garlic extract and DADS.R-S-S-R + X-SH R-SH + R-S-S-X Such exchange reactions of DPDS could inhibit fatty acidsynthase and HMG CO-A reductase. DPDS is also aliphaticdisulphide like garlic extract component, DADS but with saturatedgroups so similarly could inhibit fatty acid synthase and HMG CO-A reductase. CH3 CH3 | | (Dipropyl Disulphide) CH2 CH2 (Propyl disulphide) | | CH2 – S – S – CH2 (DPDS) Feeding DPDS significantly increases the alcohol inducedinhibition of lipoprotein lipase activity. Thus reduction in Triacylglycerol, Cholesterol and Total lipids in serum by DPDS in ethanolfed rats could at least partly be due to this effect.
  • 156. The high levels of FFA seen in rats fed ethanol fed rats werebrought to very near the normal levels by DPDS feeding (RefTable-3.6a). Thus DPDS could protect against hyperketonemia andketonuria due to heavy alcohol consumption. ( Lefevre A. etal1970, Lefevre and Lieber C. S. 1967). Feeding DPDS along with ethanol for 60 days considerablyreduces the TBARS levels in the liver. The titrable –SH groups ofliver also did not improve significantly with DPDS feeding. Theserum levels of AST, ALT and ALP in rats fed ethanol along with,were considerably lower than those of rats fed ethanol alone(Graph – 3.11a, 3.11b and 3.11c) but they were still above thelevels seen in normal control rats. Thus levels of these serumenzymes parallel the liver TBARS level. The low levels of total serum proteins and albumin seen inrats fed ethanol for 60 days showed considerable improvement byfeeding DPDS. Probably the DPDS could counteract at least partlythe alcohol induced reduction in protein biosynthesis. Thus DPDS feeding can provide considerable attenuationagainst the deleterious effects of alcohol.4.7. Hypolipidemic effects of DPDS in HLD fed rats. There is a general reduction in all the lipid levels in theserum and liver by feeding DPDS to HLD fed rats except theserum total lipids. The probable mechanism of the action of the
  • 157. DPDS may be similar to the actions of garlic oil and DADS.Metabolism of DPDS utilizes the NADPH, which could have ageneral hyperlipidemic effect. The exchange reaction of DPDSwhich is an saturated aliphatic disulphide with –SH groups ofvarious enzymes and proteins like fatty acid synthetase and HMGCO-A reductase may also contribute to the hypolipidemic effects. It is interesting to note that the FFA levels which weresignificantly low in HLD fed rats is increased by feeding DPDS.Feeding DPDS significantly increases the serum lipoprotein lipasethere by contributing to the hypolipidemic effects of thiscompound. The serum levels of AST that was high in HLD fed rats issignificantly reduced by DPDS feeding (Ref3.12a). The high levelof TBARS and low level –SH groups in the liver are brought toalmost normal levels by feeding DPDS. Thus DPDS can maintainthe membrane permeability and tissue integrity of hepatic cells inHLD fed rats, which is exhibited by reduction of serum AST levelsand reduction of TBARS levels in the liver due to DPDS (Ref3.12d and 3.12e). There is no significant change in plasma proteins, serumALT and ALP by feeding DPDS along with HLD (Ref table-3.10a,3.12b and3.12c). This protective effect of DPDS is probably due to the saturatednature of DPDS, which is metabolized as follows without
  • 158. producing any toxic compound unlike DADS, which gives toxiccompound acrolein. CH3 CH3 | | (Dipropyl Disulphide) (DPDS) CH2 CH2 (Propyl disulphide) | | CH2 - S-S - CH2 NADPH + H + NADP+ CH3 - CH2 - CH2- SH ( Propyl mercaptan) CH3 - CH2 - COOH (Propionic acid)4.8. DPDS is safer and acceptable There was no mortality when rats were fed DPDS up to 500mg /kg body weight. Feeding the optimum dose of DPDS to fastednormal rats did not increase TBARS levels in the liver or AST,ALT, and ALP levels in the serum. Feeding similar dose of DPDSto ethanol fed rats or rats fed HLD also did not increase any of theabove parameters. The results of the experiment show significantincrease in all the above parameters in garlic fed rats.
  • 159. However DPDS improves the titrable -SH groups of liver inrats fed HLD or ethanol. The TBRS levels that were high in ratsfed HLD or ethanol were also brought down by DPDS(Ref table3.11d and3.12d). Thus DPDS feeding has a general oxygen freeradical scavenging action, but in normal rats DPDS feeding doesnot reduce the titrable -SH groups or increase TBARS levels. A single dose of DPDS reduced the serum cholesterol,serum triacyl glycerols, serum FFA and liver total cholesterollevels in fasted normal rats. However the other lipid levels in theserum or liver are unaffected. Thus DPDS is safe since, its hypolipidemic effects areconsiderable in conditions like ethanol feeding or HLD feeding,but in normal rats its hypolipidemic effects are very moderate. There are no adverse histomorphological changes by feedingDPDS for 60 days along with ethanol or along with HLD (fig3.11a - 3.11e and fig. 3.12a - 3.12e). Such rats actually showedbetter preservation of tissue integrity. DPDS is water-soluble compound and is more suitable forfeeding experiments in animals compared to garlic oil. It is almostodourless and tasteless in aqueous solution of 100 ppm. A 10% solution of this compound in water has a very weaksmell and taste of garlic. DPDS is soluble in water at lowerconcentration but at higher concentration it forms a stableemulsion in water where as garlic oil is not water soluble. Thus
  • 160. DPDS may be more acceptable for human use by way of taste,smell and water solubility. However DPDS is not acceptable forhuman use in the present situation, since its effectiveness andsafety in humans is yet to be established. When a large dose of ethanol was fed to rats mixed withtwice the optimum dose of DPDS many rats died. Thus a very highdose of DPDS could potentiate the ethanol toxicity in rats.
  • 161. Chapter-5Conclusions
  • 162. ConclusionsHence it is concluded that,1.Garlic extracts both AEG and HEG possess hypolipidemic action in both alcohol fed and HLD fed rats.2.This hypolipidemic action of garlic extracts may be due to their principle organo sulphur compound DADS.3.The garlic extracts as well as DADS are toxic as seen by maximum increase in serum AST, serum ALT, and serum ALP and liver TBARS levels.4.The toxicity of garlic extracts may be due to unsaturated nature of DADS, which on metabolism give a toxic compound, Acrolein.5. DADS is comparatively more toxic than garlic as the increase in serum AST, ALT and ALP is more.6.The DPDS, which is saturated disulphide, is equally hypolipidemic compared to garlic extracts and DADS.7.DPDS is effective in both alcoholic and HLD fed rats at an optimum dose of 100 mg / kg body weight.8.DPDS is well tolerated, safer and non toxic at the dosage employed in the present studies
  • 163. Chapter-6Summary
  • 164. IntroductionPlants and their medicinal uses Plants have been the major source of drugs in the Indiansystem of medicine. The earliest reference of the medicinal plantsis found in Rig Veda (3500-1800 B.C). The important works ofmedicine of later period (1000-400 B.C) namely Charaka samhitaand Susruta samhita also gives extensive description of variousmedicinal herbs. Apart from the written records, some knowledgeon the subject of medicinal plants has descended through time.Many other Asian and African countries have also reported themedicinal values of the plants and their extracts. Many medicinal plants, when subjected to scientificexperiments have yielded useful drugs, which could be taken upby the modern system of medicine. One such plant is Ammivisanga. Decoction of the dried seeds of this plant is used as adiuretic and as an antispasmodic in renal colic in Mediterraneancountries and America. Investigation on this plant showed theactive constituent to be ‘Khellin’ which was found to be effectivevasodilator with a selective action on coronary arteries (AnrepG.V.etal 1946). The scattered information on the medicinal plantsin India has been systematically organized by Kirtikar and Basu in1933 (Kirtikar K. H etal 1933) and later by Chopra in 1956(Chopra R. N. etal 1956). These works cover an extensive list ofmedicinal plants available in various parts of the country withtheir reported medicinal values. Scientific studies and clinical
  • 165. trials have confirmed the medicinal properties of many of theseplants. The Central Drug Research Institute Lucknow, India hasscreened around three thousand plant materials for a wide varietyof chemotherapeutic and pharmacological activities of whichmany have been confirmed (Dhar M. L. etal 1973, Bhakuni D. S.etal 1973, Dhawan B. N. 1977, Aswal B. S. 1984).Hypolipidemic effects of plants and their extracts Among the various biological activities of the medicinalplants, the hypolipidemic activity has been the most commonlystudied one. Extracts of various plants have been shown toproduce hypolipidemia in normal experimental animals. Some ofthe commonly studied plants are Allium sativum, Allium cepa,Momordica charantia, Ficus bengalensis, Coccinia indica etc.Active principles have been isolated from some of these plants. The plants belonging to the genus Allium group have beenextensively used as hypolipidemic agents in medicine forcenturies. Studies of the products and essential oils of garlic andonion for their physiological and therapeutic effects have beenconducted since early part of this century probably even before.But these studies were necessarily limited by the lack ofknowledge about the nature and interrelationship of the chemicalcomponents in the fresh tissue processed product or essential oils.The studies of Cavallito and Coworker (Cavallito etal 1985) andStoll and Seeback (Cavallito etal 1985) may thus be seen as
  • 166. crucial in the development of chemical and biochemical basis oftherapeutic studies.Garlic: Chemistry and uses Garlic is used widely in food and pharmaceuticalpreparations in India. It is a member of allium species and itsbotanical name is Allium sativum Lin. The botanical word alliumis derived from the celitic word, all means pungent and it betraysthe presence of a host of remarkable flavorants and odorants all ofthem having in common, one element, sulphur. Garlic has beenused for its therapeutic effects such as antidiabetic, antioxidant,antiatherogenic, anticancer as well as fibrinolytic action forcenturies. Biological actions of allium products are ascribed toorganosulphur compounds having allyl (CH2=CH-CH2) or itsisomer propenyl group (CH3-CH=CH-) (Itokayway etal 1973, J. L.Brewster etal 1990). Raw garlic contains 0.4 % by weight of“alliin” which is S-allyl cysteine sulphoxide (alliin). Allinase isthe enzyme which catalyses the conversion of S-allyl cysteinesulphoxide to allyl sulphinic acid which spontaneously changes todiallyl thiosulphinate which on warming / heating becomes DiallylDisulphide (DADS). Allicin inhibits nearly all sulphydryl enzymes but very fewnon sulphhydryl enzymes which are associated with the presenceof (-S-O-S-) group and not (-SO-), (-S-S-) or-S- groups. Such anenzyme inhibition by allicin was prevented by reducing agentslike cysteine or glutathione (Wills E. D. 1956).
  • 167. Following pathway shows the reactions involved in theformation of DADS. CH2=CH-CH2-S-CH2-CH-COOH | O NH2 Allyl-L-Cysteinyl Sulphoxide (Alliin) CH3COCOOH (Pyruvic acid) Allinase NH3 CH2=CH-CH2-SH O Allyl sulphinic acid (2molecules) 2H2O CH2=CH-CH2-S-S-CH2-CH=CH2 O Diallyl thio sulphinate (allicin) Warming / Distillation CH2 CH2 || || CH CH | | CH2 – S – S – CH2 Diallyl Disulphide (DADS)
  • 168. Hypolipidemic effects of garlic Augusti and Mathew (Augusti etal 1973) first reported thatan aqueous extract of garlic has hypolipidemic action on normalrats. Later it was showed that allicin also reduced serum and tissuecholesterol and triglycerides (Augusti etal 1974). Mirhadi and others (Mirhadi etal 1991) reported thatsupplementation of garlic to rabbits fed cholesterol rich dietsuppressed the increased levels of cholesterol in plasma, aorta andliver. Total lipids, phospholipids and free fatty acids in aorta andliver. Gebhardt (Gebhardt etal 1991) found that culture of rathepatocytes on incubation with water soluble extracts of garlicpowder diminished cholesterol biosynthesis and its export in to themedium. Pure alliin alone or after incubation with alliinase (thatproduces allicin) in concentration corresponding to its content inthe extracts does not exert any inhibition. HMG COA reductaseactivity is significantly inhibited by garlic extracts (DiallylDisulphide (DADS) a component of garlic extract). Fatty acidsynthetase is the only enzyme, inhibited by alliin even at higherconcentrations. Thus alliin does not seem to be of majorsignificance in the cholesterol biosynthesis (Kumar etal 1991). Bordia and Verma (Bordia etal 1978, 1980) reported thatsupplementation of garlic to rabbits fed with a hypercholesteremicdiet showed significant decreases in LDL and VLDL andsignificant increase in HDL levels. In another report by Lau etal(Lau etal, 1983) an increased HDL levels was demonstrated in rats
  • 169. fed freeze-dried garlic powder making up 2% of an atherogenicdiet. Jain (Jain 1978) compared the effects of garlic and onion inrabbits fed a diet containing cholesterol at 0.5 % g / day. Thegroup supplemented with garlic juice equivalent to 0.25 g of garlic⁄ day showed approximately 20 % of the cholesterol level of thecontrol group after 16 weeks. Using garlic oil, Jain and Komar (Jain etal 1978) showedthat the cholesterol lowering effect was dose tested. Rabbits werefed with a diet of 2g⁄day of cholesterol for 16 weeks.Administration of garlic oil at the dose of 0.25, 0.5 and 1g ⁄ dayresulted in 6.2 %, 21 % and 30 % reduction in serum cholesterol. According to Benjamin Lau (Lau B. H. S. etal 1987) kyolic,an odour modified garlic product when given to patients havingelevated cholesterol level (220-440mg⁄dl) increased the cholesterollevel and triglycerides level for the first two months, from thethird month onwards a significant drop in serum lipids began, bysix months, normal levels of lipids reached by 65 % of thesubjects. The initial rise in serum lipids may be due to the shift ofthe lipid deposits from the tissues in to the blood stream (Changetal 1980, Bordia A. 1981, Chi M. S. 1982, Jain R. C. 1975,Nakumura H. etal 1971, Krichevsky D. etal 1980). On continued garlic consumption the excess lipids werebroken down and finally excreted from the body. Similarly aninitial rise occurred with LDL ⁄ VLDL before significant reduction
  • 170. followed. HDL steadily rose after the first month. According toKrichevsky (Kritohevsky etal 1991) garlic can lower serum lipidlevels in rats and significantly reduce the severity of cholesterol-induced atherosclerosis in rabbits. Qureshi etal (Qureshi A. A. etal1983) reported that the odorless water soluble component of garlicwas equally effective as garlic in lowering blood cholesterol andtriglycerides levels. Shoetan et al (Shoetan A. etal 1984) reported that whenalcohol mixed with garlic oil was fed to rats on a high fat diet noincrease of tissue lipids was observed. Several animal studies have demonstrated that componentsof garlic inhibit synthesis by liver cells (Kritchevsky D. etal 1980,Kritchevsky etal 1991, Qureshi A. A. etal 1983). On feedinggarlic to rats decreased the activity of several important enzymesinvolved in the synthesis of lipids not only in the liver but also inadipose tissues such as fat pads (Chang etal 1980).Toxic effects of garlic: The overuse and misuse of either oils or aqueous extracts ofgarlic often produces certain undesirable effects. As compared tothe studies conducted on the benefits of garlic, there are relativelyfew studies on the toxicity of these vegetables. The studies ofDr.Osamu Imada (Osamu Imada etal 1990) indicated that the orlaLD (50) values (mg/kg body weight) in mices for various garliccomponents were as follows.
  • 171. Garlic components LD (50) Values (mg/Kg Body weight) Males FemalesAllicin 306 363Diallyl disulphide 145 130S- ally mercapto cysteine 600 922Diallyl sulphide 2029 1089S-allyl 8890 9390 Further the same authors (Osamu Imada etal 1990) showedthat raw garlic causes extensive edema, bleeding and ulceration offore-stomach mucosa, reduction of red blood cell count andhematocrit and increase of reticulocytes and anemia (Joseph, P.K.etal 1989). The administration of Diallyl disulphide and S-allyl-L-cysteine at sub acute levels caused hematological disorders,increased serum transaminases and alkaline phsophotase andblood urea levels. It was observed when 100mg/Kg body weightgarlic oil fed to 24hours fasted rats, the rats died of pulmonaryoedema (Joseph, P. K. etal 1989). Further studies conducted by Kashinath (Kashinath R. T.1993 established that the toxic effects of garlic may be due to theproduction of acrolein, a metabolite of DADS which is a toxicproduct (Reynold E. F. 1993).
  • 172. CH2 CH2 || || CH CH | | (Diallyl Disulphide) (DADS) CH2 – S – S – CH2 NADPH + H + NADP+ CH2 = CH – CH2 – SH ( Allyl mercaptan) CH2 = CH – CHO (Acrolein) The acrolein probably triggers the production of freeradicals and causes damage to the tissues.Ethanol and its metabolic effects: Ethanol or ethyl alcohol (C2H5OH) generally known asalcohol is commonly used organic solvent in the laboratory.Although a trace amount of ethanol can be synthesizedendogenously (McManus etal 1966,Iwata K. 1972) ethanol isprimarily exogenous compound, which can be readily absorbedfrom gastro intestinal tract [GIT]. Only 2-10 % of the absorbeddose can be eliminated through the kidneys and lungs, the restmust be oxidized in the body and most of this occurs in the liver.The liver is the only organ capable of metabolizing significantamount of ethanol and magnitude of gastrointestinal ethanolmetabolism has been reported to be negligible. Since liver is themajor organ of ethanol induced toxicity many of the derangements
  • 173. of hepatic functions have been attributed to the products of ethanoloxidation rather than to ethanol perse. (Liber C. S. 1984, OrregoH. 1981, Sorrel M. E. etal 1979). Although ethanol perse has been shown to alter membranefluidity and in this case impaired function in a variety of organs,the susceptibility of the liver to the toxic action of ethanolindicates that metabolism of ethanol in this organ likely plays akey role in alcoholic liver injury (Rubin E. etal 1982). Despite the vast number of studies reported concerning theeffects of ethanol on liver, the mechanism by which ethanolexhibits its hepato toxic effects is still an enigma. The followingmechanisms are proposed to explain the pathogenesis of alcoholicliver injury. Most of the absorbed ethanol is degraded by oxidativeprocess primarily in the hepatocytes first to acetaldehyde and thento acetate.CH3CH2OH CH3CHO CH3COOH Ethanol Acetaldehyde Acetate The acetaldehyde formed, covalently binds to hepatocellularmacromolecules especially to proteins and there by altershepatocellular structure and function ultimately resulting in theliver injury (Liber C. S. 1984, Orrego H. 1981). The hepatocytes contain three main pathways for themetabolism of ethanol, each located in a different sub cellularcompartment. They are,
  • 174. 1.Alcohol dehydrogenase pathway of the cytosol or soluble fraction of the cell.2.Microsomal ethanol oxidizing system (MEOS) located in the endoplasmic reticulum, and3.Catalase degradative route located in peroxisomes. In the first step, ethanol is oxidized to acetaldehyde in theliver mainly by the enzyme, alcohol dehydrogenase (ADH).Catalase and mixed function dehydrogenase - microsomal ethanoloxidizing system, MEOS, which accounts for the 25% ethanoloxidation (Racker etal 1949). ADH is an NAD+ dependent enzyme of the cell sap(cytosol) with an optimum pH of 10-11 and catalyses theconversion of ethanol to acetaldehyde. In ADH mediated oxidationof ethanol, hydrogen is transformed from substrate to cofactorNicotinamide Adenine Dinucleotide [NAD+] resulting in theconversion of its reduced form NADH. AlcoholCH3CH2OH + NAD+ CH3CHO +NADH +H+ dehydrogenase This results in the generation of reducing equivalent in thecytosol in the form of NADH and H+ with concomitant depletionof NAD+.
  • 175. The altered redox state, in turn is responsible for a variety ofmetabolic abnormalities. Enzyme activities related to ethanol oxidation and drugmetabolism are frequently altered, these changes may be producedeither directly by enzyme induction or suppression or indirectly bythe shift in reducing equivalents NAD+ / NADH.Biochemical and metabolic effects of ethanol: The oxidation of ethanol results in the transfer of hydrogento NAD+, which is reduced to NADH. This excess reducingequivalent of NADH reflects itself in red ox potenicial of thecytosol of some metabolites such as pyruvate and lactate, asmeasured by changes in the lactate and pyruvate ratio. The red ox changes associated with oxidation of ethanolresults in increased lactate level resulting from either decreasedhepatic utilization of lactate derived from extra hepatic tissues ordepending on the metabolic state of liver increased hepatic lactateproduction (Krebs 1967). As consequences lactate level rises inblood resulting in hyperlacticidemia and lactic acidosis[Daughaday W. H etal 1962). In addition to acidosis the hyper lactiacdemia also hasclinically significant effects on uric acid metabolism. The rise inblood lactate decreases urinary uric acid out put, which leads to anincrease in uric acid level, i.e. hyperuricemia (Lieber etal 1962).Various hepatotoxic agents result in an increased breakdown of
  • 176. liver nucleioproteins and enhance the release of uric acid into the blood. Both hyper uricemia and hyper lactacidemia play a role in the aggravation or precipitation of gouty attacks traditionally associated with alcoholism. Aims and objectives of the study Garlic, a member of Allium species, is used in food and pharmaceuticals in India as well as in other parts of the world. It is claimed that garlic has antidiabetic, antioxidant, antiatherogenic, anticancer and fibrinolytic effects. These beneficial effects of garlic may be due to its organosulphur compounds, which consist of either allyl or propenyl groups. The predominant sulphur compound in garlic is Diallyl Disulphide (DADS). Consumption of garlic and its extracts, as stated in earlier part of this thesis, have certain biochemical toxic effects like increase in transaminases, increase in urea, creatinine as well as increase in tissue thiobarbutaric acid reactive substances (TBARS). Hence it is necessary to have much care while using or consuming garlic and its products. In accordance with the information given so far in the present thesis regarding the effects of garlic and its components, the present work is under taken to study the effects of garlic extracts as well as synthetic disulphide specifically pertaining to,1.Anti cirrhotic effects of garlic extracts (both aqueous i.e. AEG and Hexane methanol extract i.e. HEG) in chronic ethanol fed rats.
  • 177. 2.Antihyperlipidemic effects of garlic extracts (both aqueous i.e. AEG and Hexane methanol extract i.e. HEG) in high lipid diet (HLD) fed rats. 3.Anti cirrhotic effects of Diallyl disulphide (DADS) and Dipropyl disulphide (DPDS) in chronic ethanol fed rats. 4.Antihyperlipidemic effects of Diallyl disulphide (DADS) and Dipropyl disulphide in HLD fed rats. Materials and Methods: Preparation of garlic extracts: Aqueous extract of garlic (AEG): One-part of fresh garlic bulbs were crushed with three parts of water (w/w) in a waring blender. It is filtered through a gauge cloth one ml of this filtrate was considered equivalent to an aqueous extract of 250 mg garlic. This was prepared fresh each time. Hexane methanol extract of garlic (HEG): One part of fresh garlic bulbs was homogenized in enough (w/w) methanol. It was filtered through gauge cloth. The filtrate was collected separately. The garlic pulp was soaked in enough methanol for a day. Again it was filtered and filtrates collected in the same container. This extraction was repeated for three days
  • 178. and the filtrates were collected. All the filtrate was combined andmethanol was distilled off from the extract. Methanol free extractwas treated with diethyl ether and ether soluble fraction wasseparated in a separating funnel. This extraction was repeatedthree times. The entire ether fraction was combined and ether wasdistilled off. The left over oil was treated with aqueous methanoland was stored at 25°C for four days. It was treated with hexaneand hexane layer was separated from aqueous layer. This was alsorepeated for 3 times. This was reduced to 1/3 by evaporatinghexane at 45-60°C and the left over oil was and stored inrefrigerator for feeding. This contains Diallyl disulphide, Diallyltrisulphide, Diallyl tetra sulphide, Allicin, Dithiens. The maincomponent is Diallyl disulphide.Experimental animals Healthy adult male albino rats of 3 to 4 months old andweighing 100-150 g were selected from the stock of colonies ofthe animal house of Biochemistry Department, Dr. B.R. AMC,Bangalore were used for experiments. They were divided into 30groups of six rats each. They were given normal stock lab dietadlibitum until and unless specified.To establish the effective optimum hypolipidemic dose of AEG as well asHEG Group-1: Normal rats fed stock lab diet and were given intragastrically using stomach
  • 179. tube, 30ml normal saline per kg body weight every day for 60 days. Water was given adlibitum.Group-2: Normal rats fed stock lab diet and were given intragastrically using stomach tube 30ml 30 % alcohol per kg body weight every day for 60 days. And water was replaced by 10 % alcohol and was given adlibitum. Group -3: Normal rats were given high lipid diet (HLD) adlibitum for 60 days. Water was given adlibitum.Group-4 to Group-7: Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with different doses of AEG - 1g, 3g, 5g, 10g per kg body weight respectively everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.Group-8 to Group-10: Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with different doses of HEG - 0.1g, 0.2g, 0.3g in 30 ml warm
  • 180. water per kg body weight respectively everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.To study the hypolipidemic and other relevant biochemical effectsincluding toxic effects of optimum dose of AEG as well as HEG inchronic alcohol fed ratsGroup-1, Group-2 rats served as normal group and control group.Group-11: Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with 5g of AEG per kg body weight everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.Group-12: Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with 0.2g of HEG in warm water per kg body weight everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.
  • 181. To study the hypolipidemi and other relevant biochemical effectsincluding toxic effects of optimum dose of AEG and HEG in high lipiddiet (HLD) fed ratsGroup-1 and Group-3 rats served as normal and control group.Group-13: Normal rats were given high lipid diet (HLD) adlibitum and were given intragastrically using stomach tube 3g of AEG per kg body weight, everyday for 60 days and water was given adlibitum.Group-14: Normal rats were given high lipid diet (HLD) adlibitum and were given intragastrilcally using stomach tube 0.2g of HEG in 30 ml warm water per kg body weight everyday for 60 days. Water was given adlibitum.To establish the effective optimum hypolipidemic dose of Diallyldisulphide (DADS) as well as Dipropyl disulphide (DPDS)Group-1 and Group-2 rats served as normal and control group.Group-15 to Group-18: Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with different doses of DADS-50mg, 100mg, 150 mg and 200
  • 182. mg per kg body weight everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.Group-19 to Group 22: Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with different doses of DPDS 50mg, 100mg, 150mg and 200mg per kg body weight everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.To study the hypolipidemic and other relevant biochemical effectsincluding Toxic effects of optimum dose of DADS and DPDS in chronicalcohol fed rats:Group-1 and Group-2 rats served as normal and control group.Group-23: Normal rats fed stock lab diet and were given intragastrically using stomach tube 30 ml 30% alcohol per kg body weight, along with 100mg DADS in 30 ml normal saline everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.Group-24: Normal rats fed stock lab diet and were given intragastrically using stomach
  • 183. tube 30 ml 30% alcohol per kg body weight, along with 100mg DPDS in 30 ml normal saline everyday for 60 days and water was replaced by 10% alcohol and was given adlibitum.To study the hypolipidemic and other relevant biochemical effectsincluding Toxic effects of optimum dose of DADS and DPDS in HLD fedratsGroup-1 and Group-3 rats served as normal and control group.Group-25: Normal rats were given high lipid diet (HLD) adlibitum and were given, intragastrically using stomach tube 100mg DADS in 30 ml normal saline per kg body weight, everyday for 60 days and water was given adlibitum.Group-26: Normal rats were given high lipid diet (HLD) adlibitum and were given intragastrilcally using stomach tube 100mg DPDS in 30ml normal saline per kg body weight every day for days and water was given adlibitum.To study the biochemical effects of feeding garlic extracts (AEG andHEG), DADS and DPDS alone to normal rats Group-1 rats served as normal.
  • 184. Group-27: Normal rats fed stock lab diet and were given intragastrically using stomach tube, 3g of AEG per kg body weight in 30ml normal saline everday for 60 days. Water was given adlibitum.Group-28: Normal rats fed stock lab diet and were given intragastrically using stomach tube 0.2g HEG in 30ml normal saline per kg body weight every day for 60 day. Water was given adlibitum.Group-29: Normal rats fed stock lab diet and were given intragastrically using stomach tube 100mg DADS in 30ml normal saline per kg body weight every day for 60 days. Water was given adlibitum.Group-30: Normal rats fed stock lab diet and were given intragastrically using stomach tube 100mg DPDS in 30ml normal saline per kg body weight every day for 60 days. Water was given adlibitum.
  • 185. After the feeding period of 60 days the rats of Group-1 toGroup-30 were anesthetized and sacrificed. Blood was collected inheparinised beakers. The liver tissue was separated and put intopre weighed clean sterile beakers. Blood samples were used for estimation of Total lipid (TC),Tri Glyceride (TAG), Phospholipid (PL), Free fatty acids (FFA),Estresified fatty and (EFA), Total protein (TP) Albumin (Alb)Uric acid (UA), vit.C as well as Aspartate transaminase (AST),Alanine transaminase(ALT), Lipoprotein lipase (LPL) andalkaline phosphatase (ALP). A part of liver tissue was homogenized with chloroformmethanol (1:1 v/v) and extracts were used for TC, TL, TAG andPL. Another part of liver was homogenized with 10% TCA andextracts were used for thiobarbutaric acid reactive substance(TBARS) analysis. Third part of the liver was homogenized with phosphatebuffer (PH 7.4) and extracts were used to estimate total -SH group. Fourth part of the liver was immediately fixed in bufferedformalin and preserved for histological studies.
  • 186. Results and Discussion The results of the experiments of feeding both AEG andHEG extracts of garlic in chronic alcohol fed rats shows that garlicextracts exhibit sufficient hypolipidemic effects in these rats.These effects may be due to the principle garlic organo sulphurcompound DADS. This action of DADS may be due toNADH/NADPH lowering effects as the DADS possibly mayutilize NADH/NADPH for its catabolism. Similar hypolipidemic results were observed when DADS isdirectly employed.Biochemical toxicity of garlic extracts and DADS The results of the various other experiments conducteddemonstrate that feeding garlic extracts or DADS induces a raisein plasma transaminases as well as liver TBARS levels with aconcomitant increase in tissue total –SH groups. This is possiblymay be due to the production of free radicals, as the possibleproduct of DADS is Acrolein that is, a toxic substance. Similar findings were seen in experiments conducted withHLD fed rats. Feeding HLD induces nutritional hyperlipidemia aswell as fatty liver. Both garlic extracts and DADS significantlyinhibit this. However similar biochemical toxic effects were alsoobserved in HLD fed rats.
  • 187. The hypolipidemic action of garlic extracts and DADS inchronic alcohol fed rats and HLD fed rats is due to the decrease inNADH/NADPH level as DADS is catabolised probably usingNADH /NADPH. Hence it may be generally concluded that the hypolipidemicaction of both garlic extracts and DADS is due to its NADH/NADPH lowering effect where as biochemical toxic effect maybe due to the production of Acrolein.Lipotropic and anti cirrhotic effects of DPDS Thus in order to retain the beneficial actions of garlicdisulphide (DADS) but devoid of toxic effects a saturateddisulphide Dipropyl disulphide (Propyl disulphide-DPDS) hasbeen employed. The results of feeding 100 mg/kg body weightshows a significant hypolipidemic effect both in chronic alcoholas well as HLD fed rats. This may be possible due to reduction ofNADH / NADPH levels, (Adamu I. Joseph P. K. Augusti K. T.1982, Black S. 1962) as DPDS may utilize NADH/NADPH for itscatabolism similar to any other disulphide. DPDS being a saturated disulphide may under go sulphhydryl exchange reactions with tissue proteins and enzymessimilar to any other disulphide (Adamu I. etal 1984)R1-S-S-R1 + R2SH ---------------- R1-S-S-R2 + R1SH
  • 188. Such a possible sulphhydryl exchange reaction with HMGCO-A reductase, fatty acid synthase, Glycerol-3 phosphatedehydrogenase etc may slow down or inhibit these enzymes thereby decreasing the synthesis of fat and cholesterol. It is known thatAllicin and probably DPDS may under go such exchange reactionwith HMG CO-A reductase. CH3 CH3 | | (Dipropyl Disulphide) (DPDS) CH2 CH2 (Propyl disulphide) | | CH2 - S-S - CH2 NADPH + H + NADP+ CH3 - CH2 - CH2- SH ( Propyl mercaptan) CH3 - CH2 - COOH (Propionic acid)DPDS is more safer:Feeding DPDS either in chronic alcohol fed rats or HLD fed ratsdoes not cause any significant raise in plasma transaminases andliver TBARS levels as well as tends to maintain –SH groups ascompared to either garlic extracts given or DPDS given group.This may be due to the saturated nature of DPDS and themetabolic product is propionic acid, which is non toxic.
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  • 219. Fig 3.11. Normal group ; Photomicrograph with normal hepatrocytes. (HE x 400)Fig 3.11a Alcohol fed control group Photomicrograph of alcoholic cirrhosis showing fibrous septa. (HE x 400)
  • 220. Fig 3.11.b. Alcohol + AEG fed – Photo Micro Graph with mild focal Microvascular Fatty Change. (HE x 400)Fig 3.11.c. Alcohol + HEG fed – Photo Micro Graph with diffuse moderate Fatty Change. HE x 400
  • 221. Fig 3.11.d Alcohol + DADS fed – Photo Micro Graph with focal Micro vesicular Fatty Change, otherwise hepatocytes are normal. (HE x 400)Fig 3.11.e Alcohol + DPDS fed – Photo Micro Graph with normal structure of hepatocytes. (HE x 400)
  • 222. Fig 3.12. HLD fed Control group photo micrograph showing fatty changes with lipogranuloma formation (HE x 400) Fig 3.12.a. HLD + AEG fed – Photo Micro Graph with microvescicular fat vacuoles. (H x 400)
  • 223. Fig 3.12.b. HLD + HEG fed – Photo Micro Graph with Microvescicular fatty change. (HE x 400)Fig 3.12.c. HLD + DADS fed – Photo Micro Graph with diffuse hydropic change. (HE x 400)
  • 224. Fig 3.12.d. HLD + DPDS fed – Photo Micro Graph with normal hepatocytes (HE x 400)