Among the various potential therapeutic applications of garlic, its antilipidemic effects has been well demonstrated in studies of humans and animals [C. C. Gardner et. al, Atherosclerosis 154,213-220, (2001); Liu et. al, Lipids 35,197-203, (2000) and L. A.

Simons et. al, Atherosclerosis 113,219-225, (1995) ]. Allicin (diallylthiosulfinate) is reported to be one of the most active constituents of garlic. Due to its unstable nature, allicin disallows its chemical modification to achieve a potent analogues. Allicin readily undergoes transformation to a volatile diallyldisulphide. The precursor of allicin, i. e.

Alliin is known to be effective in treating hepatic diseases and fatigue. [Nagai Katsuji, Pat No. JP 56127312].

There has been also report of various active sulphur compounds from garlic, namely, Alliin, Allicin, Ajoene, diallyldisulphide, diallyldisulphide, allylmercaptan and S- allylcysteine [J. G. Dausch et. al, Preventive Medicine 19,346-361 (1990); Y. Itokawa et. al, J. Nutr. 103,88-92, (1973) and R. Gebhardt and H. Beck, Lipids 31,1269-1276 (1996) ] This opens up the avenue for the synthesis of analogues of diallyldisulphide moiety with greater stability and activity in the reduction of serum lipids and this is of immense interest in the development of agents to combat cardiovascular risk factors.

Objects of the Invention The main object of the invention is to provide a novel diallyldisulphide analogues exhibiting antilipidemic and antioxidant activity.

Another object is to provide pharmaceutical compositions containing novel diallyldisulphide compounds.

Yet another object of the invention is to provide an effective and economical process for the preparation of novel diallyldisulphide compounds.

Summary of the Invention Accordingly, the present invention relates to novel diallyldisulphide analogues of general formula (I), exhibiting antilipidemic and antioxidant activity.

R-CH=CH-CH2-S-S-CH2-CH=CH-R Formula I Wherein R=C6HnXp [when n = 3 to 5, p = 2 to 0; X= H, Cl, Br, F, CF3, CH3, CN, COOH, NO2, NH2, OH, OC", H2m+1 (m = 1 to 8)] Description of the Invention Accordingly, the invention relates to novel active diallyldisulphide analogues which are represented by general formula I as shown hereinbelow: R-CH=CH-CH2-S-S-CH2-CH=CH-R Formula I Wherein R=C6HnXp [when n = 3 to 5, p = 2 to 0; X= H, Cl, Br, F, CF3, CH3, CN, , COOH, N02, NH2, OH, OCn, H2m+1 (m = 1 to 8)] The preferred diallyldisulphide derivatives are: R-CH = CH-CH2-S-S-CH2-CH = CH-R (Formula I) Wherein R=C6H5 ; C6H4NO2 (4); C6H3 (NO2) 2 (2,4) ; C6H4COOH (4); C6H4CN (4); C6H3 (COOH) 2 (2,4) ; C6H3 (CN) 2 (2,4) ; C6H2 (NO20 3 (2,4, 6); C6H3NO2 (4) COOH (2); C6H3NO2 (2) COOH (4); C6H4 (CF3) (4) or C6H4CF3 (2) The novel diallyldisulphide compound and its analogues are found to be less toxic than known drugs and exhibit activity equipment to or greater than known drugs.

The process for the preparation of diallyldisulphide compounds comprises the steps of : (a) treating 2-mercapto ethanol with liquid bromine in presence of a suitable aqueous inorganic base in a halogenated hydrocarbon solvent at a temperature of about 0°C, (b) separating the organic phase and extracting the aqueous phase with a halogenated hydrocarbon solvent, (c) drying the organic layer over anhydrous sodium sulphate and evaporating the organic layer to yield bis (2-hydroxyethyl) bisulphide, (d) treating the compound of step (c) under stirring with hydrohalogenic acid and conc. sulphuric acid at an ambient temperature? ?? ? for 15 to 30 hours followed by heating for 2 to 4 hours in a steam bath, (e) separating the upper layer of step (d), washing with 10 % aqueous alkali carbonate solution, drying the organic layer over anhydrous sodium sulphate and evaporating the organic layer to yield bis (2-bromoethyl) disulphide, (f) reacting the product of step (e) with triaryl phosphine in dry dimethyl formamide at reflux temperature for 4-6 hours, (g) cooling the reaction mixtures of step (f) to room temperature and diluting with hexane to yield the required phosphonium bromide salt of bis (2- homothyl) disulphide, (h) treating the compound of step (g) with a benzaldehye or substituted benzaldehyde derivative in the presence of alkali metal alkoxide in the corresponding alcohol at reflux temperature for 15 to 30 hours, and (i) pouring the reaction mixture of step (h) onto ice and the precipitated solid is filtered, washed and dried to yield the corresponding final diallyldisulphide analogue and if desired, converting the analogues into their pharmaceutically acceptable salts.

In one embodiment of the invention, the stirring in step (c) is carried out at an ambient temperature for 24 hours followed by heating for 3 hours.

In an another embodiment of the invention, the benzaldehyde used in step (h) is unsubstituted or substituted with suitable funcational group selected from cyano, carboxyl, nitro, trifluoromethyl, amino, methoxyl. or combination thereof.

In another embodiment of the invention, the diallyldisulphide analogue is as active and two times less toxic than the standard drug.

In still another embodiment of the invention, the protective effective dose of the diallyldisulphide analogue is 20 mg/kg of body weight for antilipidemic activity and 20 mg/kg of body weight for antioxidant activity.

In yet another embodiment of the invention, the acceptable additives are selected from the group of nutrients which are pharmaceutically acceptable carrier.

In still another embodiment of the invention, the novel diallyldisulphide analogues may be used in the form of tablets, capsules, syrup, powder, ointment, injectable, etc.

In a further embodiment of the invention, the diallyldisulphide analogue may be administered through routes such as oral, nasal intravenous, intra-peritoneal, intramuscular, etc.

In an embodiment of the invention, the effective dose of diallyldisulphide analogues may be in the range of 20 mg/kg of body weight.

Yet another embodiment of the invention relates to novel pharmaceutically acceptable salts comprising the novel diallyldisulphide analogues, said salts may be hydrochloride, hydrobromide, maleate, citrate, sodium or potassium, etc.

The preparation of diallyldisulphide derivatives starting from 2-mercaptoethanol is represented by Scheme I. The details of the reaction conditions employed are referred to in Examples 1 to 4.

OH-CH2-CH2SH HOCH2-CH2-S-S-CH2-CH20H (I) # Br [Ph3P+CH2CH2-S-S-CH2CH2P+Ph3]Br # BrCH2-CH2-S-S-CH2-CH2Br <BR> <BR> (III)<BR> R-CH=CH-CH2-S-S-CH2-CH=CH-R (IV) Wherein R=C6HnXp [when n = 3 to 5, p = 2 to 0; X= H, Cl, Br, F, CF3, CH3, CN, COOH, NO2, NH2, OH, OCmH2m+l (m = 1 to 8)] Scheme I

The invention is further illustrated with reference to the following Examples and should not be construed to limit the scope of the invention: Example 1 Synthesis of P-nitrophenyl substituted diallyldisulphide Step (a): Conversion of 2-mercaptoethanol (I) to Bis [2-hydroxy ethyl] Disulphide (II) Procedure 40ml Dichloromethane, 3ml (40mM) 2-Mercaptoethanol (I) and 10% aqueous KHCO3 were added to a round bottom flask. The flask was immersed in ice, and 2ml Bromine (99%) 40mM in Dichloromethane was added to the reaction vessel. The organic phase was separated and the aqueous phase was extracted with dichloromethane. The organic phases were combined and dried with anhydrous Na2S04. The solvent was evaporated yielded the pure disufide (lg, 33.8%) II.

Yield lg % Yield 33.8% T. L. C.

Solvent system 20: 80: : Ethyl acetate: Hexane Rf-0.13 IR (thin film) (Major peaks) 3330.0 cm''OH str.

2925. 8cm~l C-H str.

696. 0cm'' C-S str.

NMR 'H NMR (300MHz ; CDCl3) : 8 2.88 [t, 2x2H (a), J=1.05Hz] ; 2.99 [s, 2xlH (OH) ]; 3.89 [t, 2H (b), J=1.05Hz) Step (b): Conversion of (II) to Bis [2-bromoethyl] Disulfide (III)

Procedure 70ml of 48% HBr was taken in a 500ml 3 necked flask, fitted with a stirrer, condenser & dropping funnel. The flask was cooled in an ice bath and 46ml of conc. H2SO4 was added to HBr with stirring. To the resulting ice cold solution, 2g of compound II was added dropwise. The reaction mixture was left on stirring for 24hours at room temperature. After this the reaction mixture was heated for 3hours on a steam bath. Two layers separated out, 10ml of dichloromethane was added. The upper layer was taken, washed with water, 10% Na2CO3 solution and then dried over anhydrous Na2SO4. The dichloromethane was evaporated using a rotavapor and the desired compound III was obtained.

Yield-2g % Yield 55% T. L. C.

Solvent System 20: 80: : Ethyl acetate: Hexane IR (thin film) (Major peaks) 2922 cm''Csps-H str.

617 cm''C-S str.

563 cm-1 C-Br str.

444 cm~l-S-S-str.

NMR lHNMR (300MHz ; CDC13) : 8 3.10 [t, 2x2H (a), J = 1. 3Hz); 3.62 [t, 2x2H (b), J=1. 3Hz) Step (c): Conversion of (III) to the analogous Phosphonium bromide (IV) Procedure 2.8g (0. 01M) of Compound III and 5.5g (0.02M) of Triphenyl phosphine (PPh3) were taken in a round bottom flask. 15ml of dry dimethylformamide (DMF) was added to the reactants. The reaction mixture was refluxed with stirring for 5hours. The mixture was allowed to cool to room temperature and 15ml of hexane was added. The desired phosphonium salt precipitated out. The solution was filtered, washed with hexane and dried. White crystals of compound IV were obtained.

Yield-5.6g % Yield 71.7 Melting point of white crystals of compound IV-130°C T. L. C.

Solven System 20: 80: : Ethyl acetate: Hexane Rf 0.35 IR IR (KBr pellets) 3053 cm~l-Csp2-H str.

691 cm~l-C-S-str.

NMR Spectroscopy 'H NMR (300MHz ; CDC13) : 8 2.61 (t, 2x2H (a), J=0.29Hz) ; 2.86-3. 10 [m, 2x2H (b) ] ; 7.40-7. 50 (m, 24H, aromatic protons); 7.50-7. 63 (m, 6H, aromatic protons) Step (d): Conversion of IV to the p-nitrophenyl substituted diallyldisulfide (V) Procedure 0.0174g of Lithium ethoxide (LiOEt) (25mM) and 50ml of ethanol were taken in a dry round bottom flask. The flask was flushed with Argon gas 0.779g (1mM) of the phosphonium salt (IV) in 10ml of super dry ethanol was added to the Lithium ethoxide formed by the addition of lithium in superdry ethanol 0.37g (25mM) p-nitrobenzaldehyde dissolved in 5ml of superdry ethanol and 3ml of THF was added to the reaction mixture.

The reaction mixture was refluxed with stirring for 24hours. The reaction mixture was poured into ice, the final compound (V) precipitated out. The compound was filterted and dried.

Yield 200 mg % yield 60.6 Melting point of dry compound V-140°C T. L. C.

Solvent System 20: 80 :: Ethyl acetate: Hexane Rf 0.4 IR IR KBr pellet 3055 cm-1 -Csp2-H str.

1583 cl'-N = O str. (anti) 1433 cm-1 -N = O str. (sym) 690. 5cm-1 -C-S- str.

NMR 'H NMR (CDCL3 + DMSO): S 4.75 [d, 2x2H (a), J=1. 10Hz] ; 7.4-7. 6 [m, 2x4H (b, c &aromatic protons) ] ; 7. 6-7. 8 (m, 2x2H aromatic protons) Example II : Conversion of compound IV of example I to p-aminophenyl substituted diallyldisulfide (VI)

Procedure 0.0174g of Lithium ethoxide (LiOEt) (25mM) and 50ml of ethanol were taken in a dry round bottom flask. The flask was flushed with Argon gas 0.779g (1mM) of the phosphonium salt (IV) in 10ml of super dry ethanol was added to the Lithium ethoxide formed by the addition of lithium in superdry ethanol 0.6g (50mM) p-aminobenzaldehyde was dissolved in 5ml of superdry ethanol and 3ml of THF was added to the reaction mixture. The reaction mixture was refluxed with stirring for 24hours. After completion, the reaction mixture was concentrated under vacuum. The residue was dissolved in water and extracted with chloroform (2 X 50ml). The organic layer was dried over anhydrous Na2SO4 and conc. under reduced pressure. The yellow solid obtained was finally recrystallized from chloroform-hexane (75: 25). The compound was filtered and dried.

Yield 220 mg % yield 65.7 Melting point of dry compound VI-150°C T. L. C.

Solvent System 20: 80: : Ethyl acetate: Hexane Rf 0. 514.

IR IR KBr pellet 3435.97 cm~l =N-H str. (anti) 3054. 34 cm~l-Csp2-H str.

2951. 92 cm~l-Csp3-H str NMR 1H NMR (Cl3) ; S 2.26 (s, 2x2H,-NH2) ; 2.74 [d, 2x2H (a), J=1.14Hz] ; 7.2-7. 4 [m, 2x3H (b, c & aromatic protons) ] ; 7.4-7. 6 (m, 2x2H aromatic protons), 7.85 (s, 2xlH aromatic protons) Example III : Step IVc Conversion of compound IV of example I to p-methoxyphenyl substituted diallyldisulfide (VII) Procedure 0.0174g of Lithium ethoxide (LiOEt) (25mM) and 50ml of ethanol were taken in a dry round bottom flask. The flask was flushed with Argon gas 0.779g (lmM) of the phosphonium salt (IV) in 10ml of super dry ethanol was added to the Lithium ethoxide formed by the addition of lithium in superdry ethanol 0.68g (50mM) p- methoxybenzaldehyde was dissolved in 5ml of superdry ethanol. After completion, the reaction mixture was concentrated under vacuum. The residue was dissolved in water and extracted with chloroform (2 X 50ml). The organic layer was dried over anhydrous Na2S04 and conc. under reduced pressure. The yellow gummy residue was chromatographed on silica gel using hexane-Ethylacetate as eluent. The compound was finally recystallized from Ethylacetate-hexane (80: 20). The compound was filtered and dried.

Yield 240mg % yield 77.8 Melting point of dry compound VII-155°C T. L. C.

Solvent System 20: 80: : Ethyl acetate: Hexane Rf 0.542 IR IR KBr pellet 3435cm~l O-H str. (phenolic impurity)

3047. 97 cmi'-Csp2-H str 2923. 40 cmi'-Csp3-H str.

1654.17 cmi'-C=C-str.

691. 20 crri'-C-S-str.

477.83 cm~l-S-S-str.

NMR 'H NMR (CDCl3) : # 3.75 (d, 2x2H, J=1. OHz,) ; 3.81 (s,-OCH3, 2x3H); 6.81- 6.94 [m, 2x2H (b, c) ] ; 7.45 (d, 2x2H aromatic protons, J=0. 51Hz) ; 7.76 (d, 2x2H aromatic protons, J=0. 51Hz) Example IV: Conversion of compound IV of example I to phenyl substituted diallyldisulfide (VIII).

Procedure 0.0174g of Lithium ethoxide (LiOEt) (25mM) and 50ml of ethanol were taken in a dry round bottom flask. The flask was flushed with Argon gas 0.779g (lmM) of the phosphonium salt (IV) in 10ml of super dry ethanol was added to the Lithium ethoxide formed by the addition of lithium in superdry ethanol 0.53g (50mM) p-benzaldehyde dissolved in 5ml of superdry ethanol. The reaction mixture was refluxed with stirring for 24hours. After completion (TLC), the reaction mixture was concentrated under vaccum.

The residue was dissolved in water and extracted with chloroform (2 X 50ml). The organic layer was dried over anhydrous Na2S04 and conc. under reduced pressure. The yellow

solid obtained was finally recystallized from chloroform-hexane (75: 25). The compound was filtered and dried.

Yield 190 mg % yield 69.3 Melting point of dry compound VIII-220°C (Decomp. ) T. L. C.

Solvent System 20: 80: : Ethyl acetate: Hexane Rf 0. 428 IR IR KBr pellet 3435. 67cm~l O-H str. (hydroxylic impurity/moisture) 3055. 83 cm~l-Csp2-H str.

2927. 08 cm-1 -Csp3-H str.

1685.41 cm-1 -C=C=str.

691.44 cm~l-C-S-str.

427.07 cm~l-S-S-str.

NMR 'H NMR (CDCl3) : 2.87 [d, 2x2H (a), J=lHz] ; 7.49-7. 51 [m, 2x5H (b, c& aromatic protons]; 7.68-7. 75 (m, 2x2H aromatic protons) BIOLOGICAL ACTIVITY Antilipidemic activity assay : Data pertaining to this activity is depicted in Table I.

1. Materials and methods Material-Garlic (Allium sativum) was bought from the local market. All other chemicals and reagents used were of highest analytical grade available commercially. U. V. spectrophotometeric studies were done using model Shimadzu UV-1601. NMR studies were done using Bruker spectro spin 300 MHz instrument.

2. Animals Adult male rats of Wistar strain (n=25) with body weight range 200-250g obtained from Animal House Facility at A. C. B. R. University of Delhi, were used in the present study.

They were maintained in an air-conditioned room, and were provided with standard food pellets and tap water ad libitmn.

3. Method of preparation of garlic clove polar and thiosulphinate fractions After homogenization of garlic cloves in water at 10 ml/g, the thiosulphinates (Allicin extract) were extracted with two volumes of chloroform, quickly rotary evaporated at ambient temperature, and redissolved in water at 2 mg/ml. All homogenates, fraction and compounds were kept at 4°C while in use and at-20°C for storage.

Planning of Experiment Experiment on Normocholesterolemic rats Rats (250-300g) were divided in seven equal groups. Five rats in each group.

GROUP-1 Orally administered equivalent amount of saline, treated as control.

GROUP-2 20mg/kg. b. wt of the Diallyl thiosulphinate fraction of garlic (Allicin extract) was orally administered for 5days.

GROUP-3 20mg/Kg. b. wt of Lovastatin was orally administered to the rats.

GROUP-4 20mg/Kg. b. wt. of Bis [4-nitro phenyl allyl] disulphide was orally administered.

GROUP-5 20mg/Kg. b. wt. of Bis [4-amino phenyl allyl] disulphide was orally administered.

GROUP-6 20mg/Kg. b. wt of Bis [4-methoxy-phenyl allyl] disulphide was orally administered.

GROUP-7 20mg/Kg. b. wt. of Bis [phenyl allyl] disulphide was orally administered Experiment on hypercholesterolemic rats Rats (150-200g) were divided into eight groups. Five rats in each group.

GROUP-8 Orally administered equivalent amount of saline, treated as control GROUP-9 Rats were fed with 5% cholesterol in their diet for one week.

GROUP-10 Rats were fed with 5% cholesterol in diet for one week, along with oral administration of 20mg/Kg. b. wt. diallyl thiosulphinate fraction of garlic (Allicin extract).

GROUP-11 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Lovastatin (20mg/Kg. b. wt.).

GROUP-12 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Bis [4-nitro phenyl allyl]'disulphide (20mg/Kg. b. wt.).

GROUP-13 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Bis [4-amino phenyl allyl]'disulphide (20mg/Kg. b. wt.).

GROUP-14 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Bis [4-methoxy phenyl allyl] disulphide (20mg/Kg. b. wt.).

GROUP-15 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Bis [phenyl allyl]'disulphide (20mg/Kg. b. wt.) 5. Tissue preparation At the end of the experimental period, after overnight starvation animals were anesthetized using chloroform. Blood was drawn from retroorbital sinus using capillary tubes, into dried test tubes, which were kept at an angle of 45° for ten minutes at room temperature and then for an hour at 4°C. After that the blood was centrifuged at 800 x g for 10 min. to get serum separated from cellular clot. The animals were immediately dissected to remove their tissues, which were washed in ice-cold saline (0.85% NaCI), and extraneous material was removed. For biochemical studies, approximately lg of tissue was kept for estimation of lipids and the remaining was homogenized in Potter-Elvejem type glass homogenizer in 0.1 M Potassium phosphate buffer (pH-7.4) having 0.25M sacrose to give a 20% homogenate, according to the method of Hogeboom and Umbelt et al.

Ref: 1. Hogeboom, G. H. Methods in Enzymology (Colowick, S. P. and N. O. Kaplar eds. ); Academic Press, New York, 1,16, (1955) 2.. Umbeit, W. W. , Burries, R. H. and Stauffer, S. F. Burgers Publishing Co.

Minneapolis; III Ed. 10, (1957).

6. Biochemical estimations 6a. HMG-CoA reductase HMG-CoA reductase activity was determined by the method of Venugopala Rao et al (1975). Equal volumes of the 10% tissue homogenate (i. e. Ig of tissue/lOml of saline arsenate sol. ) and diluted perchloric acid (50ml of HCIO4/L of water) were mixed. This was allowed to stand for 5 min. and centrifuged (600g, 10 min. ). In case of Mevalonate estimation, 1.0 ml of supernatant was treated with 0.5 ml of freshly prepared hydroxylamine hydrochloride reagent (equal volumes of hydroxylamine hydrochloride reagent (2 mol/L) and water) and after 5 min., 1.5 ml of ferric chloride reagent was added to the same tube and the tube was vortexed.

In case of HMG-CoA estimation 1.0 ml of supernatant was treated with 0.5 ml of Alkaline hydroxylamine hydrochloride reagent [equal volume of hydroxylamine hydrochloride reagent (2 mol/L of water) and sodium hydroxide solution (4.5 mol/L of water) ]. And after 5 min. 1.5 ml of ferric chloride reagent was added and the tube was vortexed. Readings were taken after 10 min. , at 540 nm vs. A similarly treated saline arsenate blank. The ratio of HMG-CoA/Mevalonate was taken as an index of HMG-CoA reductase activity.

Ref.: Venugopala Rao A and Ramakrishnan S. , Clin. Chem. 21,10, 1543-1525 (1975) 6b. Isolation of lipids For estimation of lipds the tissue was weighed. Approximately 1 g of tissue was homogenized with 10 ml of methanol, then 20 ml of chloroform was added and process continued for a further 2 min. After filtering, the solid was washed once more with chloroform (20 ml) and once with methanol (10 ml), the combined filtrates were transferred to the measuring cylinder and one quarter of the total volume of the filtrate 0.88% KCI in water was added, the mixture was shaken thoroughly and allowed to settle.

The upper layer was removed by aspiration, one quarter of the volume of the lower layer of water-methanol was added. The lower layer contains the purified lipid.

6c. Estimation of total cholesterol For quantitative estimation of total cholesterol the method of Zlatkis et al. (1953) and Hanley (1957). A known amount of (200 pl) the sample from the total lipid in CHC13 was taken and evaporated to dryness. To the dried sample, 5 ml of 0.05% FeCl3 in CH3COOH were added. The contents were mixed using a vortex mixer, 2 ml of Conc. Sulphuric acid was added to each of the tubes, and then vortexed and allowed to stand for 20-30 minutes.

The absorbance was recorded at 560 nm. For standard, Cholesterol (1 mg/ml of CH3COOH) was used.

Ref.: Zlatkis, A, Zak, B. and Boyle, A. J. , J. Lab. Clin. Med. 41,486, (1953) 6d. Estimation of Triglycerides Thin Layer Chromatography (TLC) was used to fractionate the neutral lipids silica gel G was used as an adsorbent. A known amount (-100go) of the total lipid, in duplicate, was applied on silica gel treated plates. Tristearin (100 mg/dl) was used as a standard. The plates were developed upto 13-15 cm from the original in chamber lined with sufficient amount of saturated solvent system petroleum ether (60-80), solvent ether, and acetic acid (80: 20: 2). The plates were dried and the spots made visible by exploring the plates to iodine vapors in a closed chamber. The fraction was then scrapped off the plates and the

glyceride concentration was estimated by the method of Van Handel and Zilversmit. The glyceride was saponified with 0.5 ml of 0.1 N Alcoholic KOH at 70°C for 20 minutes. 200 gl of 0.4 N H2SO4 was added and placed in boiling water bath for 10-15 min. After that 50 ul of 0.05 N sodium metaperiodate was added. Excess Na104 was neutralized with 0.1 ml of 0.5 M NaAs02. A brown coloration developed after the addition of 5 ml of 0.108% chromotropic acid in 50% H2S04. Again heated in boiling water bath for 30-40 minutes.

Then the samples were cooled and 3 ml of distilled water were added. Readings were taken at 570 nm.

Ref.: VanHandel, E. and Zilversmit, D. B. , J. Lab. Clin. Invest. 50,152 (1957).

Antioxidant acitvity assay: Data pertaining to this activity is depicted in Table II.

6e. Reduced glutathione Reduced glutathlone was determined by the method of Jollow et al (1974). 1.00 ml of Post Mitochondrial Supernatant (PMS) obtained after centrifugation at 10,000 x g. was precipitated with 1.0 ml of sulfosalicylic acid (4%). The samples were kept at 4°C for at least one hour and then subjected to centrifugation at 1200xg for 15 minutes at 4°C. The assay mixture contained 0.1 ml of filtered aliquot, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB [5.5"-Dithiobis (2-nitro-bezoic acid)] (40 mg/10 ml of phosphate buffer, 0.1 M, pH 7.4) in a total volume of 3.0 ml. The yellow color developed was read immediately at 412 nm.

Ref.: Jollow J. D. , Mitchell R. J. , Zampaglione N, and Gillette R. J; Pharmacology, 11,151- 169 (1974).

6f. Glutathione reductase activity Glutathione reductase activity was assayed by the method of Carlberg and Mannervik (1975) as modified by Mohandas et al (1984). The assay system consisted of 1.65 ml phosphate buffer (0.1 M, pH 7.6), 0.1 ml NADPH (0.1 mM), 0.1 ml EDTA (0.5 mM), 0.05 ml oxidized glutathione (1 mM) and 0.1 ml PMS (10% w/v) in a total volume of 2.0 ml.

Glutathione reductase activity was calculated in terms of NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 x 10'Mcm''.

Ref.: Carlberg J, and Mannervik B. , J. Biol. Chem. 250,14, 5475-5480 (1975).

6g. Catalase activity Catalase activity was assayed by the the method of Claiborne (1985). The assay mixture consisted of 1.95 ml phosphate buffer (0.05 m, pH 7.0), 1.0 ml hydrogen peroxide (0.019M), and 0.05 ml PM (10% w/v) in a total volume of 3.0 ml. Changes in absorbance

were recorded at 240 nm. Catalase activity was calculated in terms of nmol H202 consumed/min/mg protein.

6h. Glutathione-S-transferase activity Glutathione-S-transferase activity was measured by the method of Habig et al. (1974) The reaction mixture consisted of 1.425 ml phosphate buffer (0.1 M, pH 6.5), 0.2 ml reduced glutathione (1 mM), 0. 025ml CDNB (1-Chloro-2, 4-dinitrobenzene) (1 mM) and 0.30 ml PMS (10% w/v) in a total volume of 2.0 ml. The changes in absorbance were recorded at 340 nm and the enzyme activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 x 103 M~lcm~l.

Ref.: Habig, W. H. , Pabst, M. J., and Jaokby, W. B. J. Biol. Chem. 249,7130-7139 (1974).

6i. Lipid Peroxidation Lipid Peroxidation level was measured by the method of Yagi et. al. 0. 05ml of the blood was taken in 1.0 ml of physiological saline and centrifuged at 800g for 10min. 0. 5ml of supernatant (0. 02ml of serum) was added in 0. 5ml of 10% phosphotungstic acid and mixed. After standing at room temperature for 5min, the mixture was centrifuged at 800g for 10min. The sediment was suspended in 4. 0ml of distilled water, and 1. 0ml of TBA reagent was added. The reaction mixture was heated for 60min. at 95° in an oil bath. After cooling with tap water, 5.0 ml of n-butanol was added and the mixture was shaken vigorously. After centrifugation at 800g for 15min, the n-butanol layer was taken for fluorometrically measured at 553nm with 515nm excitation. Tetraethoxypropane was used as standard.

Ref : Yagi, K. (1979) Biological damage imposed by Oxygen. Methods in Enzymology.

10 (5), 328-331.

6j. Estimation of Protein Protein was estimated by the method of Lowry et al (1951). 0.1 ml of tissue homogenate was diluted to 1 ml with water and 5 ml alkaline copper sulphate reagent containing sodium carbonate (2%), CuS04 (1%) and sodium potassium tartrate (2%) was added.

After 10 minutes, 0.5 ml of Folin's reagent was added, and incubate the mixture for 30 min. at room temperature, the blue color developed and the absorbance was read at 660 nm. Bovine serum albumin (0.1 mg/ml) was used as a standard.

Ref.: Lowry, O. H. , Rosebrough, N. J., Farr, A. L. and Randall, R. J. , J. Biol. Chem. , 193, 265-275 (1951). Biological Activities of Bis [4-amino phenyl allyl] disulphide (Compound VI) and Bis [4- methoxy phenyl allyl] disulphide (Compound VII) Effect of Compound [ (VI, VII), 20mg/Kg. b. wt. ], Lovastatin (20mg/kg. b. wt. ) and Allicin (Garlic extract) given. for 5 days on Lipid profile of Wistar rats

Cholesterol Control Allicin Lovastatin Compound VI Compound 20mg/Kg. b. 20mg/Kg. b. wt. VII wt. (Mean (Mean S. E. ) 20mg/kg. b. wt S. E.) (Mean S. E.) Liver mg/g. 5.60 0. 86 5.00 0. 52 3.41 0. 789 5.39 ~0. 12 5.45 ~1. 77 wet tissue (Mean S. E.) % Decrease 10.7 39.1 3.7 2.67 Serum 32. 3 8. 65 28. 8 8. 6 22. 4 ~6. 58 30.2 5. 40 31. 35 ~2. 08 mg/dl (Mean S. E.) % 0 Decrease 10.8 30.6 6.5 2. 9 Triglyceride Contr Allicin Lovastatin Compound VI Compound VII ol 20mg/Kg. b. wt. 20mg/Kg. b. wt. 20mg/kg. b. wt Liver mg/g. 16. 9 9.3 ~0. 452 11.19 ~0. 834 15.56 ~0. 330 15.84 0. 608 wet. Tissue 1.04 (Mean S. E.) % Decrease 44 33. 7 8. 2 5. 9

HMG-CoA Reductase Control Allicin Lovastatin Compound VI Compound VII 20mg/Kg. b. 20mg/Kg. b. wt. 20mg/kg. b. wt. wt Ratio of index 2.09 2. 800 3.22 2.139 2.12 of HMG-CoA reductase % increase 33. 9 54. 06 2.34 1.4 Effect of Compound [(VI, VII), 20mg/Kg. b. wt. ], Lovastatin (20mg/kg. b. wt. ) and Allicin (Garlic extract)

given for 5 days on Oxidative stress in Wistar rats Reduced Glutathione Organ Control Allicin Lovastatin Compound VI Compound (Mean S. E.) (Mean S. E. ) 20mg/Kg. b. wt. 20mg/Kg. b. wt. VII (Mean S. E.) (Mean S. E. ) 20mg/kg. b. wt. (Mean S. E.) Liver* 302. 92 77. 9 469.88 85. 17 474.41 47. 9 346.79 59. 59 235.23 ~50. 5 Spleen 1 126.77 ~42. 4 208. 58 45. 54 315.48 80. 29 178. 115 33. 4 107.32 ~21. 6 * nmol of GSH/mg. protein

Glutathione Reductase Organ Control Allicin Lovastatin Compound VI Compound VII (Mean S. E. ) (Mean S. E. ) 20mg/Kg. b. wt. 20mg/Kg. b. wt. 20mg/kg. b. wt. (Mean~S. E. ) (Mean S. E. ) (Mean S. E. ) Liver* 17.9~5 22.82~8. 7 25. 17. 7 20. 57. 3 17. 33. 83 Spleen* 5. 6~0. 66 13. 0~4. 3 14. 0~4. 73 6. 8~2. 07 5.1~0. 4 * nmol NADPH oxidised/min/mg protein Glutathione S-transferase Organ Control Allicin Lovastatin Compound VI Compound VII (Mean (Mean S. E. ) 20mg/Kg. b. wt. 20mg/Kg. b. wt. 20mg/kg. b. wt. S. E.) (Mean S. E.) (Mean S. E.) (Mean S. E.) Liver* 27.0~8.3 32.3~2.8 33.7~5. 0 26.8~3.0 29.5~9.6 Spleen* 14. 54. 9 19. 2~6.7 21. 18~3.2 15. 5~2.39 12. 073. 5 * nmol of CDNB conjugate formed/min/mg protein Catalase Organ Control Allicin Lovastatin Compound VI Compound VII (Mean S. E. ) (Mean S. E. ) 20mg/Kg. b. wt. 20mg/Kg. b. wt. 20mg/kg. b. wt. (Mean S. E.) (Mean S. E.) (Mean S. E.) Liver* 8.75 ~1. 38 11.8 0. 187 9. 11 1. 20 8.94 ~1. 089 6.689 ~1. 326 Spleen* 2.82 ~0. 404 4.24 1. 089 4.37 ~0. 347 3. 49 ~0. 67 2. 14 ~0. 212 * jmo) of H2°2 consumed/min./g. wet weight tissue Lipid Peroxidation Control Allicin Lovastatin Compound VI Compound (Mean S. E.) (Mean S. E.) (Meant S. E.) VII Serum* 277. 38 127.60 ~2. 48 169.76 ~20. 8 222.98 ~0. 72 275.55 ~2. 48 3.25 * nmol of MDA conjugate formed/ml of Serum Biological Activities of Bis [4-nitro phenyl allyl] disulphide (Compound V) and Bis [ phenyl allyl] disulphide (Compound VIII) Effect of Compound [ (V, VIII), 20mg/Kg. b. wt. ], Lovastatin (20mg/kg. b. wt. ) and Allicin (Garlic extract) given for 5 days on Lipid profile of Wistar rats Cholesterol Control Allicin Lovastatin Compound V Compound 20mg/Kg. b. w 20mg/Kg. b. w VIII t. (Mean t. (Mean 20mg/kg. b. wt. S. E. ) S. E.) (Mean S. E.) Liver mg/g. 8. 06~2. 33 6.81 ~1. 24 4.78 ~0.031 4.86 0. 075 7.89 ~0. 082 wet tissue (Mean S. E.) % 15.6 40.6 39.8 3.2 Decrease Serum 47. 75 ~9. 55 43.9 8. 94 43.95 6. 67 36.9 4. 66 44. 9 7. 14 mg/dl (Mean S. E.) % 8. 06 26. 7 23.3 5.99 Decrease Triglyceride Control Allicin Lovastatin Compound V Compound 20mg/Kg. b. wt. 20mg/Kg. b. wt. VIII 20mg/kg. b. wt. Liver mg/ g. 13.0 1. 426 7.130 ~2. 53 8.78 ~1. 54 10.88 ~0. 974 12. 4 ~0. 939 wet. Tissue (Mean S. E.) % Decrease45. 15 34. 6 16.15 6. 9 HMG-CoA Reductase Control allicin Lovastatin Compound V Compound 20mg/Kg.b. wt. 20mg/Kg.b.wt. VIII 20mg/kg.b.wt. Ratio of index of HMG - CoA 1.981 2.603 3.129 3.07 2.0152 reductase % increase in 31.46 58.03 55.05 1.51 ratio

Effect of Compound [ (V, VIII), 20mg/Kg. b. wt. ], Lovastatin (20mg/kg. b. wt. ) and Allicin (Garlic extract) given for 5 days on Oxidative stress in Wistar rats Reduced Glutathione Organ Control Allicin Lovastatin Compound V Compound (Mean S. E. ) (Mean S. E. ) 20mg/Kg. b. wt 20mg/Kg. b. wt ( VIII (Mean S. E.) Mean S. E.) 20mg/kg. b. wt. (Mean~ S. E.) Liver* 501.46~176. 67 601.38 131. 60 705. 02 70. 63 824.57 45. 19 503.28 41. 9 Spleen* 141. 51 20.35 281.37 41.72 231.44 20.1 406.76 92.89 161.99 24. 41

* nmol of GSH/mg. protein Gluathione reductase Organ Control Allicin Lovastatin Compound V Compound VIII (Mean (Mean S. E. ) 20mg/Kg. b. wt. 20mg/Kg. b. wt. 20mg/kg. b. wt. S. E. ) (Mean~ S. E.) (Mean S. E.) (Mean S. E.) Liver* 8. 57 2. 02 10. 5~1. 9 13. 9~3. 8 14. 3 0. 313 9. 4~1. 29 Spleen* 1.85~0. 68 3.41 ~1. 33 5. 9~0.9 6.43~1.75 2. 62~0. 315 * nmol NADPH oxidised/min/mg protein Gluatathione-S-transferase Organ Control Allicin Lovastatin Compound V Compound VIII (Mean (Mean S. E. ) 20mg/Kg. b. wt. 20mg/Kg. b. wt. 20mg/kg. b. wt. S. E. ) (Mean S. E.) (Mean S. E.) (Mean S. E.) Liver* 3. 49 1. 96 7.02 ~0. 2 6.53 ~0 33 7.32 ~1. 24 3.62 ~1. 3 Spleen* 1.44~0.4 1. 86 ~0. 65 2. 08 0. 24- 2. 14~0. 6 1. 80 + 0 3

* nmol of CDNB conjugate formed/min/mg protein Catalase Organ Control Allicin Lovastatin Compound V Compound (Mean S. E. ) (Mean S. E. ) 20mg/Kg. b. wt. 20mg/Kg. b. wt VIII (Mean S. E.) (Mean S. E. ) 20mg/kg. b. wt. (Mean S. E.) Liver* 4.95 ~0. 71 7. 301 2. 91 5.217 ~0. 057 7.65 ~0. 602 3.44 ~0. 34 Spleen* 1. 68 ~0. 152 2. 86 ~0. 229 2. 29 ~0. 45 2. 14 ~0. 66 1. 337 ~0. 202 * cymol of H202 consumed/min./g. wet weight tissue Lipid Peroxidation Control Allicin Lovastatin Compound V Compound VIII (Meant S. E. ) (Mean S. E.) (Meant S. E.) Serum* 244.81~5.18 120.59~3.99 143.84~16.4 127.53~26.6 241.50~6.34 * nmol of MDA conjugate formed/ml of Serum.

Biological Activities of Bis [4-amino phenyl alkyl I disulphide (Compound VI) and Bis [4-methoxy phenyl allyl] disulphide (Compound VII) Effect of Compound [ (VI, VII), 20mg/Kg. b. wt. ], Lovastatin (20mg/kg. b. wt. ) and Allicin (Garlic extract) given for 5 days on Lipid profile of Mild Hypercholesterolemic Wistar rats (administered 5% cholesterol in their diet) Cholesterol Triglyceride Liver mg/g. % Serum mg/dl % Increase Liver mg/g. % Increase wet tissue Increase (Mean S. E. ) wet. Tissue (Mean (Meant S. E.) S. E.) Control 2.66 ~0. 16 38.62 ~2. 25 10. 66 0.04 Hyperchole 4.96 0. 035 86. 4 63. 25 2. 25 63. 77 15. 63 0.163 46.62 sterolemic rats Cholesterol Hypercholest Allicin Lovastatin Compound VI Compound VII erolemic rats 20mg/Kg. b. w 20mg/Kg. b. wt. 20mg/kg. b. wt. t. (Mean (Mean S. E.) (Mean S. E.) S. E.) Liver mg/g. 4.96 ~ 3 59 ~ 2. 84 ~0. 03 4.805 4.9 ~0. 030 wet tissue 0.035 0.160 0.045 (Mean S. E.) % Decrease 27.6 42.3 3.125 1.2 Serum mg/dl 63.25 50. 25 ~ 43. 5 1. 0 59. 76 ~3. 01 61.95 ~4. 45 (Mean S. E. ) 2.25 1.25 % Decrease 20.5 31.23 5.5 2.05

Triglyceride Hyperchol Allicin Lovastatin Compound Compound esterolemic 20mg/Kg. b. VI VII rats wt. 20mg/Kg. b. 20mg/kg. b. wt. wt. Liver mg/g. 15.63 ~ 9.44~0. 1 9. 86 0.163 14.58 ~ 14.85~ wet. Tissue 0.163 93 0. 148 0.029 (Mean S. E.) % Decrease 39. 6 36. 91 6.71 4. 91 HMG-CoA Reductase Control Hypercholesterolemic rats Ratio of index of HMG-CoA 1. 625 1. 995 reductase % decrease in ratio 22. 7 HMG-CoA Reductase Contr Allici Lovastatin Compound Compound ol n 20mg/Kg. b. wt VI VII Ratio of index 1.625 2.075 2.20 1.747 1.685 of HMG- CoA reductase % increase in 27. 6 35.38 7.50 3.69 ratio

Effect of Compound [ (VI, VII), 20mg/Kg. b. wt. ], Lovastatin (20mg/kg. b. wt. ) and Allicin (Garlic extract) given for 5 days on Oxidative stress in Wistar rats Reduced Glutathione Organ Hypercholest Allicin Lovastatin Compound Compound erolemic rats (Mean~ 20mg/Kg. b. wt VI VIII S. E.). (Mean 20mg/Kg. b. wt 20mg/kg. b. wt. S. E.). (Mean (Mean S. E.) S. E.) Liver* 402.93 ~0. 484 461.49 483.90 ~265. 95 431.05 ~46. 6 416.65 14. 73 6.34 Spleen* 143.29~12.81 158.12~ 162.78~1.24 154.50~18,58 153.011~ 27.61 31.45

* nmol of GSH/mg. protein Glutathione Reductase Organ Hypercholeste Allicin Lovastatin Compound VI Compound rolemic rats (Mean 20mg/Kg. 20mg/Kg. VII S. E. ) b. wt. b. wt. 20mg/kg. b. wt (Mean S. E.) (Mean S. E.) (Mean S. E.) Liver* 11.11~1. 7 12.09 ~0.9 12. 32 ~0.53 11. 78 ~0. 58 11.25 ~0. 53 Spleen* 3. 75 ~0. 26 4. 47 ~0. 18 5. 62 ~0. 8 4. 01 ~0.26 3. 70 ~0. 58 * nmol NADPH oxidised/min/mg protein Glutathione S-transferase Organ Hypercholesterol Allicin Lovastatin Compound VI Compound emic rats (Mean S. E. ) 20mg/Kg. 20mg/Kg. VII b. wt. b. wt. 20mg/kg. b. wt (Mean S. E.) (Mean S. E.) (Mean S. E.) Liver* 8.885 tO. 184 11.7 ~0. 58 12. 5 _ 0. 97 9.27 ~0. 19 8.81 ~0. 136 Spleen* 4. 18 ~0. 908 4.74 ~0. 115 4. 56 ~0. 29 4.23 0. 145 4. 04 ~0. 294

* nmol of CDNB conjugate formed/min/mg protein Catalase Organ Hypercholesterol Allicin Lovastatin Compound VI Compound VII emic rats (Mean S. E.) 20mg/Kg. b. wt. 20mg/Kg. b. wt. 20mg/kg. b. wt. (Mean S. E. ) (Mean S. E. ) (Mean S. E. ) Liver* 5. 47 ~0. 20 6.39 ~0. 135 5.79 ~0. 97 5. 67 ~1. 204 5.160 ~1. 204 Spleen* 1. 12 ~0. 086 2.66 ~0. 086 2.23 ~0. 172 1. 806 ~0. 087 1.49 ~0. 34 * cymol of H202 consumed/min./g. wet weight tissue

Lipid Peroxidation Hypercholester Allicin Lovastatin Compound VI Compound VII olemic rats (Mean S. E.) (Mean S. E.) Serum* 300. 08 ~1. 36 229. 87 ~3. 79 248.86~5. 05 279.92 ~21. 95 286.88 ~1. 68 * nmol of MDA conjugate formed/ml of Serum

Biological Activities of Bis [4-nitro phenyl allyl] disulphide (Compound V) and Bis [ phenyl allyl] disulphide (Compound VIII) Effect of Compound [ (V, VIII), 20mg/Kg. b. wt. ], Lovastatin (20mg/kg. b. wt. ) and Allicin (Garlic extract) given for 5 days on Lipid profile of Mild Hypercholesterolemic Wistar rats

Cholesterol Hypercho Allicin Lovastatin Compound V Compound VIII lesterolem 20mg/Kg. b. wt 20mg/Kg. b. wt. 20mg/kg. b. wt. ic rats. (Mean (Mean S. E.) (Mean S. E.) S. E.) Liver mg/g. 4.96 ~ 3. 59 0. 160 2.84 ~0. 03 3.15 ~0. 03 4.86 ~0. 070 wet tissue 0.035 (Mean S. E.) % Decrease 27.6 42.27 36.49 2.0 Serum 63. 25 t 43 5 1. 0 50. 25 1. 25 44. 15 4. 90 60. 95 ~4. 25 mg/dl 2. 25 (Mean S. E.) % Decrease 20. 5 31. 23 30.19 3. 63 Triglyceride Hypercholester Allicin Lovastatin Compound V Compound VIII polemic rats 20mglKg. b. wt. 20mg/Kg.b.wt 20mg/kg.b. wt. Liver 15.63 ~0. 163 9.44 ~0. 193 9. 86 ~0. 163 14.85 ~0. 029 14.19 ~0. 029 'nez wet. Tissue (Mean~ S. E.) % 39. 6 36. 91 9. 21 4.9 Decrease

HMG-CoA Reductase Control Allicin Lovastatin Compound V Compound VIII 20mg/Kg. b. wt 20mg/Kg. b. wt. 20mg/kg. b. wt Ratio of 1.625 2.075 2.20 2.53 1.721 index of HMG- CoA reductase % increase 27.6 35.38 55.69 5. 90 Effect of Compound [ (V, VIII), 20mg/Kg. b. wt. ], Lovastatin (20mg/kg. b. wt. ) and Allicin (Garlic extract) given for 5 days on Oxidative stress in Wistar rats Reduced Glutathione Organ Hyperch Allicin Lovastatin Compound V Compound VIII olesterol (Mean S. E. ) 20mg/Kg. b. wt. 20mg/Kg. b. wt. 20mg/kg. b. wt. (Mean S. E. ) (Mean~S. E. ) (Mean~S. E. ) rats Liver* 402.93 461.49 ~6. 34 483.90 ~26. 95 486.97 ~20. 97 406.01 ~49. 86 0. 484 Spleen* 143. 29~ 158.01~27. 61 162. 78 ~1. 24 170. 86~23. 3 146. 80 4. 31 12. 81 * nmol of GSH/mg. protein Glutathione Reductase Organ Hypercholes Allicin Lovastatin Compound V Compound terolemic (Mean 20mg/Kg. b. wt. 20mg/Kg. b. wt. VIII rats S. E.) (Mean S. E. ) (Mean S. E. ) 20mg/kg. b. wt. (Mean S. E. ) Liver* 11. 11~1. 7 12. 09_0. 9 12. 32 0. 53 12. 94 1. 3 11.28 ~0. 14 Spleen* 3.75 ~ 0.26 4.47~0.18 5.62~0.8 5. 74_0. 68 3. 85_0. 64 * nmol NADPH oxidised/min/mg protein Glutathione S-transferase Organ Hypercholeste Allicin Lovastatin Compound V Compound<BR> <BR> <BR> <BR> rolemic rats (Mean S. E. ) 20mg/Kg. b. wt. 20mg/Kg. b. wt. VIII<BR> (Mean S. E.) (Mean S. E.) 20mg/kg. b. wt.<BR> <BR> <P> (Mean~S. E. ) Liver* 8.885 ~0. 184 11.7 ~0. 58 12. 5 ~0. 97 13.2 ~0. 29 8. 74 ~0. 277 Spleen* 4. 18 0. 908 4. 74 0. 115 4.56 ~0.29 5.02 0. 185 4 17 ~0. 92 * nmol of CDNB conjugate formed/min/mg protein Catalase Organ Hyperchole Allicin Lovastatin Compound V Compound VIII sterolemic (Mean 20mg/Kg. b. wt. 20mg/Kg. b. wt. 20mg/kg. b. wt. rats S. E. ) (Mean S. E.) (Mean S. E.) (Mean S. E.) Liver* 5.47 ~0. 20 6.39 ~0. 135 5.79 ~0. 97 8.65 ~1. 089 5.24 ~0. 258 Spleen* 1. 12~0 086 2. 66 + 0. 086 2. 23~0.172 1.94~0.68 1. 66~0. 74 * µmol of H2O2 consumed/min. /g. wet weight tissue Lipid Peroxidation Hypercholes Allicin Lovastatin Compound V Compound terolemic (Mean S. E.) (Mean S. E. ) VIII rats Serum* 300. 08 ~1 36 229. 87 3. 79 248.86 ~5. 05 238.29 15. 80 299. 63 ~2. 1 * nmol of MDA conjugate formed/ml of Serum Toxicological studies Effect of single dose of synthesized compounds on Wistar rats. Blood Urea Creatinine Uric acid Alanine Aspartate Alklaline Nitrogen mg/dl mg/dl transferase transferase Phosphatase mg/dl (GPT) IU (GOT) IU KA Units Normal 5-29 0.20-0. 80 1.20-7. 5 18-30 46-81 14-32 Range Control 10. 45~1. 41 0. 715~0. 075 1. 36~0. 10 22~3 41 6 30. 24~ 18.16 Compou 7. 65~0. 15 0. 4 0. 06 2. 93~0. 59 21~3. 5 51 1 29. 55~12. 5 ndV Compou 12. 96~0. 07 0. 55~0. 28 1. 88~0. 43 26. 5~1. 5 51. 5 2. 50 40. 6~5. 18 nd VI Compou 7. 43~0. 65 0. 595 0. 145 2. 31~0. 32 26.5~0. 5 49~4 45. 62~9 nd VII Compou 12. 39~1. 05 0. 58~0. 06 1. 65~0. 13 17. 5~0. 5 51 6 34. 01 0. 55 nd VIII

Effect of administration of five doses of synthesized compounds on Wistar rats. BUN mg/dl Creatinine Uric acid Alanine Aspartate Alkaline mg/dl mg/dl transferase transferas Phosphatase (GPT) IU e KA Units (GOT) IU Normal 5-29 0.20-0. 80 1.20-7. 5 18-30 46-81 14-32 Range Control 10. 8~1. 89 0. 79~0. 04 1. 45~0. 11 22~4 48. 5 2. 5 45. 59~5. 81 Compound 6. 48~0. 015 0. 8 0. 04 1. 01~0. 02 15. 5 0. 50 49~1 48~8 v Compound 14.64 ~1. 23 0.84 ~0. 175 2.21 ~0. 14 21 2 37 ~7 46.88 ~6. 68 VI Compound 8. 48~0. 59 0. 495 0. 06 1. 25~0. 27 23 2 29. 5~4. 5 25.21 ~9. 39 VII Compound 7. 37~0. 31 0. 57~0. 05 2. 14~0. 16 27~1 39~3 33. 6~12. 5 VIII