Title of Invention	" A PROCESS FOR THE PREPARATION OF NOVEL DIALLYLDISULPHIDE DERIVATIVES."
Abstract	The present invention relates to a process of preparation of novel diallylsulphide derivatives of general Formula 1. The said derivatives are having antilipidemic and antioxidant activity.

Full Text

Field of the Invention The present invention relates to a process of preparation of novel diallyldisulphide analogues of general formula I having antilipidemic and antioxidant activity, equivalent to or greater than the available standard drugs, such analogues having general Formula I is represented by the structure shown here below: (Formula Removed) Formula 1 Wherein R=C6HDXp[when n = 3 to 5, p = 2 to 0; X= H, CI, Br, F, CF3, CH3, CN, COOH, N02, NH2, OH, OCmH2m+1(m = 1 to 8)] Background of the Invention Garlic is known for its medicinal and nutritional values for a very long time 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 etal, Atherosclerosis. 113, 219-225, (1995)]. Allicin (diallylthiosulfinate) is reported to be one of the most active constituents of garlic. Due to its unstable characteristic, allicin disallows its chemical modification to achieve a more potent analogue. Allicin readily undergoes transformation to the volatile diallyldisulphide The precursor of allicin, i.e. Allnn is known to be effective in treating hepatic disease and fatigueness [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 compounds 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 process for preparing novel diallyldisulphide analogues having antihpidemic and antioxidant activity Another object of the invention is to provide an effective and economical process for the preparation of novel diallyldisulphide compounds, and their pharmaceutically acceptable salts thereof. Still another object of the invention is to provide usage of the novel compounds belonging to basic diallyldisulphide moiety and their pharmaceutically acceptable salts.. Still yet another objective is to provide the ;process of preparation of diallyldisulphide analogues using the starting material 2-mercaptoethanol involving four steps of reactions. Summary of the Invention Accordingly, the present invention provides a process of preparation of novel diallyldisulphide analogues of general formula (I), having antihpidemic and antioxidant activity equivalent or greater than the standard available drugs and also associated with less toxicity. The said diallyldisulphide analogues having following structural formula (Formula Removed) Formula I Wherein R=C6HnXp[when n = 3 to 5, p = 2 to 0; X= H, CI, Br, F, CF3, CH3, CN, COOH, N02, NH2, OH, OCmH2m+1(m = 1 to 8)] Description of the Invention The invention essentially relates to novel active diallyldisulphide analogues which are represented by general formula 1 obtained by following synthetic route (Scheme I, p.6) using different substituted benzaldehyde as one of the reagents in the sequential steps of the synthetic route adopted. (Formula Removed) Formula I Wherein R=C6HnXp[when n = 3 to 5, p = 2 to 0; X= H, CI, Br, F, CF3, CH3, CN, COOH, N02, NH2, OH, OCmH2m+i(m = 1 to 8)] In an embodiment of the present invention, preferred diallyldisulphide derivatives as represented by the structural formula (I) and pharmaceutically acceptable salts thereof are: (Formula Removed) (Formula I) Wherein R=C6H5; C6H4N02(4); C6H3(N02)2(2,4); C6H4COOH(4), C6H4CN(4); C6H3(COOH)2(2,4), C6H3(CN)2(2,4); C6H2(N02)3(2,4.6). C6H3N02(4)COOH(2); C6H3N02(2)COOH(4); C6H*(CF3)(4) orC6H4CF3(2) In an embodiment of the present invention provides a pharmaceutical composition providing antilipidemic and antioxidant activity to a subject, said composition comprising an effective amount of diallyldisulphide denvatives of formula I or their pharmaceutically acceptable salts, and pharmaceutically acceptable additive. Another embodiment, the diallyldisulphide derivative containing pharmaceutical composition may be administered systemically or orally Still another embodiment, said composition is administered for the pathological conditions selected from high cholesterol levels and other cardiovascular risk factors Yet another embodiment, the subject is selected from animals and mammals. Yet another embodiment, ED50 value of active diallyl disulphide derivatives is in the range of for hypolipidemic activity 20 mg/kg body weight and antioxidant activity 20 mg/kg body weight. In yet another embodiment, the said composition containing diallyl disulphide derivatives are administered to the subject in combination with a pharmaceutical^ acceptable additive, earner, diluent, solvent, filtei. lubricant, excipient, binder or stabilizer In yet another embodiment, wherein the preferred dosage of the composition is in the range of 20-mg/kg body weight. Therefore, the present invention relates to a process for the preparation of diallyldisulphide derivatives, the said process comprising steps of: (a) treating 2-mercapto ethanol with liquid bromine in presence of a suitable aqueous inorganic base in a halogenated hydrocarbon solvent around 0°C, (b) separating the organic phase and extracting the aqueous phase with 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 cone, 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-bromoethyl) 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 represented by general formula (I) and optionally, converting the analogues into their pharmaceutically acceptable salts. In one embodiment of the invention, the stirring in step © is earned 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 either unsubstituted or substituted with functionl groups selected from nitro, cyano, carboxyl, tnfluoromethyl, amino, methoxyl or combinations thereof. In yet another embodiment of the invention, the present invention provides a novel diallyldisulphide analogue having antilipidemic and antioxidant activity One more embodiment of the invention provides that .still another embodiment of the invention, is to provide an effective and economical process for the preparation of diallyldisulphide analogues. 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 earner. 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, said subject comprising of administration of effective amount of diallyldisulphide analogue 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. In an embodiment of the invention, the novel diallyldisulphide compounds are charactensed by m.p., IR, UV, NMR and MS data 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 invention provides a novel antilipidemic and antioxidant molecules, a process of preparation and their method of testing. It particularly relates to the development of a novel analogue of diallyldisulphide, having antilipidemic and antioxidant activity and process for its preparation. 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. (Formula Removed) Wherein R=C6HnXp[when n = 3 to 5, p = 2 to 0; X= H, CI, Br, F, CF3, CH3, CN, COOH, N02, NH2, OH, OCmH2m+i(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) (Scheme Removed) 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"1 OH str. 2925.8cm"' C-H str. 696.0cm"1 C-S str. NMR 'H NMR (300MHz; CDC13): δ 2.88[t, 2x2H(a), J=l 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) (Scheme Removed) 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 cone. 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% NaiC03 solution and then dried over anhydrous NaiS04 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"1 Csp3 - H str. 617 cm"1 C-Sstr. 563 cm"1 C-Br str. 444 cm"1 -S-S- str. NMR 1HNMR (300MHz; CDCI3): δ 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) (Scheme Removed) Procedure 2.8g (0.01M) of Compound m and 5.5g (0.02M) of Triphenyl phosphine (PPh3) were taken m 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"1 -Csp2-H str 691cm"1 -C-S-str NMR Spectroscopy 1H NMR (300MHz; CDC13): δ 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) (Scheme Removed) rocedure 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 (ImM) of the phosphonium salt (IV) m 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-Hstr. 1583 cm"1 -N = O str.(anti) 1433 cm*1 -N = O str. (sym) 690.5 cm'1 -C-S- str. NMR 1H NMR (CDC13 + DMSO): δ 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) (Scheme Removed) 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 (ImM) 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 Na2S04 and cone, under reduced pressure. The yellow solid obtained was finally recrystallized from chloroform-hexane (7525). 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 RfO.514. IR IR KBr pellet 3435.97 cm"1 =N - H str.(anti) 3054.34 cm"1 -Csp2-Hstr 2951.92 cm"1 -Csp3-Hstr NMR 1H NMR (CDCl3); δ 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, 2x1 H aromatic protons) Example III: Step IVc Conversion of compound IV of example I to p-methoxyphenyl substituted diallyldisulfide (VII) (Scheme Removed) 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 (ImM) 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 cone, 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 the dry compound VII-155°C T.L.C. Solvent System 20:80 Ethyl acetate Hexane Rf 0.542 IR IR KBr pellet 3435cm"1 O-H str. (phenolic impurity) 3047.97 cm"' -Csp2 -H str 2923.40 cm"1 -Csp3 -H str. 1654.17 cm"1 -C=C-str. 691.20 cm"1 -C-S-str. 477.83 cm"1 -S-S-str. NMR 'H NMR (CDC13): δ 3.75(d, 2x2H,J=1.0Hz,); 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 diatlyldisulfide (VIII). (Scheme Removed) 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 (ImM) of the phosphomum 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 cone, under reduced pressure. The yellow solid obtained was finally recystallized from chloroform-hexane (75:25) The compound was filtered and dned. Yield 190 mg; percentageyield 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"1 O-H str. (hydroxylic impurity/moisture) 3055.83 cm"' -Csp2-Hstr. 2927.08 cm"1 -Csp3 -H str 1685.41 cm"1 -C=C-str. 691.44 cm"1 -C-S-str 427.07 cm"1 -S-S-str. NMR *H NMR (CDC13): 82.87[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. spectrophotometer^ 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 libitum 3. Method of preparation of garlic clove polar and thiosulphinate fractions After homogenization of garlic cloves in water at 10 ml/g, the thiosulphinates (Alhcin 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. 4. 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 thiosulphmate fraction of garlic (Alhcin 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 thiosulphmate fraction of garhc (Alhcin 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-ammo 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] v disulphide (20mg/Kg.b.wt) 5. Tissue preparation At the end of the experimental penod, 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 centnfuged at 800 x g for 10 mm. 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% NaCl), 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., Burnes, R.H. and Stauffer, S.F. Burgers Publishing Co. Minneapolis; HI 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. lg 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 mm and centnfuged (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 hydroxylamme hydrochloride reagent (2 mol/L) and water) and after 5 mm., 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 rrun., 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 Ramaknshnan 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% KC1 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 u,l) the sample from the total lipid in CHCh was taken and evaporated to dryness To the dned sample, 5 ml of 0 05% FeCh in CH3COOH were added The contents were mixed using a vortex mixer, 2 ml of Cone. 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 (~100µl) of the total lipid, in duplicate, was applied on silica gel treated plates. Tnsteann (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 glycende concentration was estimated by the method of Van Handel and Zilversmit. The glycende was saponified with 0.5 ml of 0.1 N Alcoholic KOH at 70°C for 20 minutes. 200µl of 0.4 N H2SO4 was added and placed in boiling water bath for 10-15 mm After that 50 µl of 0.05 N sodium metapenodate was added. Excess NalO4 was neutralized with 0.1 ml of 0.5 M NaAsO2. A brown coloration developed after the addition of 5 ml of 0.108% chromotropic acid in 50% H2SO4. 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 glutathione was determined by the method of Jollow et al (1974) 1 00 ml of Post Mitochondnal Supernatant (PMS) obtained after centnfugation at 10,000 x g. was precipitated with 1.0 ml of sulfosahcylic acid (4%). The samples were kept at 4°C for at least one hour and then subjected to centnfugation 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). f. 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 lO^M'cm'1 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 H2O2 consumed/mm/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 (l-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 'cm"1. 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 centnfuged at 800g for lOmin 0.5ml of supernatant (0.02ml of serum) was added in O.Sml of 10% phosphotungstic acid and mixed. After standing at room temperature for 5min, the mixture was centrifuged at 800g for lOmin.The sediment was suspended in 4.0ml of distilled water, and 1.0ml of TBA reagent was added. The reaction mixture was heated for 60mm.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 fluorometncally 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 mm. 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.], Lovastatm (20mg/kg.b.wt.) and Allicin(Garlic extract) given for 5 days on Lipid profile of Wistar rats Cholesterol (Table Removed) Triglyceride (Table Removed) HMG-CoA Reductase (Table Removed) Effect of Compound [(VI, VII),20mg/Kg.b.wL], Lovastatin (20mg/kg.b.wt.) and Allicin(GarIic extract) given for 5 days on Oxidative stress in Wistar rats Reduced Glutathione (Table Removed) • nmol of GSH / mg.protem Glutathione Reductase (Table Removed) Glutathione S-transferase (Table Removed) • nmol of CDNB conjugate formed /min/ mg protein Catalase (Table Removed) *µmol of H2O2 consumed/min /g wet weight tissue (Table Removed) * 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, Vm),20mg/Kg.b.wt.], Lovastatin (20mg/kg.b.wt.) and Allicin(Garlic extract) given for 5 days on Lipid profile of Wistar rats Cholesterol (Table Removed) Triglyceride (Table Removed) Giuathione reductase (Table Removed) * nmol NADPH oxidised/mm/ mg protein Gluatathione -S-transferase (Table Removed) * nmol of CDNB conjugate formed /min/ mg protein Catalase (Table Removed) • µmol of H2O2 consumed/min. / g. wet weight tissue (Table Removed) * nmol of MDA conjugate formed / ml of Serum. 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 Mild Hypercholesterolemic Wistar rats (administered 5%cholesterol in their diet) (Table Removed) Cholesterol (Table Removed) Triglyceride (Table Removed) HMG-CoA Reductase (Table Removed) HMG-CoA Reductase (Table Removed) (Table Removed) Effect of Compound [(VI, VII),20mg/Kg.b.wL], Lovastatin (20mg/kg.b.wt) and Allicin(Garlic extract) given for 5 days on Oxidative stress in Wistar rats (Table Remov (Table Removed) (Table Removed) * 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 (Table Removed) HMG-CoA Reductase (Table Removed) Effect of Compound [(V, VIII), 20mg/Kg.b.wt], Lovastatin (20mg/kg.b.wL) andAllicin(GarIic extract) given for 5 days on Oxidative stress in Wistar rats (Table Removed) * nmol of GSH / mg.protem Glutathione Reductase (Table Removed) Catalase (Table Removed) • µmo] of H2O2 consumed/mm. / g. wet weight tissue (Table Removed) Effect of single dose of synthesized compounds on Wistar rats. (Table Removed) Effect of administration of five doses of synthesized compounds on Wistar rats. (Table Removed) We claim: 1. A process for the preparation of diallyldisulphide derivatives, the said process comprising steps of: (a) treating 2-mercapto ethanol with liquid bromine in presence of a suitable aqueous inorganic base in a halogenated hydrocarbon solvent around 0°C, (b) separating the organic phase and extracting the aqueous phase with 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 cone, 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-bromoethyl) 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 derivative represented by general formula (I) and optionally, converting the derivative into their pharmaceutically acceptable salts in a manner as herein described. 2. A process as claimed in claim 1 wherein in step (a) and (b), the halogenated hydrocarbon is selected from a group consisting of carbon tetrachloride, methylenechloride, dichloromethane and /or chloroform. 3. A process as claimed in claim 1 wherein in step (c), the stirring is carried out at an ambient temperature for 24 hours followed by heating for 3 hours. 4. A process as claimed in claim 1 wherein in step (d), the hydrohalogen acid is selected from a group consisting of hydrochloric, hydrobromic and hydroiodic acid, preferably hydrobromic acid. 5. A process as claimed in claim 1 wherein in step (d), the stirring at ambient temperature is carried out for 24 hours followed by heating for 3 hours. 6. A process as claimed in claim 1 wherein in step (e), the alkali carbonate used is selected from sodium or potassium carbonate, preferably sodium carbonate. 7. A process as claimed in claim 1 wherein in step (f), the triaryl phosphine is triphenyl phosphine. 8. A process as claimed in claim 1 wherein in step (h), the benzaldehyde used is unsubstituted or substituted with a functional groups. 9. A process as claimed in claim 1 wherein in step (h), the substituted benzaldehyde is selected from electron withdrawing or donating group or combination thereof, preferably mono or disubstituted benzaldehyde. 10. A process as claimed in claim 1 in step (h), the refluxing is performed for 24 hours. 11. A process of claim 9 wherein the substituted benzaldehyde used consists of substitution with functional groups selected from a group consisting of nitro, cyano, carboxyl trifluoromethyl, methoxyl, amino, or combination thereof. 12. A process for the preparation of diallylsulphide derivatives substantially as herein described with reference to the Examples and Scheme I mentioned in the specification. 13. A process for the preparation of diallylsulphide derivatives substantially as herein described with reference to the foregoing examples.

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