Title of Invention

"NOVEL DIALLYLDISULPHIDE COMPOUNDS HAVING ANTILIPIDEMIC AND ANTIOXIDANT ACTIVTITY"

Abstract The present invention relates to novel diallyldisulphide analogues of general formula I having antilipidemic and antioxidant activity, equivalent to or greater than the available standard drugs, a process of preparation of such analogues and pharmaceutical composition containing such analogues. Formula I is represented by the structure shown here below:
Full Text NOVEL DIALLYLDISULPHIDE COMPOUNDS HAVING ANTILIPIDEMIC AND ANTIOXIDANT ACTIVITY
Field of the Invention
The present invention relates to novel diallyldisulphide analogues of general formula I having antilipidemic and antioxidant activity, equivalent to or greater than the available standard drugs, a process of preparation of such analogues and pharmaceutical composition containing such analogues. Formula I is represented by the structure shown here below:
R-CH=CH-CH2-S-S-CH2-CH=CH-R Formula 1
Wherein R=C6HnXp[when n = 3 to 5, p = 2 to 0; X= H, Cl, Br, F, CF3, CH3, CN, COOH, N02) NH2, OH, OCmH2m+i(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 et.al, Atherosclerosis 113, 219-225, (1995)]. Allicin (diallylthiosulfmate) 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. Alliin 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 liallyldisulphide 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 having antilipidemic and antioxidant activity. This is the first report of this class of compound in the literature.
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..
Yet another object of the invention is to provide a method for testing the novel compounds for antilipidemic and antioxidant activities.
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 a novel lead diallyldisulphide analogue which is two times less toxic and more active than the standard compound Lovastatin.
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.
Still another objective is to provide m.p., ER, UV, NMR and MS data of intermediates and final diallyldisulphide analogues.

Summary of the Invention
The present invention relates to novel diallyldisulphide analogues of general formula (I), having antilipidemic and antioxidant activity equivalent or greater than the standard available drugs and also associated with less toxicity, a process of preparation and its pharmaceutical composition of
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, OCmH2m+i(m = 1 to 8)]
Description of the Invention
The invention essentially relates to novel active diallyldisulphide analogues which are represented by general formula I 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.
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, OCmH2m+i(m = 1 to 8)]
In an embodiment of the invention, diallyldisulphide derivatives are represented by the structural formula I and pharmaceutically acceptable salts thereof..
R-CH = CH - CH2-S-S-CH2-CH = CH-R
(I)
Wherein R=C6H5, C6H5NO2, R=C6H5NH2, R=C6H5OCH3 and/or R=C6H5COOH
In another embodiment of the invention, a process for the preparation of diallyldisulphide derivatives comprises 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, 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 © 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 selected from group of anisaldehyde, nitrobenzaldehyde aminobenzaldehyde, chlorobenzaldenyde etc.
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 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, 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 characterised by m.p., ER, 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.
OH-CH2-CH2SH HOCH2-CH2-S-S-CH2-CH2OH
(I)
Br'[Ph3P+CH2CH2-S-S-CH2CH2P+Ph3]Br' (HI) (II)
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, N02, NH2, OH, OCmH2m+](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 Preparation of Bis(2-hydroxy ethyl) disulphide (I)
Dichloromethane (40 ml, 2-mercaptoethanol (3 ml, 40 mM) and 20% aqueous
potassium bicarbonate is taken in a round bottom flask. The flask is immersed in an
ice bath, and bromine (2 ml, 40 mM) in dichloromethane is added dropwise to the
reaction vessel. The organic phase is separated and the aqueous phase is extracted
with dichloromethane. The organic phases are combined and dried over anhydrous
sodium sulphate. The solvent is evaporated to yield the pure disulphide (I, 1 g).
Yield 33.8%.
TLC (solvent system: Ethyl acetate Hexane 2: 8): Rf- 0.13
IR (Thin film) (Major peaks): 3330 cm'1 (OH str), 2925 cm'1 (CH str.), 696 cm'1 (CS
str)
'H NMR (300MH,, CDC13): 5 2.90(m, 4H, 2xS-CH2); 2.99(s,2H, 2xOH); 3.90 (m,
4H, 2xCHj-O)

Example 2 Preparation of Bis(2-bromoethyl) disulphide (II)
48% hydrobromic acid in water (70 ml) is taken in a 500 ml 3 necked flask, fitted
with a stirrer, condenser and dropping funnel. The flask is cooled in an ice bath and
concentrated sulphuric acid is added to hydrobromic acid with stirring the resulting
ice cold solution, compound II (2g) is added dropwise. The reaction mixture is stirred
for 24 hours at room temperature. After this, the reaction mixture is heated for 3
hours on a steam bath. Two layers separated out. Dichloromethane (10 ml) is added.
The upper layer was taken, washed with water followed 10% Na2CO3 solution and
then dried over anhydrous Na2S04. The dichloromethane is evaporated using a
rotavapor and the desired compound III (2 g, 55%)is obtained. Yield - 2g % Yield
55%
T.L.C. (Solvent System; 20:80: Ethyl acetate: Hexane)
IR (thin film) (Major peaks) 2922 cm'1 (C-H str;); 617 cm"1 (C-S str); 563 cm'1 (C-Br
str); 444 cm"1 (-S-S str)
'HNMR (300MHZ; CDC13); 8 3.10 (t,4H, J = 7.59HZ, 2xS-CH2); 3.62(t,4H, J=6.65HZ,
Example 3 Preparation of Phosphonium bromide analogue (III)
Compound II (2.8 g, 0.01 M) and triphenyl phosphine (5.5 g, 0.02 M) taken in a round bottom flask. Dry dimethyl formamide (15 ml) is added to the reactants. The reaction mixture is refluxed under stirring for 5 hours. The mixture is allowed to cool to room temperature and hexane (15 ml) is added. The desired phosphonium salt precipitated out. The solution is filtered, washed with hexane and dried. White crystal of compound III is obtained. Yield - 5.6 g; % Yield 71.7; Melting point -130°C
T.L.C. (Solvent System 20:80; Ethyl acetate; Hexane) R,-0.35 IR (KBr. pellets) 3053 cm"1 (C-H str); 691 cm"1 (-C-S- str)
'HNMR (300MHZ; CDC13); 5 1.66 (s,4H, 2xS-CH2), 2.61(t,4H, J = 5.4 Hz, 2xPh3PfCH2); 7.40-7.75 (m,30H, phenyl ring protons)

Example 4 Preparation of Bis (p-nitrophenyl)-diallyldisulphide (IV)
Lithium ethoxide (0.0174 g, 25 mM) and ethanol (50 ml) is taken in a dry round
bottom flask. The flask was flushed with argon gas and phospohonium salt (III)
(0.779 g, 1 mM) in super dry ethanol (10 ml) is added, p-nitrobenzaldehyde
dissolved in superdry ethanol (5 ml) and tetrahydrofuran (3 ml) is added to the
reaction mixture. The reaction mixture is refluxed under stirring for 24 hours. The
reaction mixture is then poured on to eye, the final compound (IV) precipitates out.
The compound is filtered and dried. Yield 200 mg; % yield 60.6; Melting point
140°C
TLC (Solvent system 20:80 : Ethyl acetate: Hexane) Rf 0.4
IR (KBr pellet): 3055 cm"1 (C-H str): 1305 cm'1 (NO str)(sym) : 1583 cm'1 (N-O
str)(anti): 690.5 cm"1 (C-S str)
'HNMR (300MHZ; CDC13 + DMSO); 5 1.63 (s,4H, 2xS-CH2): 4.75 (s,4H, 2x-
CH=CH;), 1.26-1.IS (m,8 H, phenyl ring 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 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 (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.
4. Experimental Protocol
Rats were divided in five equal groups. Five rats in each group.
GROUP-1 Orally administered equivalent amount of saline orally, treated as
control. GROUP-2 Orally administered 20mg/kg.b.wt. with diallyl thiosulphinate fraction
(Allicin extract) GROUP-3 Orally administered 10 mg/kg.b.wt. with Bis(4-nitrophenylallyl)
disulphide GROUP-4 Orally administered 20 mg/kg.b.wt. with Bis(4-nitrophenylallyl)
disulphide GROUP-5 Orally administered 20 mg/kg.b.wt. with Lovastatin
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% NaCl), and extraneous material was removed. For biochemical studies, approximately Ig 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 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/1 Oml of saline arsenate sol.) and diluted perchloric acid (50ml of HCICVL 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 •eagent was added to the same tube and the tube was vortexed.
r 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% 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-methanoi was added. The lower layer contains the purified lipid.

6c. Estimation of total cholesterol
Por quantitative estimation of total cholesterol the method of Zlatkis et al. (1953) and Hanley (1957). A known amount of (200 u]) 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 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 rag/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 (~100ul) 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 (al of 0.4 N H2SO4 was added and placed in boiling water bath for 10-15 min. After that 50 |al of 0.05 N sodium metaperiodate was added. Excess Nal04 was neutralized with 0.1 ml of 0.5 M NaAsOa. A brown coloration developed after the addition of 5 ml of 0.108% chromotropic acid in 50% HaSO-j. 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 Mitochondrial Supernatant (PMS) obtained after centrifugation at 10,000 x g.

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 RJ; Pharmacology, 1 1, 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 10~3M~1cm'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 Claiborae (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 H2C»2 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 (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"
Ref.: Habig, W.H., Pabst, M.J., and Jaokby, W.B.J. Biol. Chem. 249, 7130-7139 (1974).
6i.Lipid peroxidation
The assay for microsomal lipid peroxidation was done following the method of Wright et al (1981), The reaction mixture in a total volume of 1.0 ml contained 0,58ml phosphate buffers (0.1M, pH7.4), 0.2ml microsome obtained from the homogenate (10%w/v), 0.2ml ascorbic acid (lOOmM), and 0.02 ml ferric chloride (lOOmM). The reaction mixture was incubated at 37°C in a shaking water bath for one hour. The reaction was topped by the addition of 1 ml of TCA (10%). Following addition of 1.0 ml of TBA (0.67%), all the tubes were placed in a boiling water bath for a period of twenty minutes. In the end, tubes were shifted to crushed ice bath and then centrifuged at 250xg for 10 minutes. The amount of malondialdehyde formed in each of the samples was assessed by measuring the optical denisity of the supernatant at 535 nm using a spectrophotometer against a reagent blank. The results were expressed as nmol MDA formed/hour/gm of tissue at 37°C by using molar extinction coefficient of 1.56 x 105M"Wl.
Wright, J.R., Colbe H.D. & Miles, P.R. Arch. Bio Chem. Biophys. 1981, 206, 296-304
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%), CuSO4 (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.K., Rosebrough, N.J., Fair, A.L. and Randall, R.J., J. Biol. Chem., 193,265-275(1951).

Biological Activities of Bis [4-nitro phenyl allyl] disulphide
Effect of Compound (10 mg/Kg. b. wt.,20mg/Kg.b.wt), Lovastatin
(20 mg/Kg.b.wt.) and Allicin (Garlic extract) each given for 2 days on Lipid
profile of Wistar rats
Table I Cholesterol
(Table Removed)

Triglyceride

(Table Removed)
HMG-CoA Reductase (Table Removed)
Effect of Compound (10 rag/Kg, b. wt., 20mg/Kg.b.wt.), Lovastatin ^20 mg/Kg.b.wt.) and Allicin (Garlic extract) given for 2 days on Oxidative stress
in Wistar rats
Reduced Glutathione
Table II


(Table Removed)

(Table Removed)

(Table Removed)
(,imol of HiOi consumcd/min./g.wet weight tissue
},ipid Peroxidation

(Table Removed)
* nmol of MDA conjugate formed / hour /g. of tissue at 37°C











We Claim:
1. Novel diallyldisulphide compound(s) having Formula (I) and their
pharmaceutically acceptable salts thereof, said Formula (I) has the structure
represented here below:
(Formula Removed)
Wherein R=C6HnXp [where n= 3 to 5, p = 1 to 2; X = Cl, Br, F, CF3, CH3, CN, COOH, NO2, NH2, OH, OCmH2m+1 (m=l to 8)
2. The compound as claimed in claim 1, wherein R=C6H4NO2 (4); C6H3(NO2)2 (2,4); C6H4COOH(4); C6H4CN(4); C6H3(COOH)2(2,4); C6H3(CN)2(2,4); C6H2(NO2)3(2,4,6); C6H3NO2(2)COOH(4); C6H4(CF3)(4) or C6H4CF3(2).
3. The compound as claimed in claims 1 and 2, wherein the pharmaceutically acceptable salts are selected from hydrochloride, hydrobromide, citrate, maletae, fumarate, camphorsulphonic acid, sodium or potassium.
4. A pharmaceutical composition comprising an effective amount of compound of formula I as claimed in preceding claims along with pharmaceutically acceptable additives, for providing antilipidemic and antioxidant activity to a subject.
5. The composition as claimed in claim 4, wherein the pharmaceutically acceptable salts are additive, carrier, diluent, solvent, filter, lubricant, excipent, binder or stabilizer.
6. Novel diallyldisulphide compound(s) having Formula (I) and their pharmaceutically acceptable salts thereof, and a pharmaceutical composition, substantially as herein described with reference to the foregoing examples.

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Patent Number 245820
Indian Patent Application Number 1154/DEL/2001
PG Journal Number 05/2011
Publication Date 04-Feb-2011
Grant Date 02-Feb-2011
Date of Filing 16-Nov-2001
Name of Patentee DEPARTMENT OF SCIENCE AND TECHNOLOGY
Applicant Address TECHNOLOGY BHAVAN, NEW MEHRAULI ROAD, NEW DELHI, 110016. INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 MANISHA TIWARI DR. B.R. AMBEDKAR CENTER FOR BIOMEDICAL RESEARCH, UNIVERSITY OF DELHI-110007, INDIA.
2 MEENAKSHI SHARMA DR. B.R. AMBEDKAR CENTER FOR BIOMEDICAL RESEARCH, UNIVERSITY OF DELHI-110007, INDIA.
3 RAMESH CHANDRA DR. B.R. AMBEDKAR CENTER FOR BIOMEDICAL RESEARCH, UNIVERSITY OF DELHI-110007, INDIA.
PCT International Classification Number C01B 17/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA