FIELD OF THE INVENTION:
The present invention relates to a novel compound useful for the treatment & prevention of atherosclerosis from the plant Trigonella foenum-graecum and a process for its preparation.
The condition of atherosclerosis is associated with the accumulation of LDL -derived cholesterol in the intima of susceptible arteries with the development of fatty streaks. The liver plays a central role in whole body cholesterol homeostasis, as it is the site for cholesterol metabolism and neutral sterol elimination. Besides, it is the regulator of plasma cholesterol concentrations through the synthesis of VLDL and catabolism of LDL via the apo B, E (LDL) receptor. Thus changes in hepatic receptor activity produce changes in plasma LDL level.
The drugs used for the treatment and prevention of atherosclerosis retard or combat the disease by enhancing hepatic cholesterol utilization by its degradation into bile acids or by secondary mechanisms, such as an increase in the catabolism of LDL via the LDL receptor.
We, in our publication in the Journal titled "Atherosclerosis " (An ELSEVIER Publication) vol 123, (1996) 235 - 241, have already proved the influence of the alcoholic extract of the plant Crataegus oxycantha (berry part of the plant) in up-regulating the LDL receptor activity in rats. A Finland company is marketing this extract in the name "Cretemax", a heart tonic, quoting our paper.
PRIOR ART KNOWLEDGE:
A number of studies have shown that saponins from different sources such as Jamaican bitter yam, mung beans & Soya, lower cholesterol levels and act as anti-atherosclerotic agents in a variety of animals including humans. Reference in this context may be made to West Indian Medical Journal, vol 54(2), (2005), 97-

101 and British Journal of Nutrition, vol 88(2002), 8287-8292. Besides, the ethanol extract of de-fatted fenugreek seeds inhibited taurocholate and deoxycholate absorption in vitro, in a dose - dependent manner in everted intestinal sacs. Saponins also reduce the more harmful LDL-cholesterol selectively in the serum of rats, gerbils and humans. Although saponin induced accelerated metabolism of cholesterol in the liver may account for the decrease in the serum cholesterol level, Soybean and Lucerne saponins were found to be not singularly responsible for the hypocholesterolemic effect but act in conjunction with the hypocholesterolemic diet to reduce the serum cholesterol level.
STRUCTURE OF SAPONIN :
Saponins consist of a sugar moiety usually containing glucose, galactose, glucuronic acid, xylose, rhamnose or methyl pentose, glycosidically linked to a hydrophobic aglycone (sapogenin), which may be triterpenoid or steroid in nature. The aglycone may contain one or more unsaturated C-C bonds. The oligosaccharide chain is normally attached at the C3 position, but many saponins have an additional sugar moiety at the C26 or C2s position (bidesmosidic). The general structure of saponins is given below


Taking the lead from the above information we undertook studies on the plant Trigonella foenum-graecum to find out whether this plant contains saponins, which can act as an anti - atherosclerotic agent. Our in vivo studies resulted in the findings that the alcoholic extract of the powder of the seeds of the plant Trigonella foenum-graecum has excellent anti-atherosclerotic potential .We have observed that the up regulation (namely increase in the number of LDL receptors in the liver plasma membrane) of hepatic LDL receptors, the greater influx of plasma LDL and VLDL into liver and the reverse cholesterol pathway via bile acid excretion as the mechanisms by which the alcoholic extract acts as an antiatherosclerotic agent.
Therefore, the main objective of the present invention is to provide a novel compound falling within the class of sapogenin from the plant Trigonella foenum-graecum that is useful for the treatment and prevention of atherosclerosis.
Another objective of the present invention is to provide a process for the isolation of a new compound falling within the class of sapogenin from the plant Trigonella foenum-graecum that is useful for the treatment and prevention of atherosclerosis
Accordingly, the present invention provides a new compound falling within the class of sapogenin that is useful for the treatment and prevention of atherosclerosis from the plant Fenugreek (Trigonella foenum- graecum).
Accordingly, to another embodiment of the present invention there is provided a process for the preparation of a new compound falling within the class of sapogenin that is useful for the treatment and prevention of atherosclerosis from Fenugreek (Trigonella foenum- graecum).
A novel saponin was isolated from the n-Butanol extract of the seeds of Fenugreek (Trigonella foenum- graecum). It consists of a hydrophobic aglycone (sapogenin) with sugar moieties linked to it. This novel compound shares a

common structural feature with saponins and possesses differential glycosylation pattern that makes it unique among other known saponins.
Accordingly, there is provided a process for the preparation of a the novel compound falling within the class of sapogenin that is useful for the treatment and prevention of atherosclerosis from Fenugreek (Trigonella foenum-graecum) which comprises..
(i) Finely powdering the seeds of the plant Fenugreek and subjecting the
powder to phase separation in water and ethyl alcohol
(ii) treating the fraction isolated from step (i) with isobutanol-butanol
(iii) evaporating the eluate obtained in step (ii) to dryness
(iv) suspending the dried product obtained in step (iii) in ethyl alcohol
(v) subjecting the ethyl alcohol extract to thin layer chromatography
employing dehydrating reagents and anisyl aldehyde in sulfuric acid and .
(vi) separating the third fraction containing the novel compound by
extracting the fraction with ethyl alcohol
The structure and chemical nature of the fractions were determined through NMR and IR spectroscopy. The NMR data showed a basic pattern that resembles the saponin scaffold. The saponins were also subjected to hydrolysis to characterize the glycosidic moieties that showed a novel pattern. Glycosidase treatment was also performed to separate and characterize novel glycosidic moieties.
This novel saponin was tested against its anti-atherosclerotic activities. Our study revealed that an up-regulation of LDL receptors in the liver plasma membranes and a greater influx of plasma LDL and VLDL into the liver. It also promoted the reverse cholesterol pathway via bile acid secretion.
Experiments, in vitro, employing everted - sac technique showed that the ethanol extract had the ability to inhibit taurocholate and deoxycholate absorption in a dose dependent manner. Besides the reduction in the plasma cholesterol levels

ranged from 17 % to 25% and low liver cholesterol was observed. It also lowered total plasma cholesterol without altering triglycerides.
The in vitro bioactivity of this novel saponin was studied using LDL isolated from human plasma by density gradient centrifugation. The pure LDL fraction was treated with the novel sapogenin prepared by the process described above. Thereafter the resulting mixture was exposed to Cu2+ It was'observed that Cu2+ did not oxidize the LDL. It was concluded therefore that the novel saponin had anti atherosclerotic properties.
The exceptional efficacy of the novel saponin isolated by us from Fenugreek by alcohol (isobutanol) extraction process is partially attributed to its antioxidant and LDL receptor upregulation activities. Although reverse cholesterol mechanism alone could potentially lower the risk of atherosclerosis, combined with antioxidant activities, the Fenugreek extract suppresses the pathophysiological course of atherosclerosis at its incipient level, thus making it a most promising preventive and therapeutic drug.
The antiatherosclerotic nature of the new saponin of the present invention appears to be a glycosylation dependent property. Differential glycosylation identified through NMR and IR spectroscopy on the hydrophobic aglycone (saponin) scaffold confers specificity to LDL resistance to oxidation and promotes LDL receptor activities. This novel saponin might also act by up-regulating glucose-6—phosphate dehydrogenase activity and might protect biomembranes (Lipid peroxidation) under oxidant-laden environments.
ADVANTAGE OF THE INVENTION:
• The new compound is a potential drug for the treatment and prevention of atherosclerosis and is a safe herbal drug
• The plant is abundantly available in the country and in all seasons
• The process of extraction is simple, economical & environmentally friendly
• The new compound can be made available to the needy persons at reasonable and affordable price.

We Claim:
1. A novel compound falling within the class of sapogenin that is useful for
the treatment and prevention of atherosclerosis from the plant Fenugreek
(Trigonella foenum-graecum).
2. A process for the preparation of a novel compound falling within the class
of sapogenin that is useful for the treatment and prevention of atherosclerosis
from Fenugreek (Trigonella foenum-graecum) which comprises:
(i) Finely powdering the seeds of the plant Fenugreek and subjecting the
powder to phase separation in water and ethyl alcohol (ii) treating the fraction isolated from step (i) with isobutanol-butanol (iii) evaporating the eluate obtained in step (ii) to dryness (iv) suspending the dried product obtained in step (iii) in ethyl alcohol (v) subjecting the ethyl alcohol extract to thin layer chromatography
employing dehydrating reagents and anisyl aldehyde in sulfuric acid and . (vi) separating the third fraction containing the novel compound by extracting
the fraction with ethyl alcohol
3. A novel compound falling within the class of sapogenin that is useful for the treatment and prevention of atherosclerosis from the plant Fenugreek (Trigonella foenum-graecum) substantially as herein described.
4. A process for the preparation of a novel compound falling within the class of sapogenin that is useful for the treatment and prevention of atherosclerosis from the plant Fenugreek (Trigonella foenum-graecum) substantially as herein described.