US20050281901A1

US20050281901A1 - Composition to enhance HDL cholesterol and to decrease intima-media thickening in animals and humans and a method for its preparation

Info

Publication number: US20050281901A1
Application number: US11/111,798
Authority: US
Inventors: Benny Antony
Original assignee: Arjuna Natural Extracts Ltd
Current assignee: Arjuna Natural Pvt Ltd
Priority date: 2003-03-03
Filing date: 2005-04-22
Publication date: 2005-12-22
Also published as: US7780996B2; US20150104532A1; US20130309339A1; US9757423B2; US10434129B2; US8945634B2; WO2004078190A1; EP1638585A1; US20110014309A1; US20170333507A1; US8158167B2; EP1638585A4; JP2006514670A; US8455020B2; US20070196525A1; US20120195990A1; AU2003241146A1

Abstract

A method of producing a product to correct hypercholesterolemia comprising pulping fruits of Emblica Officinalis with demineralized water to create a slurry. The slurry is treated with pectinase. The slurry is filtered to create a solution. The solution is concentrated to create a product.

Description

TECHNICAL FIELD

This is a Continuation of International Application PCT/IN2003/000137, with an international filing date of Apr. 3, 2003 and claiming priority of India Patent Application 169/MAS/2003 filed on Mar. 3, 2003, both of which documents are incorporated herein by reference.
This invention relates to a product (nutritional supplement) to correct dyslipidemia in animals and humans and to combat intima-media thickening thus affording significantly improved therapeutic and prophylactic cardioprotection, the said composition being an extract of Emblica officinalis, Gaertn (Euphorbiaceae) prepared without using any organic solvent and without resorting to any chemical processing steps.

BACKGROUND OF THE INVENTION

E. officinalis (Amla) fruit is one of the key constituents of the celebrated Ayurvedic preparation, Chyavanaprash, used in India for thousands of years as a vitalizing and rejuvenating health tonic. The low molecular weight hydrolyzable, gallo- and ellagi tannins (Ghosal, S. et al., Indian J. Chem., 1996, 35B, 941-48) of the fruit provide multi-pronged benefits arising out of their antioxidative, hypocholesterolemic, immunomodulating and HMG CoA reductase inhibitive properties. While its LDL-cholesterol lowering property has been described in published literature, the more desirable property of enhancing HDL cholesterol, as described in the present invention, was not noted before. We have also now surprisingly found that E. officinalis fruit extract reduces intima-media thickening in experimental animals. Thus the beneficial effects of the present inventive composition goes beyond the simple correction of LDL cholesterol levels, as achieved in prior art.
Coronary heart disease (CHD) continues to be the major cause of premature death in most developed and developing countries. A low level of HDL cholesterol is the second most important risk factor for CHD, as demonstrated in numerous clinical and epidemiological studies (Gorden, D. and Rifkind, H. M., N. Engl. J. Med., 1989, 321:1311-1315; Brewer, Jr., H. B., New Engl. J. Med., 2004, 350:1491-94) and HDL has emerged, during the past decade, as a new potential target for the treatment of cardiovascular diseases. The key role of HDL as a carrier of excess cellular cholesterol in the reverse cholesterol transport pathway is believed to provide protection against atherosclerosis. In reverse cholesterol transport, peripheral tissues, for example, vessel-wall macrophages, remove their excess cholesterol through the ATP-binding cassette transporter 1 (ABCA1) to poorly lipidated apolipoprotein A-I, forming pre-β-HDL. Lecithin-cholesterol acyltransferase then esterifies free cholesterol to cholesteryl esters, converting pre-β-HDL to mature spherical α-HDL.
HDL cholesterol is transported to the liver by two pathways: 1) it is delivered directly to the liver through interaction with the scavenger receptor, class B, type I (SR-BI); 2) cholesteryl esters in HDL are transferred by the cholesterol ester transferase protein (CETP) to very-low-density-lipoproteins (VLDL) and low-density lipoproteins (LDL) and are then returned to the liver through the LDL receptor. HDL cholesterol that is taken up by the liver is then excreted in the form of bile acids and cholesterol, completing the process of reverse cholesterol transport (Brewer, H. B. Jr., Arterioscl. Thromb. Vasc. Biol., 2004, 24:387-91). HDL is believed to have the ability to remove cholesterol from macrophages, thus preventing the formation of foam cells.
A second beneficial role of HDL in atherosclerosis is in protecting LDL from oxidation (Navab, M. et al, Circulation, 2002, 105:290-92). Unlike normal LDL, oxidized LDL is readily taken up by macrophage scavenger receptor SR-A or CD36 resulting in the formation of foam cells. Foam cells are a major component of the early atherosclerotic lesion. Further, HDL may slow the progression of lesions by selectively decreasing the production of endothelial cell-adhesion molecules that facilitate the uptake of cells into the vessel wall (Barter, P. J., et al, Curr. Opin. Lipid, 2002, 13:285-88). HDL may also prolong the half-life of prostacycline and preserve its vasodilatory effect (Mackness, M. I. et al, Atherosclerosis, 1993, 104:129-35).
Several lines of evidence support the concept that increasing the HDL level may provide protection against the development of atherosclerosis. Epidemiologic studies have shown an inverse relation between HDL cholesterol levels and the risk of cardiovascular disease. Increasing the HDL cholesterol level by 1 mg may reduce the risk of cardiovascular disease by 2 to 3 percent. Overexpressing the apo-A-I gene in transgenic mice and rabbits and infusing complexes consisting of apo A-I and phospholipids into hyperlipidemic rabbits increase HDL cholesterol levels and decrease the development of atherosclerosis (Brewer, H B, Jr., loc. cit). In humans, infusing either of these complexes or pro-apo-A-I results in short term increase in HDL cholesterol, biliary cholesterol and fecal cholesterol loss, reinforcing the concept that elevating the HDL cholesterol level decreases the risk of cardiovascular disease.
More than 40 percent of patients with myocardial infarction have low HDL-C as a cardiac risk factor. (Genest, J. J., et al, Am. J. Cardiol., 1991, 67:1185-89). In the prospective and multicentric European Concerted Action on Thrombosis and Disabilities (ECAT) Angina Pectoris Study, Bolibar et al (Thromb. Haemost., 2000, 84:955-61) identified low HDL-C and low apoA-I as the most important biochemical risk factors for coronary events in patients with angiographically assessed CHD. By convention, the risk threshold value of HDL-C has been defined as 35 mg/dL (0.9 mmol/L) in men and 45 mg/dL (1.15 mmol/L) in women [Expert panel on detection, evaluation and treatment of high blood cholesterol in adults. The second report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation and treatment of high blood cholesterol in adults (Adult Treatment Panel II). Circulation. 1994; 89:1329-1445)]. Because of interaction, the strength of the association between HDL-C and cardiovascular risk depends on the presence of additional risk factors. Therefore, threshold values are higher in men with diabetes mellitus or hypercholesterolemia or in the presence of multiple risk factors (von Eckardstein A, and Assmann G. Curr Opin Lipidol. 2000; 11:627-637). Low HDL-C has been identified as the most frequent familial dyslipoproteinemia in patients with premature myocardial infarction (Genest, J. J. Jr., Circulation. 1992; 85:2025-2033). Finally, in the Helsinki Heart Study (Manninen, V. et al, Circulation. 1992; 85:37-45) and the High-Density-Lipoprotein Cholesterol Intervention Trial of the Department of Veterans Affairs (VA-HIT) study (Rubins, H. B. et al, N Engl J. Med. 1999; 341:410-418), increases of HDL-C on treatment with gemfibrozil were correlated with the prevention of CHD events. Thus, HDL-C has become an important component of algorithms to assess the global cardiovascular risk of patients and also a target for therapeutic intervention and for the definition of treatment goals.
Strategies to correct dyslipidemia in atherosclerosis generally involve diet and/or drugs. The threshold serum total cholesterol and LDL cholesterol concentrations above which diet and drug therapy should be initiated, as well as the goals of therapy, have been defined by the National Cholesterol Education Program (JAMA, 1993,269:3015-23). The target serum LDL-C is <160 mg/dl (4.3 mmol/l) for patients with no risk factors or only one risk factor for CHD; <130 mg/dl (3.4 mmol/l) for patients with 2 or more risk factors and less than 100 mg/dl (2.6 mmol/l) for those with CHD. Persons with diabetes also fall into the third category. A reasonable target for triglyceride concentration is 200 mg/dl or less; higher values are associated with a doubling of the risk of cardiovascular disease when serum cholesterol concentration exceeds 240 mg/dl or when the LDL-C/HDL-C ratio exceeds 5:1.
A number of studies have shown that reducing serum LDL-C below the target levels does not necessarily result in proportional reduction in the risk of CHD [(The Scandinavian Simvastatin Survival Study Group. Randomized trial of cholesterol lowering in 4444 patients with coronary heart disease, Lancet, 1994, 344:1383-89; Shepherd, J. et al, N. Engl. J. Med., 1995, 333:1301-7; Sachs, F. M. et al, N. Engl. J. Med., 1998, 315:1001-9; Circulation, 1998, 97:1446-52; The West of Scotland Coronary Prevention Study Group, Circulation, 1998, 97:1440-45; Pederson, T. R., Circulation, 1998, 97:1453-60] because of the attenuation of the cholesterol-heart disease relation at lower serum cholesterol concentrations (Grundy, S. M., Circulation, 1998, 97:1436-39).
Dietary treatment of hyperlipidemia is a necessary foundation for drug treatment. Depending on the degree of hyperlipidemia, the Step I and Step II diets can be introduced sequentially. The Step II diet contains no more than 30% of calories from fat, less than 7% of calories from saturated fatty acids and less than 200 mg of cholesterol per day. In long term studies, the Step II diet decreased serum LDL-C concentrations 8-15% (Knopp, R. H., et al, JAMA, 1997, 278:1509-15; Walden, C. E., Arterioscl. Thromb. Vasc. Biol., 1997, 17:375-82; Denke, M. A., Arch. Intern. Med., 1995, 156:17-26). Diets more restricted in fat than the Step II diet result in little additional reduction in LDL-C, raise serum TG concentration and lower HDL-C.
The point to note, from the above, is that reducing LDL-C alone is of little value in reducing the risk of CHD. Further, diets meant for reducing LDL-C may reduce HDL-C to a similar degree (Hunninghake, D. B. et al, N Engl. J. Med., 1993, 328:1213-19; Schaefer, E. J., et al, Arterioscl. Thromb. Vasc. Biol., 1995, 15:1079-85); Stefanick, M. L., N. Engl. J. Med., 1998, 339:12-20).
Drug therapy is resorted to when the desired effects are not achieved with diets alone. Statins are the most popular among the lipid lowering drugs. These drugs lower serum LDL-C concentrations by upregulating LDL-receptor activity as well as reducing the entry of LDL into the circulation. The maximal reductions achieved with a statin ranges from 24-60%. Statins also reduce the serum TG levels; but they are often insufficient. Statins are ineffective in the treatment of patients with chylomicronemia. Adverse effects of statins include, gastrointestinal upset, muscle aches and hepatitis. Rarer problems include myopathy (muscle pain with serum creatine kinase concentrations more than 1,000 Upper litre), rashes, perpheral neuropathy, insomia, bad or vivid dreams and difficulty in sleeping or concentrating (Abramowica, M., Med. Lett., 1996, 38:67-70; Vgontzas, A. N. et al, Clin. Pharmacol. Ther., 1991, 50:730-37; Roth, T. et al, Clin. Cardiol., 1992, 15:426-32; Partinen, M. et al, Am. J. Cardiol., 1994, 73:876-80). Other lipid-lowering drugs include bile acid-binding resins (e.g, cholesteramine and colestipol), nicotinic acid, and fibrates.
Drug therapy is not recommended for premenopausal women and men under 35 years of age unless they have serum LDL-C concentrations of more than 220 mg/dl (5.7 mmol/l), because their immediate risk of heart disease is low [Summary of the second report of the National Cholesterol Education Program (NCEP): expert panel on detection, evaluation and treatment of high blood cholesterol in adults, JAMA, 1993, 269:3015-23].
Thus, diets alone or in conjunction with lipid lowering drugs fail to yield the desired goal of safe lipid lowering. However, this goal is acheivable with the present inventive composition containing the active principles of Emblica officinalis Emblica has been in safe use in India for thousands of years as component of Ayurvedic preparations. The composition offers the twin benefits of reducing the harmful LDL cholesterol and enhancing the desirable HDL cholesterol.
Further, the composition was found to reduce the intima-media thickening of the arteries in experimental animals which is an added benefit. Such an effect has not been observed before.
The increased thickness of intima plus media of the carotid artery is associated with the prevalence of cardiovascular diseases and a number of studies have shown a positive association between cardiovascular risk factors and carotid intima-media thickness (IMT)(O'Leary, D. H. et al., Stroke, 1992, 22:1156-63; 1992, 23:1752-60; New Engl. J. Med., 1999, 340(1):14-22; Howard, N. et al, Ann. Int. Med., 1998, 128(4):262-69); Zureik, M. et al, Stroke, 1999, 30:550-55; del Sol, A. I. et al, Stroke, 2001, 32:1532-38). Howard et al (loc.cit.) makes the following statement: For each 0.03 mm increase per year in carotid arterial IMT, the relative risk for non-fatal myocardial infarction or coronary death was 2.2(95% CI, 1.4-3.6) and the relative risk for any coronary event was 3.1. Absolute IMT was also related to risk for clinical coronary events. Absolute thickness and progression in thickness predicted risk for coronary events beyond that predicted by coronary arterial measures of atherosclerosis and lipid measurements. A growing number of epidemiological studies and clinical trials use IMT as an early marker of systemic atherosclerosis (Zanchetti, A., et al, J. Hypertens., 1998, 16:949-61; MacMahon, S. et al, Circulation, 1998, 97:178-90: Borhani, N. O. et al, JAMA, 1996, 124:548-56; Hodis, H. N. et al, Ann. Intern. Med., 1996, 124:548-52).
IMT is increasingly being used in clinical trials as surrogate end point for determining the success of interventions that lower risk factors for atherosclerosis. To distinguish early atherosclerotic plaque formation from thickening of the intima-media, the following consensus has evolved (Touboul, P. J. et al, Mannheim Intima-Media Thickness Consensus. on Behalf of the Advisory Board of the 3rd Watching the Risk Symposium 2004, 13th European Stroke Conference, Mannheim, Germany, May 14, 2004, Cerebrovasc. Dis., 2004, 18(4):346-49): Plaque is defined as a focal structure that encroaches into the arterial lumen of at least 0.5 mm or 50% of the surrounding IMT value or demonstrates a thickness of ≧1.5 mm as measured from the media-adventitia interface to the intima-lumen interface. Standard use of IMT measurements is recommended in all epidemiological and interventional trials dealing with vascular diseases to improve characterization of the population investigated.
Endothelial vasodilator dysfunction and carotid IMT are two indicators of subclinical cardiovascular disease. In a study of a large, community-based cohort of young adults (aged 24-39 years), Jounala et al (Circulation, 2004, 110(18):2918-23) found that IMT was inversely associated with endothelium-dependent brachial artery flow-mediated dilation (FMD). The number of risk factors was correlated with increased IMT in subjects with evidence of endothelial dysfunction. In a related study, FMD and glyceryl trinitrate-induced endothelium-independent vasodilation (GTN) were measured in the brachial artery. IMT of the common carotid artery and insulin sensitivity were also measured. There was a significant positive relation between insulin resistance, as measured by steady-state glucose levels, and IMT. Insulin resistance was negatively correlated with both FMD and GTN. This indicates that both FMD and GTN were also negatively correlated with IMT (Suzuki, M. et al., Am. J. Hypertens, 2004, 17(3):228-32). Similarly, in a study on 252 healthy adults, IMT was significantly greater in subjects with subclinical aortic valve sclerosis (Yamamura, Y., et al, Am. J. Cardiol., 2004, 94(6):837-39). To determine whether IMT is related to an increased risk of cardiovascular event after percutaneous coronary angioplasty (PTCA), IMT was measured within 2 days following PTCA in 88 patients (mean age 62 years) in another study. A common carotid IMT>0.7 mm was associated with an increased risk of cardiac events after PTCA (Lacroix, P. et al., Int. Angiol., 2003, 22(3):279-83).
Low HDL cholesterol was associated with increased IMT independent of other risk factors in healthy subjects from families with low HDL cholesterol (Alagona, C., et al, Eur. J. Clin. Invest., 2003, 33(6):457-63). Conversely, increased HDL cholesterol was negatively correlated with IMT (Blankenhorn, D. H., et al, Circulation, 1993, 88(1)20-28; Bonithon-Kopp, C. et al, Arterioscl. Thromb. Vasc. Biol, 1996, 16:310-16).
A statistically significant IMT greater than 0.8 mm was associated with coronary artery disease with an odds ratio of 2.4 in Indian subjects (Jadhav, U. M. and Kadam, N. N., Indian Heart. J, 2001, 53(4):458-62). The same authors also found (J. Assoc. Physicians India, 2002, 50:1124-29) a statistically significant association of microalbuminuria with IMT and coronary artery disease in diabetic patients.
In a large population-based of 6943 subjects, carotid IMT and aortic calcification were found to be the strongest predictors of stroke (Hollander, M. et al., Stroke, 2003, (10):2368-72).
From the foregoing, the importance of IMT in cardiovascular disease management is amply evident. Fortunately, IMT is modifiable. Various synthetic drugs, for example, colestipol plus niacin (Blankenhorn, D. H., Circulation, 1993, 88(1):20-28), candesartan (Igarashi, M. et al, Hypertension, 2001, 38(6):1255-59), simvastatin (Detmers, P. A., et al, Circulation, 2002, 106(1):20-23), rampamycin with tacrolimus or cyclosporin (Weller, J. R., et al, Br. J. Surg., 2002, 89(11):1390-95), calcium channel blockers (Wang, J. G. and Staessen, J. A., J. Am. Soc. Nephrol., 2002, 13 Suppl:S208-15), sulfated oligosaccharide PI-88 (Francis, D. J., et al, Circ. Res., 2003, 92(18):70-77), fluvastatin (Ye, P. et al, Chin. Med. Sci. J, 2000, 15(3):140-44), lovastatin (Furberg, C. D., et al, Circulation, 1994, 90:1679-87), chemically modified tetracycline (Islam, M. M., et al, Am. J. Pathol., 2003, 163(4): 1557-66) reduce IMT. There are very few natural products which are reported to suppress intimal thickening. Thus, the finding that Emblica extract can reduce IMT assumes great significance.
The present inventive composition thus offers much improved cardioprotection than any other similar product, natural or synthetic, with the added benefit of its time-tested safety.
Ghosal has disclosed a process for preparation of an extract of Emblica officinalis (U.S. Pat. No. 6,124,268) and a few more by the same inventor for various applications of the preparation, such as, stabilization of vitamin C (U.S. Pat. No. 6,235,721), inhibiting platelet aggregation (U.S. Pat. No. 6,290,996) and antioxidant to block free radical process (U.S. Pat. No. 6,362,167). In none of the above patents, the hypocholesterolemic action, and more specifically, its HDL enhancing property has been described. The present inventive preparation also materially differs in composition from that described by Ghosal. The extraction process described by Ghosal involves treating the fruit pulp with water containing 1% sodium chloride which was then left at room temperature for 12 hours followed by keeping the mixture at 10° C. for 3 days and thus is very time-consuming, costly and tedious. Further, he uses sodium chloride solution for extraction and apparently, the salt remains in the final preparation. This may not be desirable, given the adverse affects of salts for patients with hypertension which is closely associated with cardiac diseases. Further, going by the examples given in the said Ghosal patent, the content of active principles, which he calls as the antioxidant fraction, is less than 4% in the final preparation. Thus, a more commercially attractive process would be highly desirable.

BRIEF DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides for a preparation with standardized contents of bioactive principles suitable for reduction of the harmful LDL cholesterol, and more importantly, the enhancement of the beneficial HDL cholesterol and surprisingly, the reduction of intima-media thickening in experimental animals. Though this has not been proven in humans, it is expected such results may be achieved in human beings as well because IMT is inversely associated with HDL concentration and any intervention to increase serum HDL-C level would reduce IMT.
The objective of the present invention is to provide a safe natural product for treatment of lipid disorders connected with coronary heart disease and stroke without the disadvantages associated with the use of synthetic lipid lowering agents such as statins. More specifically, the invention aims at providing a composition which lowers the harmful LDL cholesterol and at the same time increasing the beneficial HDL cholesterol contents. Low HDL cholesterol is considered as the second most important predictor of coronary heart disease (CHD).
A second objective of the invention is to provide for a composition for potential reduction of intima-media thickening observed in heart patients and which is considered as an important marker and predictor of CHD.
A further objective of the present invention is to provide an easy and economical process for the commercial preparation of E. officinalis extract offering the benefits described above.
These and further objectives of the invention will be apparent from the detailed description of the invention given below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows Table 7; and
FIG. 2 shows Table 8.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a safe natural product for treatment of lipid disorders connected with coronary heart disease and stroke without the disadvantages associated with the use of synthetic lipid lowering agents such as statins. More specifically, the invention aims at providing a composition which lowers the harmful LDL cholesterol and triglycerides and at the same time increasing the beneficial HDL cholesterol contents. Low HDL cholesterol is considered as the second most important predictor of coronary heart disease (CHD) risk. Further, though limited to animal studies, the composition was found to prevent smooth muscle cell proliferation and to reduce the intima-media thickening associated with the process.
The present composition uses the extract of fresh fruits of Emblica officinalis, a tree that occupies a prime position in Ayurvedic preparations for its rejuvenating, vitalizing properties and above all its time-tested safety record. The extract is processed as detailed below to contain a minimum amount of active constituents which are believed to be the low molecular weight hydrolyzable tannins, especially, emblicanin A.
In one embodiment of the process, the invention provides for a composition to correct lipid disorders associated with coronary heart disease, namely high LDL cholesterol and triglycerides and low HDL cholesterol in blood.
In another embodiment of the invention a composition is provided for reducing the intima-media thickness which is increasingly being used as a marker for heart diseases. Very few natural products have been reported to possess this capability.
In yet another embodiment, the invention provides for a process for the commercial scale preparation of such a composition without the use of any organic solvent and without any chemical treatment or additives and thus possesses much superior properties than any of the lipid lowering agents known.
Accordingly, the pulp obtained from fresh amla fruits was treated with a pectinase enzyme for a sufficient length of time at room temperature. The slurry was filtered to yield a clear filtrate which was then spray-dried to get the composition as a dry, free-flowing powder.
The product was then encapsulated in hard gelatin capsules to contain 500 mg of the spray-dried powder which supplies a minimum of 10 wt % of emblicanin A.
Acute and sub-acute toxicities of the products were tested in mice and rats, respectively. Up to a dosage level of 10 g/kg body weight produced no adverse effects such as increased motor activity, tremors, clonic convulsions, piloerection, muscle spasm, hyperesthesia, ataxia, sedation, hypnosis and analgesia in mice. No mortality was recorded in 72 hours. Sub-acute toxicity studies at a dosage level of 2 g/kg body weight for 3 months produced no toxic effects in rats.
The hypercholesterolemic properties of the composition was tested in rabbits. Rabbits were made hypercholesterolemic by oral feeding of cholesterol for 4 months. At the end of 4 months, the treatment groups were administered with the inventive composition for an additional 4 months. Body weight measurements, haematological parameters and lipid profiles of the animals were determined at regular intervals. A near-reversal of the hypercholesterolemic conditions were observed in these animals. There was also reduced activity of the cholesterol-synthesizing enzyme HMG CoA reductase and surprisingly, the thickness of intima plus media also were reduced to normal levels.
The lipid lowering properties of the composition were further tested in hypercholesterolemic human volunteers. The results were in general agreement with those observed in the animal studies. More importantly, there was significant increase in the beneficial HDL cholesterol levels. Recent research has indicated that increasing the HDL cholesterol level is even more important than reducing the LDL cholesterol level.
These and other details of the present invention are explained in more detail in the following non-limiting examples.

EXAMPLE 1

Five hundred kilograms of fresh amla fruits were pulped with an equal quantity of demineralized water and the slurry was treated with 2 wt % of pectinase enzyme under stirring at room temperature for 6 h and then filtered to yield 310 litres of the extract with a solids content of 4.8%. This solution was then concentrated below 60° C. to obtain a slurry with a solids content of 15.2%. This was then spray-dried (inlet temperature 180° C., outlet temperature 90° C.) to obtain 13.5 kg of a free flowing powder. The emblicanin A content of this preparation was 10.2%.

EXAMPLE 2

Toxicity Studies

Acute Toxicity
Healthy albino mice of either sex, having body weight 20-25 g were used. They were housed in clean polypropylene cages with food and water available ad libitum. After acclimatization for one week, their body weights were recorded and were divided into 8 groups of 6 each. Group A served as control and the remaining 7 groups were kept as experimental group. The experimental animals were supplied 200 mg, 400 mg, 600 mg, 800 mg, 2.5 g, 5 g and 10 g/kg of amla extract, respectively, orally after an overnight fasting. Animals were observed continuously for the first 6 h and mortality was recorded for 72 hours.
Amla extract up to a dosage level of 10 g/kg body weight produced no adverse effects such as increased motor activity, tremors, clonic convulsions, piloerection, muscle spasm, hyperesthesia, ataxia, sedation, hypnosis and analgesia. No mortality was recorded in 72 hours.
Sub Acute Toxicity
Thirty healthy male Sprague-Dawly rats weighing 200-250 g were used for the present study. They were housed in polypropylene cages (38×23×10 cms) with 5 animals per cage and maintained under standard housing conditions (room temperature 24-27° C. and humidity 60-65%) with 12-h light and dark cycle. The food in the form of dry pellets and water were available ad libitum.
The animal experiments were conducted according to internationally followed ethical standards and approved by the ethics committee of the Little Flower Hospital and Medical Research Centre, Angamaly, Kerala, India.
The animals were divided into 5 groups of 6 each. Group A served as control while groups B, C, D and E were fed orally a standardized extract of Emblica extract at dosages of 200 mg, 500 mg, 1.0 g and 2.0 g every day for 3 months. Body weights were recorded each week. At the end of 3 months blood samples were collected and analyzed for RBC, WBC, haemoglobin (Hb) and lymphocytes. Blood sugar, serum cholesterol, total protein, aminotransferases (SGOT, SGPT) and alkaline phosphatase were estimated by well-established standard methods. Cholesterol contents of liver and heart were estimated.
At the end of the study, all animals were sacrificed and the various organs and tissues were isolated for detailed examination. The main observations were:

- 1. All animals in the control and experimental groups showed a steady increase with weight and were in the normal range.
- 2. Haematological and biochemical parameters of both the control and experimental groups were in the normal range. The inventive extract was found to enhance the RBC, WBC counts and haemoglobin in the experimental group to a moderate degree. Three months of treatment produced a decrease in blood sugar, serum cholesterol as well as cholesterol in the heart and liver and a moderate increase in serum total protein.
- 3. Levels of the enzymes SGOT, SGPT and ALP were in the normal range indicating amla extract had no hepatotoxic effect.
- 4. There was a significant decrease in the HMG CoA reductase activity in the experimental group in a dose-dependent manner.
- 5. The lumen of aorta, myocardial cells, nephrotic tissues, hepatocytes, spleen tissue and tissues of the adrenal gland appeared normal on microscopic examination.
- 6. Oral feeding of amla extract up to a dose of 2 g/kg for 3 months does not produce any toxic effect.

EXAMPLE 3

Male NZ white rabbits weighing 1.3-1.6 kg were individually caged and fed a normal standard diet. After an acclimatization period, they were divided into 4 groups of 5 animals each. One group (Group A) served as control and groups B 1, B2 and B3 served as experimental groups. The experimental groups were made hypercholesterolemic by feeding 100 mg cholesterol along with the diet daily for 4 months. After 4 months, Group B 1 was kept as untreated hypercholesterolemic control and the remaining two groups (B2 and B3) were fed orally with amla extract in the dosage of 10 mg and 20 mg/kg/day, respectively, for additional 4 months. Body weights of animals were recorded every 15 days.
Before starting the experiment, fasting blood was collected from all animals for estimation of serum total cholesterol, LDL cholesterol (LDL-C), HDL cholesterol (HDL-C) and triglycerides (TG). Blood samples were also analyzed for haematological parameters (RBC, WBC, haemoglobin (Hb) and lymphocytes). These analyses were repeated every month.
At the end of 8 months, all animals were sacrificed and liver, aorta, spleen, heart, liver and kidney were isolated and examined for gross macroscopic changes and thereafter fixed in 10% formalin for histological studies. Tissue cholesterol of liver, kidney, spleen and heart were estimated. A part of the liver was homogenized for estimating HMG CoA reductase and mevalonate.

One-way ANOVA with repeated measures was used to statistically analyze the variance over a period of time. Inter-group comparisons were also made using the same method. Punnet multiple comparison test was used to compare the baseline values with periodically observed values. Post ANOVA comparison in inter-group analyses was performed by using Turkey-Kramer multiple comparison test. Paired t-test was used to compare the biochemical parameters both before and after the experiment. The results are given in the following Tables (1-6).

TABLE 1


Haematological and Biochemical Parameters

		Group B1 (Hyper-
	Group A	cholesterolemic
Parameters	(Control)	control)	Group B2	Group B3

RBC	5.57 ± 0.27	5.63 ± 0.25	5.67 ± 0.21	5.68 ± 0.17
(millions/mm²)
WBC	7.63 ± 0.45	7.2 ± 0.1	7.66 ± 0.21	7.57 ± 0.32
(′000/mm²)
Lymphocytes	2.44 ± 0.33	2.17 ± 0.08	2.27 ± 0.04	2.61 ± 0.23
(′000/mm²)
Hb	14.6 ± 1.21	13.59 ± 1.0	15.56 ± 1.23	14.4 ± 1.11
(g/dl)
Blood sugar	110.6 ± 6.50	118.83 ± 4.75	102 ± 4.0	104 ± 4.0
(mg/dl)
Total protein	5.56 ± 0.77	5.63 ± 0.40	5.73 ± 0.30	6.2 ± 0.2
(g/dl)
Liver-Chol.	9.43 ± 0.20	14.63 ± 0.51*	9.53 ± 0.10**	9.56 ± 0.18**
(mg/g)
Heart-Chol.	7.31 ± 0.07	8.51 ± 0.20*	7.50 ± 0.10**	7.43 ± 0.10**
(mg/g)
Kidney-Chol.	5.58 ± 0.10	6.6 ± 0.18*	5.56 ± 0.08**	5.67 ± 0.01**
(mg/g)
Spleen-Chol.	3.40 ± 0.10	3.80 ± 0.12	3.42 ± 0.02	3.32 ± 0.05
(mg/g)
HMG CoA to	1.03 ± 0.15	0.90 ± 0.2	1.53 ± 0.6**	1.33 ± 0.06**
Mevalonate
ratio

*significant increase (P < 0.05)
**Significant decrease (P < 0.05)

TABLE 2


Serum Cholesterol

Serum Cholesterol concentration (mg/dl)

Group	0 Month	4 Month	5 Month	6 Month	7 Month	8 Month

A	55.8 ± 5.0	56.66 ± 2.7	53.38 ± 3.25	53.33 ± 3.3	54.23 ± 0.9	52.8 ± 1.8
B1	50 ± 2.72	229.16 ± 3.2	220.33 ± 2.8	200.83 ± 3.2	185.31 ± 1.9	164.28 ± 3.6
B2	48.33 ± 4.3	228.13 ± 1.9	163.56 ± 5.8	88.89 ± 5.1	74.57 ± 3.4	63.33 ± 2.89
B3	52.49 ± 3.2	230.83 ± 3.2	177.96 ± 4.4	95.0 ± 1.9	72.88 ± 2.0	58.33 ± 2.89

TABLE 3


LDL Cholesterol

Serum LDL Cholesterol (mg/dl)

Group	0 Month	4 Month	5 Month	6 Month	7 Month	8 Month

A	39.22 ± 4.91	39.72 ± 2.59	38.6 ± 5.82	36.51 ± 4.09	37.64 ± 0.53	36.62 ± 1.56
B1	32.38 ± 1.80	195.73 ± 4.2	187.36 ± 1.8	169.79 ± 3.6	154.1 ± 3.2	136.22 ± 4.3
B2	32.4 ± 4.68	193.7 ± 2.2	137.5 ± 5.2	67.54 ± 5.61	54.26 ± 2.71	43.03 ± 2.27
B3	33.57 ± 3.35	195.86 ± 2.4	153.76 ± 5.2	73.82 ± 2.90	53.41 ± 1.43	39.25 ± 2.12

TABLE 4


VLDL Cholesterol

Serum VLDL Cholesterol (mg/dl)

Group	0 Month	4 Month	5 Month	6 Month	7 Month	8 Month

A	8.28 ± 0.76	8.43 ± 0.75	8.08 ± 0.44	8.27 ± 0.46	7.95 ± 0.44	7.95 ± 0.44
B1	8.96 ± 0.45	26.94 ± 0.89	25.19 ± 1.16	22.3 ± 1.09	20.26 ± 1.14	19.73 ± 0.9
B2	8.79 ± 0.45	26.56 ± 0.77	17.69 ± 1.41	12.53 ± 0.46	10.25 ± 0.4	9.07 ± 0.46
B3	8.96 ± 0.45	26.36 ± 0.79	17.12 ± 0.97	12.2 ± 0.40	9.42 ± 0.39	8.53 ± 0.46

TABLE 5


HDL Cholesterol

Serum HDL Cholesterol (mg/dl)

Group	0 Month	4 Month	5 Month	6 Month	7 Month	8 Month

A	8.33 ± 0.43	8.47 ± 0	8.19 ± 0	8.33 ± 0.45	8.33 ± 0.12	8.24 ± 0.08
B1	8.75 ± 0.83	9.32 ± 0.98	7.78 ± 0.82	8.75 ± 0.84	8.94 ± 1.06	8.33 ± 0.13
B2	8.81 ± 1.42	8.05 ± 0.85	8.19 ± 0	8.89 ± 0.96	10.05 ± 1.76	11.24 ± 1.07
B3	8.81 ± 0.76	8.47 ± 1.20	8.61 ± 0.83	8.75 ± 0.84	10.05 ± 1.44	10.62 ± 1.07

TABLE 6


Serum Triglycerides

Serum Triglycerides (mg/dl)

Group	0 Month	4 Month	5 Month	6 Month	7 Month	8 Month

A	41.34 ± 3.85	42.15 ± 3.92	40.38 ± 2.22	41.33 ± 2.83	39.74 ± 2.72	39.74 ± 2.72
B1	44.61 ± 2.11	134.84 ± 4.4	125.96 ± 5.8	111.53 ± 5.4	121.94 ± 4.3	98.67 ± 4.62
B2	43.84 ± 0.61	133.33 ± 2.1	88.47 ± 2.15	62.67 ± 2.31	51.28 ± 2.22	45.33 ± 2.3
B3	44.61 ± 2.1	132.55 ± 3.3	88.57 ± 4.84	61.0 ± 2.2	47.11 ± 1.93	42.67 ± 2.3

Results in the above tables reveal the dramatic effect of feeding amla extract on the lipid profiles of hypercholesterolemic animals. All the parameters returned almost to their original levels after 4 months of amla treatment. This unprecedented result strongly suggests that with continued treatment hypercholesterolemia could be completely reversed, at least in experimental animals.
This reversal of hypercholesterolemia was further supported by the results of histological examination of the aorta of the animals. Aortic strips of control groups were normal with normal intima, media and adventia. So was the case with those of amla-treated hypercholesterolemic rabbits, while there were smooth muscle cell proliferation, fatty infiltration and foam cell formation in the untreated hypercholesterolemic animals. Hepatocytes of all animals appeared normal.
Reduced HMG CoA reductase activity was noted in the amla extract-treated groups (data not shown). The activity of this key enzyme was reduced by 30 and 56%, respectively, in animals of Group B2 and B3.

EXAMPLE 4

Human Studies

Hypercholesterolemic subjects (total cholesterol>240 mg/dl, LDL Cholesterol>130 mg/dl) of either sex were selected for the study. Patients having valvular heart disease, congestive heart disease and diabetes and patients taking lipid lowering drugs were excluded from the study. A total of 70 patients were enrolled. They were divided into control group (20 patients) and intervention group (50). They were briefed about the study and written consents were taken before commencement of the study. Before commencement of the study blood samples were collected from each patient. The intervention group were advised to take amla extract in the form of 500 mg hard gelatin capsules in the dosage of 2-O-2 after meals. The study period was 3 months. Lipid profiles were determined at the end of each month. Results are given in Tables 7 and 8, shown in FIGS. 1 and 2.
The foregoing embodiment and advantages are merely exemplary and are not to be construed as limiting the present invention. The present teaching can be readily applied to other types of apparatuses. Also, the description of the embodiments of the present invention is intended to be illustrative, and not to limit the scope of the claims, and many alternatives, modifications, and variations will be apparent to those skilled in the art.

Claims

1. A method of producing a product to correct hypercholesterolemia comprising:

pulping fruits of Emblica Officinalis with demineralized water to create a slurry;

treating the slurry with pectinase;

filtering the slurry to create a solution; and

concentrating the solution to generate the product.

2. The method of claim 1, wherein the product is a free flowing powder obtained by spray drying a concentrate.

3. The method of claim 1, wherein the product is prepared without using any organic solvent and without subjecting to any chemical treatment at any stage.

4. The method of claim 1, wherein the product does not contain any preservative or additive.

5. The method of claim 2, wherein the powder does not contain any preservative or additive.

6. A product to correct hypercholesterolemia comprising:

an extract of fruits of Emblica Officinalis.

7. The product of claim 6, wherein the product is a concentrate.

8. The product of claim 7, wherein the product is a powder.

9. The product of claim 6, wherein the product is prepared without using any organic solvent and without subjecting to any chemical treatment at any stage.

10. The product of claim 6, wherein the product does not contain any preservative or additive.

11. The product of claim 6, comprising at least 10 percent by weight of emblicanin A.

12. A method of reducing serum total cholesterol levels comprising administering effective doses of the product of claim 6.

13. A method of reducing at least one of serum LDL and VLDL cholesterol concentrations comprising administering effective doses of the product of claim 6.

14. A method of enhancing HDL cholesterol levels comprising administering effective doses of the product of claim 6.

15. A method of reducing triglyceride to correct dyslipidemia comprising administering effective doses of the product of claim 6.

16. A method of preventing smooth muscle cell proliferation and reducing intima-media thickening comprising administering effective doses of the product of claim 6.

17. A method of reducing HMG CoA reductase activity comprising administering effective doses of the product of claim 6.