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

Abstract

A method of producing a product to correct hypercholesterolemia including pulping fruits of Emblica officinalis with demineralized water to create a slurry. The slurry is treated with pectinase. The pectinase-treated slurry is filtered to create a solution. The solution is concentrated to create a product. A product having an extract of Emblica officinalis for prophylactic and for therapeutic treatment of coronary diseases, atherosclerosis, hypothyroidism and hyperthyroidism.

Claims

1. A method of decreasing inflammation in a human in need thereof comprising administering to said human an effective amount of an extract of Emblica officinalis, the extract prepared by a method 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 extract of Emblica officinalis.

2. A method of decreasing CRP level in a human subject in need thereof comprising administering to said human subject an effective amount of an extract of Emblica officinalis, the extract prepared by a method 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 extract of Emblica officinalis.

3. The method of decreasing CRP level of claim 2, wherein a decrease in CRP level ranges from about 4.2% to about 88% following administration of the extract of Emblica officinalis.

4. The method of decreasing CRP level of claim 2, wherein a decrease in CRP level ranges from about 20% to about 60% following administration of the extract of Emblica officinalis.

5. The method of decreasing CRP level of claim 2, wherein a decrease in CRP level ranges from about 25% to about 45% following administration of the extract of Emblica officinalis.

6. The method of decreasing CRP level of claim 2, wherein a decrease in CRP level is about 4.2% following administration of the extract of Emblica officinalis.

7. The method of decreasing CRP level of claim 2, wherein a decrease in CRP level is about 88% following administration of the extract of Emblica officinalis.

8. The method of decreasing CRP level of claim 2, wherein a CRP level ranges from about 1 mg CRP per liter to about 4.5 mg CRP per liter following administration of the extract of Emblica officinalis.

9. A method of decreasing CRP level in a human subject having coronary artery disease comprising administering to said human subject an effective amount of the extract of Emblica officinalis to the human subject, the extract prepared by a method 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 extract of Emblica officinalis.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows Table 7, which provides the effect of product on hematological parameters in human patients; and

(2) FIG. 2 shows Table 8, which provides effect of product on lipid profile in hypercholesterolemic patients.

DETAILED DESCRIPTION OF THE INVENTION

(3) The three important components of coronary artery diseases are lipids, inflammation and immunity (for example, see Binder C J, et al. Nature Med, 2002, 8:1218-26). What initiates the atherosclerotic process is not entirely clear, and there are probably several different pathogenic processes that can elicit localized inflammatory responses in the artery. One prime candidate is minimally oxidized LDL, and late forms of oxidized LDL (ox-LDL). Once trapped in the artery wall by binding to extracellular proteoglycans, a key event in atherogenesis (Williams K J and Tobas I, Curr Opin Lipidol, 1998, 9:471-74; Skalen K, et al. Nature, 2002, 417:750-54), LDL is oxidized by mechanisms still unknown or undergoes other types of modifications, such as non-enzymatic glycation, enzymatic degradation, aggregation, or a combination of these, all of which results in alteration of the ‘self’. These modifications makes the modified LDL and leads to both cellular and humoral responses (Horkko, S, et al. Free Radio Biol Med, 2000, 28:1771-79. In addition, the oxidation, of LDL generates oxidized lipids that are toxic, proinflammatory and pro-atherogenic (Pratico D, Trends Cardiovasc Med, 2001, 11:112-16). Oxidized phospholipids can induce artery wall cells to secrete chemotactic molecules (chemokines), activate endothelial cells to express adhesion molecules, and induce expression of growth factors that facilitate the transformation of monocytes to macrophages and stimulate the proliferation of smooth muscle cells (Berliner J A, et al. Trends Cardiovasc Med, 2001, 11:112-16; Marathe G K, et al. Trends Cardiovasc Med, 2001, 11:139-42). Macrophages, a central mediator in innate and adaptive immunity, are essential in lesion initiation and progression (Glass C K and Witztum J L, Cell 2001, 104:503-16; Hansson G K, et al. Circ Res, 2002, 91:281-91). Once activated they initiate oxidation of LDL and rapidly take up oxLDL through specific scavenger receptors leading to foam cell formation (Witztum J L, et al. trends Cardiovasc Med, 2001, 11:93-102). This is a key event in disease progression. Activated macrophages also secrete a variety of pro-inflammatory molecules that affect lesion progression and plaque stability.

(4) The present invention provides for a natural product that has been found to directly influence two of the three components, namely lipid disorder and inflammation, of coronary artery diseases discussed above, with potential for modulating the third component, namely immunity, as well, it would be difficult to assess the involvement of amla in modulating the immune system component, but earlier studies have clearly shown its immunomodulating properties (Nemmani K V et al. Indian J Exp Biol, 2002, 40:282-87; Muruganandam A V, et al. Indian J Exp Biol, 2002, 40:1150-60; Bhattacharya A, et al. Indian J Exp Biol, 2002, 40:1161-63; Sai Ram, M, et al. J Ethnopharmacol, 2002, 81:5-10; Phytother Res, 2003, 17:430-33; Bhattacharya S K et al. Indian J Exp Biol, 2000, 38:945-47).

(5) Thus, one embodiment of the disclosed amla product modulates all the important components of coronary artery diseases to induce regression of the disease. Further, one embodiment of the disclosed amla product reduces the fasting blood sugar levels, a confounding factor in atherosclerosis. The potential of the disclosed amla product in inducing regression is shown by the reduction in intima-media thickness in experimental animals.

(6) The present invention provides for a safe natural product for treatment of disorders connected with coronary artery diseases, such as coronary heart disease, stroke and peripheral artery disease, without the disadvantages associated with the use of synthetic lipid lowering agents such as statins.

(7) 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. One embodiment of the disclosed amla product thus corrects dyslipidemia associated with coronary artery diseases.

(8) One embodiment of the disclosed amla product further reduces inflammation which is an integral part of coronary artery diseases, as evidenced by reduction in CRP levels. Since CRP is a predictor of future coronary events, reduction in CRP by the disclosed amla product indicates that the amla product directly affords reduction in risks of clinical events such as myocardial infarction and stroke.

(9) Furthermore, one embodiment of the disclosed amla product reduces the blood sugar levels in diabetics who are at an elevated risk of heart diseases and corrects dyslipidemia associated with diabetes.

(10) One embodiment of the amla product was also found to correct thyroid dysfunction (both hypo- and hyperthyroidisms) which has a direct effect on heart and vascular systems, and now recognized as a risk factor for cardiovascular diseases.

(11) 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.

(12) 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.

(13) The present composition uses the extract of fresh fruits 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. Amla is known as an immunomodulator and this property also is believed to be acting in concert with the observed effects to further induce remission of the disease. 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.

(14) 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.

(15) In another embodiment, the invention provides for a composition to reduce inflammation associated with coronary artery diseases, as evidenced by the reduction in blood CRP levels.

(16) In a further embodiment, the invention provides for a composition to reduce blood sugar levels which has a confounding effect on heart diseases.

(17) In another embodiment, the invention provides for a composition to correct thyroid dysfunctions.

(18) In another embodiment of the invention a composition is provided for reducing the major risk factors associated with coronary artery diseases and induce remission of the disease as evidenced by reduction in 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.

(19) 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.

(20) 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.

(21) 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.

(22) In one embodiment, the invention provides a product having an extract of fruits of Emblica Officinalis, wherein the extract is prepared without using any organic solvent and without subjecting to any chemical treatment at any stage.

(23) In one embodiment, the invention provides a method of producing a product including:

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

(25) treating the slurry with pectinase to form a pectinase-treated slurry;

(26) filtering the pectinase-treated slurry to create a solution; and

(27) concentrating the solution to generate the product.

(28) In one embodiment, the invention provides a method of producing a product to correct hypercholesterolemia in a human.

(29) 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.

(30) The hypocholesterolemic properties of the composition were 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.

(31) 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.

(32) The composition was further tested in apparently healthy human volunteers, for its effect on blood CRP levels and fasting blood sugar levels, in addition to its effect on the lipid profiles. The composition showed positive benefits in these aspects as well. Blood CRP level is a marker for systemic inflammation and a predictor of future cardiac events. Blood sugar has a confounding effect on the disease.

(33) The product can be administered to a human for a method of reducing serum cholesterol levels. The product can be administered to a human for a method of reducing at least one of serum. LDL and VLDL cholesterol concentrations. The product can be administered to a human for a method of enhancing HDL cholesterol levels. The product can be administered to a human for a method of reducing triglyceride to correct dyslipedemia. The product can be administered to a human for a method of preventing smooth muscle cell proliferation and reducing intima media thickening. The product can be administered to a human for a method of reducing HMG CoA reductase activity.

(34) The product can be administered to a human for a method of correcting dyslipedemia. The product can be administered to a human for a method of reducing inflammation. The product can be administered to a human for a method to reduce fasting sugar levels in the blood. The product can be administered to a human for a method of therapeutic and prophylactic cardioprotection. The product can be administered to an atherosclerotic human patient for a method to induce regression of the atherosclerotic process, whereby dyslipedemia, inflammation and blood sugar levels are also corrected. The product can be administered to a human patient having coronary artery diseases to induce regression of atherosclerotic process. The product can be administered to a patient having coronary artery diseases to correct lipid abnormalities. The product can be administered to a patient having coronary artery diseases to reduce serum total cholesterol. The product can be administered to a patient having coronary artery diseases to reduce VLDL cholesterol. The product can be administered to a patient having coronary artery diseases to reduce LDL cholesterol. The product can be administered to a patient having coronary artery diseases to reduce triglyceride concentration. The product can be administered to a patient having coronary artery diseases to elevate beneficial HDL cholesterol. The product can be administered to a patient having coronary artery diseases to reduce inflammation associated with coronary artery disease. The product can be administered to a patient having coronary artery disease to reduce C-reactive protein. The product can be administered to a human to correct hypothyroidism. The product can be administered to a human to correct hyperthyroidism.

(35) In one embodiment, the invention provides for a method of producing a product to correct hypercholesterolemia in a human by pulping fruits of Emblica Officinalis with demineralized water to create a slurry. The slurry is treated with pectinase to form a pectinase-treated slurry. The pectinase-treated slurry is filtered to create a solution. The solution is concentrated to generate the product.

(36) These and other features of the present invention are explained in more detail in the following non-limiting examples.

Example 1

(37) 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 liters 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 hydrolysable tannin content of this preparation was at least 30%. The emblicanin A content of this preparation was 10.2%.

Example 2

Toxicity Studies

(38) Acute Toxicity

(39) 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.

(40) 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.

(41) Sub Acute Toxicity

(42) 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.

(43) 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.

(44) 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.

(45) 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

(46) 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 B1, B2 and B3 served as experimental groups. The experimental groups were made hypercholesterolemia by feeding, 100 mg cholesterol along with the diet daily for 4 months. After 4 months, Group B1 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.

(47) 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.

(48) 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 HMO CoA reductase and mevalonate.

(49) 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).

(50) TABLE-US-00001 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/mm.sup.2) WBC 7.63 ± 0.45 7.2 ± 0.1 7.66 ± 0.21 7.57 ± 0.32 (′000/mm.sup.2) Lymphocytes 2.44 ± 0.33 2.17 ± 0.08 2.27 ± 0.04 2.61 ± 0.23 (′000/mm.sup.2) 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)

(51) TABLE-US-00002 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

(52) TABLE-US-00003 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

(53) TABLE-US-00004 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

(54) TABLE-US-00005 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

(55) TABLE-US-00006 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 

(56) 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.

(57) 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.

(58) 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 Study 1

(59) 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 2-0-2 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.

Example 5

Human Study 2

(60) Twenty two apparently healthy human volunteers aged 26 to 76 years consumed 500 mg of amla product, referred to as Amlamax, per day. None of the participants had a history of myocardial infarction, stroke or coronary revascularization. Their physical attributes such as height, weight and blood parameters were analyzed before study and 3 months after consumption of AmlaMax. These data are given in Table 9. The participants were arbitrarily divided into three groups, namely those between ages 26 to 45, 46 to 60 and those above 60 years. Response to Amlamax with respect to the studied parameters were impressive (Table 10), with only one non-responder each for total cholesterol and LDL cholesterol. There were two non-responders to fasting blood sugar reduction, three did not respond to TG and CRP while 5 persons did not show the expected increase in their HDL profiles. The extent of response among responders (Table 11) were as follows: Total cholesterol: (−) 4.6 to 32.3% ⇓ (mean 13.6%) LDL cholesterol: (−) 4.9 to 41.9% ⇓ (mean 17.4%) HDL Cholesterol: (+) 2.2 to 44.8% ⇑ (mean 15.8%) Triglycerides (TG): (−) 1.4 to 62.9% ⇓ (mean 27.2%) CRP: (−) 4.2 to 65.0% ⇓ (mean 48.1%) FBS: (−) 1.1 to 28.5% ⇓ (mean 10.8%)

(61) The magnitude of change generally follows the extent of abnormality in the respective starting values. For example there were two participants with very high TG levels of 534 and 350, respectively. These two persons showed a decrease of 50.4% and 62.9%, respectively, after AmlaMax treatment. Similarly the patient showing the highest enhancement of 44.8% in HDL cholesterol had the lowest HDL cholesterol to start with. The most significant changes were noted in CRP levels which responded to AmlaMax very well. Here again the person who responded the maximum (88.4% reduction) had the highest starting CRP value of 12.0 which was reduced by AmlaMax treatment to the normal range of 1.4 mg/L. These results should be considered significant because such gross abnormalities are normally difficult to correct by drug treatment. The results on fasting blood sugar (FBS) are also noteworthy. FBS recorded the least mean changes in the participants, because most of them had near-normal FBS values. There was only one diabetic patient (FBS above 140 mg/dl (entry No. 1) (Tables 9 and 11)). This patient also recorded the highest reduction in FBS. AmlaMax did not produce random reduction in blood sugar irrespective of the starting value (as would a normal drug do) but only in those cases where such a correction is needed and thus does not lead to hypoglycemia, a problem observed with many drugs for hyperglycemia. One is reminded of one of the three attributes of an ‘adaptogen’ (Rasayana in Ayurveda): It must cause only minimal disorders in the body's physiological functions. In other words, AmlaMax corrects only where correction is required, rightly justifying its position as an adaptogen, and in sharp contrast with common medications.

(62) In agreement with the above observation, AmlaMax also modulated thyroid function, correcting both hyper- as well as hypothyroidisms. Among the 22 participants involved in the study, there were two persons (one male and one female) with overt hypothyroidism with TSH levels of 79.96 and 111.02 mU/L. These values after 3 months of AmlaMax treatment were 38.96 and 76.79 mU/L, respectively and are expected to improve further to normal values on continued treatment. Similarly, there was one woman patient with overt hyperthyroidism with TSH level less than 0.01 mU/L which got corrected to normal level (1.32 mU/L) after 3 months of AmlaMax consumption. These results indicate the normalizing effect of AmlaMax on thyroid functions in patients with such disorders.

(63) TABLE-US-00007 TABLE 9 Effect of the amla product on Lipid profile, CRP and FBS in Healthy Human Volunteers Lipid Profile CRP FBS Chol TG VLDL LDL HDL (mg/l) (mg/dl) Group 0 Mo 3 Mo 0 Mo 3 Mo 0 Mo 3 Mo 0 Mo 3 Mo 0 Mo 3 Mo 0 Mo 3 Mo 0 Mo 3 Mo A 235 200 229 190 46 38 151 125 38 37 2.4 2.3 225 162 (Age 275 188 534 265 — 53 — 93 29 42 1.6 2.0 125 94 25-45 Yr) 310 276 192 170 38 34 238 204 34 38 4.0 4.5 74 71 (n = 13) 230 211 167 110 33 22 156 147 41 42 3.5 2.8 85 76 230 212 158 150 32 30 157 142 41 40 2.8 1.0 100 94 270 212 250 250 50 50 173 124 38 47 3.8 2.5 95 94 220 165 196 130 39 26 147 100 34 39 1.9 2.1 80 76 240 229 273 180 55 36 147 150 38 43 2.0 3.2 95 100 235 212 294 185 59 37 142 135 34 40 3.0 1.5 92 76 220 176 142 130 28 26 136 99 51 56 4.0 1.9 93 82 235 212 350 130 70 26 121 147 39 44 3.0 1.3 110 97 245 224 175 175 35 35 172 147 38 42 2.6 1.1 85 82 260 176 158 120 32 24 184 107 44 45 12 1.4 102 76 Group B 210 188 92 70 18 14 144 126 48 48 4 1.4 108 147 (Age 275 241 271 210 54 42 180 159 41 40 2 1.6 95 88 45-60 Yr) 180 152 192 120 38 24  92 86 42 50 3.8 2.4 75 71 (n = 6) 245 224 100 90 20 18 175 164 40 46 3.0 2.9 95 82 285 253 142 140 28 28 219 182 38 43 4.5 1.8 100 88 235 259 273 200 55 40 142 181 38 38 4.2 1.0 98 97 Group C 220 153 117 80 23 16 163 90 34 47 4.0 1.2 95 88 (Age 220 200 242 250 48 50 138 110 34 40 3.7 1.9 90 88 >60 Yr) 245 224 167 100 33 20 174 165 38 39 2.9 1.2 125 109 (n = 4) Chol = Total cholesterol; VLDL = very low density lipoproteins; LDL = low density lipoproteins; HDL = high density lipoproteins; TG = Triglyceride; CRP = C-reactive protein; FBS = Fasting blood sugar; Mo = Months after treatment with AmlaMax

(64) TABLE-US-00008 TABLE 10 Response of Healthy Subjects to Treatment with the product No of Non- Parameter responders Cholesterol 1 (4.5%) LDL chol 1 (4.5%) HDL chol 5 (22.7%) TG 3 (13.6%) CRP 4 (18.18%) FBS 2 (9.1%) Total number of subjects treated = 22

(65) TABLE-US-00009 TABLE 11 Extent of Response to Treatment with the product in Healthy Human Subjects HDL C- Fasting Chol- Tri- LDL chol- reactive blood Subject esterol glycerides cholesterol esterol protein sugar 1 −15% −18.0% −27.2% −2.6% −4.2% −28.0% 2 −31.6 −50.4 — +44.8 +11.1 −24.8 3 −11 −11.5 −14.3 +11.7 +12.5 −4.1 4 −8.3 −34.2 −5.8 +2.4 −20.0 −10.6 5 −8.3 −5.1 −9.6 −2.5 −64.3 −6.0 6 −21.5 0.0 −28.4 +23.6 −34.3 −1.1 7 −25.0 −33.7 −32.0 +14.7 +10.5 −5.0 8 −4.6 −36.1 +2.0 +13.1 +60.0 +5.2 9 −9.8 −36.9 −4.9 +17.6 −50.0 −17.4 10 −20.0 −8.5 −27.2 +9.8 −52.5 −11.9 11 −9.8 −62.9 +21.5 +12.8 −56.7 −11.9 12 −8.6 0.0 −15.5 +10.5 −57.3 −3.6 13 −32.3 −24.1 −41.9 +2.2 −88.4 −25.5 14 −10.5 −24.0 −12.5 0.0 −65.0 +38.6 15 −12.4 −23.4 −11.7 −2.5 −20.0 −7.4 16 −15.6 −37.5 −6.6 +19.0 −35.9 −5.4 17 −8.6 −10.0 −16.9 +15.0 −4.4 −13.7 18 −11.3 −1.4 −6.3 +13.1 −60.0 −12.0 19 +10.2 −26.8 +27.4 0.0 −76.2 −1.1 20 −30.5 −31.7 −44.8 +38.2 −70.0 −7.4 21 −9.1 +5.4 −20.3 +17.6 −48.7 −2.3 22 −8.6 −40.2 −5.2 +2.6 −58.7 −12.8 Mean (−)13.6 (−)27.2 (−)17.4 (+)15.8 (−)48.1 (−)10.8 Change* *Among responders

(66) 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.