Emblica officinalis plant extracts and uses thereof

FIELD OF THE INVENTION

The present invention pertains to nutritional or pharmaceutical compositions comprising extracts or concentrates of plants and the mixtures thereof belonging to Emblica sp. with specific reference to Emblica officinalis. The present invention further relates to extracts which are isolated from the fruits of Emblica officinalis plant, the preparation of such extracts and the medicaments containing said extracts. The invention further relates to screening and characterization of extracts for their activity in preventing, mitigating, or treating cardiovascular disorders to achieve the lowest possible cardiac risk. Furthermore, the invention relates to the use of the extracts as a supplement or a medicament useful in the treatment/alleviation or prevention of Cardiovascular disease.

BACKGROUND OF INVENTION

Cardiovascular disease is group of diseases affecting heart and blood vessels. Increased incidence of cardiovascular disease in the recent decades can be attributed to the lifestyle and diet as the disease has dietary basis (Bogin, 1997; Hamilton et al., 1988). The cardiovascular disease is one of the main causes of mortality in human beings, which needs major breakthrough discoveries from natural products that can be helpful in preventing risks of cardiovascular disease.

Classification of Cardiovascular disease

Cardiovascular disease is broadly categorized into - Coronary heart disease, high blood pressure, heart stroke, heart failure and vascular inflammation. Coronary heart disease refers to the lack of blood supply to the myocardium (heart muscles) due to the accumulation of cell debris containing cholesterol and fatty acids, fibrous connective tissue and Calcium, which are collectively called atheromatous plaques. High blood pressure (hypertension) is a medical condition wherein the blood pressure reading rises to 140/90 mmHg or above. Prolonged hypertension increases the risks of heart attacks and heart failure. Stroke results in the poor supply of blood to the brain and are due to either blockage or rupturing of the blood vessels that connects the brain. Stroke is further classified into four types, cerebral thrombosis and cerebral embolism (blockage of blood vessels) and cerebral and subarachnoid haemorrhages (rupture of blood vessels). Normal blood pressure with respect to cardiovascular risk should be close to 120/80 mm Hg.

The Etiology of Cardiovascular disease

Over the past three decades, great progress has been made in identifying and correcting risk factors for cardiovascular disease (CVD), such as smoking, high blood pressure and an elevated plasma level of total cholesterol and low-density lipoprotein (LDL) cholesterol. This has led to a significant reduction in the incidence of CVD in several countries. Despite this encouraging result, however, CVD is still the leading cause of death in many nations. A further decline in cardiovascular morbidity and mortality could be achieved by employing a wide, multiple risk factor approach. Many studies opined that risk factors for cardiovascular disease largely consist of dietary basis and also on lifestyle. Despite the information available on risk factors, studies are being undertaken to understand the complexity of biomolecules involved and preventive mechanisms. Studies showed that increased intake of saturated fats leads to the increase in level of low-density lipoprotein (LDL), which enhances the risk of CVD (Bankson et al., 1993). The increased LDL also leads to the formation of atheromatous plaques, subsequently blocking the blood vessels and also increases calcium accumulation in walls of blood vessels (Hattersley, 1993). Low high-density lipoprotein (HDL) cholesterol levels, often associated with elevated plasma triglycerides, play a significant role in the risk of developing CVD (Regnstrom, et al., 1990). This is observed to be one of the most common forms of CVD (Steinberg et al., 1989). Reduced thyroid function increases the incidence of CVD owing to its higher adrenalin production that degenerate the blood vessels.

Hypertension is classified by etiology as being either primary (essential, idiopathic) or secondary. Primary hypertension accounts for 90-95% of cases of hypertension, while the remaining 5-10% of cases are the result of secondary hypertension. Evidence suggests varying combinations of many potential interacting factors causes the disease. Patients with primary hypertension do not appear to share any one, or a specific combination of, suspected etiologic factors. Some of the potential etiologic factors for primary hypertension include:

• Sympathic nervous system dysfunction and/or hyperactivity.

• Renin-angiotensin system defects.

• Sodium transport defects.

• Intracellular sodium and calcium defects.

Other factors have been implicated as either predisposing or contributing to the development of primary hypertension. These include obesity, excessive alcohol use, cigarette smoking, excessive salt intake, stress, and physical inactivity. No clearly established genetic pattern has been established for primary hypertension. However, blood pressure levels appear to have strong familial tendencies.

Etiologies of secondary hypertension include: renal vascular disease (atherosclerotic, thrombotic, embolic stenosis or obstruction, fibromuscular hyperplasia, etc.).

Hypertension is the most important etiologic factor for cardiovascular disease. In adults, systolic blood pressure elevations are usually considered to be more a determinant of cardiovascular risk than are diastolic blood pressure elevations. Hypertension accelerates the development and progression of atherosclerosis (leading to peripheral and coronary vascular insufficiency), and subsequently increases the patient's risk for myocardial infarction. Hypertension also causes left ventricular hypertrophy, which may result in congestive heart failure, ventricular arrhythmias, myocardial ischemia, and sudden death. Hypertension is a major etiology for both dissecting and atherosclerotic aortic aneurysms, and also acts as an exacerbating factor in the progression of these conditions. Retinal vascular narrowing, hemorrhages, exudates, and papilledema are also consequences of hypertension.

Inflammatory vascular diseases are initiated and perpetuated by the interaction of immune cells with cells of the affected vessel wall. This is directed by a network of chemical messengers, which, in a state of vascular health, exist as balanced but opposing forces. The detection of vascular inflammation and monitoring of this activity have long been attempted in systemic vasculitis, and, more recently, in atherosclerosis. Markers of vascular inflammation used thus far have been of limited value; few provide both adequate sensitivity and specificity for any particular disease

Inflammatory markers are predictors of recurrent CVD and death in different settings, including the short-term risk, long-term risk, and risk after revascularization procedures such as percutaneous coronary intervention (PCI), including the risk of restenosis. Several Inflammatory markers which seem to have predictive abilities for Cardiovascular Risk are high-sensitivity C- reactive protein [hs-CRP], serum amyloid A [SAA], white blood cell [WBC] count and fibrinogen. Preferably, only the acute-phase reactants (fibrinogen and CRP) and WBC count have widely available assays. hs-CRP consistently predicts recurrent myocardial infarction independent of troponins, which suggests it is not merely a marker for the extent of myocardial damage. It also may be useful in the estimation of prognosis in patients who need secondary preventive care, such as those with stable coronary disease or acute coronary syndromes (risk of restenosis) and those who have undergone PCI. Elevated hs-CRP levels also seem to predict prognosis and recurrent events in patients with stroke and peripheral arterial disease (Pearson, et.al, 2007).

Treatment

Lifestyle modifications with diet therapy include reduced intake of saturated fats, trans-fatty acids, and plant stanol/sterols and viscous fiber and a good exercise program helps to prevent or delay further damage to the cardiovascular system.

Statin drugs, Aspirin and Anticholesterol drugs, such as Mevacor, Cholestyramine, and Clofibrate are prescribed to control the risks of high cholesterol and reduce the chances of strokes and heart attacks (Lita Lee, 2005). Statin drugs and Anticholesterol drugs do lower cholesterol but does not eliminate the risk of heart attacks. Omega-3-fatty acids, carotenoids, vitamins, minerals and some of the herbal drugs are suggested for preventing cardiovascular risks.

Statins work by blocking cells from making cholesterol. Unfortunately, these drugs also stop cells from making the nutrient co-enzyme QlO (co-Q10). This nutrient helps to release energy from food and to protect cells from harmful compounds. Statins decrease levels of co-Q10 in rats and several studies in people also suggest the same effect. A sustained decrease in co-Q10 may explain the appearance of some side effects associated with statins like tiredness, muscle pain and muscle wasting.

Besides these drugs, angiograms, bypass surgery, and angioplasty are a big business. Over one million heart angiograms are performed each year for a total annual cost of over ten billion dollars. But based upon extensive analysis, it appears that most of this money is wasted. The use of expensive surgery is physically invasive and traumatic for the heart patient, and upon evaluation of case histories has been shown to be five to ten times more deadly than the disease, and in many instances unnecessary!" [As reported in: Graboys, et. al. 1987 Journal of the American Medical Association; Graboys, et. al. 1992 Journal of the American Medical Association; Coronary Artery Surgery Study (CASS) 1984 New England Journal of Medicine; CASS study, Alderman, et. al. 1982 and 1990 Circulation (Journal)].

The inclusion of botanicals in a nutritional approach presents an inexpensive means of achieving the goal of cost effective management of the said disease condition. However, the effects of the nutritional strategies recommended today are rather modest. Thus, research into novel nutritional strategies preventing cardiovascular risks is needed.

The treatment of cardiovascular disorders is a particularly promising area for botanicals. Most botanicals derive their effectiveness from a mixture of active molecules, acting in concert. Multiple agents attacking multiple targets simultaneously present decided advantages over conventional drugs, which are each based on one compound that produces one action. Plants hold the power to keep the increasing prevalence of cardiovascular disorders, in check, prompting the search and trial of plant

extracts to develop a whole new category of natural products. Comprehensive studies on the components of the herb that are responsible for certain indications need to be undertaken to obtain effective medications from this therapy. Also, not necessarily all the ayurvedic extracts are non-toxic.

The use of herbs has shown positive effects on the cardiovascular system. Dry fruits reduce cholesterol and lower the risk of genetic coronary disease due to its rich source in alpha-linolenic acid (ALA). Flavonoids rich fruits and peels have the potential to reduce the risks of cardiovascular disease. (Donald R. Yance, 2005)

Emblica officinalis, also known by its Indian name AmIa, is one of the most celebrated herbs in the Indian traditional medicine system, Ayurveda. The fruit has been used for thousands of years by Ayurvedic physicians who refer to it as the "sustainer" and "the fruit where the goddess of prosperity resides." It has a reputation as a powerful rejuvenating herb ("Sattwic") and increases "OJAS". AmIa is used in many of the indigenous medicinal preparations against a variety of diseases. It is used in Ayurveda as a cardiotonic.

Amalaki fruit contains a series of diterpenes referred to as the gibberellins, as well as the triterpene lupeol, flavonoids (e.g. kaempherol-3-O-β-Dglucoside, quercetin-3-O-β-Dglucoside), and polyphenols (e.g. emblicanin A and B, punigluconin and pedunculagin). Also present are the phyllantine and zeatin alkaloids, and a number of benzenoids including amlaic acid, corilagin, ellagic acid, 3-6-di-O-galloyl-glucose, ethyl gallate, 1,6-di-O-galloyl-β-Dglucose, 1-di-O-galloyl-β- Dglucose, putranjivain A, digallic acid, phyllemblic acid, emblicol, and alactaric acid. The fruit is a rich source of ascorbic acid-Vitamin C.

Emblica provides a broad-based approach to preventing cardiovascular damage through its powerful cholesterol regulating and multi-faceted antioxidant properties. Two of its most exciting effects are its ability to reduce cholesterol and LDL (bad cholesterol) levels, and improve glucose tolerance and blood sugar regulation. This is especially important because both high blood cholesterol and high blood sugar are risk factors for the development of cardiovascular disease.

Flavanoids which had significant inhibitory effect on hepatic 3-hydroxy-3-methylglutaryl- Coenzyme A (HMG CoA) reductase activity brings the reduction in the absorbtion of cholesterol which is the primary factor responsible HDL increase and ultimately this enhances the reverse transport of circulating cholesterol. The tannoid principles (emblicanin-A- and emblicanin-B-) of fruits of E. officinalis have been reported to exhibit antioxidant activity in vitro and in vivo.

In the light of the volatile components that are having a significant role on HDL increase could prove as a pharmacological tool for the treatment of cardiovascular diseases.

This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

PRIOR ART

US Patent application, 2007/0196525 AI entitled "Composition to enhance HDL cholesterol and to decrease intima-media thickening in animals and humans and a method for its preparation" claims to the 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.

However, the same is nowhere in relation to the application of the instant invention, where it actually works on the application of the extract of Emblica officinalis for improvement of cardiovascular health by monitoring the up-regulation of Apo Al markers.

US granted patent 6290996 entitled "Method of inhibiting blood platelet aggregation" claims to the method of inhibiting blood platelet aggregation in humans which comprises administering an extract blend of the fruit of the Emblica officinalis plant to control said aggregation, suitably in a dose amount of about 50-500 mg/day. However, the aforementioned citation does not show any relevant similarity with the application of the present invention, wherein it specifies the usage of a non-toxic plant extract to improve the cardiovascular health by monitoring the up-regulation of Apo Al markers.

A PCT application bearing the application no. WO 2004/078190 Al entitled "A process and technique to elevate serum high-density lipoprotein claims to the invention" relates to an innovative and path breaking technique that elevates high-density lipo protein (HDL) serum and enhances its efficacy to prevent accumulation of Cholesterol in the tissues. The inventive step is also epitomized in an Eco-friendly method and manner of extracting a standardized and efficacious vegetable phytosterol of Embilica officinalis dispensable in free flowing ingestible granules.

However, the given invention is nowhere in relation to the application of the instant invention, where it actually works on the application of the plant extract for improvement of cardiovascular health by monitoring the up-regulation of Apo Al markers.

A PCT application bearing the application no. WO 2008/1 18010 Al entitled "Composition for the treatment of diabetes mellitus and metabolic syndrome" relates to the field of pharmacology. More specifically, the invention relates to a composition for the treatment of diabetes mellitus and/or the metabolic syndrome. Even more specifically, the invention relates to a herbal composition for treating the same. In one of its embodiments, the invention provides a composition comprising a) an anti hyperglycaemic agent b) an anti inflammatory agent c) an anti hyperlipidemic agent d) an anti oxidant agent e) a gastro-intestinal agent, wherein said activities are provided by parts or extracts of Curcuma longa, Gymnema sylvestre, Momordica charantia, Emblica officinalis, Trigonella foenum graecum and at least one species of Terminalia.

However, the same is nowhere in relation to the application of the instant invention, where it actually works on the application of plant extract for improvement of cardiovascular health by monitoring the up-regulation of Apo Al markers.

A PCT application bearing the application no., WO 2008/080162 A2 entitled Anti- cholesterolemic compounds and methods of use relates to the invention provides novel compounds with hypocholesteremic activity from crude Embilica officinalis (EO) extracts and methods of use. The invention also provides nutraceuticals. The invention claims to the method for preventing or treating an elevated blood lipid level-related disease or disorder in a subject comprising administering to the subject an effective amount of one or more gallic acid derivatives, thereby preventing or treating an elevated blood lipid level-related disease or disorder in the subject.

However, the aforementioned citation does not shows any relevant similarity with the application of the present invention, wherein it specifies the usage of a non-toxic plant extract to improve the cardiovascular health by monitoring the up-regulation of Apo Al markers.

US Patent application, US 2004/0121028 Al entitiled "Plant based agents as bioavailability / bioefficacy enhancers for drugs and nutraceuticals" relates to the isolation and preparation of an active fraction from plant Cuminum cyminum, its further purification and standardization as chemically defined entity with their intended use as drug bioavailability enhancer for the drugs belonging to therapeutic categories such as antimicrobial, antifungal, anti-viral, antitubercular, antileprosy, anti-inflammatory, antiarthritic, cardiovascular, antihistamines, respiratory distress relieving drugs, immunosuppressants, anti-ulcerogenic, anti-cancer, CNS drugs, corticosteroids, nutraceuticals in compositions to be administered orally/parenterally, topically, inhalations (including nebulizers), rectally, vaginally in human beings and/or veterinary conditions. The invention also claimed in the claim 22, wherein herbal formulation is selected from the group comprising of

echinacea, tinospora cordifolia, picrorrhiza kurroa, emblica ribes, asparagus racemosus, terminalia chebula and centella asiatica.

However, the given invention is nowhere in relation to the application of the instant invention, where it actually works on the application of plant extract for improvement of cardiovascular health by monitoring the up-regulation of Apo Al markers.

European Patent bearing the Application No. EP 1 176971 Bl entitiled "Use of phyllanthus for treating chronic inflammatory and fϊbrotic processes relates to the use of Phyllanthus for preventing or treating the propagation of connective tissue". The aim of the invention is to maintain the level of reduced glutathione, to inhibit the lipopolysaccharide (LPS)-induced nitrogen monoxide synthesis (NOS) and to inhibit the expression of the cyclooxygenase (COX-2) protein.

However, the aforementioned citation does not shows any relevant similarity with the application of the present invention, wherein it specifies the usage of a non-toxic plant extract to improve the cardiovascular health by monitoring the up-regulation of Apo Al markers.

OBJECT OF THE PRESENT INVENTION

The principle object of the present invention is to obtain an active extract and bioactive fraction from different parts of Emblica officinalis plant.

Another object of the invention is to develop a process for isolating bioactive fraction from Emblica officinalis using aqueous, alcoholic and/or hydro-alcoholic and organic solvent, the preparation of such extracts.

Still another object of the present invention is to study the activity of the Emblica plant extract for its HDL upregulation, triglyceride inhibition, total cholesterol inhibition, free radical scavenging activity and other cardio vascular related disorders.

Yet, another object of the invention is to obtain a composition comprising active principles of Emblica officinalis, and the use of these extracts and constituents for the preparation of nutritional and nutraceutical application.

Still another object of the present invention is to provide Emblica plant extract, which is easily and safely administrable to children and adults.

Still another object of the present invention is to provide Emblica plant extract capable of treating cardiovascular diseases in more than one mode of action.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides Emblica officinalis plant extract for management of Cardiovascular disease and/ or related disorders in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of standardized Emblica officinalis plant composite extract, optionally along with pharmaceutically acceptable additives, to the subject; and a process for enhancing cardiovascular health properties of a Emblica officinalis plant extract, said process comprising steps of (a) size-reducing plant parts to obtain powder; (b) extracting the bioactives with a solvent and/or combination of solvents by heating at temperature ranging from 21° to 105° C to obtain a mixture; (c) clarifying the mixture to arrive at clear liquid; (d) concentrating the clear liquid to achieve a concentrated extract; (e) solubilizing the concentrated extract in a solvent and re-concentrating it to obtain further concentrated extract, followed by drying the treated extract to obtain the plant bioactives which have significant role in reducing cardiovascular risk. The invention further provides for uses of the extract to manufacture a medicament for multiple therapeutic uses, as well as other healthful benefits.

BRIEF DESCRIPTION OF THE ACCOMPAJSfYING DRAWINGS

Figure 1: Metabolite profile of the extract at 254nm of A VDHC09Eo( 105)08(100)

Figure 2:Comparative overlay of AVDHC09Eo( 105)08(100) extract.

Figure 3: Metabolite fingerprinting of AVDHC09Eo( 105)08(100) extract scanned from 200 to 700 nm using Metagrid™ software.

Figure 4: is a Bar graph showing effect of AVDHC09Eo( 105)08(100) on the cell viability in HepG2 cells at 24 hrs of treatment.

Figure 5: is a Bar graph showing effect of AVDHC09Eo( 105)08(100) extract on ApoAl release in HepG2 cells at 24 hrs of treatment.

Figure 6: is a Bar graph showing effect of AVDHC09Eo( 105)08(100) extract on Triglyceride levels in HepG2 cells at 4 hrs of treatment.

Figure 7: is a Bar graph showing effect of AVDHC09Eo( 105)08(100) extract on Total Cholesterol levels in HepG2 cells at 4 hrs of treatment.

Figure 8: is a Bar graph showing effect of AVDHC09Eo( 105)08(100) extract on HDL cholesterol levels in HepG2 cells at 4 hrs of treatment.

Figure 9: is a graph showing Antioxidant effect of AVDHC09Eo( 105)08(100) extract

DETAILED DESCRIPTION OF THE INVENTION

The present invention is in relation to a method of treating cardiovascular disorder and/ or related disorders in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of Emblica plant extract, optionally along with pharmaceutically acceptable additives, to the subject.

In another embodiment of the present invention, wherein the subject is an animal or human being.

In yet another embodiment of the present invention, wherein the Emblica plant extract is having ApoAl enhancing activity.

In still another embodiment of the present invention, wherein the related disorders comprise increased level of LDL, decreased level of HDL, increased cholesterol triglyceride level, hypertentsion, atherogenesis, sclerosis, hypercholestrolaemia, hypocholestrolaemia, angina, vascular inflammation, stroke, and myocardial infarction.

In still another embodiment of the present invention, wherein the additives are selected from a group comprising granulating agents, binding agents, lubricating agents, disintegrating agents, sweetening agents, coloring agents, flavoring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents and spheronization agents.

In still another embodiment of the present invention, wherein the Emblica extract is formulated into dosage forms selected from a group comprising tablet, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion in hard or soft gel capsules, syrups, elixirs, phyotceuticals and neutraceuticals.

In still another embodiment of the present invention, wherein the Emblica plant extract is non-toxic and free of adverse effects.

In still another embodiment of the present invention, wherein the Emblica plant extract can be administered to subjects in combination with agents selected from a group comprising synthetic drugs, plant extracts and food stuffs.

In still another embodiment of the present invention, there is provided a process for enhancing therapeutic properties of a Emblica plant extract, said process comprising steps of (a) size- reducing plant parts to obtain powder; (b) extracting the powder with a solvent and/ or combination of solvents by heating at temperature ranging from 21° to 105° C to obtain a mixture; (c) clarifying the mixture to arrive at clear liquid; (d) concentrating the clear liquid to achieve a concentrated extract;

(e) solubilizing the concentrated extract in a solvent and re-concentrating it to obtain further concentrated extract; and (f) drying the treated extract to obtain Emblica plant extract.

In another embodiment of the present invention, wherein the size reduction is done either manually or mechanically to achieve powder particle size ranging from 20 U to 80 # mesh size.

In yet another embodiment of the present invention, wherein the parts of plant are selected from a group comprising root, shoot, leaf, fruits and seeds or the whole plant.

In still another embodiment of the present invention, wherein the solvent used for extraction could be aqueous, organic and/or combinations thereof.

In still another embodiment of the present invention, wherein said solvents and/ or combination of solvents are selected from a group comprising water, buffer, cell media, dilute acid, dilute bases, methanol, ethanol, n-propanol, isopropanol, 2-butanol, and terbutanol.

In still another embodiment of the present invention, wherein the powder is extracted with a solvent at a ratio ranging from 1 : 3 to 1 : 50 or 100%.

In still another embodiment of the present invention, wherein the powder is extracted with a solvent preferably at a ratio of about 1 :10.

In still another embodiment of the present invention, wherein the extraction of powder with solvent and/ or combination of solvents is brought about by stirring for a time period preferably ranging from 2-3 hours.

In still another embodiment of the present invention, wherein the extraction of powder with solvent and/ or combination of solvents is brought about by stirring preferably for a time period of about 2.5 hours.

In still another embodiment of the present invention, wherein the extraction of powder with solvent and/ or combination of solvents is brought about by heating preferably at specific temperatures of about 21° to 105° C.

In still another embodiment of the present invention, wherein said clarification is achieved by filtration or centrifugation.

In still another embodiment of the present invention, wherein said concentration method is selected from a group comprising but not limiting to soxhlation, rotary evaporation, distillation, centrifugal vacuum evaporation and lyophilisation.

In still another embodiment of the present invention, wherein said solubilization of concentrated extract is carried out in a solvent selected from a group comprising water, ethyl acetate, diethyl ether, hexane, dichloromethane, butyl alcohol, ether, acetone and/ or combination thereof.

The present invention provides a plant extract with HDL enhancing activity. In particular, the invention provides an Emblica plant extract with HDL increasing activity. Furthermore, the invention provides a non-toxic Emblica plant extract capable of treating cardiovascular disorders in more than one mode of action. Therefore, the invention provides a non-toxic Emblica extract with a better efficacy in the treatment of cardiovascular disorders. Still, the invention provides an Emblica plant extract which is easily and safely administrable to children and adults. Still further, the invention provides an Emblica plant extract, which is non-toxic, patient compliant, shows better efficacy and effectively in the treatment of cardiovascular. The invention provides also the method of preparation of a non-toxic, patient compliant, improved and effective Emblica plant extract.

The present invention is in relation to efficacy of the bioactive component of the Emblica plant extract for therapeutic use in the mitigation of cardiovascular and other related disorders, wherein said extract from Emblica officinalis optionally along with healthful or for nutritional and nutraceutical applications.

In one aspect of the invention, there is a provided a prophylactic method for preventing the occurrence of a disease state in a mammal which comprises administering to the said mammal an effective non-toxic amount of an extract from Emblica officinalis as defined herein in the preparation of a comestible (foodstuff) for prophylaxis against the occurrence of cardiovascular disease. Preferably the mammal is human and the said extract comprises a single extract from a plant part of Emblica officinalis or a combination of extracts there from as detailed herein. Thus the present invention further relates to extracts, which may be isolated from fruits of the Emblica officinalis plant, the preparation of such extracts, medicaments comprising such extracts, and the use of these extracts and constituents for the preparation of a medicament.

In another aspect of the instant invention, Pre preparation of raw material is carried out by cleaning the plant material to remove any contaminant from the plant material, free of dust etc. followed by powdering raw materials using a pulverizer and passing through the mesh to get uniform size of powder.

In another aspect of the present invention, extracts are isolated from fruits of Emblica offcinalis, using conventional inorganic and organic solvent extraction and supercritical fluid extraction technology alone and/or in combination. Generally, extracts of the invention capable of functioning in a prophylactic or therapeutic manner as outlined herein can be extracted from any Emblica officinalis plant, depending on the end purpose that is required of the extract.

In one of the embodiments of the present invention there is provided a process for preparing extracts of the invention from plant parts of Emblica officinalis that comprises:

• Obtaining plant material from one or more parts of the plants.

• Obtaining an extract from the plant material by contacting the plant material with an aqueous, an ethanolic or an organic solvent, or a combination thereof, optionally for a defined period of time thereby providing one or more plant extracts.

• Removing the plant material from the supernatant.

• Optionally, lyophilizing said supernatant.

• Analyzing the plant extracts for efficacy and enhancement of ApoAl levels against cardiovascular disorders.

• Selecting plant extracts having one or both of these activities.

The choice of selected plant material may be of any type but is preferably the fruits of the Emblica officinalis plant.

The solvent extraction process may be selected from direct types such as extraction from plant parts in reflux extractor apparatus or in flasks at room temperature or at higher temperature with polar and/or non-polar solvent(s). Typically, the extraction process is as outlined herein. In another embodiment of the invention, the compositions for preventing, treating, or managing cardiovascular diseases and related disorders, comprises of direct composite extract of plant species with alcohol, water and hydroalcohol solvent and successive extract of solvents from non-polar to polar range. The compositions/medicaments may contain a pharmaceutically acceptable carrier, excipient, or diluent.

In another embodiment of the invention, the HPLC profiles and Mass spectrums of direct and successive solvent extracts of Emblica officinalis plant parts are provided thereby giving each extract an identity of itself.

In yet another embodiment of the present invention, wherein the plants selected for the isolation of therapeutically relevant extracts/molecules to be used in the treatment of cardiovascular disorders, are subjected to both targeted and non-targeted screening procedures. The targeted screening procedures, which feature a comprehensive metabolite profiling of multitudes of phytoextracts, facilitates the creation of a metabolite grid.

The successive extraction from plant extract is carried out using soxhlet extractor. The solvents used, are based on their sequential polarity starting from non-polar to polar, wherein, various classes of metabolites will be extracted viz. petroleum ether (phytosterols, fixed oils and fats), benzene (fixed oils and fats), chloroform (alkaloids), acetone (phytosterols, phenolics and tannins) ethanol (alkaloids, carbohydrates, glycosides, phytosterols, saponins, phenolics, tannins, proteins and amino acids) and water (alkaloids, carbohydrates, glycosides, saponins, phenolics, tannins, proteins,

amino acids, gums and mucilage) at 65 ⁰ C. These fractions are lyophilized and stored in amber colored bottles at 4 ⁰ C.

High Performance liquid chromatography (HPLC) analysis is done for all the extracts extracted using Shimadzu and Waters HPLC systems. All the samples are run for HPLC analysis four or five times for reproducibility and best three reproducible runs are subjected for overlay and MetaGrid™ analysis.

The extracted fractions are subjected to HPLC using μ bondapak C 18 column (Waters Alliance 2695 Separation Module and Shimadzu LC 20AT) to separate the constituent metabolites. The fractions are eluted using a combination (80:20, 60:40, 50:50, 40:60, 20:80) of methanol: water / acetonitrile: water. The gradient run is also carried out wherever required. 5- lOul of sample is injected with flow rate of 1 ml/min and HPLC run is performed for about 30 minutes. The detection is done on photodiode array and the results are analysed with the help of Millennium™ software.

In yet another embodiment of the present invention, the analysis of output of LC-PDA and LC/MS/MS is done using both LC-Solution and Analyst software of Applied Biosystems along with the script that is developed by Avesthagen to determine the molecular weight of chemical compounds by ionizing, separating and measuring molecular ions according to their mass-to-charge ratio (m/z).

Extraction and resolution of components present in complex phyto-extracts is an area that has for long been plagued by lack of standard operating procedures that permit adequate standardization and quantitative estimation of metabolites at a comprehensive level. The metabolite fingerprinting methods commonly employed in the herbal industries are focused on less than 10 metabolites that occur in significant proportions (> 0.1%) within a given extract that may contain several thousand metabolites. A metabolite profiling approach that is solely focused on major constituents alone can not address the batch to batch variations observed in bioactivity especially in situations where the minor constituents (< 0.01 %) are responsible for the observed bioactivity. Further, there is poor coverage of the metabolite diversity which is typical of plant extracts by most of the commercially available standard metabolites detection techniques and often occasional variations in degree of interaction of metabolite constituents with a given chromatographic matrix resulting in concomitant changes in the retention time is encountered. Hence, to overcome this, in instant invention, a novel algorithm has been used that enables metabolite fingerprinting of multi-constituent plant extracts.

This metabolite profiling approach allows simultaneous profiling of >90% resolvable constituents of a phytoextracts (with respect to each given set of chromatographic column matrix and associated run parameters) across 50 different wavelength windows in the UV-Visible range (190-700 nm) of the absorption spectrum. The chromatographic data analysis system used in the instant

invention enables the analyst to focus on the development of metabolite resolution methodologies that capture the chemodiversity of complex phytoextracts even in the absence of information pertaining to the identities of the individual metabolite components. Further, the linear representation of all validated data points generated using instrument specific data acquisition software allows the runtime and absorption maxima based visualization of all resolved constituents. The Novel algorithm searches for all retention times that are within the tolerance range provided by the user that accommodates for possible variations in retention time of similar/ identical constituents across multiple runs of the same extract or multiple runs of different extracts that contain the said constituents. Also, this approach facilitates bioactivity-guided fractionation to a great extent since it allows the correlation of observed bioactiviry with specific sections of the chromatographic spectrum that represent a fraction of the total metabolite content.

The invention further describes the biotherapeutic potential of various extracts of Emblica officinalis as described above, by studying their performance in cell based assay models. The modulation of a major protein of HDL - ApoA-I in human hepatoblastoma cell line (Hep G2) has been studied. In one of the embodiments of the present invention, there is provided the results of the mammalian cell based efficacy tests conducted by growing Human hepatoblastoma cell line (Hep G2), in a flask with Eagle's Minimum Essential Medium (EMEM) containing 10% Fetal Bovine Serum (FBS), 1% glutamine-penicillin-streptomycin and 1% fungizone in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air. It is further subcultured and when cell become 80% confluent they are subjected to treatment with the said bioactive. The incubation is followed by estimating levels of bio-markers for Cardiovascular disorder like apolipoprotein AI (Apo AI), total cholesterol, HDL cholesterol, triglyceride levels, Angiotensin converting enzyme (ACE), endothelial Nitric oxide synthase (eNOS), C-Repactive protein (CRP), Tumor Necrosis Factor (TNF-alpha), Interlukins (IL-6) between the bioactive treated and untreated sets.

In another aspect of the invention there is provided a method for treating a disease in a mammal, which comprises administering to the said mammal an effective non-toxic amount of at least an extract from Emblica officinalis as defined herein. Preferably, the mammal is a human being. The skilled addressee will appreciate that "treating a disease" in a mammal means treating, that is to say, alleviating symptoms of the disease and may also mean managing a disease in the sense of preventing such a disease state either advancing i.e. getting worse or becoming more invasive, or slowing down the rate of advance of a disease.

The compositions/medicaments may contain a pharmaceutically acceptable carrier, excipient, or diluent. The compositions can be included as unit dosage suitable for parenteral, oral, or

intravenous administration to a human. Alternatively, the compositions are dietary supplements, food compositions or beverage compositions suitable for human or animal consumption.

It will be readily understood that the components of the present invention, as generally described herein, could be arranged and designed in a wide variety of different configurations. Thus, the following more detailed description of the embodiments of the system and method of the present invention is not intended to limit the scope of the invention, as claimed, but is merely representative of the presently preferred embodiments of the invention.

The instant invention herewith states in greater details an extraction procedure for the improvement of basic therapeutic activity of any given plant extract. It discloses unique procedures for preparation of improved plant extracts with increased efficacy. The plant extracts so produced by making use of the procedures laid down hereunder act in unprecedented modes of action towards the treatment of cardiovascular disorder. Plant material suitable for preparation of the plant extract for inclusion of the therapeutic composition of the invention is derived from a potential plant administered to a person suffering from cardiovascular disorders, which results in increasing the HDL levels of the patient. Administration of the composition to the patient both prevented and treated incidences of clinical cardiovascular disorder.

In accordance with a further embodiment of the present invention, the potential plant is a member of the family Euphorbiaceae. In another embodiment of the invention, the potential plant is a member of the genus Emblica. It will be readily apparent to one skilled in art that other extracts capable of potential positive effect on cardiovascular health could be isolated using similar techniques from other known Emblica species and also from a wide range of plants i.e., potential plants. The potential plants include all species of the family Euphorbiaceae, including terrestrial, aquatic or other plants that can be subjected to standard extraction procedures such as those described herein in order to generate an extract that can be tested for its therapeutic abilities. The present invention is directed to an herbal medicinal composition comprising the foregoing plant extracts that can be administered to a person suffering from CVD which results in increasing the HDL level of the patient.

AmIa is a deciduous tree, small to medium in size, the average height being 5.5 meters; its bark is usually light brown to black, coming off in thin strips or flakes, exposing the fresh surface of a different colour underneath the older bark; the average girth of the main stem is 70 cm; in most cases, the main trunk is divided into 2 to 7 scaffolds very near the base. Leaves are 10 to 13 mm long, 3 mm wide, closely set in a pinnate fashion, making the branches feathery in general appearance. The leaves develop after the fruit-set. Flowers are unisexual, pale green, 4 to 5 mm in length, borne in leaf-axils in clusters of 6 to 10; staminate flowers, tubular at the base, having a very small stalk, gamosepalous,

having 6 lobes at the top; stamens 1 to 3, polyandrous, filaments 2 mm long; pistillate flowers, fewer, having a gamopetalous corolla arid a two-branched style; both staminate and pistillate flowers are borne on the same branch, but the staminate flowers occur towards the apices of small branches. Fruits are fleshy, almost depressed to globose. 2.14 cm in diameter, 5.68 g in weight, 4.92 ml in volume and are primrose yellow in colour. The stone of the fruit is six-ribbed, splitting into three segments, each containing usually two seeds; seeds 4-5 mm long, 2 to 3 mm wide.

As used herein, "extract" refers to a concentrated preparation of the essential constituents of the medicinal plant. Typically, an extract is prepared by drying and powderizing the plant. Optionally, the plant, the dried plant or the powderized plant may be boiled in solution. The extract as used herein may be used in liquid form, or it may be mixed with other liquid or solid medicinal herbal extracts. Alternatively, this medicinal herbal extract may be obtained by further precipitating solid extracts from the liquid form.

As used herein the term "composition" means any administrable form of the herbal extract given alone or in combination with other herbal, ayurvedic or pharmaceutical composition. The term includes but is not restricted to tablets, capsules, lozenges, creams, lotions, suspensions, oils and the like.

As used herein the term, "related disorders" means disorders related to cardiovascular health, which include but are not limited to hypertension, atherogenesis, sclerosis, hyper and hypocholesterolaemia, angina, vascular inflammation and stroke.

As used herein the term, "potential plants" includes plants from which extracts effective against cardio vascular disorders can be extracted out. The term comprises of plants like Momordica sp., Berberis sp, Gymnema sp, Terminalia sp.

As used herein the term "herbal composition" means an extract containing different compounds from the same plant or from different plants in various combinations

The invention is further elaborated with the following examples. However, these examples should not be construed to limit the scope of the invention.

Extraction of the plant material by solvent extraction process:

The plant material employed in the extraction process can be the entire potential plant, or it can be one or more distinct tissues from the plant for example, leaves, seeds, roots, stems, flowers, fruits or various combinations thereof but preferably the root of the plant. The plant material may also be treated prior to extraction, for example, by drying, freezing, lyophilizing, or some combinations thereof. If desired, the plant material can be fragmented and/or homogenized by some means such that a greater surface area is presented to the solvent. For example, the plant material can be crushed

or sliced mechanically, using a grinder or other device to fragment the plant parts into small pieces or particles, or the plant material can be frozen in liquid nitrogen and then crushed or fragmented into smaller pieces.

The solvent used for the extraction process can be aqueous, alcoholic or organic, or a combination thereof. In one embodiment of the present invention, plant material is extracted with an aqueous solvent. Examples of suitable solvents include but are not limited to water, buffers, cell media, dilute acids or bases and the like.

In an alternate embodiment of the invention, the plant material is extracted with hydro alcoholic solvent. Examples of suitable alcoholic solvents include, but are not limited to methanol, ethanol, n-propanol, iso-propanol, 2-butanol, ter-butanol, and combinations thereof.

Various extraction processes are known in the art and can be employed in the methods of the present invention. The extract is generally produced by contacting the solid plant material with a solvent with adequate mixing and for a period of time sufficient to ensure adequate exposure of the solid plant material to the solvent such that inhibitory activity present in the plant material can be taken up by the solvent.

The solvent extraction process may be selected from direct and successive extraction types such as extraction from plant parts in soxhlet apparatus or in flasks at room temperature or at higher temperature with polar and/or non-polar solvent (s). Regardless of the number of extraction processes, each extraction process typically is conducted over a period of time between about 6 hours to 24 hours at room temperature. Adequate contact of the solvent with the plant material can be encouraged by shaking the suspension.

The liquid fraction is then separated from the solid (insoluble) matter resulting in the generation of two fractions: a liquid fraction and a solid fraction, which is the potential extract. Separation of the liquid and solid fractions can be achieved by one or more standard processes known to those skilled in art.

The potential extracts obtained thereof may be concentrated and solubilised in an appropriate solvent preferably ethyl acetate. Examples of various other organic solvents include but are not limited to, di-ethyl ether, hexane, heptane, dichloromethane, ethyl acetate, butyl alcohol, ether, acetone and the combinations thereof.

The purified extracts or partially purified extracts are concentrated by solvent removal from the original extract and/or fractionated extract, and/or purified extract. The techniques of solvent removal are known to those skilled in the art and include, but are not limited to rotary evaporation,

distillation (normal and reduced pressure), centrifugal vacuum evaporation (speed vac), and lyophilisation.

The extract referred to herein can be produced by any of the two procedures stated hereunder. The procedures laid down herewith are general procedures alterable with variations known in the art to one skilled in the art. These may not in any way be treated as restrictive to the instant invention.

The technology of the instant application is further elaborated with the help of following examples. However, the examples should not be construed to limit the scope of the invention.

Extraction of Emblica officinalis plant parts was carried out with alcohol, water and hydroalcohol solvent in reflux extractor apparatus or at room temperature under agitation followed by lyophilization under vacuum.

Brief description of Extract Nomenclature:

AVD-Avesthagen, HC-HDL cholesterol, plant genus and species,(temperature) of the extraction, solvent code(04-ethanol, 08-water), (% of solvent used)

Example 1:

Extraction of Emblica officinalis

Process A: Reflux Extraction

Extraction of Emblica officinalis plant parts was carried out with alcohol, water and hydroalcohol solvent in reflux extractor apparatus or at room temperature under agitation followed by lyophilization under vacuum.

Powdered Emblica officinalis plant material was weighed into the round bottom flask. Various concentrations of alcohol, water and hydro-alcohol was added in to the round-bottomed flask and placed on the mantle along with few (3-4) ceramic chips. The reflux condenser was then placed on the flask. Cold water was allowed to circulate continuously in the condenser from the tap. The mantle was switched on and the temperature was set to the boiling point of the solvent. The vapors of the solvent from the flask passed through the inlet of the extractor and condenses. The condensed (distilled) solvent thus extracting the compounds from it. This process is continuous as long as there is stable heat and water circulation. The extraction was continued for 2 hours at room temperature. After 2 hours the mantle was switched off and the water flow was stopped. After cooling the extract was collected separately and centrifuged.

The extract was concentrated by fitting the flask containing the extract with the empty soxhlet extractor body that in turn was fitted tightly with the water-cooled condenser. Continuous water flow was maintained and the flask was heated till the solvent from the flask was distilled and collected in the extractor body up to a level (One inch below the inlet). The temperature was reduced to avoid charring as the volume of the solvent reduced in the flask. The distilled solvent collected in the extractor was transferred to the solvent bottles and label appropriately. The process was continued till only very little solvent was left in the flask and no charring had occurred. Further concentration was done in the rotovapour apparatus to remove the solvent completely. The extract in the flask were swirled and were dried under vacuum. Storage and labeling of the extract was done to obtain the Extract ED.

Example 2:

Extraction of Emblica officinalis

Process B: Room Temperature Extraction

Powdered Emblica officinalis plant material was weighed into conical flask. Various concentrations of alcohol, water and hydro-alcohol was added in to the conical flask and placed under agitation at room temperature for 2 hrs. After 2 hrs, centrifuge at 4500 rpm for 15 minutes at 4 ⁰ C. After centrifugation supernatant is taken and concentration is done in rotor evaporator. Further concentration is done in lyophilizer. The % yield of the extract obtained is calculated.

The extract was concentrated by fitting the flask containing the extract with the empty soxhlet extractor body that in turn was fitted tightly with the water-cooled condenser. Continuous water flow was maintained and the flask was heated till the solvent from the flask was distilled and collected in the extractor body up to a level (One inch below the inlet). The temperature was reduced to avoid charring as the volume of the solvent reduced in the flask. The distilled solvent collected in the extractor was transferred to the solvent bottles and label appropriately. The process was continued till only very little solvent was left in the flask and no charring had occurred. Further concentration was done in the rotovapour apparatus to remove the solvent completely. The extract in the flask were swirled and were dried under vacuum. Storage and labeling of the extract was done to obtain the Extract ED.

Example 3:

Extraction of AVDHC09Eo(105)08(100)

Raw material was cut / pulverized and 100 grams of powdered plant material was weighed into the round bottom flask. The pulverized powder was contacted to an aqueous solvent in a ratio ranging from 1 :5 to 1 : 50 (powder: solvent). 1000 ml of DM water were added in to the round- bottomed flask and was placed on the mantle. The condenser was placed on the flask and cold water was allowed to circulate continuously in the condenser from a tap. The mantle was switched on and temperature was set to boiling point of the solvent. The extraction step was continued for 2 hours, after which the mantle was switched off, water flow was stopped and it was allowed to cool. The top solvent layer was taken and centrifuged at 4500 rpm for 15 mins at 4° C. The extract collected is transferred to a flask and it was concentrated by lyophilizing under vacuum. The weight of the dry extract was recorded and it was stored in an airtight glass container in a dry place at room temperature.

Yields and extract characteristics: The % yield of the extract is 52.0%. The extract is brown color powdered extract.

Example 4:

HPLC fingerprinting of AVDHC09Eo(105)08(100)

Sample Preparation:

ImI of HPLC grade water was added to lOmg of the sample and sonicated for 15mts.This was centrifuged at 13000rpm for 15mts.The supernatant was filtered using a Nylon filter (13X0.2μm) with a ImI syringe into an injection vial.

Instrument Initiation:

The instrument was switched on in the following order pump, auto sampler, column oven, PDA and then the communication bus module. The LC solution is activated, which solution controls the analytical instruments. From the Operations Menu, Analysis 1 icon is clicked to open a Data Acquisition window that displays the following fields.

Instrument Parameters:

Data Acquisition: LC stop time: 66min, Acquisition time (PDA), Sampling: 1.5625 Hz, Start Time: 0 mts, End Time: 48mts

Pump: Model: LC-20AT, Mode: Low Pressure Gradient, Total Pump flow: 1 ml/min,

Solvent B Cone: 0 ml/min, Solvent C Cone: 0 ml/min, Solvent D Cone: 0 ml/min, Maximum Pressure Limit: 380kgf/cm ²

PDA: Model: SPD-M20A, Start wavelength: 190nm, End wavelength: 800nm,Slit Width: 1.2nm Column Oven: Oven Temperature: 33 ⁰ C Controller: Model: CBM-20A, Power: On

Auto sampler: Model: SIL 2OA, Sample Rack: 1.5ml STD, Rinsing Volume: 200μl, Needle stroke: 53mm, Control Vial: 52mm, Rinsing Speed: 35μl/sec, Sampling Speed: 15μl/sec, Purge Time: 25min, Rinse Mode: Before and after aspiration, Rinse dip time: 10 sec

Auto purge: Mobile Phase A: 5 min, Mobile Phase B: 5 min, Mobile Phase C: 5 min, Mobile Phase D: 5 min, Auto sampler: 5 min, Total Pump Flow: 1 ml/min

After checking all the above mentioned parameters the inlets from every solvent system were purged with the respective solvents for five minutes to clear the tubes of any air bubbles. Further the column was washed with HPLC grade water (100%) for 30mts and then equilibrated with the initial run conditions of the corresponding LC time program.

Run Specifications:

Column Used: Atlantis, C 18; 5μm; 4.6X250mm Solvent A: 0. 1%TFA in water Solvent B: 0. 1%TFA in Acetonitrile Solvent C: 100% Methanol Solvent D: 100% Water Injection Volume: 20μl LC Time Program: Flow rate: 1 ml/min

The extract was injected at a constant volume of 25μl and its metabolite profiling monitored at 254nm. Table 1 provided below represents the peak information of the said extract at 254 nm. A comparative profiling as represented in Figure 2 was performed between the runs/ extract to check the reproducibility of the results. Typical HPLC chromatogram using the above said protocols and conditions for the extract AVDHC09Eo (105) 08(100) is provided in Figure 1.

Table 1: Peak information of AVDHC09Eo (105) 08(100) extract at 254 nm

Table 2: Summary of percentage conservation of AVDHC09Eo (105) 08(100) extract scanned from 200 to 700 nm using Metagrid™ software

Constituents and conservation profiles of AVDHC09Eo (105) 08(100) extract scanned from 200 to 700 nm using Metagrid™ software, as shown in Table 2 and Figure 3 revealed that 90-100% of conservation at the wavelength of 550/580nm, 80-90% conservation at the wavelength of 210/220/230/240/250/260/270/290/560/590/600/610 nm, 70-80% conservation at the wavelength of 300/370/420/440/460/530/540/630/650/660/680/690/700 nm, 60-70% conservation at the wavelength of 380/390/400/410/450/470/490/500/510/640/670 nm, and 25-60% of conservation at the 430/480 nm.

Example 5:

Cell based assay for Apo Al activity:

Lipids are transported throughout the body by complex structures called lipoproteins. Lipoproteins are classified into five major density classes: chylomicrons, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Associated with these lipoproteins, at least five major apolipoproteins have been described and have been labeled A through E. The principle apolipoproteins of HDL are the A apolipoproteins, constituting nearly 90% of the protein mass. Thus, apo A-I has been found to be more powerful as a marker in some studies for coronary disease than other HDL components (eg. cholesterol). Apo A-I enhances fibrinolysis and antiplatelet activity, thus possibly having an antithrombotic role in coronary disease prevention. Studies have indicated that the increased apoA-I gene expression in apoE-deficient mice markedly suppressed atherosclerosis, further supporting a protective role for apoA-I.

The composite extracts were taken forward for efficacy validation using HepG2 cells based assay system. This step is for estimating bioactivity of the composite extracts to modulate levels of apoAI.

ApoAl a major protein of HDL, initiates cholesterol efflux and thereby facilitates removal of excess tissue (e.g. arterial) cholesterol by the process of reverse cholesterol transport.

Human hepatoblastoma cell line (Hep G2) was grown in a T-75 flasks with 15 ml of EMEM containing 10% FBS, 1% glutamine-penicillin-streptomycin and 1% fungizone in a humidified incubator at 37 ⁰ C in an atmosphere of 5% CO ₂ and 95% air. Subcultures when cell become 80% confluent by trypsinization with PBS containing 0.5 mmol/L EDTA.

The cells were plated in 96 well plates at a concentration of 3x106 cells/ml and grown until they attained 75% to 80% confluence. A dose response curve of gemfibrozil (0 to 400 μmol/L) / herbal extracts on apoA-I secretion for varying times (24 hours) was prepared and at the termination of the incubation, 50-μL of cell supernatant was taken to estimate the apoA-1 concentration by an enzyme-linked immunosorbent assay (ELISA)

After termination of the reaction, the cell supernatant was removed and washed twice with PBS and the cells were lysed either with 100 μL of 1% Triton X 100 or with 0.1% SDS. 10 μl of the sample was taken and added to 250 μl of Bradford reagent and the reading was taken at 590 nm.

Calculation was done according to the kit protocol as ng /μg of cellular protein and the graphs were generated using Graphpedprism Software.

Apo Al analysis of AVDHC09Eo(105)08(100)

AVDHC09Eo (105) 08 (100) did not show any cytotoxic effect on HepG2 cells at all the studied doses (0.1 to 500 μg/ml) at 24 hrs of treatment. The results obtained from cell assay on the said extract is stated in Tables 3 and represented in Figure 4.

AVDHC09Eo (105) 08 (100) did not show any significant increase in the Apo Al level at lowest dose of 0.1 μg/ml. But at doses of 1-10 μg/ml, a significant increase in the ApoaAl level was observed with 1.2-1.4 fold increase when compared to that of the positive control (Gemfibrozil) exhibiting 1.3 fold increase followed by no increase at higher doses of 100-500 μg/ml The results is stated in Tables 4 and represented in Figure 5.

Thus the result seems to suggest that AVDHC09Eo (105) 08 (100) extract is a good extract in elevating the level of ApoAl in human hepatoblastoma cell line (HepG2) at lower doses.

Table 3: Effect of AVDHC09Eo (105) 08 (100) water extract on ApoAl levels in HepG2 (Human hepatoblastoma) cells at 24 hrs of treatment

* - The sample was diluted in water, sterilized though a 0.22 μ filter and used for the assay. The experiment was terminated at 24 hrs.

** - Results are the mean ± SD of triplicates per sample. AVDHC09Eo (105) 08 (100) is compared to the cell control while Gemfibrozil has been compared respective Ethanol control.

*** - P < 0.001, ** - P < 0.01

Example 6:

Cell based assay for Triglyceride Level inhibition

Effect of The Plant Extracts on Triglyceride And Cholesterol Level Of Hepg2 Cells

The estimation of Triglycerides involves the following enzymatic reactions:

LPL Triglycerides → Glycerol + FFA

GK

Glycerol + ATP → Glycerol-3-phosphate + ADP

GPO Glycerol-3-phosphate + O2 → DHAP + H2O2

POD

2H2O2 + 4-AAP → Quinoneimine dye + 4H2O

Absorbance of the Quinoneimine dye measured at 505nm is directly proportional to triglycerides concentration

Triglyceride assay:

The supernatant was taken and the absorbance was measured at 5O5nm. The cells were plated in 12 well plates at a concentration of 2.5x105 cells/ml. The cells were grown until they attained 75% to 80% confluence. The old media was discarded and fresh media containing 10%LPDS was added and the plates were incubated for 24 hrs. The old media is removed and fresh media is added again followed by the addition of the herbal extracts / positive control and incubated the plate for 4 hrs. The supernatant is removed and the cells are washed with PBS. To this is added 100 μl of 1% Triton X 100. For Processing, the cells are lysed and 500 μl of triglyceride monoreagent is added by mild vortexing and spun at 13,000rpm for 1 min. The supernatant taken and the absorbance is measured at 505nm. Cell based assay for Triglyceride inhibition by the extract of AVDHC09Eo(105)08(100)

AVDHC09Eo( 105)08(100) extract exhibited a statistically significant inhibition in triglyceride level (39%) at highest concentration of 500 μg/ml when compared to that of simvastatin showing 33% as shown in Figure 6 and Table 4.

Table 4: Effect of AVDHC09Eo(105)08(100) on triglyceride level in HepG2 (Human hepatoblastoma) cells at 4 hrs of treatment

Example 7:

Cell based assay for Triglyceride Level inhibition

The estimation of cholesterol involves the following enzymatic reactions:

CE Cholesterol esters — » Cholesterol + FFA

CHOD

Cholesterol + O2 → Cholest-4-en-3-one + H2O2

POD 2H2O2 + phenol + 4-AAP → Quinoneimine dye + H2O

Absorbance of the Quinoneimine dye measured at 5O5nm is directly proportional to total cholesterol concentration.

For HDL cholesterol: LDL, VLVL and chylomicron fractions are precipitated by addition PEG. After centrifugation the HDL fractions remain in the supernatant and is determined by CHOD- PAP method.

Cell line: Name (Passage number): HepG2 cells (P/N - 23) Seed density: number of cells/well: 2.5 X 105 cells/ml/well for triglyceride

1 X 105 cells/ml/well for cholesterol AVDHC09Eo( 105)08(100) was tested at concentrations of 0.1, 1, 10, 100 and 500 μg/ml.

Cholesterol assay:

The cells are plated in a 12 well plate at a concentration of 1 x105 cells/ml and grown until they attained 75% to 80% confluence. The old media is discarded and fresh media containing 10%LPDS is added and the plates are incubated for 48 hrs. The old media is removed and fresh media is added along with the herbal extracts / positive control to the plates and incubated for 4 hrs. The supernatant is removed and the cells are washed with PBS and 100 μl of 1% Triton X 100 is added.

Processing for total cholesterol is done by lysing the cells by sonication at 30 amp for 20 sees and 500 μl of cholesterol monoreagent is added by mild vortexing. After mixing it well the lysed cells are incubated at 37° C for 10 mins or at 15-30° C for 30 mins and the absorbance is measured at 505nm.

Processing for HDL is done by lysing the cells by sonication at 30 amp for 20 sees and equal volume of precipitating reagent is added. After mixing it well, the lysed cells are incubated at 15-30° C for 10 mins followed by centrifugation for 15 mins at 2000 rpm at room temperature. 100 μl of supernatant is taken and 500 μl of cholesterol monoreagent is added and mixed well followed by incubation at 37° C for 10 mins or at 15-30° C for 30 mins and the absorbance is measure at 5O5nm.

Cell based assay for total cholesterol inhibition and up regulation of HDL cholesterol by the extract AVDHC09Eo(105)08(100)

AVDHC09Eo( 105)08(100) did not show any effect on total cholesterol level at lower doses of 0.1-100 μg/ml, however significant inhibition was observed in total cholesterol level with 31% inhibition at 500 μg/ml as presented in table 5 and Figure 7. A significant increase in HDL cholesterol level was observed at lower doses of 1-10 μg/ml with 26-27% increase, but no effect at higher doses as shown in Table 6 and Figure 8.

This result suggests that AVDHC09Eo( 105)08(100) is an active extract for reducing triglyceride level at higher doses but HDL cholesterol at lower doses.

Table 5: Effect of AVDHC09Eo(105)08(100) on total cholesterol level in HepG2 (Human hepatoblastoma) cells at 4 hrs of treatment

Table 6: Effect of AVDHC09Eo( 105)08(100) on HDL cholesterol level in HepG2 (Human hepatoblastoma) cells at 4 hrs of treatment

Example 8:

DPPH Assay for Free radical scavenging potential of the Emblica water extracts

The DPPH molecule is characterized as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole and has violet in color under this state. Up on DPPH reduction by any hydrogen donor, reduced DPPH losses violet color and turn to pale yellow color. The reduction in violet color is directly proportional to DPPH reduction. The reduction reaction, therefore intended to provide the link with the reactions taking place in an oxidizing system, such as the auto-oxidation of a lipid or other unsaturated substance and the DPPH molecule is thus intended to represent the free radicals formed in the system.

DPPH Assay of AVDHC09Eo(105)08(100):

In the present invention the isolated extracts were used to estimate its free radical scavenging potency relative to ascorbic acid by using Calorimetric-DPPH method (Polterait O. (1997) Anti Oxidants and free-radical Scavengers of Natural origin Current Org. Chem. 1. 415-440). The DPPH assay of the extract AVDHC09Eo( 105)08(100) is performed in triplicate within the range of lOμg/ml to 50 μg/ml. The Emblica water extract isolated from the fruits of the plant showed IC50 range at 39.3μg/ml concentration. The result is represented as a graph in figure 9.

Example 9:

Modes of administration

For administration to a mammal, the therapeutic composition can be formulated as a pharmaceutical or naturopathic formulation such as phytoceuticals or nutraceuticals, for oral, topical, rectal or parenteral administration or for administration by inhalation or spray. The phytoceutical or naturopathic formulation may comprise the one or more plant extracts in dosage unit formulations containing the conventional non-toxic physiologically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrathecal, intrasternal injections or infusion techniques.

The pharmaceutical or naturopathic formulations may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs. The therapeutic compositions of the invention may be formulated as phytoceuticals, or nutraceuticals. Phytoceuticals may optionally comprise other plant-derived components and can therefore be delivered by such non-limiting vehicles as teas, tonics, juices or syrups. Nutraceuticals contemplated by the present invention may provide nutritional and/or supplemental benefits and therefore be delivered, for example as foods, dietary supplements, extracts, beverages or the like. Phytoceutical and nutraceuticals can be administered in accordance with conventional treatment programs and/or may be a part of the dietary or supplemental program.

Formulations intended for oral use may be prepared according to methods known in art for the manufacture of pharmaceutical compositions and may contain one or more agents selected from the group of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide palatable preparations.

Tablets contain the active ingredient in admixture with suitable non-toxic physiologically acceptable excipients including, for example, inert diluents, such as calcium carbonate, lactose,

calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch, or alginic acid, binding agents, such as starch, gelatine or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets can be uncoated, or they may be coated by known techniques in order to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

Various additives or carriers can be incorporated into the orally delivered pharmaceutical naturopathic formulations or the invention. Optional additives of the present composition include, without limitation, phospholipids, such as phosphatidyl glycerol, phosphotidyl inositol, phosphotidyl serine, phosphotidyl choline, phosphotidyl ethanolamine as well as phosphatidic acids, ceramide, cerebrosides, sphingomyelins and cardiolipins.

Pharmaceutical or naturopathic formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the active ingredient is mixed with water or an oil based medium such as peanut oil, liquid paraffin or olive oil.

A syrup may be made by adding the active extract to a concentrated, aqueous solution of a sugar, for example sucrose, to which may also be added any necessary ingredients. Such accessory ingredient (s) may include flavorings, an agent to retard crystallisation of the sugar or an agent to increase the solubility of any other ingredients, such as polyhydric alcohol for example glycerol or sorbitol.

Oily suspensions may be formulated by suspending the plant extract(s) in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or acetyl alcohol. Sweetening agents and/or flavoring agents may be added to provide palatable oral preparations. These formulations can be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation suitable for an aqueous suspension by the addition of water provide the active ingredient in admixture with dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents, sweetening, flavoring and coloring agents may also be present.

In a further aspect of the invention there is provided a comestible, that is to say, a food stuff comprising at least an extract of the invention, typically in dried form, such as in a lyophilised form.

The skilled addressee will appreciate that such comestibles may contain more than one extract of the invention and may be used. Such foodstuffs may be used in a prophylactic manner and

may contain further extracts having a similar function to the first added extract or further added extracts may be added that have a different prophylactic function. Thus a foodstuff could either comprise extracts that provide for a comestible having a single functional aspect, or a comestible may have a multi-functional prophylactic effect against two or more disease types. It is thought that a multi-functional role could be assigned to pharmaceutical formulations comprising two or more extracts possessing dissimilar therapeutic or prophylactic properties designed either for prophylaxis or for the treatment of more than one disease(s) in a mammal, particularly in a human.

The type of foodstuff or comestible to which at least an extract of the invention may be added includes any processed food such as confectionaries, baked products including breads such as loafs, and flat breads such as pitta bread, naan bread and the like, cakes, snack foods such as muesli bars, compressed dried fruit bars, biscuits, fruits and vegetable juices, water, aerated drinks, such as carbonated soft drinks and non-aerated drinks such as squashes, soya milk, rice milk and coconut milk and the like, pastas, noodles, vegetables, seed and nut oils, fruited oils such as sunflower oil, rapeseed oil, olive oil, walnut, hazelnut, and sesame seed oil and the like.