BACKGROUND OF INVENTION

Cardiovascular disease is a group of diseases affecting heart and blood vessels (arteries). 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.

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). Flavonoid rich fruits and peels have the potential to reduce the risks of cardiovascular disease (Donald R. Yance, 2005).

The alkaloids in the bark and root bark of Berberis aristata are berberine, berbamine, aromoline, karachine, palmatine, oxyacanthine and oxyberberine. Cicero etal. in 2007 stated that Berberine and a combination of Berberine is a useful support to diet and life style changes to correct dyslipidemias and to reduce cardiovascular risk in subjects with moderate mixed dyslipidemias. In their study they used Berberine and a combination of
berberine with policosanol, red yeast extract, folic acid and astaxanthin, which were
orally administered in a clinical trial. BERB and COMB significantly reduced TC , LDL ,
ApoB and TG and increased HDL. During this trial no adverse effects of liver were
observed.

Several studies have also been undertaken to test the cardioprotective effects of Berberine
over a period of time. The phytochemical and pharmacological studies carried out have
aimed at isolation and structural identification of an attractive, new cardiotonic agent
from Berberis aristata fruit. The above-referred studies are supportive to the suggestion
that Berberine may play a very successful role in cardio protection and more specifically
in controlling cholesterol with its ability to bring about an increase in HDL levels and
ApoAl.

Other cardioprotective functions provided by the nutrients in tomatoes may include the
reduction of low-density lipoprotein (LDL) cholesterol, homocysteine, platelet
aggregation, and blood pressure (Willcox et al.2003).
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:

The related art of interest describes different process for obtaining the extract of Berberis aristata and thereby describing the utility of the extract in different ways but none of citations disclose the present invention. There is a vital necessity to obtain Berberis extract, which is not only safe but also effectively applicable in curing and preventing cardiovascular disease and its related disorders. The related art will be discussed in order of obvious and noticeable significance to the present invention.

The US patent application bearing the application no. US2005229339A,entitled, "Methods and compositions for the treatment of hyperlipidemia" describes the method and composition containing berberin or beberine derivated compound for the treatment of hyperlipidemia, elevated cholesterol and cardiovascular disease in the mammalian
subject.

However the present invention relates to the treatment and improvement of cardiovascular health using the composite extract of Berberis aristata in terms of the
HDL-up regulation using Apo Al marker.Therefore not relevancy to the present
invention.

A PCT application bearing the application no: WO2007IB51109A,entitled, "oral formulation with beneficial cardiovascular effects, comprising berberine" describes about the formulation of the oral administration in form of tablets or powder for extemporaneous use,able to exert the beneficial effect on cardiovascular system and having eulipidaemic activity, cholesterol-lowering and triglyceride-lowering activity, antioxidant activity, and a protective action on the vasal endothelium, comprising berberine in combination with a policosanol and/or red yeast and preferably containing an antioxidant, such as astaxanthm and/or folic acid.

But the present invention relates to the use of the whole composite extract of Berberis aristata for the treatment/prevention of the cardiovascular disease by checking the extract efficacy to increase the HDL-c level in mammalian subject. Therefore, the given citation showing no relevancy with the present invention.

A PCT application, WO2007CA194A, entitled "combinations of botanical extracts for promoting cardiovascular health" describes about the lipid-lowering agent having the same mechanism as berberin or pharmacologically acceptable salts of berberin. The present invention invention deals with the Berberis composite extract in HDL-c regulation by monitoring the Apo Al marker.Therefore, the given PCT citation does not showing relevancy with the present invention.

A PCT application, bearing the application no. W01983US729A, entitled "compounds, compositions and method of treatment for improving circulatory performance" describes about the compound containing protoberberin alkaloid or its salts useful for increasing the contractility of the mammalian heart by showing the positive inotropic effect accompanied by at least one the following symptoms: improved aortic blood flow, decrease in heart rate, decrease in systemic arterial pressure, reduction of afterload of the left ventricle, decrease in preload of the left ventricle, decrease in calculated vessel peripheral resistance, decrease in mean arterial pressure and freedom of arrhythmias. However the present invention deals with the involvement of the Berberis composite extract for the prevention/treatment of cardiovascular disease by increasing the HDL-c.Thus the given citations does not showing relevancy with the present invention.

OBJECT OF THE PRESENT INVENTION

The principle object of the present invention is to provide an active extract and bioactive fraction obtained from different parts of Berberis aristata plant.

Another object of the invention is to provide a process for isolating bioactive fraction from Berberis aristata using aqueous, alcoholic and/or hydro-alcoholic and organic solvent, the preparation of such extracts, evaluating bioenhancing/bioavailability of Berberis aristata extract or bioactive fraction in combination with nutraceuticals or herbal drugs/products to evaluate the Berberis plant extracts, capable of treating cardiovascular disorders in more than one mode of action.

Yet, another object of the invention is to provide composition comprising active principles of Berberis aristata, 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 Berberis aristata plant extract, which is easily and safely administrable to children and adults.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides Berberis aristata 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 Berberis plant composite extract, optionally along with pharmaceutically acceptable additives, to the subject; and a process for enhancing cardiovascular health properties of a Berberis 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 ACCOMPANYING DRAWING

Figure 1: Metabolic profile of AVDHC 29Ba(65)04(80) extract

Figure 2: Comparative overlay of the triplicate run of AVDHC29Ba (65)04(80) extract

Figure 3: Metabolic fingerprinting of AVDHC29Ba(65)04(80) extracts scanned from 200nm to 700nm using Metagrid™ software.

Figure 4: Effect of AVDHC29Ba(65)04(80) extract on the cell viability in HepG2 cells at 24hrs of treatment.

Figure5: Effect of AVDHC29Ba(65)04(80) extract on the Apo Al release in the Hep G2 cells at 24 hrs of the treatment.

Figure 6: DPPH free radical scavenging activity of AVCVD29Ba(65)04(80) extract.

DETAILED DESCRD7TION OF THE INVENTION

The present invention is in relation to efficiency of the bioactive component of the plant extract for therapeutic use, wherein said extract from Berberis aristata 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 Berberis aristata 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 berberis aristata 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 Berberis aristata plant, the preparation of such extracts, medicaments comprising such extracts, and the use of these extracts and constituents for the preparation of a medicament.

Some of the embodiments of the present invention will include pharmacognostic evaluation of the botanicals as per the conventional pharmacopoeial standards for ascertaining the quality, purity and efficacy of the herbs. These tests will comprise determination of physico-chemical standards like total ash, water and alcohol soluble extractives, foreign organic matter, moisture content and screening of the plant material for total microbial count (Total Bacterial Count and Total Fungal Count) in order to meet the most stringent quality regulations. Chromatographic finger printing of the herbs for their general profile or marker compounds using Thin Layer Chromatography will also form part of this study.

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

In some of the embodiments of the present invention there is provided a process for preparing extracts of the invention from plant parts of Berberis aristata that comprises:

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

b. 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.

c. Removing the plant material from the supernatant obtained in step b.

d. Optionally, lyophilizing said supernatant.

e. Analyzing the plant extracts for efficacy and presence of inhibitory activity
against cardiovascular disorders.

f. 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 Berberis aristata 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 with solvents from non-polar to polar range. The compositions/medicaments may contain a pharmaceutically acceptable carrier, excipient, or diluent.

It will be apparent to the skilled addressee that the selection of solvent, or mixtures of solvents for each step in the isolation of extracts of the invention showing activity can be guided by results of bioassay analysis of separate fractions.

Some of the embodiments of the invention will describe the HPLC profiles and Mass spectrums of direct and successive solvent extracts of Berberis aristata plant parts thereby giving each extract an identity of itself.

The plants selected for the isolation of therapeutically relevant extracts/molecules to be used in the treatment of cardiovascular disorders, will be subjected to both targeted and non-targeted screening procedures. The ongoing-targeted screening procedures, which feature a comprehensive metabolite profiling of multitudes of phytoextracts, are envisaged in the study to facilitate the creation of a metabolite grid.

The successive extraction from plant extract will be carried out using soxhlet extractor. The solvents used, will be 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°C. These fractions will be lyophilized and stored in amber colored bottles at 4°C.

NOMENCLATURE OF THE EXTRACT ID:
AV-Avesthagen, D-Danone, HC-HDL cholesterol, plant genus and species,(temperature) of the extraction, solvent code(04-ethanol,08-water), (% of solvent used).

Phytochemical investigations will be also carried out on these extracts using various tests like Mayer's and Dagendorfs tests for alkaloids; Molisch, Fehling and Benedict tests for carbohydrates; Lieberman Buchard's test for phytosterols and triterpenes; spot test for fixed oils and fats; Ferric chloride and Lead acetate test for phenolic compounds and tannins; Ninhydrin and Biuret tests for protein and aminoacids; alcoholic precipitation followed by Molisch test for gum and mucilages.

The extracted fractions will be subjected to HPLC using u bondapak C i8 column (Waters Alliance 2695 Separation Module) 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 will also be carried out wherever required. 5- lOul of sample is injected with flow rate of 1 ml/min and HPLC run will be performed for 30 minutes. The detection is done on photodiode array and the analysis of the results will be done with the help of Millennium™ software.

In some embodiments of the present invention, the analysis of output of LC-PDA and LC/MS/MS will be 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).

The invention further describes the biotherapeutic potential of various extracts of Berberis aristata as described above, by studying their performance in cell based assay models.

In some of the embodiments of the present invention, mammalian cell based efficacy tests are 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), l%glutamine-penicillin-streptomycin and 1% fungizone in a humidified incubator at 37°C in an atmosphere of 5% C02 and 95% air. It is further subcultured when cell become 80% confluent they are subjected to treatment with bioactive under investigation. The incubation is followed by estimating levels of bio-markers for Cardiovascular disoreder like apolipoprotein AI (Apo AT), total cholesterol, HDL cholesterol, triglyceride levels, Angiotensin converting enzyme (ACE), endothelial Nitric oxide synthase (eNOS), C-Reactive 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 Berberis aristata 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.

EXTRACTION OF Berberis aristata:
Extraction of Berberis aristata 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.

Example 1: Extraction Process A: Reflux Extraction

Powdered Berberis aristata 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 ID.

EXAMPLE 2: PROCESS B: ROOM TEMPERATURE EXTRACTION

Powdered Berberis aristata 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°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.

Calculations:

Calculate the percentage yield of the extract with respect to the initial weight of the plant material taken before extraction.

% Yield = wt. of lyophilized extract (after drying) * 100 Wt. of dry Plant material (initial)

Example 3: Extraction Procedure Of AVDHC29Ba (65) 04(80):

100ml of the powdered plant material is taken in the round bottom flask and 1000ml of 80% ethanol is added to it. The extraction is started and continued for 2 hours at the boiling point of the solvent. After 2 hours of extraction, the solvent containing the extract is centrifuged at 4500 rpm for 15 minutes at 4°C temperature. The supernatant is taken and is concentrated in the rotor-vapour. Remaining drying and concentration of the extract is done by lyophilizing under vaccum. The dry weight of the extract is taken and the powdered dried extract is kept in an airtight glass container at room temperature.

Calculations:

Calculate the percentage yield of the extract with respect to the initial weight of the plant material taken before extraction.

% Yield = wt. of lyophilized extract (after drying) * 100 wt. Of dry Plant material (initial

Yield and charectoristics:

% of yield is 12.0 and Greenish brown colored powder.

Example 4: HPLC fingerprinting of extracts:

HPLC fingerprinting of AVDHC29Ba (65) 04(80):

Sample Preparation:

1ml of 100% methanol was added to lOmg of the sample and sonicated for 15mts.This was centrifuged at BOOOrpm for 15mts.The supernatant was filtered using a Nylon filter (13X0.2um) with a lml syringe into an injection vial.

Method:

Instrument Parameters: Shimadzu LC20AT: Data Acquisition: LC stop time: 58min, Acquisition time (PDA), Sampling: 1.5625 Hz, Start Time: 0 mts, End Time: 30mts,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/cm2

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

Auto sampler: Sample rack: 1.5ml standard, Rinsing volume: 200ul, Needle stroke: 52mm, Control vial needle stroke: 52mm, Rinsing speed: 35ul/sec, Sampling speed: 15ul/sec, Purge time: 25 min, Rinse mode: Before and after aspiration, Rinse dip time: l0Sec 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, CI 8; 5um; 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: 5ul

Number of runs made: Four
Matchins runs: C28Cr(65)04(80)run_2, C28Cr(65)04(80)run_3
C28Cr(65)04 (80)run_4

Result and discussion:

The extract was injected at a constant volume of 25 ul and its metabolite profiling monitored at 254nm. A comparative profiling was performed between the runs/ extract to check the reproducibility of the results.

Table 1: Peak information of AVDHC29Ba (65) 04(80) extract at 254 nm

Table 2: Summary of percentage of conservation of AVDHC29Ba (65) 04(80) extract scanned from 200 to 700 nm using Metagrid™ software

Conclusion:

Constituents and conservation profiles of AVDHC29Ba (65) 04(80) extract scanned from 200 to 700 nm using Metagrid™ software revealed that 95-100% conservation at the wavelength of 200/220/260nm and 60-80% conservation at 210/230/240/250 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, apoA-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°C in an atmosphere of 5% C02 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 umol/L) / herbal extracts on apoA-I secretion for varying times (24 hours) was prepared and at the termination of the incubation, 50-uL 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 uL of 1% Triton X 100 or with 0.1% SDS. 10 ul of the sample was taken and added to 250 ul of Bradford reagent and the reading was taken at 590 nm.

Calculation was done according to the kit protocol as ng /ug of cellular protein and the graphs were generated using Graphpedprism Software. Cell assay for the upregulation of ApoAl was performed on all the 4 extracts. The results obtained from cell assay on these 4 extracts are stated in tables 9 to 12 and represented in figure 13 to 20.

EXAMPLE 6: CELL BASED ASSAY FOR APO Al ACTIVITY OF AVDHC29 Ba(65)04(80)

AVDHC29Ba (65) 04 (80) 80% alcoholic extract did not show any cytotoxic effect on HepG2 cells at all the studied doses (0.1 to 500 ug/ml) at 24 hrs of treatment.
AVDHC29Ba (65) 04 (80) 80% alcoholic extract did not show any statistically significant increase in the Apo Al level at lower doses of 0.1-1 ug/ml, followed by an increase at dose of 10 ug/ml with 1.2 fold increase when compared to that of the positive control (Gemfibrozil) exhibiting 1.3 fold increase. However no significant increase was observed at doses of 100-500 ug/ml. Thus the result seems to suggest that AVDHC29Ba (65) 04 (80) 80% alcoholic extract is a good extract in elevating the level of ApoAl in human hepatoblastoma cell line (HepG2) at lowest dose.

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

** - Results are the mean ± SD of triplicates per sample. AVDHC29Ba (65) 04 (80) 80% alcoholic extract is compared to the cell control while Gemfibrozil has been compared respective Ethanol control. *** - P < 0.001, ** - P < 0.01, * - P < 0.05

EXAMPLE 7: DPPH ASSAY FOR FREE RADICAL SCAVENGING POTENTIAL OF THE EXTRACT:

The DPPH molecule is characterized as a stable free radical by virtue of the derealization 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 AVDHC29Ba (65) 04 (80):

In the present invention, the isolated extracts is 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 AVDHC29Ba(65)04(80) is performed in triplicate within the range of 200ug/ml to 250 ug/ml. The Berberis 80% ethanolic extract isolated from the peel of the plant showed IC50 range at 240.8 ug/ml concentrations. The result is represented as a graph in figure 6.

We Claim:

1. A method of obtaining a plant extract from the plant capable of delaying the onset and/or management of cardiovascular disorders comprising the steps:

a. Obtaining plant material from one or more parts of the plants of claim 1.

b.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.

c. Removing the plant material from the supernatant obtained in step b.

d.Optionally, lyophilizing said supernatant.

e. Analyzing the plant extracts for efficacy and presence of inhibitory activity
against cardiovascular disorders, such as herein described.

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

2. A method according to claim 1 wherein the plant extracts are derived from the plant of
the genus Berberis

3. A method according to claim 1, wherein, the plant extracts are extracted from leaves,
seeds, roots, stems, flowers, or various combinations thereof but preferably from the roots
of the plant.

4. The plant extract AVDHC029Ba(65)08(100) extract produced by the process of claim
1.

5. A method of profiling plant extract(s) produced by the process of claim 1, comprising
steps of:

a. Preparing sample solution of plant part(s); (b) injecting the sample solution into HPLC
column; (c) detecting eluant(s) from the HPLC column; and (d) analyzing the eluant(s)
via visual interface to profile the plant extract(s).

6. The method as claimed in claim 5, wherein said profiling involves analyzing
chromatographic data, assessing efficacy and bioactivity screening of plant extract(s).

7.The method as claimed in claim 5, wherein the HPLC column is maintained at a
temperature of about 37 °C.

8.The method as claimed in claim 5, wherein said HPLC is carried by gradient technique using water, methanol and acetonitrile as mobile phase solvents.

9.The method as claimed in claim 6, wherein the chromatographic data is obtained after every second from wavelength ranging from 190 nm to 700 nm with a resolution of about 1.2 nm.

10 .The method as claimed in claim 5, wherein said HPLC comprises reverse phase liquid chromatography coupled with photo diode array detector or reverse phase liquid chromatography and photo diode array detector along with mass spectrometer.

11.The method as claimed in claim 5, wherein said visual interface allows user to pictorially visualize differences in chromatographic profile of an extract and provides unique and consistent chromatographic peaks.

12.Visual interface for displaying plant extract analysis having predetermined colored data points for depicting retention time and tolerance range as herein described along with accompanying examples and drawings.

13. A method of treating Cardiovascular disease and/or related conditions thereof in a mammal in need thereof, said method comprising step of administering pharmaceutically acceptable amount of Berberis plant extract, optionally along with pharmaceutically acceptable additives, to the said mammal.

14. The method of treating as claimed in claim 13, wherein the Berberis plant extract is having free radical scavenging efficacy.

15. An herbal composition for therapeutic use, the composition comprising extracts of Berberis aristata species.

16. A composition of claim 15, comprising one or more plant extracts, wherein the extracts can be formulated as phytoceuticals or nutraceuticals not limiting to teas, tonics, juices, syrups and nutraceuticals providing nutritional benefits in the form of foods, beverages, supplements and the like.

17. A composition of claim 15, comprising one or more plant extracts, wherein the administration of the therapeutic composition can be formulated as pharmaceutical or naturopathic formulation such as phytoceuticals or nutraceuticals, for oral, topical, rectal, parenteral administration or for administration by inhalation or spray.

18. A composition of claim 15, comprising one or more plant extracts, wherein 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.

19. The composition of claim 15, wherein the pharmaceutically acceptable additives are selected from a group consisting of 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.

20. An herbal composition comprising of Berberis plant extract, the method of treating cardiovascular disorders substantially as herein described along with accompanying examples and figures.