Citrus reticulata plant extracts and uses thereof

FIELD OF THE INVENTION

The present invention pertains to the nutritional or pharmaceutical compositions comprising extracts or concentrates of plants and the mixtures thereof belonging to Citrus sp. with specific reference to Citrus reticulata. The invention further relates to extracts which are isolated from the peel of Citrus reticulata 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, extenuating, or treating cardiovascular health disorder 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 diseases.

BACKGROUND OF INVENTION
Cardiovascular disease is 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 adrenahn 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 usefial 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 xmdergone 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
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. Further, Statins, like all drugs, are associated with concomitant side effects including severe muscle damage (rhabdomyolysis) and other related symptoms like muscle pain and/or weakness, fever, dark urine, nausea and vomiting. In extreme cases, this can also lead to kidney damage.

Statins work by blocking cells from making cholesterol. Unfortunately, these drugs also stop cells from making the nutrient co-enzyme QIO (co-QlO). This nutrient helps to release energy from food and to protect cells from harmful compounds. Statins decrease levels of co-QlO in rats and several studies in people also suggest the same effect. A sustained decrease in co-QlO 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)].

Further, Lifestyle modifications with diet therapy including reduced intake of saturated fats, trans-fatty acids, and plant stanol/sterols and viscous fiber and a good exercise program are recommended to prevent or delay further damage to the cardiovascular system.
Many scientific studies validate the effect diet and supplements can have for the body to heal damages to the cardiovascular system. Lifestyle changes can also make a big difference. 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.

There are several studies suggesting the use of herbs with 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)

PRIOR ART
The related art of interest describes different process for obtaining the extract of Citrus reticulata, and thereby describing the utiHty of the extract in different ways but none of citations disclose the present invention. There is a vital necessity to obtain Citrus reticulata 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.

A US patent bearing Application No: US2002187618A, entitled "Cardiovascular promotion and maintenance composition " relates to the invention where it describes about the composition for to assist in the achievement of optimal cardiovascular health. The composition addresses various concerns including low antioxidant status, low levels of essential fatty acids, magnesium, potassium, and elevated levels of homocysteine. It is designated to treat and prevent heart disease and stroke. Citrus bioflavonoid is used as a part of the composition.

Whereas the present invention deals with the efficacy of the composite extract of Citrus reticulata in order to increase the HDL-cholesterol in the blood serum. Moreover lipoprotein Apo Al is used as the marker to regulate the up regulation of HDL-cholesterol. Therefore, the citation given does not have any relevancy with the application of the instant invention.

A US patent Application bearing the Application No: US2002208487A, entitled "Cardiovascular health enhancement with soy fortified citrus juice compositions" relates to the method of treating individuals to favorably modify cardiovascular health risk indicators by consuming an effective concentration of a soy fortified citrus treatment composition comprising single strength citrus juice and soy protein, wherein said health-enhancing maimer raises the HDL cholesterol level, lowers the LDL cholesterol level, lowers the LDL to HDL cholesterol ratio by at least 0.1, lowers the total cholesterol level and also lowers blood pressure of the individual.

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

A PCT application bearing the application no: WO2000US41784A, entitled "MODIFICATION OF CHOLESTEROL CONCENTRATIONS WITH CITRUS PHYTOCHEMICALS" relates about the invention describes about the administration of Citrus phytochemicals (Monoterpene, Terpene, Flavonoids) that raises HDL serum cholesterol levels while typically lowering the ratio of LDL to HDL serum cholesterol levels.

But the present invention relates to the function of Citrus reticulata composite extract and the process in increasing the HDL-cholesterol level by monitoring the regulation of Apo Al marker. Therefore the given citation does not show any similarity with the present invention as the present invention.

Following are some non-patented citations found w.r.t to Citrus reticulata and the related disease condition:

The given reference, entitled, "Biological effects of Hesperidin, a Citrus flavonoid (note II) Hypolipidemic activity on experimental hypercholesteromia rats" relates to the Hesperidin, the most important flavanone of Citrus sp., obtained from the solid residue of orange peel, showed antihypertensive and diuretic effects on normotensive rats and on spontaneously hypertensive rats (SHR). The application of the instant invention relates to the effectiveness and activity of a non-toxic, extract to increase the HDL-c levels in the mammalian and human system. Therefore, the citation given does not have any relevancy with the application of the instant invention.

The given reference, entitled, ''HDL-cholesterol-raising effect of orange juice in subjects with hypercholesterolemia" relates to the consumption of orange juice thereby increasing the HDL-c concentration. However, the given invention is nowhere in relation to the application of the instant invention, where it actually works on the application of a plant extract of Citrus for improvement of cardiovascular health in terras of HDL-c up regulation by monitoring the of Apo Al markers.

The given reference, entitled, "Seasonal changes in the relationship between serum concentration ofbeta-cryptoxanthin and serum lipid levels" relates to the Beta-cryptoxanthin (beta-CRX), a carotenoid pigment found especially in Satsuma mandarin (Citrus unshiu Marc.) fruit, which is mainly produced in Japan. The paper discuss about the increasing in the HDL-c and lowering the LDL-c. However, the same is nowhere in relation to the application of the instant invention, where it actually works on the application of the Citrus plant extract for improvement of cardiovascular health.

The given reference, entitled, "Reduced-calorie orange juice beverage with plant sterols lowers C-reactive protein concentrations and improves the lipid profile in human volunteers" relates to the supplementation with a reduced-calorie orange juice beverage containing plant sterols is effective in reducing CRP and LDL cholesterol and could be incorporated into the dietary portion of therapeutic lifestyle changes. The application of the instant invention relates to the effectiveness and activity of a non-toxic, extract to increase the HDL-c levels in the mammalian and human system. Therefore, the citation given does not have any relevance with the application of the instant invention.

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 Citrus reticulata plant.

Another object of the invention is to develop a process for isolating bioactive fraction from Citrus reticulata 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 Citrus plant extract for its HDL up-regulation, total cholesterol inhibition and other cardio vascular related disorders. Therefore to provide the Citrus plant extract capable of treating cardiovascular disease in more than one action.

Yet, another object of the invention is to obtain a composition comprising active principles of Citrus reticulata, 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 Citrus plant extract, which is easily and safely administrable to children and adults.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides Citrus 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 Citrus plant composite extract, optionally along with pharmaceutically acceptable additives, to the subject; and a process for enhancing cardiovascular health properties of a Citms plant extract, said process comprising steps of (a) size-reducing plant parts to obtain powder; (b) extracting the bioactive 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 bioactive 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 DRAWINGS:

Figure 1: Metabolite profile of the AVDHC28Cr (25)04(80) extract at 254 nm.

Figure 2: Comparative overlay of triplicate run of AVDHC28Cr (25)04(80) extract at 254nm.

Figure 3: Metabolite fingerprinting of AVDHC 28Cr (25)04(80) extract fi-om 200 to 700nm using MetagridTM software.

Figure 4: Metabolite profile of the AVDHC28Cr (65)04(80) extract at 254nm.

Figure 5: Comparative overlay of triplicate run of the AVDHC28Cr (65)04(80) extract at 254nm.

Figure 6: Metabolite fingerprinting of the AVDHC28Cr (65)04(80) extract scanned from 200nm to 700nm using MetagridTM software.

Figure 7: Metabolite profile of the AVDHC28Cr (65)04(20) extract at 254nm.

Figure 8: Comparative overlay of the triplicate AVDHC28Cr (65)04(20) extract at 254nm.

Figure 9: Metabolite fingerprinting of AVDHC28Cr (65)04(20) extract scanned from 200nm to 700nm using MetagridTM software.

Figure 10: Effect of AVDHC28Cr (25)04(80) on the cell viability in HepG2 cells at 24 hrs of treatment.

Figure 11: Effect of AVDHC28Cr (25)04(80) on Apo Al release in Hep G2 cells at 24 hrs of treatment.

Figure 12: Effect of AVDHC28Cr (65)04(80) on the cell viability in Hep G2 cells at 24 hrs of treatment.

Figure 13: Effect of AVDHC28Cr (65)04(80) on Apo Al release in Hep G2 cells at 24 hrs of treatment.

Figure 14: Effect of AVDHC28Cr (65)04(20) extract on the cell viability in the Hep G2 cells at 24 hrs of treatment.

Figure 15: Effect of AVDHC28Cr (65)04(20) on Apo Al release in Hep G2 cells at 24 hrs of treatment.

Figure 16: DPPH free radical scavenging activity of AVDHC28Cr (25)04(80)

Figure 17: DPPH free radical scavenging activity of AVDHC28Cr (65)04(80)
Figures: DPPH free radical scavenging activity of AVDHC28Cr (65)04(20)

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 Citrus 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 Citrus plants extract is having ApoAl and HDL cholesterol 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 Citrus reticulata 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, phyotoceuticals and neutraceuticals.

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

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

In still another embodiment of the present invention, there is provided a process for enhancing therapeutic properties of a Citrus 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 mixtxjre 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 Citrus 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 # to 80 # mesh size.

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, centrifiigal 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 a Citrus plant extract with HDL increasing activity. Furthermore, the invention provides a non¬toxic Citrus plant extract capable of treating cardiovascular disorders in more than one mode of action.

Therefore, the invention provides a non-toxic Citrus extract with a better efficacy in the treatment of cardiovascular disorders. Still, the invention provides a Citrus plant extract, which is easily and safely administrable to children and adults. Still further, the invention provides a Citrus 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 Citrus plant extract.

The present invention is in relation to efficacy of the bioactive component of the Citrus plant extract for therapeutic use in the mitigation of cardiovascular and other related disorders, wherein said extract from Citrus reticulata 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 Citrus reticulata 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 Citrus reticulata 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 Citrus reticulata 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 peels of Citrus reticulata, using conventional inorganic and organic solvent extraction and supercritical fluid extraction technology alone or in combination thereof. Generally, extracts of the invention capable of frmctioning in a prophylactic or therapeutic manner as outlined herein can be extracted from any Citrus reticulata 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 Citrus reticulata 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, triglyceride level, total cholesterol, and/or HDL cholesterol level 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 Citrus reticulata 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 mjmaging 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 Citrus reticulata 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, facilitate the creation of a metabolite grid.

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 )i 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 analyzed with the help of MilleruiiumTM software.

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 cannot 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 run-time 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 bioactivity 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 Citrus reticulata 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 humdified incubator at 37°C in an atmosphere of 5% C02 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-Reactive protein (CRP), Tumor Necrosis Factor (TNF-alpha), Interleukins (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 Citrus reticulata 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 pharraaceutically 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 Rutaceae. In another embodiment of the invention, the potential plant is a member of the genus Citrus. 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 Citrus species and also from a wide range of plants i.e., potential plants. The potential plants include all species of the family Rutaceae, 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.

Citrus is a small spiny tree with a dense top of slender branches, believe to have been introduced sometime in the eighth century A.D. from Indo-China, widely grown in India. Leaves lanceolate with prominent midrib; petioles narrowly winged or slightly margined, articulated; flowers white, single or in unbranched inflorescence; fruits medium to large, flattened or depressed globose, yellow or reddish orange, core hollow, rind thin, rind and segments easily separable, segments 10-14, pulp of exceptionally fine quality; seeds small, beaked. Though commonly designated as Mandarin, the names Mandarin and Tangerine are used more or less interchangeably to designate the whole group. Tangerine is however more commonly used in USA and is strictly applied to varieties of this group producing deep orange or scarlet fruit. The two names have got firmly established in the world markets and cannot be easily erased. The mandarin oranges can easily be separated into rather distinct groups and in fact certain investigators have proposed to recognize these groups as distinct species.

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 hypercholesterolemia, 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 fiuther 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, 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 an 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 Citrus reticulata 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 THE EXTRACT NOMENCLATURE FOR EXTRACT ID
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: PROCEDURE A REFLUX EXTRACTION
Powdered Citrus reticulata plant material was weighed into the roimd 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 vapor 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 EXTRACTION: PROCEDURE B ROOM TEMPERATURE EXTRACTION
Powdered Citrus reticulata 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, centrifiage 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 percentage yield of the extract is calculated with respect to the initial weight of the plant material taken before extraction.
Percent Yield = wt. of lyophilized extract (after drying) * 100 Wt. of dry Plant material (initial) 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 was swirled and dried under vacuum. Storage and labeling of the extract was done to obtain the Extract ID.

EXAMPLE 3: EXTRACTION PROCEDURE OF AVDHC28Cr (25) 04(80):
100ml of the powdered plant material is taken in the round bottom flask and lOOOml 80% ethanol is added to it. The extraction is started and continued for 2 hours at room temperature. 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 Extracts characteristics:
% Yield of extract is 7.2 and powdered brown color extract.

EXAMPLE 4: EXTRACTION PROCEDURE OF AVDHC28Cr (65) 04(80):
100ml of the powdered plant material is taken in the round bottom flask and 1000ml 80% ethanol is added to it. The extraction is started and continued for 2 hours at boiling point of the solvent. After 2 hours of extraction, the solvent contaiiung 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 Extracts characteristics:
%Yield of extract is 12.0 and powdered brown color extract.

EXAMPLE 5: EXTRACTION PROCEDURE OF AVDHC28Cr (65)04(20):
100ml of the powdered plant material is taken in the round bottom flask and 1000ml 20% ethanol is added to it. The extraction is started and continued for 2 hours at boiling point of the solvent. After 2 hours of extraction, the solvent containing the extract is centriftaged 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 Extracts characteristics:
%Yield of extract is 20.0 and powdered greenish brown color extract.

EXAMPLE 6: HPLC FINGERPRINTING OF AVDHC28Cr (25) 04(80):
Sample Preparation:

1ml of 100% methanol was added to l0mg of the sample and sonicated for ISmts.This was centrifuged at 13000pm for 15mts.The supernatant was filtered using a Nylon filter (13X0.2jim) with a 1ml syringe into an injection vial.

Method:
Instrument Parameters: Shimadzu LC20AT

Data Acquisition: LC stop time: 87min, Acquisition time (PDA), Sampling: 1.5625 Hz, Start Time: 0 mts.
End Time: 65mts.
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: 380kgf7cm
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: Model; SIL 20A, Sample rack: 1.5ml standard. Rinsing volume: 200|il, Needle stroke:
52mm, Control vial needle stroke: 52mm, Rinsing speed: 35|il/sec, Sampling speed: 15fil/sec, Purge time:
25 min, Rinse mode: Before and after aspiration, Rinse dip time: lOSec
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 fi-om 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; 5(4,m; 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: 10μl

L C Time Program:
Flow rate: 1ml/mn

Number of runs made: Four
Matching runs: C28Cr (25)04(80) run_2.1, C28Cr (25)04(80) run_3.1 C28Cr (25)04 (80) run_4.1

Result and discussion:
The extract was injected at a constant volume of 25^1 and its metaboUte 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 AVDHC28Cr (25) 04 (80) extract at 254 nm

Table 2: Summary of percentage conservation of AVDHC28Cr (25) 04 (80) extract scanned from 200 to 700 nm using MetagridTM software

Conclusion:
Constituents and conservation profiles of AVDHC28Cr (25) 04 (80) extract scanned from 200 to 700 nm using Metagrid™ software revealed that 80-100% of conservation at the wavelength of 250/290/330 nm, 70-80% conservation at the wavelength of 210/240/270/280/300/310/320 nm, 60-70% conservation at the wavelength of 200/260nm
And 30-50% conservation at 340/350/360/370/380/530/540/550/560/570/580/600/620 nm.

Example 7: HPLC fingerprinting of AVDHC28Cr (65) 04(80):
Sample Preparation:
1ml of 100% methanol was added to lOmg of the sample and sonicated for ISmts.This was centrifUged at 13000rpm for 15mts.The supernatant was filtered using a Nylon filter (13X0.2nm) with a 1ml syringe into an injection vial.

Method:

Instrument Parameters: Shimadzu LC20AT
Data Acquisition: LC stop time: 80min, Acquisition time (PDA), Sampling: 1.5625 Hz, Start Time: 0 mts.
End Time: 68mts,
Pump: Model: LC-20AT, Mode: Low Pressure Gradient, Total Pump flow: 1 ml/min, Solvent B Cone: 0
inl/min, Solvent C Cone: 0 ml/min, Solvent D Cone: 0 ml/rain. Maximum Pressure Limit: 380kgl7cm
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: Model: SIL 20A, Sample rack: 1.5ml standard, Rinsing volume: 200nl, Needle stroke:
52mm, Control vial needle stroke: 52iran, Rinsing speed: 35^1/sec, Sampling speed: 15fil/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: 1ml/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, CI8; 5^m; 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:
Number of runs mad Matchim 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|al and its metabolite profiling monitored at 254nm. A comparative profiling was performed between the runs/ extract to check the reproducibility of the results.

Table 3: Peak information of AVDHC28Cr (65) 04(80) extract at 254 nm

Table 4: Summary of percentage of conservation of AVDHC28Cr (65) 04(80) extract scanned from 200 to 700 nm using MetagridTM software

Conclusion:
Constituents and conservation profiles of AVDHC28Cr (65) 04(80) extract scanned from 200 to 700 nm using Metagrid™ software revealed that 85-90% of conservation at the wavelength of 240/250/260/270/280/290/300/330/340/ 350/360/370/380/390/440 nm, 80-85% conservation at the wavelength of 210/220/230/310/320/370/400/410/450 nm 70-80% conservation at the wavelength of 200/420/430/470/480/500/570/590/600/610 nm and, 50-70% conservation at 490/520/580/620/630 /640/660/670/680/690/700nra.

EXAMPLE 8: HPLC FINGERPRINTING OF AVDHC28Cr (65) 04(20):
Sample Preparation:
1ml of 50% methanol in water was added to lOmg of the sample and sonicated for ISmts.This was centrifiiged at 13000rpm for 15mts.The supernatant was filtered using a Nylon filter (13X0.2^m) with a 1ml syringe into an injection vial.

Method:
2.3.2.1. Instrument Parameters:_Shimadzu LC20AT
Data Acquisition: LC stop time: 87rain, Acquisition time (PDA), Sampling: 1.5625 Hz, Start Time: 0 mts.
End Time: 65mts.
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: 380kgf7cm
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: Model: SIL 20A, Sample rack: 1.5nil standard. Rinsing volume: 200^1, Needle stroke:
52mm, Control vial needle stroke: 52nam, Rinsing speed: 35^1/sec, Sampling speed: 15(il/sec, Purge time:
25 min. Rinse mode: Before and after aspiration. Rinse dip time: lOSec
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 Pimap 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, CI8; 5m; 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: 20 μl
LC Time Program:


Number of runs made: Four
Matching runs: C28Cr (25)04(20) run_2f, C28Cr (25)04(20) run_3g C28Cr (25)04 (20) run_4h

Result and discussion:
The extract was injected at a constant volume of 25^,1 and its metabolite profiling monitored at 254nm. A comparative profiling was performed between the runs/ extract to check the reproducibility of the results.

Table 5: Peak information of AYDHC28Cr (65) 04 (20) extract at 254 nm

Table 6: Summary of percentage conservation of AVDHC28Cr (65) 04 (20) extract scanned from 200 to 700 nm using MetagridTM software

Conclusion:
Constituents and conservation profiles of AVDHC28Cr (65) 04 (20) extract scanned from 200 to 700 nm using Metagrid™ software revealed that 95-100% conservation at the wavelength of 300/310/320/330/340/350/360/370/380/390/400/410/420/430/440/450/460/470/480/490/500/510/520/530 /540/550/560/570/580/590/600/610/620/630/650/660/670/680/690/700 nm, 90-95% conservation at the wavelength of 200/240/260/270/290 nm and 80-90% conservation at 210/250/280 nm.

APOAl CELL BASED ASSAY:
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 apoA-I.

apoA-I 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% COj 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 )xmol/L) / herbal extracts on apoA-I secretion for varying times (24 hours) was prepared and at the termination of the incubation, 50-^L 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 ^iL of 1% Triton X 100 or with 0.1% SDS. 10 nl of the sample was taken and added to 250 ^il of Bradford reagent and the reading was taken at 590 irai.

Calculation was done according to the kit protocol as ng /^g of cellular protein and the graphs were generated using Graphpedprism Software. Cell assay for the up regulation 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 9: CELL BASED ASSAY FOR APO Al ACTIVITY AVDHC28Cr(25)04(80)
EXTRACT:

AVDHC28Cr (25)04(80) did not show any cytotoxic effect on HepG2 cells at all the studied doses (0.1 to 500 i^g/ml) at 24 hrs of treatment as depicted in Figure X.
AVDHC28Cr (25) 04(80) did not show any significant increase in the Apo Al level at lower doses of 0.1-10 ng/ml. But at doses of 100-500 ^g/ml, a significant increase in the ApoaAl level was observed with 1.1-1.2 fold increase when compared to that of the positive control (Gemfibrozil) exhibiting 1.3 fold increase as depicted in Table 7 and Figure X.Thus, the result seems to suggest that AVDHC12 (a) Vv (25) 04(80) extract is a good extract in elevating the level of ApoAl in human hepatoblastoma cell line (HepG2) at higher doses.

Table 7: Effect of AVDHC28Cr (25) 04 (80) 80% alcoholic RT 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 n filter and used for the assay. The experiment was terminated at 24 hrs.
** - Results are the mean ± SD of triplicates per sample. AVDHC28Cr (25) 04 (80) 80% alcoholic extract RT is compared to the cell control while Gemfibrozil has been compared respective Ethanol control.
**-P < 0.01, *-P< 0.05

EXAMPLE 10: CELL BASED ASSAY FOR APO Al ACTIVITY AVDHC28Cr(65)04(80) EXTRACT:

AVDHC28Cr (65)04(80) did not show any cytotoxic effect on HepG2 cells at all the studied doses (0.1 to 500 ^g/ml) at 24 hrs of treatment as depicted in Figure X.

AVDHC28Cr (65) 04(80) did not show any significant increase in the Apo Al level at lower doses of 0.1-10 ng/ml. But at doses of 100-500 ^g/ml, a significant increase in the ApoaAl level was observed with 1.1-1.2 fold increase when compared to that of the positive control (Gemfibrozil) exhibiting 1.3 fold increase as depicted in Table 8 and Figure X.Thus, the result seems to suggest that AVDHC12 (a) Vv (25) 04(80) extract is a good extract in elevating the level of ApoAl in human hepatoblastorha cell line (HepG2) at higher doses.

Table.8: Effect of AVDHC28Cr (65) 04 (80) 80% alcoholic 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 )x filter and used for the assay. The experiment was terminated at 24 hrs.
** - Results are the mean ± SD of triplicates per sample. AVDHC28Cr (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

EXAMPLE 11: CELL BASED ASSAY FOR APO Al ACTIVITY AVDHC28Cr(65)04(20) EXTRACT:

AVDHC28Cr (65)04(20) did not show any cytotoxic effect on HepG2 cells at all the studied doses (0.1 to 500 ng/ml) at 24 hrs of treatment as depicted in Figure X.

AVDHC28Cr (65) 04(20) did not show any significant increase in the Apo Al level at lower doses of 0.1-10 ^.g/ml. But at doses of 100-500 |ag/ml, a significant increase in the ApoaAl level was observed with 1.1-1.2 fold increase when compared to that of the positive control (Gemfibrozil) exhibiting 1.3 fold increase as depicted in Table 9 and Figure X.Thus, the result seems to suggest that AVDHC12 (a) Vv (25) 04(80) extract is a good extract in elevating the level of ApoAl in human hepatoblastoma cell line (HepG2) at higher doses.

Table 9: Effect of AVDHC28Cr (65) 04 (20) 20% alcoholic 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 j^ filter and used for the assay. The experiment was terminated at 24 hrs.
** - Results are the mean ± SD of triplicates per sample. AVDHC28Cr (65) 04 (20) 20% alcoholic extract is compared to the cell control while Gemfibrozil has been compared respective Ethanol control.
***. p< 0.001**-P< 0.01

DPPH ASSAY FOR FREE RADICAL SCAVENGING POTENTIAL OF THE EXTRACT:

The DPPH molecule is characterized as a stable free radical by virtue of the delocalization 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.

EXAMPLE 12: DPPH ASSAY OF AVDHC28Cr (25) 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 AVDHC28Cr (25) 04 (80) is performed in triplicate within the range of 200ng/ml to 250 ^g/ml. The Citrus 80% ethanolic extract isolated from the peel of the plant showed IC50 range at 213.8 ng/ml concentrations. The result is represented as a graph in figure 16.

EXAMPLE 13: DPPH ASSAY OF AVDHC28Cr (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 AVDHC28Cr (65) 04 (80) is performed in triplicate within the range of 260jxg/ml to 320 ^g/ml. The Citrus 80% ethanolic extract isolated from the peel of the plant showed IC50 range at 290.0 ^g/ml concentrations. The result is represented as a graph in figure 17.

EXAMPLE 14: DPPH ASSAY OF AVDHC28Cr (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 AVDHC28Cr (65) 04 (20) is performed in triplicate within the range of 300|ig/ml to 350 ng/ml. The Citrus 20% ethanolic extract isolated from the peel of the plant showed 1C50 range at 335.6^g/ml concentrations. The result is represented as a graph in figure 18,

EXAMPLE: 15 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, intrastemal 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 com starch, or alginic acid, binding agents, such as starch, gelatine or acacia, and lubricatmg 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 inoshol, 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 foodtuff 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 cosmetibles may contain more than one extract of the invention and may be used. Such foodstuffs may be used in a prophylactic maimer and may contain fiirther extracts having a similar function to the first added extract or further added extracts may be added that have a different prophylactic fiinction. Thus a foodstuff could either comprise extracts that provide for a comestible having a single fimctional 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 desgined 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.

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 is derived from the plant of the genus Citrus.

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

4. The plant extracts AVDHC28Cr (25)04(80), AVDHC28Cr (65)04(80) and AVDHC28Cr (65)04(20) 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 Citrus plant extract, optionally along with pharmaceutically acceptable additives, to the said mammal.

14. The method of treating as claimed in claim 13, wherein the Citrus plant extract is having triglycerides or cholesterol decreasing activity or enhancing HDL activity, and alpha glucosidase activity.

15. An herbal composition for therapeutic use, the composition comprising extracts of Citrus reticulate 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 Citrus plant extract, the method of treating cardiovascular disorders substantially as herein described along with accompanying examples and figures.