AN EXTRACT AND A PROCESS THEREOF

FIELD OF THE INVENTION:

The present invention pertains to nutritional or pharmaceutical compositions comprising extracts or concentrates of plants and the mixtures thereof belonging to Arjuna sp. with specific reference to Terminalia arjuna. The present invention further relates to extracts, which are isolated from different parts of Terminalia arjuna plant, the preparation of such extracts and the medicaments containing the said extracts. The invention further relates to screening and characterization of extracts for their activity in the case of metabolic syndrome such as atherogenic dyslipidemia and elevated plasma glucose. Furthermore, the invention relates to the use of the extracts as a supplement or a medicament useful in the maintenance of blood sugar and blood cholesterol level.

BACKGROUND OF THE INVENTION:

Metabolic syndrome often referred as Syndrome X, is the name given to a collection of metabolic conditions; highlighted by insulin resistance, but also includes obesity (especially in the abdomen), high cholesterol, low HDL cholesterol, high triglycerides and hypertension. These symptoms are often termed prediabetic syndrome. Insulin resistance is one of the key features and dangers of this syndrome. Insulin resistance is a condition where the pancreas is able to manufacture more than enough insulin, but cells have become resistant to insulin's effects. This increases blood glucose as well as stimulates insulin-induced metabolic functions (conversion of carbohydrates to fats leading to obesity). The constant intake of refined carbohydrates, leading to dramatic spikes in blood glucose followed by dramatic spikes in insulin secretion, is the primary cause of insulin resistance and then type 2 diabetes. It is for this reason that diet is possibly the most important aspect to the treatment and prevention of type 2 diabetes and its precursor condition, Syndrome X. Metabolic syndrome has been associated with increased risks for both type 2 diabetes and Coronary Heart Disease (CVD).

Elevated glucocorticoids in the body either endogenous (that is in Cushing's syndrome) or exogenous (in form of medication) increase insulin resistance and may precipitate diabetes mellitusl. Further it may be mentioned that glucocorticoids also cause obesity, hypertension, hyperuricemia, increased plasminogen activator inhibitor-1, low HDL cholesterol along with glucose intolerance. Obese diabetics have excess of 11-beta-HSD enzyme in their visceral fat. This enzyme converts inactive cortisone to active Cortisol.

The treatment of metabolic syndrome is a particularly promising area for botanicals because the complex syndrome has many different targets. Most botanicals derive their effectiveness from a mixture of active molecules, acting in concert. Multiple agents atta cking multiple targets simultaneously present decided advantages over conventional drags, which are each based on one compound that produces one action. Plants hold the power to keep the increasing prevalence of metabolic syndrome, a collection of chronic disease symptoms, in check, prompting the search and trial of plant extracts to develop a whole new category of natural products. The instant invention presents the plant extract identified to be potentially effective against metabolic syndrome.

Indian traditional system of medicine mainly consists of plant-based raw materials. Plants produce different class of phytochemicals such as phenolics, terpenoids, alkaloids, lectins and polypeptides. Traditional medicinal plants are known to synthesis diverse set of secondary metabolites principally to combat environmental stress. Interestingly it has been discovered that these secondary metabolites are also effective in various patho-physiological conditions in humans. Plant based bio-actives have traditionally been used and scientifically proven over past years in mitigation of health related conditions like heart related disorders, diabetes, bone health, immunity enhancement, cancer to name few (Ravishankar et al., 2007).

Reactive Oxygen Species (ROS) are family of molecules, which includes molecular oxygen and its derivatives. ROS are produced as part of aerobic respiration and are known to oxidize lipids, proteins and DNA in the cells leading to patho-physiological conditions (Van Wijk et al., 2008). In the past few years research has established evidences supporting the role of oxidative damage in development and progression of human diseases such as cardiovascular disease, neurodegenerative diseases, cancer and diabetes (Van Wijk et al., 2008). Anti-oxidation system in the body neutralizes the ROS produced in the cells and prevents oxidative damage to the body under normal conditions.
Under disease condition this balance is skewed towards enhanced levels of ROS.

Oxidative stress is defined by increase in levels of ROS with/without decrease in cellular anti-oxidation defense system. It has been documented as one of the key factor in etiology of cardiovascular diseases such as atherosclerosis, heart failure and hypertension (Dusting and Triggle, 2005). Endothelium is one of the important targets for ROS. Endothelial dysfunction is being considered as an important indicator determining health of cardiovascular system (Dusting and Triggle, 2005). Further, in Diabetes, endothelial dysfunction and accelerated atherosclerosis have been reported as major cause of morbidity and mortality (Johansen et al., 2005). Plants have been found to contain a diverse set of secondary metabolites. Many of the commonly consumed plants like Vitis vinifera, Camellia senses, Curcuma longa have revealed their anti-oxidation potential; a property that may protect against cardiovascular disease (Wang et al, 2007).

The three triads of modern day life-style related disorders are cardiovascular disease, diabetes and obesity. These three disorders have been documented as closely interlinked, with one disorder increasing the risk of other, leading to metabolic syndrome. Metabolic syndrome is characterized by a group of metabolic risk factors in one person. The risk factors include abdominal obesity, atherogenic dyslipidemia, elevated blood pressure, pro-thrombotic state, pro-inflammatory state, and insulin resistance or glucose intolerance. As per American Heart Association the primary goal towards management of metabolic syndrome includes reducing risk of cardiovascular disease and type II diabetes (Grundy et al., 2006).

Terminalia arjuna, one of the well known Indian traditional plant documented in ayurveda for heart related disorders.Research have also shown its efficacy in conditions like anginal pain, hyper¬cholesterolemia and in blood pressure modulation (Bharani et al., 2002) (Nammi et al., 2003; Ram et al., 1997). Apart from these it is also reported for its anti-bacterial (Perumal Samy et al., 1998), anti-oxidation (Bajpai et al., 2005; Gupta et al., 2001), anti-carcinogenic (Nagpal et al., 2000) and hepato-protective properties (Manna et al., 2006).

The bio-therapeutic potential the extracts obtained from bark of Terminalia arjuna using different extraction protocols and conditions are described herein. The extracts were subsequently tested for anti-oxidation, anti-dyslipidemia and anti-diabetic potential. Efficacy of Terminalia arjuna extracts and its mode of action for the anti-diabetic potential has not been examined so far. This study was carried out to find a composite extract from bark of Terminalia arjuna with efficacy in dyslipidemia and diabetes management and thereby its potential in mitigation of metabolic syndrome.

OBJECT OF THE INVENTION:

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

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

Still another object of the present invention is to provide arjuna plant extract, which is easily and safely administrable to human being.

Still another object of the present invention is to provide arjuna plant extract capable of mitigation of metabolic syndrome in more than one mode of action.

SUMMARY OF THE INVENTION:

Terminalia arjuna direct and successive extracts were tested for their efficacy using cell free and cell based assays in mitigation of metabolic risk factors associated with cardiovascular disease, diabetes and thereby its potential against metabolic syndrome. Oxidative stress has been reported for its role in cardiovascular disease and diabetes (Dusting and Triggle, 2005; Johansen et al., 2005). Anti-oxidation or free radical scavenging potential of Terminalia arjuna extracts was determined by DPPH assay. Of the extracts tested 20% ethanol extract [AV016BaDi (65) 04(20)] showed best anti-oxidation potential. Management of diabetes is achieved by regulating different target sites. One of the key targets at the pre-diabetic stage in addition to lifestyle management is addressing absorption of blood sugar and thereby surges in post-prandial blood glucose. a-Glucosidase present in intestine are targets for inhibition to management surge of post-prandial glucose. a-Glucosidase inhibition assay was performed to assess the potential of Terminalia arjuna bark extracts in diabetes management. It was found that the direct alcohol and 20% ethanol extract [AV016BaDi (65) 04(20)] were found to have higher potential in inhibition of aglucosidase activity as compared to acarbose, a commercially available a-glucosidase inhibitor. Further, since direct ethanol extracts showed better anti-oxidative as well as a-glucosidase inhibition potential, cell based efficacy validation of these extracts in diabetes management was determined using muscle cell line (C2C12 cell line). It was found that 20% ethanol extracts showed increased glucose uptake. Both these extracts showed differential glucose uptake in C2C12 cells ranging from 2.8 to 5.5-fold induction as compared to control. Extracts at lower concentrations showed better glucose uptake compared to the higher concentrations tested. Insulin, used as positive control, showed 5.8-fold induction in glucose uptake.

Efficacy of Terminalia arjuna bark direct 20% ethanol extract in modulating cholesterol and triglyceride levels and thereby mitigation of dyslipidemia was tested on HepG2 cells. It was found that 20% direct ethanol extract showed increase in HDL cholesterol levels. Additionally 20% direct ethanol extract [AV016BaDi (65) 04(20)] also showed reduction in triglyceride levels.

Cell free, cell based assay and toxicity validation of 20% ethanol extract from Terminalia arjuna bark's have revealed, its potential in mitigation of metabolic syndrome. The composite extract showed efficacy in metabolic syndrome by effective management of dyslipidemia and diabetes in addition to its high anti-oxidation potential.
Sustainable production of secondary metabolites is key for effective and long-term use of bio-actives from Terminalia arjuna.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

Figure 1: Metabolic fingerprinting profile of the extract AV016BaDi (65) 04(20) at 254nm.

Figure 2: Metabolite fingerprinting of the extract AV016BaDi (65) 04(20) at 254nm scanned from
200nm to 700nm using MetagridTM Software

Figure 3: TIC of negative EMS and negative EMS spectrum of AV016BaDi (65) 04(20) extract and b.

TIC of positive EMS and positive EMS spectrum of AV016BaDi (65) 04(20)extract.

Figure 4: DPPH anti oxidant assay of the AV016BaDi (65) 04(20) extract. Figure 5:

Alpha-glucosidase, anti diabetic assays of the AV016BaDi (65) 04(20) extract.

Figure 6: Glucose uptake assay of the AV016BaDi (65) 04(20) extract in C2C12 cell lines. Figure 7: Effect of AV016BaDi (65) 04(20) extract on HDL cholesterol levels in

HepG2 cells Figure8: Effect of AV016BaDi (65) 04(20) extract on Triglycerides levels in HepG2 cells

DETAILED DESCRIPTION OF THE INVENTION:

The present invention is in relation to method of extraction, the extract and effect of plant extracts for the mitigation of metabolic syndrome and/ or related disorders in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of Terminalia arjuna plant extract, optionally along with pharmaceutically acceptable additives, to die subject.

In 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 yet another embodiment of the present invention the efficacy of extract on blood glucose levels. In still another embodiment of the present invention, wherein the

Terminalia arjuna plant extract is non toxic and free of adverse effects.

In still another embodiment of the present invention, wherein the Terminalia arjuna plant extract can be administered to subjects in combination with agents selected from a group comprising synthetic anti diabetic drugs, anti-diabetic plant extracts and food stuffs or comestible and beverage matrix . In still another embodiment of the present invention, there is provided a process for enhancing therapeutic properties of a Terminalia arjuna 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 20 to 100 °C to obtain a mixture; (c) clarifying the mixture to arrive at clear liquid; (d) concentrating the clear liquid to achieve a concentrated extract; (e) solubilizing the concentrated extract in a solvent and re-concentrating it to obtain further concentrated extract; and (f) drying the treated extract to obtain Terminalia arjuna 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 50 # to 200 # and preferably of 100 mesh size. In yet another embodiment of the present invention, wherein the parts of plant are selected from a group comprising bark, root, shoot, leaf 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 the powder is extracted with a solvent at a ratio ranging from 1: 3 to 1: 50

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 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 65 °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 soxhlation, rotary evaporation, distillation, centrifugal vacuum evaporation and lyophilisation.

In still another embodiment of the present invention, wherein said solubilization of concentrated extract is carried out in a solvent selected from a group comprising ediyl 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 Terminalia arjuna plant extract with HDL increasing activity.

Furthermore, the invention provides a non-toxic Terminalia arjuna plant extract capable of treating cardiovascular disorders in more than one mode of action. Therefore, the invention provides a non-toxic Terminalia arjuna extract with a better efficacy in the mitigation of metabolic syndrome. Still, the invention provides a Terminalia arjuna plant extract which is easily and safely administrable to children and adults. Still further, the invention provides a Terminalia arjuna plant extract, which is non-toxic, patient compliant, shows better efficacy. The invention provides also the method of preparation of a non-toxic, patient compliant, improved and effective Terminalia arjuna plant extract.
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 bark of Terminalia arjuna, 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 any Terminalia arjuna 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 Terminalia arjuna 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 against atherogenic dyslipidimia and maintenance of blood glucose level.

• 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 bark of the Terminalia arjuna 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 solvents). Typically, the extraction process is as outlined herein. In another embodiment of the invention, the compositions for preventing, treating, or managing cardiovascular diseases and related disorders, comprises of direct composite extract of plant species with alcohol, water and hydroalcohol solvent and successive extract of solvents from non-polar to polar range. The compositions/medicaments may contain a pharmaceutically acceptable carrier, excipient, or diluent.

In another embodiment of the invention, the HPLC profiles and Mass spectrums of direct extract of Terminalia arjuna plant parts are provided thereby giving each extract an identity of itself.

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

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

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

This metabolite profiling approach allows simultaneous profiling of >90% resolvable constituents of a phytoextracts (with respect to each given set of chromatographic column matrix and associated run parameters) across 50 different wavelength windows in the UV-Visible range (190-700 nm) of the absorption spectrum. The chromatographic data analysis system used in the instant invention enables the analyst to focus on the development of metabolite resolution methodologies that capture the chemodiversity of complex phytoextracts even in the absence of information pertaining to the identities of the individual metabolite components. Further, the linear representation of all validated data points generated using instrument specific data acquisition software allows the 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 Terminalia arjuna as described above, by studying their performance in cell based assay models. 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 (HepG2), in a flask with Eagle's Minimum

Essential Medium (EMEM) containing 10% Fetal Bovine Serum (FBS), 1% glutamine-penicillin-streptomycin and 1% fungizone in a humidified incubator at 37°C in an atmosphere of 5% 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, total cholesterol, HDL cholesterol, triglyceride levels, and diabetes between the bioactive treated and untreated sets.

Nomenclature Of The Extract: AV016BaDi (65) 04(20):

1. AV- first two letters represents Avesthagen.

2. Plant Name: The Plants used and in use are assigned with unique 3-digit number, 016 represents Terminalia arjuna.

3. Part of the plant /Tissue: There is a two letter ID for each plant part used. Here Ba stands for Bark.

4. Solvents: The solvents used for extraction are also assigned with two digit numbers such as 04 for Ethanol. Percentage of solvents used for extraction is given within bracket (20) for 20 % of that solvent. For example if 20% of Ethanol was used for extraction, 04(20).

5. Method of Extraction: Successive extraction is referred to as Su whereas direct extraction is referred to as Di, temperature for extraction is written in bracket. For example, Di (65) represents successive extraction at 65 °C.

Example 1: Direct Extraction. (Soxhlet based extraction from bark of Terminalia arjuna)
Powdered bark of Terminalia arjuna (100 grams) was taken into the Soxhlet extractor. The top was covered with cotton. The level of plant material was kept one inch below the vapor inlet tube. 1000 ml of extraction solvent was taken in to the round-bottomed flask and was placed on the mantle and temperature was set at 65°C. In order to prevent bubbling of the solvent few ceramic chips were added in to the flask. The extractor containing the bark powder was placed on the solvent containing round bottom flask. The extractor was then connected with the condenser and the cold water was circulated continuously in the condenser. On heating the vapors of the solvent from the round bottom flask passes through the inlet into the condenser and the extractor. The vapor gets condensed in the condenser and the solvent drips on top of the plant material and gets collected in the extractor (body), in this process metabolites get extracted from the plant material. When the extractor containing the plant material gets completely filled with solvent, the solvent along with the extracted metabolites gets drained in the solvent containing round bottom flask. This completes one cycle of extraction. The extraction was continued for 8 hours, with ~4 cycles per hour. Finally the extract collected in the round bottom flask was concentrated by vacuum lyophilization. After complete extraction with the given solvent, the plant material was air dried and taken up for extraction with the next solvent and the entire procedure was repeated. The percentage yield of the extract was calculated using following formula.

Example 2: Metabolite profiling of AV016BaDi (65) 04(20) extracts from the bark of Terminal™ arjuna

The samples for HPLC analysis were prepared by dissolving 10 mg of extract in 1 ml of methanol: water. These samples were filtered, collected in HPLC vials and 10 ul of extract was injected and subjected to separation by Waters 2695 HPLC instrument. Subsequently the metabolite profiles were analyzed using Waters Millennium32 (Waters Corporation) and Metagrid™ software developed in-house (Avesthagen).

The HPLC column used for separation was Atlantis dC18, 5µ, 4.6x250mm (Waters Corporation). The column temperature was maintained at 25° C and the solvent flow rate was set at 1.0ml per min. HPLC conditions included Gradient chromatography. Solvents used for metabolite separation were Acetonitrile (solvent A), Methanol (solvent B), Water (HPLC Grade)+ 0.1%TFA (Solvent C and D). Method standardization was done for the maximum separation of the metabolites of Terminalia arjuna direct and successive extracts. Following are the conditions that were used for separation and metabolite fingerprinting of respective extracts.

Metabolomics is defined as comprehensive metabolite profiling of a given biological system. Metabolite profiling of Terminalia arjuna was a technically challenging task due to diversity of the metabolites along with its varying concentrations. Absence of single analytical method that is capable of extracting and detecting all metabolites at once makes the task even more challenging. Currently, the most widely used technique involves reverse phase - HPLC coupled with photodiode array (PDA), with or without mass spectrometry. Till date this is the most sensitive method enabling the detection of hundreds of compounds from a given extract.

To decipher metabolite fingerprints of different Terminalia arjuna bark extracts, I have used MetaGrid™. MetaGrid™ is a reproducible analytical technique for metabolite fingerprinting, developed at Avesthagen Limited, Bangalore. This technique consists of reverse phase liquid chromatography coupled with photodiode array (PDA). The outputs of these results were analyzed using an in-house developed software; where the results are depicted in the form a metabolite signature. To obtain the metabolite fingerprint of different Terminalia arjuna extracts, individual extracts were subjected to reverse phase HPLC based separation. Each of the extract was standardized with respect to the mobile solvent system and the run conditions for maximum separation of the metabolites in composite extracts. Three HPLC runs are performed per extract to generate a metabolite profile. HPLC profiles of different extracts are represented in form of PDA chromatogram. Here, the absorption maxima of the different metabolites present in respective composite extracts are recorded from 200 to 700nm at an interval of l0 nm.

Millenium32 software (Waters Corporation) is used to generate this PDA chromatogram.

PDA chromatogram is a 3-dimensional chromatogram, where the retention times of the data points are represented by x-axis, the wave-length (nm) of analysis on z-axis and the intensity of nm absorption on y-axis.

Further, the PDA chromatogram is used as an input data for MetaGrid™ software, which in turn represents the data in form of metabolite fingerprint. Here, the x-axis represents the absorption range from 200-700nm and y-axis represents the retention time.

Additionally, the MetaGrid™ software also gives percentage conservation of metabolites between three individual HPLC runs for each extract.

Direct and successive extracts from bark of Terminalia arjuna were subjected to above mentioned HPLC based fingerprinting and MetaGrid™ analysis as shown in (Table 1). A representative PDA chromatogram of each Terminalia arjuna extract at 254nm is shown in Figure 1. Metagrid-TM profile of Terminalia arjuna direct extracts at 254nm is shown in Figure 2.

Table 1: HPLC fingerprint of extract of AV016BaDi (65) 04(20)

II Liquid chromatography Mass spectrometry (LC/MS) finger printing

The metabolites in the extract were studied by metabolomics approach. Metaboloraics is a rapidly evolving field of the comprehensive measurement of ideally all the endogenous metabolites in biological sample by using powerful technology of liquid chromatography synchronized to mass spectrometry. To obtain the most comprehensive coverage of the metabolites experimentally, we have optimized the sample extraction process and also the method for two injections one in positive polarity and the other in negative polarity.

The sample 4 mg was dissolved in 1 ml of a solvent system composed of methanol, chloroform and water (8:1:1) in a 2 ml capacity vial. The sample was extracted for 1 h in an ultrasonic bath at 20 KH and was filtered by 0.22 µx syringe filter (Millipore). The filtrate was transferred to an autosampler (SIL 20 AC, Shimadzu) maintained at 8°C.

LC-MS: A 4000 Q TRAP LC/MS/MS system (Applied Biosystems, MDS SCIEX) synchronized to ultra flow liquid chromatography (UFLC, Shimadzu, Prominence) was used for LC-MS data acquisition in both positive and negative polarity.

A 10-µl-sample injection was separated on an end capped; reverse phased, C-18, Gemini 5u. silica column (4.6 x 250 mm, Phinominex) over 30 min. The UFLC solvent system consisted of a) 0.1% formic acid in water b) 0.1% formic acid in acetonitrile. The extract was separated by a gradient of water, acetonitrile at a total flow rate of 1 ml/ min. The gradient started from 5% water to 75% acetonitrile over 20 min, remained till 25 min and suddenly made to 5% water at 26 min which was continued till 30 min. After separation the solvent from UFLC is allowed to fall into mass spectrometer. The LC-MS data acquisition was performed at a scan speed of 1000 amu/sec, in enhanced MS (EMS) type both in posetive and negative polarity using Analyst 4.2.2 software (Applied Biosystems). The EMS parameters in posetive mode were curtain gas 20; ion source gas 4000, temperature 450, GS1 50, GS2 60, ihe on, CAD gas medium, DP 40, and CE 10 where as for negative mode curtain gas 20; ion source gas -4000, temperature 350, GS1 50, GS2 40, the on, CAD gas medium, DP -35, and CE -10.
For the data data processing, The TIC of EMS was background subtracted, 10% threshold is adjusted to spectral mode, noise and isotopes removed.

A total of 772 molecular ions were covered both in positive and in negative polarity. LC-MS data showed the presence of 446 molecular ions in positive polarity and 334 molecular ions in negative polarity
( Shown in figure 3)

Example 2: DPPH anti-oxidant assay:Anti-oxidation potential of Terminalia arjuna 20% A V 016BaDi (65) 04(20) bark extracts a. Reagents used are listed below:

Methanol (Merck), Stock solution of 0.5mM 1,1- Diphenyl-2-Picryl-hydrazyl (DPPH; Sigma) was made in methanol.

Reactions were performed in 1.25 ml of methanol containing 0.5 mM freshly made DPPH and various amounts of the extract. Incubate reaction mixtures were incubated at 37 °C for 30 mins. Absorbance was

measured at 517 nm. The anti-oxidative potential was estimated using following formula:
Oxidant (DPPH) inhibitory activity (%) = [(A5nControl -A5nSample)/A5nControl}] x 100

Anti-oxidation potential of Terminalia arjuna extracts were determined by l,l-Diphenyl-2-picrylhydrazyl (DPPH) assay. DPPH assay is a rapid and widely used assay, for evaluating anti-oxidative activity of food and plant extracts (Wu et al., 2003). This method is based on the reduction of stable free radical in DPPH. DPPH in absence of any reducing agent has absorption maxima at 517 nm and gives purple coloration. As the odd electron of the radical becomes paired in presence of a hydrogen donor, the absorption at 517nm is reduced. This reduced absorption of DPPH (purple to yellow coloration) is stoichiometric with respect to the number of electrons captured.

It was found that different extracts of Terminalia arjuna have varying levels of anti-oxidative potential (Table 1). Reduction of DPPH was found to be dose dependent and is represented as 50% Inhibitory Concentration (IC50 values). IC50 value is determined by estimating the concentration of the test compound that leads to 50% inhibition in the levels of DPPH (purple coloration). Anti-oxidative potential of Terminalia arjuna 20% ethanol extract is shown in the Table 2 and Figure 4 of the specification. Ascorbic acid was used as positive control to compare the anti-oxidation potential of different extracts of Terminalia arjuna. Percentage inhibition is determined by following formula:

Table 2: DPPH-anti oxidant assay of SmartChoI [AV016BaDi (65) 04(20)] extract.
Example 4: Alpha -glucosidae inhibition assay: Diabetes management potential of

Terminalia arjuna 20% AV016BaDi (65) 04(20) bark extracts

Diabetes management potential of Terminalia arjuna bark extracts was scored by measuring their oc-Glucosidase inhibition potential as well as accounting for their glucose uptake potential.

a. Reagents used

Potassium phosphate buffer (IX): 0.1M, pH 6.8, p-nitrophenyl-a-D-glucoside (PNPG; SRL Ranbaxy): 4 mM PNPG in 0.1M potassium phosphate buffer (pH 6.8), Sodium carbonate: 0.2 M.Bovine Serum Albumin (BSA): 0.2 % BSA in IX Potassium phosphate buffer.a-Glucosidase 1.2U/ml in 0.2% BSA

Stock solution of polar extracts was prepared by dissolving them in potassium phosphate buffer. Non-polar extracts were first dissolved in small volume of ethanol (the concentration of ethanol used was not more man 4%, which did not show any inhibitory activity) and the volume was made up with potassium phosphate buffer. Concentration range of the extracts tried was from 2-100 µXg/ml. 25µ1 of a-glucosidase (1.2U/ml) was added to different concentration of the extracts at 37°C for 15 minutes.25µl of PNPG (4mM) was added to each of the tubes and the reaction was further incubated at 37°C for 15 minutes. Post incubation, stop reaction by adding 750µl of 0.2M sodium carbonate. Final volume of the reaction was 1.5ml.Finally absorbance was taken at 400nm using spectrophotometer (Biorad). c. Calculation:

a-glucosidase inhibitory activity (%) = [(A400Control -A400SampleVA400Control}] x 100

Glucosidases inhibition potential:

Diabetes mellitus is a metabolic disorder characterized by high level of blood sugar. Postprandial glucose or increase in blood glucose levels after meal is due to digestion of complex carbohydrates and absorption of glucose from the small intestine. While the liver cells and/or the peripheral organs clear the increased glucose levels, in diabetic patients it fails to do so.

a-Glucosidases are a group of enzymes present in the small intestine. They are involved in the cleavage of glycosidic bonds of dietary disaccharides and polysaccharides, converting them to simple sugar (glucose) and facilitating its absorption in small intestine. Inhibition of the a-Glucosidase activity helps to prevent the break-down of complex carbohydrates to glucose and is one of the targets for diabetes management (Gavin, 2001). The net effect of this inhibition is reduced blood glucose levels. Synthetic inhibitors of a-Glucosidase such as acarbose, voglibose and miglitol have been reported for effectively delaying the digestion and absorption of carbohydrates, thus diminishing the postprandial surge in blood glucose levels without loss of calories (Gavin, 2001; Vichayanrat et al., 2002). In this assay, commercially available a-Glucosidase (Sigma) was pre-incubated with Terminalia arjuna extracts. Post incubation glucoside linked fluorophore, p-nitrophenyl-a-D-glucoside (PNPG), was added and a-Glucosidase activity was estimated by measuring the increase in yellow colouration caused due to release of p-nitro phenyl (PNP) by cleavage of glycosidic linkage of PNPG. Levels of PNP released were spectrophotometrically determined at 400nm. Different concentrations of extracts (in the range of 2µg/ml to 100µg/ml) were used to determine IC50 of cc-Glucosidase. Commercially available acarbose (Bayer Pharmaceuticals) was used as positive control. The IC50 values of the extracts are listed in Table 3 and figure 5 of the specification.
Table 3: a-Glucosidase Inhibition Assay of SmartChol [AV016BaDi (65) 04(20)] extract

Example 5: Glucose Uptake Assay:

a. Reagents used for cell culture and assays are listed below

Dulbecco's Modified Eagle Medium (DMEM; Sigma), Eagle's minimal essential medium (EMEM; Sigma),Fetal Bovine Serum (FBS; Sigma),Calf Serum (Sigma), 1% glutamine-penicillin-streptomycin, Lipoprotein Deficient Serum (Sigma),Phosphate buffered saline (PBS): 137mM NaCl, 2.7mM KC1, lOmM Na2HP04, 2mM KH2P04; pH 7.0,Krebs Ringer Hepes (KRH) Buffer: 130 mM NaCl, 5 mM KCl, 1.3 nM CaCl2) 1.3mM MgS04) NaH2P04Triton X100 (Sigma): 1% Triton XIOO was prepared in IX PBS,0.05% trypsin containing 0.5 mmol/L EDTA (Sigma), 10 mM Insulin (Sigma), Dexamethason (Sigma),0.5 mM 3-isobutyl-lmethyl xanthine (IBMX, Sigma),0.5 mM tritiated 2-deoxy glucose ,1 mM Sodium Pyruvate, 100 mM Simvastatin, Cholesterol mono reagent (Span Diagnostics, India)

b. C2C12 cell culture:

Mouse myoblasts cell line (C2C12) was grown in a T-25 flasks with 5 ml of DMEM containing 10% FBS, 1% glutamine- antibiotic/antimycotic-sodium pyruvate in a humidified incubator at 37°C in an atmosphere of 5% C02 and 95% air. Cells were subcultured at 80% confluence by trypsinization with trypsin containing 0.5mmol/L EDTA.

Cells were plated into 96 well tissue culture plates at 3 X 104 cells per well (100 (il/well). Incubated at 37°C for 48 hrs (5% C02) so that the cells become confluent. Old growth medium was replaced by fresh growth medium containing 5% FBS and incubate the cells for 5 days. Growth medium was changed every alternate day. Growth medium was removed and KRH butter was added (100 unwell) and cell were then incubated at 37°C in incubator in presence of 5% C02 for 10 minutes. For standard insulin response, cells were incubated with insulin (positive control) at concentrations of 50 µM, 100 uM, 250 µM, 500 µM and 1000 uM (in triplicates). For screening the herbal extracts for insulin-mimicking activity, the cells were treated at different concentrations of 500ug, 100|Xg, 10|0.g, µg & 0.1u,g/well of extracts in triplicates for 20 min at 37°C and 5%C02. 0.5 mM tritiated 2-deoxy glucose (final concentration 0.0062 tBq/mM) (50 (il/well) was added in the same plate containing insulin/herbal extract and plates were incubated for 15 mins at 37°C and 5% C02. After 15 mins incubation ice-cold KRH buffer was added to stop the reaction and the plates were kept for 2 mins in the freezer. Cells were than washed with ice-cold KRH buffer. 30 ul of 1% triton X 100 was added to each well and the plates were kept at -20°C for overnight incubation. Cells were scraped and transferred to iso-black plate. 170 ul of scintillation fluid was added and mixed properly with multi channel pipette. Radioactive counts were measured using HIDEX spectrophometer. After 20 mins of incubation with extracts, remove the medium from the wells and add 100(11 of plain DMEM medium. 10(0.1/well of the WST-8 cell proliferation reagent was added and incubated for 15-20 mins at 37°C and 5%C02. Reading was taken at 450 nm.

Cell based assay were performed using C2C12 cells to determine glucose uptake potential of Terminnalia arjuna extracts. Glucose uptake was measured in differentiated C2C12 cells by determination of the uptake of 2-deoxy [3H] glucose. Terminalia arjuna direct 20% direct ethanol extract— AV016BaDi(65)04(20) at concentration of 0.1, 1, 10, 100 and 500 µg/ml were checked for efficacy in differentiated C2C12 cells. Insulin at concentration of 250 µM was used as positive control. AV016BaDi (65) 04(20) extracts showed around 5.2, 5.1, 4.7, 5.1 and 3.4 fold stimulation in glucose uptake at concentration of 0.1, 1, 10, 100 and 500 ng/ml as compared to vehicle control Insulin, standard drug, used as positive control showed 5.9 fold induction in intracellular glucose levels as compared to vehicle control (Figure 6; Table 4).

Table 4: Effect of Terminalia arjuna extracts on glucose uptake in C2C12 cells.

Example 6: Dyslipidemia management using Terminalia arjuna

Dyslipidemia management was examined by scoring for the cholesterol and triglycerides management potential of Terminalia arjuna extracts.

Example 7.A: Cholesterol management:

Higher concentrations of LDL and lower concentrations of functional HDL have been found to be associated with cardiovascular disease as these promote atherosclerosis, leading to myocardial infarction and stroke (Anonymous, 2002). Elevated serum cholesterol in the blood is caused due to several factors such as diet, abnormalities in the levels of lipoproteins, the particles that carry cholesterol in the bloodstream (LDL receptor) and due to other metabolic disorders such as diabetes and obesity. Management of dyslipidemia is mediated by either lowering the "bad cholesterol" or LDL as one approach; while other includes preferentially increase in HDL or "good cholesterol".

Dyslipidemia management potential of Terminalia arjuna direct ethanol extracts were determine using cell based assay. Human immortal, liver cell line (HepG2 cells) were used for this assay. Terminalia arjuna direct 20% ethanol extract— AV016BaDi (65) 04(20) at concentration of 0.1, 1, 10, 100 and 500 µg/ml were checked for their effect on the levels of total cholesterol, HDL cholesterol and triglyceride using HepG2 cells. Simvastatin (a commercially available cholesterol reducing statin drug; Ranbaxy Pharmaceuticals) at 100|iM was used as positive control. Determination of total cholesterol and HDL cholesterol was done using CHOD-PAP method (Huff et al., 1997).

The quantitative estimation of total-cholesterol and HDL-cholesterol using this method involves a series of enzymatic reactions. To start with cholesterol esters are hydrolysed by cholesterol esterase generating free cholesterol and fatty acids. This is followed by cholesterol oxidase, oxidizing the 3-OH group of free cholesterol leading to cholest-4-en-3-one and hydrogen peroxide. In presence of peroxidase, hydrogen peroxide couples with 4-Amminoantipyrine (4-AAP) and phenol to produce red quinonemine dye. Absorbance of this quinonemine dye is measure at 505 nm, which is proportional to amount of total cholesterol present in the sample.

20% direct ethanol extract— AV016BaDi (65) 04(20) was first tested for cell toxicity at concentration of 0.1, 1, 10, 100 and 500 ng/ml. It was found that at all the efficacy doses tested, 20% direct ethanol extract didn't show any cellular toxicity on HepG2 cells.

Cholesterol estimation assay: a. Cell culture

Human hepatoblastoma cell line (HepG2) were 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. Cells were subcultured at 80% confluency by trypsinization with PBS containing 0.5 mmol/LEDTA.
Total Cholesterol and HDL-cholesterol estimation were done on HepG2 cells using CHOD-PAP method (Huff et al., 1997). The cells were plated in 12 well plates at a concentration of lxlO6 cells/ml. They were grown until they attained 75% to 80% confluence. Post confluency the old media was replaced with fresh media containing 10% Lipoprotien deficient serum instead of regular serum. Plates were incubated for additional 48 hrs. Post incubation, old media was removed. Fresh media and the herbal extracts / positive control were added and incubated for 4 hrs. In case of control the vehicle was added in equivalent concentration. Supernatant was removed and the cells were over layered with PBS. PBS was aspirated and 100 µl of 1% Triton X 100 was added to the cells. Cells and were lysed and to it 500 of cholesterol mono reagent was added followed by mild vortexing. Tubes were then incubated for 10 mins at 37°C. Centrifuge at 13,000rpm for 1 min. Absorbance of the supernatant was taken at 505nm.
For determination of HDL

Add 100 ul of polyethylene glycol 600 to the 100 ul lysed cells. Centrifuge at 2000 rpm for 15 min. To 100 µl supernatant add 500 µl of cholesterol mono reagent was added followed by mild vortexing. Tubes were then incubated for 10 mins at 37°C.Centrifuge at 13,000rpm for 1 min. Absorbance of the supernatant was taken at 505nm.

Processing for total cholesterol

Lyse the cells by sonication at 30 amp for 20 sees and add 500 µl of cholesterol monoreagent by mild vortexing. Mix well and incubate at 37° C for 10 mins or at 15-30° C for 30 mins. Measure the absorbance at 505nm.

Triglyceride estimation assay:

a. Cell culture

HepG2 cell line were 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% C02and 95% air. The cells were sub-cultured at 80% confluent by trypsinization with PBS containing 0.5
mmol/LEDTA.

Cells were plated in 12 well plate at a concentration of lxlO6 cells/ml. They were grown until they attained 75% to 80% confluence. Post confluency the old media was replaced with fresh media containing 10% Lipoprotien deficient serum instead of regular serum. Plates were incubated for additional 24 hrs.

Post incubation, old media was removed. Fresh media and the herbal extracts / positive control were added and incubated for 4 hrs. In case of control the vehicle was added in equivalent concentration.

Supernatant was removed and the cells were over layered with PBS. PBS was aspirated and to the cells 100 µl of 1% Triton X 100 was added. Cells were lysed and 500 µl of cholesterol mono reagent was added followed by mild vortexing. Lysates were incubated for 10 mins at 37°C.Spun at 13,000rpm for 1 min. Supernatant was taken and its absorbance was measured at 505nm using spectrophotometer.

The efficacy validation of 20% direct ethanol extract— AV016BaDi (65) 04(20) in increasing HDL-cholesterol level showed, at concentration of 10 µg/ml, 100 ng/ml and 500 (µg/ml 20% direct ethanolextract lead to 1.8, 2 and 2.6 fold increase in HDL-cholesterol levels as compared to vehicle treated ontrol set. Simvastatin at concentration of 100 µM showed 1.4 fold increase as compared to the vehicle treated control (Figure 7, Table 5).

Table 5: Effect of SmartChol [AV016BaDi (65) 04(20)] extract on HDL cholesterol levels in HepG2 cells

* Represents P <0.01, epresents P <0.001, *** represents P <0.0001 as compared to the respective controls.

Indicates no statistically significant modulation in HDL cholesterol levels.

Example 6.B Triglyceride management:

Triglycerides, as major components of very low density lipoprotein (VLDL) and chylomicrons, play an important role in metabolism as energy sources and transporters of dietary fat. High levels of triglycerides in the bloodstream have been linked to atherosclerosis, thereby leading to increased risk of heart disease and stroke (Anonymous, 1998; Cole, 1997).

Terminalia arjuna 20% direct ethanol extract— AV016BaDi(65)04(20) at concentration of 0.1, 1, 10, 100 and 500 µg/ml were checked for its efficacy in modulating triglyceride levels using HepG2 cells. Simvastatin at 100µM was used as positive control. Quantitative estimation of triglycerides involves a series of enzymatic reactions. Triglycerides are hydrolysed by lipoprotein lipase leading to glycerol and fatty acids. This is followed by glycerol kinase phosphorylating glycerol leading to Glycerol-3-phosphate and Adenisone di-Phosphate. This is followed by glycerol-3-phosphate oxidase oxidizing Glycerol-3-phosphate to dihydroxyacetone phosphate and hydrogen peroxide. In presence of peroxidase, hydrogen peroxide couples with 4-AAP and phenol to produce red quinonemine dye. The absorbance of the dye is measure at 505 nm, which is proportional to amount of triglycerides in the sample. 20% direct ethanol extract— AV016BaDi (65) 04(20) showed dose dependent reduction in triglyceride levels (Fig 3.9, Table 3.5). 20% direct ethanol extract at concentration of 1 µg/ml, 10 µg/ml, 100 µg/ml and 500 µg/ml lead to 1.2, 1.3, 1.9 and 20.8 fold inhibition in triglyceride levels as compared to the control set. Simvastatin at concentration of 100 µM showed 1.6 fold inhibition compared to vehicle treated control set (Figure 8, Table 6). Table 7 shows the effect of the 20% ethanolic extract on total cholesterol level in HepG2 cells

Table 6: Effect of direct SmartChol [AV016BaDi (65) 04(20)] extract on triglyceride levels in

HepG2 cells

* Represents P <0.01, **represents P <0.001, *** represents P <0.0001 as compared to the respective controls.

— Indicates no statistically significant modulation in HDL cholesterol levels.

Table 7: Effect of SmartChol [AV016BaDi (65) 04(20)] on total cholesterol level in HepG2 cells

Note: The results are average of replicates. ***Represents P <0.0001 as compared to the respective controls.

AV016BaDi (65) 04(20) did not show any cytotoxicity at all the studied doses of 0.1, 1, 10, 100 and 500 µg/ml at 24 and 48 hrs of treatment. The extract exhibited a significant dose dependent decrease in triglyceride level at doses of 1-500 µg/ml on 24 hrs of treatment.

It exhibited a decrease in the total cholesterol level at doses of 0.1, 10 and 100 µg/ml, an increase was observed at dose of 1 µg/ml followed by no effect at 500 µig/ml.

AV016BaDi (65) 04(20) demonstrated no effect on the HDL level at lower doses of 0.1-1 jig/ml, but a statistically significant increase was observed with higher doses of 10-500 µg/ml.

Thus this result seems to suggest that the AV016BaDi (65) 04(20) is a potent extract for cardiovascular disease.

CLAIMS

We Claim:

1. A method for treating disease selected from group comprising of metabolic syndrome, cardiovascular disease and diabetes, which comprises administering to the said human being a non toxic plant extract AV016BaDi(65)04(20) from Tertninalia arjuna.

2. A method according to any one of claims 1, wherein the said treatment is a prophylactic treatment.

3. A process for enhancing therapeutic properties of a Tertninalia arjuna plant extract, 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 20 to 100 °C to obtain a mixture and used for extraction could be aqueous, organic and /or combination thereof;

(c) Clarifying the mixture to arrive at clear liquid;

(d) concentrating the clear liquid to achieve a concentrated extract; wherein the plant selected from group comprising ,bark, root, shoot, leaf ,and seeds or the whole plant;

(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 Terminalia arjuna plant extract.

4. A process according to claim 4, wherein the parts of plant are selected from a group comprising bark, root, shoot, leaf and seeds or the whole plant.

5. A process according to claim 4, wherein the solvent used for extraction is aqueous, organic and /or combination thereof.

6. An extract from Tertninalia arjuna consisting of AV016BaDi (65)04(20) having the HPLC and /or MS characteristic shown in Table 1 and Figure 3

7. A pharmaceutical composition for use in the treatment of a disease selected from the group metabolic syndrome, diabetes, cardiovascular disease comprising an extract isolated from Terminalia arjuna listed in Tables 1 - 7 in admixture with a pharmaceutically acceptable carrier.

8. A comestible /beverage composition for use in the treatment of a disease selected from the group metabolic syndrome, diabetes, cardiovascular disease comprising an extract isolated from Terminalia arjuna listed in Tables 1 - 7 in admixture with a food and beverage matrix.

9. A pharmaceutical/nutritional composition according to claim 9 for use in the treatment of metabolic syndrome, cardiovascular disease, diabetes comprising an extract isolated from Tenninalia arjuna consisting of AV016BaDi (65)04(20).

10. Use of extracts as claimed in claim 1-9, wherein the extracts as a supplement or a medicament useful in the maintenance of blood sugar, dyslipidemia and metabolic syndrome.