AN EXTRACT AND A PROCESS THEREOF (PomPlex)

FIELD OF INVENTION

The present invention pertains to nutritional or pharmaceutical compositions comprising extracts or concentrates of plants and the mixtures thereof belonging to Punica sp. with specific reference to Punica granatum (Pomegranate). The present invention further relates to extracts which are isolated from different parts of Punica granatum 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 Cardiovascular, Weight management and Cosmetics to achieve the lowest possible risk. Furthermore, the invention relates to the use of the extracts as a supplement or a medicament useful for Cardiovascular disorders, Weight management and Cosmetic applications. The extract obtained in the instant invention is also referred as Pomplex.

BACKGROUND AND PRIOR ART OF THE INVENTION

Obesity and Cardiovascular Health

Obesity is a condition of abnormal or excessive calorie (fat) accumulation in adipose tissue to the extent that health may be impaired. It is difficult to measure body fat directly but measurements of relative weight for height, such as the Body Mass Index (BMI) will indicate overweight and obesity in adults. The World Health Organization (WHO) has recognized obesity as an international health problem of staggering magnitude.

The effects of obesity on cardiovascular health and disease are many, one of the most profound of which is hypertension. Contemporary thinking concerning the link between obesity and subsequent renal failure has evolved from repeated observations of the relationship between body weight and blood pressure. It is well documented that blood pressure increases with weight gain and decreases with weight loss. In addition, there is increasing evidence that obesity may provide the impetus for sympathetic nervous system activation as well as for changes in renal structure and function.

Obesity has a strong effect on lipoprotein metabolism, regardless of ethnic group. Increased weight is a determinant of higher levels of triglycerides, elevated LDL-C, and low HDL-C. Conversely, weight loss is associated with a healthier lipoprotein profile in both men and women: triglycerides decrease, HDL-C increases, and LDL-C decreases. Changes in HDL-C levels are more pronounced in women than in men. The association between obesity and LDL-C is more complex. LDL-C concentrations increase with BMI in men, but such increases are not as pronounced in women, the elderly, and some ethnic groups. Increasing BMI is associated with small, atherogenic LDL. Furthermore, central obesity in women is associated with elevated LDL-C concentrations. Research should be directed toward understanding the relative importance of obesity-related changes in lipoproteins in predicting actual and potential CVD. There is a strong link between obesity and a generalized metabolic disorder of which insulin resistance is an indicator. It is difficult to define the precise contribution of obesity to insulin resistance, but most analyses suggest that it can account for 50% of the variance in insulin sensitivity in the general population. Insulin resistance is associated with a constellation of metabolic abnormalities, including obesity, diabetes, dyslipoproteinemia, hypertension, and atherosclerosis. It is also linked to a prothrombotic state. Because of the complex nature of insulin resistance, it is not known whether it is independently related to atherogenesis by an unknown mechanism. Future research should explore whether insulin resistance can promote atherosclerosis independently of other risk factors.

The response of various ethnic groups to insulin resistance is variable; e.g., Asian Indians are more susceptible to insulin resistance than are other ethnic groups and are at very high risk of coronary disease. The role of body fat distribution in insulin resistance is important; the key may be abdominal fat, which is highly correlated with insulin resistance. It is also necessary to consider the role of aging, exercise, diet, and genetics in insulin resistance.

Treatment

The scientific community has not yet reached consensus on viable ways to approach the problems associated with obesity. However, several lines of attack are being investigated.

Because of the complexity of the obesity problem, a multifactorial approach will undoubtedly be required. The pharmacological approach has yielded disappointing results, but promise is on the horizon regarding possible drugs to modify appetite and others that reduce absorption of foods or enhance energy expenditure. The public health approach requires a systematic education of the public about the dangers of obesity. Various health agencies could work together to promulgate such a message that would reach all population groups. There is a great need to address the social factors that contribute to obesity and to initiate efforts on a broad scale to modify these factors. Much skepticism exists regarding the possibility of achieving success in the treatment of obesity. It is important to note that many of the cardiovascular complications of obesity arise as a result of mild to moderate degrees of overweight. (Ronald M. Krauss, Mary Winston, Barbara J. Fletcher and Scott M. Grundy "Obesity : Impact on Cardiovascular Disease" Circulation 1998;98;1472-1476 ).

Role of Antioxidants

The skin our largest organ is the first line of defense separating us from the outside world. Oxygen, which is essential for most cellular processes, is highly toxic. However the cells have a multitude of natural protective systems to ensure that the destructive events, which could be unleashed by oxygen, are under very tight controls. In many ways the ageing process can be defined as being oxidized away.

The cells employ a multitude of protective mechanisms to defend themselves against oxidative stress. All the cellular components are susceptible to damage by free radicals, which diminish their efficient functioning. One of the most widely studied protective system is that of the free radical scavenging enzymes - superoxide dismutase (SOD) and peroxidases (catalase, glutathione peroxidases, lactoperoxidases etc) (Lods et.al., 2000).

SOD accelerates the spontaneous reduction of superoxide free radicals into peroxides and oxygen. It plays a critical role in the defense of cells against the toxic effects of oxygen radicals. SOD competes with nitric oxide (NO) for superoxide anion, which inactivates NO to form peroxynitrite. Therefore, by scavenging superoxide anions, SOD promotes

the activity of NO. Covalent conjugation of superoxide dismutase with polyethylene glycol (PEG) has been found to increase the circulatory half-life and provides prolonged protection from partially reduced oxygen species.

Catalase present in the peroxisomes of nearly all-aerobic cells, serves to protect the cell from the toxic effects of hydrogen peroxide by catalyzing its decomposition into molecular oxygen and water without the production of free radicals. (Lods LM., Dres C, Johnson C, Schloz DB., & Brooks GJ., The future of enzymes in Cosmetics., International Journal of Cosmetic Science., Apr 2000, VoI 22, (2), Pg 85-94).

Antioxidants prevent oxidative damage by donating electrons to free radicals. The normal metabolic processes release some free radicals that might cause oxidative damage to our body, but our body repairs most of the oxidative damage caused by these free radicals.

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 disorders; Weight management and Cosmetics are needed.

The management of cardiovascular disorders, Weight management and Cosmetics are 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 management for Cardiovascular disorders, Weight management and Cosmetics, in check, prompting the search and trial of plant extracts to develop a whole new category of natural products. Comprehensive studies on the components of the herb that are responsible for certain indications need to be undertaken to obtain effective medications from this therapy. Also, not necessarily all the Ayurvedic extracts are non-toxic.

The botanical compound Pomegranate (Punica granatum) also has shown positive effects on the Cardiovascular disorders, Weight management and Cosmetics The fruit contains a number of antioxidant constituents, with water-soluble tannins accounting for 92% of the activity. The main antioxidants in pomegranate are ellagic acid and punicalagin, a type of tannin known as an ellagitannin because of its content of ellagic acid. Punicalagin is a strong inhibitor of free radicals and lipid peroxidation and when digested releases ellagic acid. In the records on traditional medicine in India, pomegranate fruit (Punica granatum) is believed to be especially good for the heart and digestive tract. Gastroprotective activity was recently shown in rats orally administered an extract of the fruit rind. The extract also decreased lipid peroxidation and increased levels of natural antioxidants in the body, such as superoxide dismutase, catalase and others. Fed to mice with advanced atherosclerosis, pomegranate juice reduced atherosclerotic lesion size in parallel with a reduction in serum lipid peroxidation. Preliminary trials of pomegranate juice also suggest that it may indeed be beneficial to healthy heart function.

Recent clinical studies have shown that cholesterol levels are significantly improved in people who consume pomegranate fruit juice daily. After daily consumption of pomegranate juice for one year, blood serum showed an increase in total antioxidant status of 130%, the oxidation of lipids in serum decreased by 60%, and serum paraoxonase activity increased by 83%. Paraoxonase is an HDL (high-density lipoprotein-cholesteroty-associated enzyme that protects lipids from oxidation. After 3 years, lipid peroxidation in their serum showed a further reduction of 16% and paraoxonase activity increased to 91%. Healthy subjects consuming pomegranate juice for less than 2 weeks have also shown significantly decreased rates of lipid peroxidation and increased serum paraoxonase levels.

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

OBJECTS OF THE INVENTION

The main object of the present invention is to obtain an extract of punica species.

Another object of the present invention is to obtain an extract from Punica granatum.

Yet another object of the present invention is to obtain a process for obtaining extract from Punica species.

Still another object of the present invention is to develop a method of preventing and/ or managing cardiovascular diseases and weight management using the extract of Punica species.

STATEMENT OF THE INVENTION

Accordingly, the present invention relates to an extract of Punica species optionally along with pharmaceutically acceptable additives; a process for obtaining extract from Punica species, said process comprising steps of: (a) powdering plant material of punica species; (b) extracting and refluxing the powder with solvents to obtain extract; and (c) concentrating the extract followed by drying to obtain the extract of Punica species; and a method of preventing and/ or managing cardiovascular diseases and weight management in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of extract of Punica species optionally along with pharmaceutically acceptable additives to the subject.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

Figure 1: Process Flow Diagram for Pomplex 70%

Figure 2: Process Flow Diagram for Pomplex 50%

Figure 3 (a): Metabolite profile of Pomplex 50% extract at 254nm (b): Comparative overlay of three replicates of Pomplex 50% extract at 254nm

Figure 4 (a): Total Ion chromatogram and LC-MS spectra (Enhanced mass scan + ve mode) of Pomplex 50% (b) Total Ion chromatogram and LC-MS spectra (Enhanced mass scan - ve mode) of Pomplex 50%

Figure 5: Enhanced Product Ion Mass spectra of mass 169.10 -Gallic acid

Figure 6: Enhanced Product Ion Mass spectra of mass 177.20 - Feruladehyde

Figure 7: Enhanced Product Ion Mass spectra of mass 193.00 - Methyl Caffeic acid

Figure 8: Enhanced Product Ion Mass spectra of mass 285.0 - Luteolin

Figure 9: Enhanced Product Ion mass spectra of mass 285.00 -'Kaempferol

Figure 10: Enhanced Product Ion Mass spectra of mass 302.30 - Ellagic acid (301.5)

Figure 11: Enhanced Product Ion Mass spectra of mass 302.30 - Delphinidin

Figure 12: Enhanced Product Ion Mass spectra of mass 333.1- Galloylglucose

Figure 13: Enhanced Product Ion Mass spectra of mass 353.0 - Chlorgenic acid

Figure 14: Enhanced Product Ion Mass spectra of mass 463.20 - Ellagic acid glucopyranoside

Figure 15: Enhanced Product Ion Mass spectra of mass 465.20 - Delphinidin -o-glucoside

Figure 16: Enhanced Product Ion Mass spectra of mass 601.30 - Gallagic acid

Figure 17: Enhanced Product Ion Mass spectra of mass 781.30 - Punicalin

Figure 18: Eanced Product Ion Mass spectra of mass 1083.40 - Punicalagin

Figure 19 (a) Effect of Pomplex 50% on cell viability of HepG2 cells at 4hrs of treatment (b) Effect of Pomplex 70% on cell viability of HepG2 cells at 4hrs of treatment

Figure 20 (a) Effect of Pomplex 50% on cell viability of HepG2 cells at 24hrs of treatment; (b) Effect of Pomplex 70% on cell viability of HepG2 cells at 24hrs of treatment

Figure 21 (a) Effect of Pomplex 50% on cell triglyceride levels in HepG2 cells at 4hrs of treatment; (b) Effect of Pomplex 70% on cell triglyceride levels in HepG2 cells at 4hrs of treatment

Figure 22 (a) Effect of Pomplex 50% on total cholesterol levels in HepG2 cells at 4hrs of

Treatment; (b) Effect of Pomplex 70% on total cholesterol levels in HepG2 cells at 4hrs of treatment

Figure 23 (a) Effect of Pomplex 50% on HDL cholesterol levels in HepG2 cells at 4hrs of treatment; (b) Effect of Pomplex 70% on HDL cholesterol levels in HepG2 cells at

4hrs of treatment

Figure 24 (a) Effect of Pomplex 50% on ApoAl levels in HepG2 cells at 24hrs of treatment; (b) Effect of Pomplex 70% on ApoAl levels in HepG2 cells at 24hrs of treatment

Figure 25 (a) Group Mean Values of Plasma Triglyceride on Day 0, 16 and Day 29 (b)

Percentage variation of Plasma Triglyceride

Figure 26 (a) Group Mean Values of Plasma Total cholesterol Day 0, 16 and Day 29; (b)

Percentage variation of Plasma Total cholesterol

Figure 27 (a) Group Mean Values of Plasma HDL -C on Day 0, 16 and Day 29; (b)

Percentage variation of Plasma HDL-C

Figure 28 Group Mean Weekly Body weight

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an extract of Punica species optionally along with pharmaceutically acceptable additives.

In another embodiment of the present invention, said Punica species is Punica granatum.

In still another embodiment of the present invention, said 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, said extract is formulated into cosmetics and 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 and elixirs.

The present invention also relates to a process for obtaining extract from Punica species, said process comprising steps of:

(a) powdering plant material of punica species;

(b) extracting and refluxing the powder with solvents to obtain extract; and

(c) concentrating the extract followed by drying to obtain the extract of Punica species.

In still another embodiment of the present invention, said punica species is Punica granatum.

In still another embodiment of the present invention, said plant materials are selected from a group comprising leaves, seeds, roots, stems, flowers and combinations thereof, preferably the fruit peels.

In still another embodiment of the present invention, said extract is optionally centrifuged followed by lyophilization to obtain the extract of Punica species.

In still another embodiment of the present invention, said solvents are selected from a group comprising water, alcohol and combinations thereof.

The present invention also relates to a method of preventing and/ or managing cardiovascular diseases and weight management in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of extract of Punica species optionally along with pharmaceutically acceptable additives to the subject.

In still another embodiment of the present invention, the subject is animal including human being.

The present invention provides an active standardized extract of Pomegranate obtained from the fruit peels of Punica granatum plant. The extract obtained in the instant invention is also referred as Pomplex.

The present invention also provides for the reproducibility of plant material extractions from batch to batch variation of bioactive, which is used to ensure the quality of bioactive. The preparation of such extracts, evaluating bioenhancing/bioavailability of Punica granatum extract or bioactive fraction in combination with nutraceuticals or herbal drugs/products to evaluate the Punica plant extracts, capable in management of cardiovascular disorders, weight management and cosmetics in more than one mode of action.

The invention involves analyzing various physical, chemical and microbial parameters and Cell-based assay to monitor the extract for their cell viability, triglyceride levels, total cholesterol levels, HDL cholesterol levels, and Apo Al level.

In another embodiment, the invention provides composition comprising active principles of Punica granatum, and the use of these extracts and constituents for the preparation of nutritional and nutraceutical application.

In another embodiment, the invention provides Punica granatum plant extract, which is easily and safely administrable to children and adults.

Accordingly, the present invention provides Punica granatum plant extract for the management of cardiovascular disorders, weight management and cosmetics in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of standardized Punica granatum plant composite extract, optionally along with pharmaceutically acceptable additives, to the subject; and a process for management of cardiovascular markers and antioxidant activities of a Punica granatum plant extract, said process comprising steps of (a) size-reducing plant parts to obtain powder; (b) extracting the bioactives with a solvent and/or combination of solvents by heating at temperature ranging from 21° to 105° C to obtain a mixture; (c) clarifying the mixture to arrive at clear liquid; (d) concentrating the clear liquid to achieve a concentrated extract; (e) solubilizing the concentrated extract in a solvent and re- concentrating it to obtain further concentrated extract, followed by drying the treated extract to obtain the plant bioactives; (f) the reproducibility of plant material extractions from batch to batch were tested using in-house metabolite fingerprinting QA/QC method based on HPLC equipped with PDA, which takes care the batch-to-batch variation of bioactives in more precise way and is used to ensure the quality of bioactives; (g) Cell Based Assays: Pomplex 50% and 70% was tested for its efficacy in hepatocytes (HepG2) cell lines. The extract was monitored for their cell viability, triglyceride levels, total cholesterol levels, HDL cholesterol levels, and Apo Al level. The invention further provides for uses of the extract to manufacture a medicament for multiple therapeutic uses, as well as other healthful benefits.

The present invention is in relation to efficiency of the bioactive component of the plant extract for therapeutic use, wherein said extract from Punica granatum 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 Punica granatum as defined herein in the preparation of a comestible (foodstuff) for prophylaxis on cardiovascular disorders, weight management and cosmetics. Preferably the mammal is human and the said extract comprises a single extract from a plant part of Punica granatum or a combination of extracts there from as detailed herein. Thus the present invention further relates to extracts, which may be isolated from fruit peels of the Punica granatum plant, the preparation of such extracts, medicaments comprising such extracts, and the use of these extracts and constituents for the preparation of a medicament.

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

In another aspect of the present invention, extracts are isolated from fruit peels of the Punica granatum, 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 Punica granatum plant, depending on the end purpose that is required of the extract.

In some of the embodiments of the present invention there is provided a process for preparing extracts of the invention from plant parts of Punica granatum that comprises: 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 for cardiovascular diseases, weight management and cosmetics. f. Selecting plant extracts having one or both of these activities.

The choice of selected plant material may be of any type but is preferably the fruit peels of the Punica granatum plant.

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

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

Some of the embodiments of the invention will describe the HPLC profiles and Mass spectrums of direct and successive solvent extracts of 70% and 50% Punica granatum plant parts thereby giving each extract an identity of itself.

The plants selected for the isolation of therapeutically relevant extracts/molecules to be used in the management of cardiovascular diseases, weight and cosmetics, will be subjected to both targeted and non-targeted screening procedures. The ongoing-targeted screening procedures, which feature a comprehensive metabolite profiling of multitudes of phytoextracts, are envisaged in the study to facilitate the creation of a metabolite grid. The extracted fractions is subjected to HPLC for metabolite fingerprinting and In some embodiments of the present invention, the analysis of LC/MS/MS is done using Analyst software 1.4.2 of Applied Biosystems along with the script that is developed by Avesthagen to determine the molecular weight of chemical compounds by ionizing, separating and measuring molecular ions according to their mass-to-charge ratio (m/z).

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

In some of the embodiments of the present invention, mammalian cell based efficacy tests are conducted by growing Human hepatoblastoma cell line (Hep G2), in a flask with Eagle's Minimum Essential Medium (EMEM) containing 10% Fetal Bovine Serum (FBS), 1% glutamine-penicillin-streptomycin and 1% fungizone in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air. It is further subcultured when cell become 80% confluent they are subjected to treatment with bioactive under investigation. The incubation is followed by estimating levels of efficacy in glucose uptake 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 Punica granatum as defined herein. Preferably the mammal is a human being. The skilled addressee will appreciate that "treating a disease"

in a mammal means treating, that is to say, alleviating symptoms of the disease and may also mean managing a disease in the sense of preventing such a disease state either advancing i.e. getting worse or becoming more invasive, or slowing down the rate of advance of a disease.

The compositions/medicaments may contain a pharmaceutically acceptable carrier, excipient, or diluent. The compositions can be included as unit dosage suitable for parenteral, oral, or intravenous administration to a human. Alternatively, the compositions are dietary supplements, food compositions or beverage compositions suitable for human or animal consumption.

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

Example 1: Extraction of Punica εranatum:

Extraction of Punica granatum 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. The detailed process is given below:

Reflux Extraction

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

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

Room Temperature Extraction

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

The process flow diagram for PomPlex 70% and PomPlex 50% is shown in Figure 1 and

2 respectively.

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)

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: Metabolic fingerprinting of P omplex 50%

Metabolite finger printing was done using HPLC (Shimadzu) for metabolite profile ans and also to check the reproducibility of the extract.

Instrument initiation

Instrument was switched on and connected to the data station through communication bus module. Each line was purged with the respective solvents for five minutes. Further the column was washed with 100% methanol for 30 minutes and then equilibrated with the initial run conditions of the corresponding LC time program.

Instrument parameters

Data acquisition:

LC stops time: 65 mins

Acquisition time (PDA)

Sampling: 3.125 Hz

Start time: Omins

End time: 45mins

Time constant: 0.64sec Column used: Atlantis dc 18 ,5μm 4.6x250mm.

Mobile phase:

Mobile phase A: HPLC grade water with 0.1%TFA

Mobile phase B: 0.1% TFA acidified acetonitrile LC time program :

Pump

Mode: Low pressure gradient Total pump A flow: ImI/ min Solvent B Cone: 1 Solvent C cone: 0 Solvent D Cone: 0 Max pressure limits: 5405 Controller (CBM 20)

Power: On

Auto sampler (SIL 20A)

Sample rack: 1.5ml standsrd

Rinsing volume: 200μl

Needle stroke: 52mm

Control vial needle stroke: 52mm

Rinsing speed: 35μl/sec

Sampling speed: 15μl/sec

Purge time: 25 min

Rinse mode: Before and after aspiration

Rinse dip time: lOSec Auto purge

Purge order

Mobile phase A: 5 mins

Mobile phase B: 5 mins

Mobile phase C: 5 mins

Mobile phase D: 5 mins

Auto sampler: 5 mins

Total pump A flow: 1ml/ min

Samples analyzed : Pomplex 50%

Run conditions

No of runs performed for each sample: 5

Injection volume: lOμl

LC time program used: Pomplex MD3

Sample preparation

lOmg of the extract was weighed out into sterile eppendorf and lOOμl of DMSO was added and kept a side for some time (15min) and made it to ImI with 100% methanol and sonicated for 15 minutes to ensure that the extract dissolves completely. The extract was centrifuged at 13,000rpm for 15 minutes at room temperature and the supernatant was filtered through 0.2μ filters and collected into sterile recovery vials before analysis was performed. The data was collected from 190-700nm. HPLC analysis peaks of Pomples 50% at 254nm.

The extract was injected at a constant volume of 10μl and its metabolite profiling monitored at 254nm (Figure 3a). A comparative profiling was performed between the runs/ extract to check the reproducibility of the results ( Fig.3b)

Example -3 LC-MS/MS analysis

The analysis of the metabolites present in Pomplex 50% was done by LC/MS/MS (Applied Biosystems MDS SCIEX 4000 Q Trap MS/MS attached to UFLC Shimadzu, Prominence) and the data analysis was done using Analyst software 1.4.2 of Applied Biosystems to identify metabolites. The process is given below

1. Materials: ,

1.1. HPLC grade Acetonitrile (JT Bakers)

1.2. MiUi-Q H ₂ O (18.3ωM)

1.3. Formic acid (Fluka)

1.4. Centrifuge (Eppendorf refrigerated high speed centrifuge, 5804)

1.5. Pipettes (Eppendorf) 2. Equipment:

2.1. Applied Biosystems MDS SCIEX 4000 Q Trap MS/MS

2.2. Shimadzu LC20AB Pump

2.3. Shimadzu SIL20AC Autosampler

2.4. Shimadzu CTO20AC Column oven

2.5. Shimadzu HPLC (prominence), HPLC inserts, bottles

2.6. Eppendorf refrigerated high speed centrifuge, 5804

2.7. Vortex 3. Standard Operation Procedure (SOP)

• 1 mg of Pomplex 50% sample(s) were weighed and transferred to 1.5 graduated vial(s). 1 ml of methanol and 1 ml of methanol, water (0.1%DMSO) (9:1) were added to respective vials.The contents of the vials were mixed thoroughly for 5 min. by a vortex following this; the vials were placed in a sonicator bath for 1 hour.The vials were then centrifuged for 15 min at 14000 rpm and 4 ⁰ C to remove any suspended particles.

• The extracts were filtered through a 0.2-μ-syringe filter, the clarified extracts were carefully transferred into respective autosampler vials (1.5 mL capacity, Shimadzu, Prominence)

• The Pomplex 50%™-extracts and the blank sample loaded autosampler vials were inserted into an autosampler (SIL20AC) attached to UPLC (Shimadzu, Prominence). The temperature of the autosampler was maintained at 8 ⁰ C throughout the experiment.

• The samples were eluted from UFLC by a binary gradient through a 5 μ particle size RP- 18 column, (4.6 mm D x 250 mm xL) held at 4O ⁰ C in a temperature controlled column oven (CTO 20AC) at a flow rate of 1 ml/min over 30.01 min. The gradient system consisted of 0.1% aqueous formic acid (A) and 0.1% formic acid in acetonitrile (B). The gradient was programmed to attain 75% (B) over 20 min, remains same till 25 min and decreases instantly to 5% at the end of 26 min. The 5% (B) remains till 30 min and the UFLC stops at 31.01 min.

• The HPLC eluent was further directed into mass spectrometer (Applied Biosystems MDS SCIEX 4000 Q Trap MS/MS) by applying a splitter.

• The Mass spectrometer was operated by attaching a splitter in an EMS positive and negative polarity mode with ion spray voltage 2750, source temperature 35O ⁰ C, vacuum 4.6 ^~5 Torr, curtain gas 20, Collision Energy (CE) 5.00, Collision Energy spread (CES) 10.000, GSl 40, GS2 60, collision energy 5 and declusturing potential of 35. The turbo ion source was set at 1000 amu/ s with the

interface heater 'on', 967 scans in a period and LIT fill time 20 m sec and dynamic LIT fill time on.

• The acquisition of EPI by LC-MS/MS- The enhanced product ion and MS/MS was performed at LC flow rate of 1 mL min ^"1 over a period of 30.01 min, in splitter-attached mode. The MS was operated both in positive and negative polarity mode. For posetive polarity mode the curtain gas was set to 20, Collision Energy 40, CES 10, ion spray voltage was set at 4000.00 GSl 40, GS2 60 with interface heater and the dynamic fill time on.

• For negative polarity mode the curtain gas was set to 20, Collision Energy -40, CES 10, ion spray voltage was set at -4000.00, temp 400.00, GSl 40, GS2 60 with interface heater and the dynamic fill time on.

The total number of molecules obtain in positive ion mode were 198 (Figure 4a)and in negative ion mode were 107 (Figure 4b) Enhanced product ions were generated and few of the molecules identified are Gallic acid, Feruladehyde, Methylcaffeic acid, Luteolin, Kaempferol, Ellagic acidm Delphinidn, Galloyl glucose, ellagic acid,glucopyranoside, Delphinidin -o-glucoside, Gallagic acid, Punicalin, Punicalagin, (Figure 5-18.).

Example 4: Cell Based Assays

Viability Assay :

PomPlex 50% was studied on human hepatoblastoma cell line (HepG2) for cell viability test. PomPlex 50% did not show any cytotoxicity at all the studied doses of 0.1-500 μg/ml in HepG2 cells at 4 hours. (Figure 19a)

PomPlex 70% did not show any cytotoxicity at all the studied doses of 0.1-500 μg/ml in HepG2 cells at 4 hours of treatment. (Figure 19b)

Effect on Cell viability of HepG2 at 24 hrs treatment:

Pomplex 50% did not show any cytotoxicity at all the studied doses of 0.1-500 μg/ml in HepG2 cells at 24 hours of treatment. (Figure 20a)

Pomplex 70% did not show any cytotoxicity at all the studied doses of 0.1-500 μg/ml in HepG2 cells at 24 hours of treatment. (Figure 20b)

Effect on triglyceride level in HepG2 cells:

PomPlex 50% demonstrated a statistically significant dose dependent inhibition (7-30%) in triglyceride level at doses of 10-500 μg/ml when compared to Simvastatin showing 12% inhibition at 4hrs of treatment .(Figure 21a)

PomPlex 70% demonstrated a statistically significant dose dependent inhibition (22-84%) in triglyceride level at doses of 0.1-500 μg/ml when compared to Simvastatin showing 29% inhibition, at 4hrs of treatment (Figure 21b)

Effect on total cholesterol level in HepG2 cells:

Pomplex 50% exhibited no effect on the total cholesterol level at lower doses of 0.1-10 μg/ml, however on treatment at higher concentrations of 100-500 μg/ml showed 38-42% inhibition in total cholesterol level when compared to simvastatin showing 48% inhibition at 4 hrs of treatment (Fig.22a)

Pomplex 70% exhibited no effect on the total cholesterol level at all the studied doses of 0.1-500 μg/ml at 4 hrs of treatment. (Figure 22b)

Effect on HDL cholesterol in HepG2 cells:

Pomplex 70% demonstrated a statistically significant increase in HDL cholesterol level at higher doses of 10-100 μg/ml showing 81-152% increase when compared to Simvastatin showing 106% increase.(Fig23a)

Pomplex 50% no increase in HDL cholesterol level when compared to Simvestatin showing 108% increase.(Figure 23b)

Effect on Apo Al level in HepG2 Cells:

PomPlex 70% showed statistically significant increase in Apo Al level at higher doses of 10-100 μg/ml with 1.1-1.3 fold increase when compared to Gemfibrozil showing 1.56 fold increase at 200 μM concentrations (Figure 24a).

Pomplex 50% showed statistically significant increase in Apo Al level at higher doses of lOμg/ml with 1.3 fold increase when compared to Gemfibrozil showing 1.56-fold increase at 200 μM concentration (Figure 24 b).

Example 3 : In vivo efficacy screening in Sprague Dawley rats

Preliminary Efficacy of Pomplex 50% For Lipid Lowering Activity In Sprague Dawley Rats

Cardiovascular disease, including atherosclerosis, is the most common cause of mortality and morbidity worldwide. Although several factors, such as life style, a diet high in saturated fat and cholesterol, family history age, hypertension and diabetes mellitus, have been reported to cause heart failure.

The underlying primary cause of cardiovascular disease is believed to be atherosclerosis, a progressive multifactorial disease of arterial wall. Central to the pathogenesis of atherosclerosis is deposition of cholesterol in the arterial wall. Lipoproteins are involved in this process including cholesterol carried by very low-density lipoproteins (VLDL), remnant lipoproteins and low-density lipoproteins (LDL). High levels of LDL and low levels of HDL are important coronary risk factors.

Fortunately, a direct relationship between cholesterol level and atherosclerosis has been established in animal models. Animals consuming diets high in saturated fat and cholesterol develop elevated LDL-C levels and atherosclerosis and intimal lesions that progress from fatty streaks to ulcerated plaques resembling those of human atherosclerosis. These animal studies support the idea that lowering the cholesterol concentrations reduces the incidence of coronary artery disease events.

Objective

The purpose of this study was to evaluate the efficacy of extract Pomplex 50% in reducing triglyceride, total cholesterol and increasing high-density lipoprotein-cholesterol (HDL-C). The cholesterol powder and extract were administered orally to male Sprague Dawley rats for a minimum of 28 consecutive days. Lipid metabolism markers like HDL-C, plasma triglyceride and total cholesterol were determined.

MATERIALS AND METHODS

Test Article Test Article

Identification: Pomplex 50%

Lot/Batch No. : L2804083

Physical Description: Brown color fine dry powder

Composition/Purity: As per analysis certificate

Storage Conditions: Room temperature

Stability: TBD (To be determined)

Vehicle for cholesterol and cholic acid:

Identification: Coconut oil

Lot/Batch No.: KL.6E

Physical Description: light yellowish with slightly viscous

Storage Conditions: Room temperature

Vehicle for extract and Atorvastatin:

Identification: Distilled water

Lot/Batch No.: 8596

Physical Description: Colorless

Storage Conditions: Room temperature

Dose Preparation

Extract formulation was freshly prepared on each day prior dosing (As per SOP of Avesthagen AGTPL/PCSE/SOP/005).

Accountability and Disposition

Unused test article was retained for use on related future studies. Test System (Animals and Animal Care) Description

Species: Rat

Breed/Strain: Sprague Dawley

Total Number: Sixty

Gender: Male

Age Range: 10 - 11 weeks

Body Weight Range: 225 - 325 grams

Animal Source: Bred and reared at Bioneeds.

Experimental History: Purpose-bred and experimentally naive at the onset of the study.

Identification: Cage card and body markings.

Rationale for Choice of Species and Number of Animals

Rat is one of the rodent species accepted and used as a hypercholesterolemic animal model studies, based upon the substantial amounts of published historical data. The number of animals in each group has been used to get statistically significant result.

Husbandry

Housing: Animals are housed three per cage in compliance with committee for the purpose of care and supervision of experiments in animals (CPCSEA) guidelines. The room in which the animals are kept is documented in the study records. No other species are kept in the same room.

Lighting: 12 hours light and 12 hours dark, except when room lights are turned on during the dark cycle to accommodate blood sampling or other study procedures, if any.

Room Temperature: 17 to 24°C Relative Humidity: 40-60% Food: No contaminants are known to be present in the certified diet at levels that would be expected to interfere with the results of this study. Analysis of

the diet is limited to that performed by the manufacturer, records.

Water: Aquaguard water ad-libitum, to each animal in polypropylene bottles with stainless steel sipper tubes. Fructose 10% is added to the water for all the groups except for vehicle control. No contaminants are known to be present in the water at levels that would be expected to interfere with the results of this study.

Acclimation: Study animals were acclimatized to the polypropylene cage housing for a minimum of 7 days prior to their first day of dosing.

Pre-study Health Screen and Selection Criteria

A member of the veterinary staff assessed all animals received for this study for their general health. During the acclimation period, each animal were observed at least once daily for any abnormalities or for the development of infectious disease. Only animals that were determined by the veterinary staff and/or study director to be suitable for use were assigned to this study. Any animal considered unacceptable for use in this study was replaced with animals of similar age and weight before the initiation of the experiment.

Assignment to Study Groups

Animals were assigned to study groups by in-house randomization program designed to achieve similar group mean body weights.

Human Care of Animals

Treatment of animals is in accordance with the study protocol, which adheres to the regulations outlined in the CPCSEA and the conditions specified in the "Guide for the Care and Use of Laboratory Animals".

No alternative test systems exist which have been adequately validated to permit replacement of the use of live animals in this study. Every effort has been made to obtain the maximum amount of information, while reducing to minimum

number of animals required for this study. The assessment of pain and distress in study animals and the use or non-use of pain alleviating medications is in accordance with standard operating procedure, criteria for assessing pain and distress in laboratory animals. The study would have been terminated in part or whole for humane reasons, if unnecessary pain occurred. To the best of our knowledge, this study is not unnecessary or duplicative.

Cardiovascular disease refers to the class of diseases that involve the heart or blood vessels. Cardiovascular disease is a complex and multifactorial disease and is characterized by multiple factors. Epidemiologic studies have identified these as elevated serum or plasma lipids (cholesterol and triglyceride), increased plasma fibrinogen and coagulation factors, increased platelet activation, alterations in glucose metabolism. Increased HDL levels are negatively correlated with cardiovascular disease. Normalization of abnormal lipids and lipoproteins, hypertension, inhibition of platelet aggregation, and an increase in antioxidant status are believed to improve cardiovascular disease. In the present study the ability of the extract in lowering the total cholesterol, triglyceride and increasing the levels of HDL-C in hypercholesterolemic animal model rat was investigated.

Procedure

1. Study Design:

Male Sprague Dawley rats in the age group of 10 - 11 weeks were acclimatized for seven days. Rats were assigned to test and control groups as mentioned in the Table 1. AU the groups were gavaged with cholesterol powder and cholic acid (CP & CA) in cocoanut oil. Standard drug and extracts at three different dose levels were administered immediately after CP & CA, as assigned in the table.1 for 28 consecutive days. Blood samples (ImI) were collected on day 0, day 16 and day 29 for analysis of HDL-C, plasma triglyceride and total cholesterol. Rats were sacrificed at the end of the study period. Gross necropsy findings were recorded.

2. Group assignments

Table 1

*Dose level of Cholesterol powder (CP) & cholic acid (CA)

3. Dosing

Route: Oral administration.

Frequency: Once daily for a minimum of 28 consecutive days.

Procedure: Doses were administered through oral gavage for 28 consecutive days. Each animal received a specific (7.5 ml/kg) dose based upon its most recent body weight.

4. Justification for Route, Dose Levels and Dosing Schedule

The oral route was chosen, as it is the intended route of administration in humans. Dose levels were selected based upon the results of previously conducted cell assays and in vitro results and the maximum active ingredient that can be accommodated in the food product or nutraceutical. The frequency of dosing is the convention for general efficacy studies and supports the safety of the product under its intended clinical use.

5. In-Life Observations and Measurements

Mortality/Morbidity Frequency: Twice daily (a.m. and p.m.) on Days 1 to Day 28. Each animal is observed for evidence of death or impending death.

Clinical Observations

Frequency: Daily and after dosing, each animal is observed for evidence of death or impending death. Body Weight: Weekly

Food Consumption: Weekly

6. Biochemical parameter Evaluation

a. Blood collection

Rats were anaesthetized using carbon-di-oxide (CO ₂ ) inhalation anaesthetic and blood was collected by the retro orbital plexus method with heparin(10 IU/ml of blood) as anticoagulant (as per SOP of Avesthagen AGTPL/PCSE/SOP/007) and plasma was separated and stored at 4 ⁰ C till further analysis plasma triglyceride, total cholesterol, and HDL-C . Blood collection was done on day 1, 16 and 29.

7. Plasma Biochemical parameter assay procedure.

a. Determination of HDL Cholesterol: HDL Cholesterol was analyzed using "Erba Smart lab Random Access Batch Analyzer /Erba EC5 Plus Analyzer"(Transasia Bio-Medicals Ltd., India) using standard turbidimetric immunoassay methodology. b. Determination of Plasma Total Triglyceride: Total plasma Triglyceride was analyzed using "Erba Smart lab Random Access Batch Analyzer /Erba EC5 Plus Analyzer"(Transasia Bio-Medicals Ltd., India) using standard enzymatic methodology. c. Determination of Plasma Total Cholesterol: Total plasma Cholesterol was analyzed using "Erba Smart lab Random Access Batch Analyzer /Erba EC5 Plus analyzer"(Transasia Bio-Medicals Ltd., India) using standard enzymatic methodology.

8. Statistical Analysis

Descriptive statistics was performed on each collected variable (Body weight and different biochemical parameters). Statistical significance between different groups

was evaluated by student t test using GRAPH PAD PRISM (Version 5) at the level of significance (p < 0.05).

III. RESULTS

Plasma Triglyceride (Table 2)

Table 2

*p<0.05 vs. CC (Cholesterol control)

Animals treated with atorvastatin at 30mg/kg body weight were evidenced with a significant decrease (p<0.05) in triglyceride level on day 29 when compared with cholesterol control group (Figure 25a).

Atorvastatin at 30mg/kg body weight exhibited 57% percent variation in triglyceride, where as cholesterol control group exhibited a higher variation of 225 % on day 29 when compared to their own base line (day 0) (Figure 25b).

Pomplex 50% at the doses of 18, 35, 70 and 140-mg/kg bd-wt showed 146, 98, 88 and 149% variation in triglyceride respectively where as cholesterol control group exhibited a higher variation of 225 % when compared with their own day 0 base line.

Total Cholesterol (Table 3)

Table 3

Animals treated with Atorvastatin at 30mg/kg body wt exhibited total cholesterol (TC) variation of 74% where as cholesterol control group exhibited a higher variation of 110% on day 29 when compared to their own day 0 base line (Figure 26 a).

Pomplex 50% at the doses of 18, 35, 70 and 140 mg/kg bd-wt showed 64%, 82%, 80% and 106% variation in total cholesterol respectively, where as cholesterol control group exhibited a higher variation of 110 % when compared with their own day 0 base line (Figure 26 b).

High Density Lipoprotein Cholesterol (HDL-C) -Table 4

Table 4

* p<0.05 vs. CC (Cholesterol control) ** p<0.01 vs. CC (Cholesterol control)

*** p<0.001 vs. CC (Cholesterol control)

Atorvastatin at 30mg/kg bd-wt was evidenced with a significant increase (P< 0.001) in HDL-C on day 29 when compared with the cholesterol control group.

Animals treated with Atorvastatin at 30mg/kg body wt exhibited -15 % variation in HDL-C. Where as, cholesterol control group exhibited a variation of -32 % on day 29 when compared to their own base line (day 0).

Pomplex 50 % at the doses of 18, 35, 70 and 140 mg/kg bd-wt were evidenced with a significant increase (P< 0.01, P< 0.01, PO.05 and P<0.01) in HDL-C respectively when compared to cholesterol control group on day 29 (Figure 27a).

Pomplex 50 % at the doses of 18, 35, 70 and 140 mg/kg bd-wt showed 6, -16, -28 and -14 % variation in HDL-C respectively where as cholesterol control group exhibited a variation of- 32% when compared with their own base line (day 0) (Figure 27b).

Body weight (Table 5)

Table 5

Pomplex 50% did not show any significant variation in the body weights of animals at all dose levels when compared with the body weights of cholesterol control and atorvastatin groups on day 28 (Figure 28).

Mortality

Mortality (19 animals) observed in this study was spontaneous and incidental and could not be attributed to extract treatment.

Clinical Signs

Following dosing few animals showed dullness during the initial period due to the stress of handling and dosing. All the animals were normal and did not show any abnormal clinical signs.

IV. DISCUSSION AND CONCLUSION

Normally, most of the cholesterol in the body serves as a structural element in the walls of cells, whereas much of the rest is in transit through the blood or functions as the starting material for the synthesis of bile acids in the liver, steroid hormones in endocrine cells or vitamin D in the skin. Increased cholesterol concentrations in plasma are, however, a cause of coronary atherosclerosis and increase risk of coronary artery disease (CAD). Several studies have indicated that diet therapy or drug therapy that lowers cholesterol mainly LDL cholesterol levels can reduce subsequent CAD-associated morbidity and mortality.

Triglyceride lowering in rats correlates with efficacy in LDL animal models. Rats can be used to assess potential lipid-lowering effects of HMG-CoA reductase inhibitors in preclinical studies, and that such data correlates with the efficacy found in larger rodent animal models (guinea pigs and rabbits) in which LDL is the major cholesterol- transporting lipoprotein. Using this paradigm, we have chosen to use rat as an animal model for lipid lowering. It is possible that direct inhibition of hepatic lipoprotein secretion (production) represents a common mechanism that exists across rodent species. (Brian R. Krause et al Atherosclerosis 117 (1995) 237-244.

In the present study, we investigated the cardioprotective action of extract Pomplex 50% on hypercholesterolemia. Rats gavaged with the cholesterol powder and cholic acid (suppression of 7α hydroxylase, rate limiting enzyme in bile acid synthesis) showed increase in plasma concentrations of cholesterol while it also caused a tendency towards reduction in HDL-C as compared to baseline. Saturated fatty acids, mainly myristic, lauric and palmitic acids occurring in coconut oil were used as a vehicle for gavaging cholesterol and cholic acid. However, it is assumed that a high level of saturated fat in addition to cholesterol is required in the rat model for induction. 10 % Fructose was supplemented in drinking water to induce hypertriglyceridaemia (J. Nutrition, 94: 68 H.Bar-on and Y.stein), which was substantially evident with high plasma triglyceride level when compared to baseline.

Animals treated with Atorvastatin at 30mg/kg bd-wt lowered plasma triglyceride, total cholesterol and increased HDL-C when compared with the cholesterol control group.

Pomplex 50% at 18, 35,70 and 140 mg/kg bd-wt showed a decrease in percent variation of triglyceride by 79%, 127%, 137% and 79% respectively when compared to the animals treated with cholesterol control.

Animals treated with Pomplex 50% at 18, 35, 70 and 140 mg/kg bd-wt decreased total cholesterol percent variation by 46, 28, 30 and 4 respectively when compared to cholesterol control group.

Pomplex 50% increased the HDL-C levels significantly (p<0.05) when compared with cholesterol control group at all the dose levels on day 29.

Pomplex 50 % at the doses of 18, 35, 70 and 140 mg/kg bd-wt showed 6, -16, -28 and -14 % variation in HDL-C respectively where as cholesterol control group exhibited a variation of - 32%.

Pomplex 50% did not show any significant variation in the body weights of the animals at any of the dose levels tested when compared with cholesterol control and atorvastatin group on day 28.

Pomplex 50% lowered plasma triglyceride, total cholesterol and increased HDL-C at 18 mg/kg and 35mg/kg. However, 35mg/kg can be considered as better dose to investigate further, as it was comparable with the standard drug Atorvastatin. The findings of this study are encouraging and Pomplex 50% can be considered as a potential bio-nutrition candidate. Further studies in LDL animal models might give more insight into the biological activity of the extract.

Modes of administration:

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

The pharmaceutical or naturopathic formulations may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs. The therapeutic compositions of the invention may be formulated as phytoceuticals, or nutraceuticals.

Phytoceuticals may optionally comprise other plant-derived components and can therefore be delivered by such non-limiting vehicles as teas, tonics, juices or syrups. Nutraceuticals contemplated by the present invention may provide nutritional and/or supplemental benefits and therefore be delivered, for example as foods, dietary supplements, extracts, beverages or the like. Phytoceutical and nutraceuticals can be administered in accordance with conventional treatment programs and/or may be a part of the dietary or supplemental program.

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

Tablets contain the active ingredient in admixture with suitable non-toxic physiologically acceptable excipients including, for example, inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch, or alginic acid, binding agents, such as starch, gelatine or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets can be uncoated, or they may be coated by known techniques in order to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

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

Pharmaceutical or naturopathic formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules

wherein the active ingredient is mixed with water or an oil based medium such as peanut oil, liquid paraffin or olive oil.

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

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

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

In a further aspect of the invention there is provided a comestible, that is to say, a 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 manner and may contain further extracts having a similar function to the first added extract or further added extracts may be added that have a different prophylactic function. Thus a foodstuff could either comprise extracts that provide for a comestible having a single functional aspect, or a comestible may have a multi-functional prophylactic effect against two or more disease types. It is thought that a multi-functional role could be assigned to pharmaceutical formulations comprising two or more extracts

possessing dissimilar therapeutic or prophylactic properties 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, dairy products such as yoghurts, milk and milk-based products such as custards, cream, cheese, butter and creme fraiche, simulated dairy food product such as Elmlea products, 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, and frozen confectionaries such as ice cream, iced yoghurts and the like.