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 Garcinia sp. with specific reference to Garcinia mangostana. The present invention further relates to extracts which are isolated from different parts of Garcinia mangostana plant, the preparation of such extracts and the medicaments containing said extracts The invention further relates to screening and characterization of extracts for their activity in preventing, mitigating, free radicals that can freely react with and destroy healthy cells, to treat inflammatory diseases and skin care. Furthermore, the invention relates to the use of the extracts as a supplement or a medicament useful in the treatment/alleviation or prevention of age related complications and skin care. The extract obtained in the present invention is also referred as Xenomax.

BACKGROUND AND PRIOR ART OF THE INVENTION

An antioxidant is a substance that, when present at low concentrations compared to those of an oxidizable substrate significantly delays or prevents oxidation of that substrate (Halliwell, B.1990, 1995). Antioxidants get their name because they combat oxidation. They are substances that protect other chemicals of the body from damaging oxidation reactions by reacting with free radicals and other reactive oxygen species within the body, hence hindering the process of oxidation. During this reaction the antioxidant sacrifices itself by becoming oxidized. However, antioxidant supply is not unlimited as one antioxidant molecule can only react with a single free radical. Therefore, there is a constant need to replenish antioxidant resources, whether endogenously or through supplementation.

Inflammation is a local reaction, often beneficial, of living tissue against an irritant substance. This reaction is mainly produced by phagocytic activity of the mesodermic cells. In this reaction, however, may participate not only changes in the vascular system, but also the chemical action of the blood plasma and tissue fluids (Metchnikoff 1968, Barbara Plytycz & Rolf Seljelid 2003). Inflammation is a rather stereotypical response to a variety of stimuli, such as tissue injury and/or infection, and involves both localized and systemic effects.

The skin our largest organ is the first line of defence 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.

Markers for antioxidant property

The term "antioxidant" nutritional agent has been applied to a number of specific nutrients. To become chemically stable, free radicals take electrons from other molecules in the body, a process that causes cell damage (oxidative damage). 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 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. Antioxidants are commonly found in two forms: enzymatic and non-enzymatic. One of the most widely studied protective system is that of the free radical scavenging enzymes which are enzymatic antioxidants -superoxide dismutase (SOD) and peroxidases (catalase, glutathione peroxidases, lactoperoxidases etc) (Lods et.al., 2000 Apr).

The nonenzymatic antioxidants include the lipid-soluble vitamins, vitamin E and vitamin A or provitamin A (beta-carotene), and the water-soluble vitamin C and GSH. Vitamin E has been described as the major chain-breaking antioxidant in humans. Because of its lipid solubility, vitamin E is located within cell membranes, where it interrupts lipid peroxidation and may play a role in modulating intracellular signalling pathways that rely on Reactive Oxygen Intermediates (ROI). Vitamin E can also directly quench ROI, including oxygen ion, hydroxide and oxygen.

1. 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. Coyalent 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.

2. 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.

3. Glutathione peroxidase reduces both hydrogen peroxide and free organic hydroperoxides.

Markers for Inflammation

Inflammation is far more complex than might first appear from the simple description given above and is a major response of the immune system to tissue damage and infection, although not all infection gives rise to inflammation. Inflammation is also diverse, ranging from the acute inflammation associated with S. aureus infection of the skin (the humble boil), through to chronic inflammatory processes resulting in remodeling of the artery wall in atherosclerosis; the bronchial wall in asthma and chronic bronchitis, and the debilitating destruction of the joints associated with rheumatoid arthritis.

Many of the drugs used in the treatment of inflammatory conditions, predate our current understanding of the biochemical processes involved in the disease. Traditionally, the standard treatments for rheumatoid arthritis has been to use a non-steroidal anti-inflammatory drug (NSAID), such as aspirin, for pain relief and to use corticosteroids or even disease-modifying anti-rheumatic drugs in an attempt to reduce other symptoms of the disease.

Inflammatory vascular diseases are initiated and perpetuated by the interaction of immune cells with cells of the affected vessel wall. A network of chemical messengers, which, in a state of vascular health, exist as balanced but opposing forces directs this. The detection of vascular inflammation and monitoring of this activity have long been attempted in systemic vasculitis, and, moie recently, in atherosclerosis. Markers of vascular inflammation used thus far have been of limited value; few provide both adequate sensitivity and specificity for any particular disease Inflammatory markers are predictors of recurrent CVD and death in different settings, including the short-term risk, long-term risk, and risk after revascularization procedures such as percutaneous coronary intervention (PCI), including the risk of restenosis. Several Inflammatory markers which seem to have predictive abilities for Cardiovascular Risk are high-sensitivity C-reactive protein [hs-CRP], serum amyloid A [SAA], white blood cell [WBC] count fibrinogen and inhibition of cyclooxygenase (COX). Preferably, only the acute-phase reactants (fibrinogen and CRP) and WBC count have widely available assays. hs-CRP consistently predicts recurrent myocardial infarction independent of troponins, which suggests it is not merely a marker for the extent of myocardial damage. It also may be useful iu the estimation of prognosis in patients who need secondary preventive care, such as those with stable coronary disease or acute coronary syndromes (risk of restenosis) and those who have undergone PCI. Elevated hs-CRP levels also seem to predict prognosis and recurrent events in patients with stroke and peripheral arterial disease (Pearson,et.al., 2007).

C. Treatment

The inclusion of botanicals in a nutritional approach presents an inexpensive means of achieving the goal of cost effective management of the said conditions. However, the effects of the nutritional strategies recomme ided today are rather modest. Thus, research into novel nutritional strategies preventing, mitigating, free radicals that can freely react with and destroy healthy cells, to treat inflammatory diseases and skin care is needed.

The above-mentioned conditions are 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. Comprehensive studies on the components of the herb that are responsible for certain indications need to be undertaken to obtain effective medications from this therapy. Also, not necessarily all the ayurvedic extracts are non-toxic.

The use of herbs has shown positive effects on inflammation, skin care, free radical scavenging, oxidation of human low-density lipoprotein (LDL), and an inhibition of TBARS formation in LDL.

Mangosteen is an evergreen, cultivated tree (6-25 m tall) with a pyramidal crown and straight trunk. The leaves are leathery, shiny, opposite, elliptical or oblong (7-13 cm by 15-25 cm), yellow-green on the underside and olive-green above. The flowers are unisexual female, paired or solitary, thick and fleshy, yellow-green with edges of a reddish color or pink. The fruits are smooth, globose berries (4-7 cm wide) which turn a dark purple when ripe. The fruit flesh is white, five-lobed, juicy and sweet (Richards, 1990;Verheij and Coronel, 1991). They are regarded as the world's best-tasting fruit (Barrett, 1994) with a flavor reminiscent of strawberry and grape or pineapple and peach (Du and Francis, 1977).

Although two of the main xanthones in the fruit hulls, a-mangostin and y-mangostin, produce significant in vitro antioxidant activity, the considerably more active y-mangostin (Feng et al., 2004) is found in much smaller concentrations than the major xanthone, a-mangostin.

y-Mangostin is a more potent in vitro antioxidant than a-mangostin and is therefore the main antioxidant xanthone of the fruit hulls. Feng et al. (2004 op cit) reported that against the DPPH (l,l-diphenyl-2-picrylhydrazyl) radical, the EC50 of a-mangostin was 496.97 micrograms/mL in vitro, whereas that of y-mangostin was 6.49 micrograms/mL. Fan and Su (1997) found stronger antioxidant activity from y-mangostin compared to a-mangostin in a number of assays. In the ferric thiocyanate method, the order of potency was as follows: y-mangostin > BHA > a-tocopherol > a-mangostin > control. y-Mangostin also showed strong activity in scavenging superoxide anion whereas a-mangostin was inactive and comparable to BHA. In DPPH radical scavenging activity, the activity of y-mangostin was comparable to that of BHA whereas a-mangostin was inactive. Tested for hydrogen peroxide scavenging activity, y-mangostin was more potent than BHA whereas a-mangostin showed only weak activity. The authors noted that because the only structural difference between the two forms of mangostin is found in the methyl group, they presumed that "the adjacent hydroxy groups on C-6 and C-7 of xanthone contribute to strong antioxidative activity" (Fan and Su. 1997).

In albino mice of either sex using the carageennan-induced hind paw edema test, a model of acute inflammation. One hour prior to inducing hind paw edema, an extract of the powdered fruit hulls prepared with boiling water and dried was orally administered to the mice after a 6-hour fast. A dose-dependent reduction in edema was found from doses of 1, 2, and 4 g/kg p.o., which inhibited edema by 5% to 35%. However, using doses ranging from 1-8 g/kg p.o., the ED50 was calculated to be 5.51 g/kg which over half the LD50 (9.37 g/kg) and indicates a low level of safety to achieve anti-inflammatory activity, at least in the carageenan-induced edema model (Pongphasuk et al., 2005).

Because y-mangostin inhibited the production of PGE2 from arachidonic acid, it was tested for direct in vitro activity against trie cyclooxygenases COX-1 and COX-2. y-Mangostin concentration-dependently and competitively inhibited both COX-1 and -2 activities (IC50 approx. 0.8 ^M and 2 [xM, respectively), directly binding to the cyclooxygenases. It was concluded that by directly inhibiting COX, y-mangostin reduces the generation of prostaglandins (Nakatani et al., 2002).

The antifungal activity of several xanthones isolated from fruit hulls of G. mangostana (collected from Tamil Nadu, India) and some derivatives of mangostin against Fusarium oxysporum f.sp. vasinfectum, Alternaria tenuis [A. alternata] and Drechslera oryzae [Cochliobolus miyabeanus] was evaluated. The natural xanthones inhibited the growth of all the fungi. Substitution in the A and C rings modified the bioactivities of the compounds [Geetha et al; Gopalakrishnan et al]. Extracts of Gacinia mangostana mangostana showed inhibitory effects against the growth of Staph, aureus NIHJ 209p and some of the components had activity against methicillin-resistant Staphylococcus aureus (MRSA). One active isolate, a-mangostin, a xanthone derivative, had a minimum inhibitory concentration (MIC) of 1.57-12.5 ug/ml. Other related xanthones were also examined to determine their anti-MRSA activity. The strong in-vitro antibacterial activity of xanthone derivatives against both methicillin-resistant and methicillin-sensitive S. aureus suggested the compounds might find wide pharmaceutical use [Iinuma et al]. The technical data and scientific studies confirm that this extract is an excellent choice for antibacterial, antifungal and anti-inflammatory effects on the skin. These are exactly the conditions encountered in acne-prone skin where soaps, creams and washes ideally suit the use of the extract (Dweck data).

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.

OBJECT OF THE INVENTION

The main object of the present invention is to obtain an extract of Garcinia species. Another object of the present invention is to obtain an extract from Garcinia mangostana. Yet another object of the present invention is to obtain a process for obtaining extract from Garcinia species.

Still another object of the present inversion is to develop a method of treating inflammation using the extract of Garcinia species.

SUMMARY OF THE INVENTION

The present invention relates to an extract of Garcinia species optionally along with pharmaceutically acceptable additives; a process for obtaining extract from Garcinia species, said process comprising steps of: (a) powdering plant material of Garcinia 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 Garcinia species; a method of treating inflammation, said method comprising step of administering pharmaceutically acceptable amount of extract of Garcinia species optionally along with pharmaceutical^ acceptable additives to the subject.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

Figure 1: Process Flow Diagram for Xanomax 1%
Figure 2: Process Flow Diagram for Xanomax 10%
Figure 3: Process Flow Diagram for Xanomax 20%
Figure 4: Process Flow Diagram for Xanomax 40%
Figure 5: Process Flow Diagram for Xanomax 95%
Figure 6: Metabolite profiling of Xanomax 10% monitered at 254nm
Figure 7: Comparative profiling of Xanomax 10% to check reproducibility
Figure 8: Metagrid Profile of Xanomax 10%
Figure 9: DPPH Activity of Xanomax 20%
Figure 10: Effect of Xanomax 20% on cell viability in WS-1 cells at 24 Hrs treatments

DETAILED DESCRIPTION OF THE INVENTION

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

In another embodiment of the present invention, said Garcinia species is Garcinia mangostana.

In yet 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 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 Garcinia species, said process comprising steps of:

(a) powdering plant material of Garcinia 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 Garcinia species.

In still another embodiment of the present invention, said Garcinia species is Garcinia mangostana.

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 dried fruit rind.

In still another embodiment of the present ii ention, said extract is optionally centrifuged followed by lyophilization to obtain the extract of Garcinia 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 treating inflammation, said method comprising step of administering pharmaceutically acceptable amount of extract of Garcinia 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 is in relation to efficiency of the bioactive component of the plant extract for therapeutic use, wherein said extract from Garcinia mangostana optionally along with healthful or for nutritional and pharmaceutical applications.

It relates to an active extract and bioactive fraction obtained from different parts of Garcinia mangostana plant.

In one embodiment, the present invention provides a process for isolating bioactive fraction from Garcinia mangostana using aqueous, alcoholic and/or hydro-alcoholic and organic solvent, the preparation of such extracts, evaluating bioenhancing/bioavailability of Garcinia mangostana extract or bioactive fraction in combination with nutraceuticals or herbal drugs/products to evaluate the Garcinia mangostana extracts, capable of testing antioxidant, anti-inflammatory and skin care property in more than one mode of action.

In another embodiment, the present invention provides a composition comprising active principles of Garcinia mangostana, and the use of these extracts and constituents for the preparation of nutritional and nutraceutical application.

In another embodiment, the present invention provides Garcinia mangostana plant extract, which is easily and safely administrable to children and adults.

The present invention provides Garcinia mangostana plant extract for testing antioxidant, anti-inflammatory and skin care property in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of standardized Garcinia plant composite extract, optionally along with pharmaceutically acceptable additives, to the subject; and a process for enhancing the above said properties of a Garcinia 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. Plant material extractions from batch to batch were tested using in-house metabolite fingerprinting QA/QC method based on HPLC equipped with PDA. Followed by drying the treated extract to obtain the plant bioactives, which have significant role in Antioxidant, anti-inflammatory and skin care property. The invention further provides for uses of the extract to manufacture a medicament for multiple therapeutic uses, as well as other healthful benefits.

In one aspect of the invention, there is 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 Garcinia mangostana as defined herein in the preparation of a comestible (foodstuff) for prophylaxis against the occurrence of inflammation, skin disorders or any of the age related disorders. Preferably the mammal is human and the said extract comprises a single extract from a plant part of Garcinia mangostana or a combination of extracts there from as detailed herein. Thus the present invention further relates to extracts, which may be isolated from the dried fruit rind of the Garcinia mangostana 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 Ceynt 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 dried fruit rind of the Garcinia mangostana, 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 Garcinia mangostana plant, depending on the end purpose that is required of the extract.

In one of the embodiment fruit rind extract of G. mangostana containing 1% Alpha-mangostin with 5% Flavonoids showing free radical scavenging and antimutagenic activity

In one of the embodiment fruit rind extract of G. mangostana containing 10% Alpha-mangostin with 25% flavonoids showing free radical scavenging and antimutagenic activity

In one of the embodiment fruit rind extract if G. mangostana containing 20 % of Alpha-mangostin with 40% flavonoids showing free radical scavenging and antimutagenic activity

In one of the embodiment fruit rind extract of G. mangostana containing 40% Alpha-mangostin with 95% flavonoids showing free radical scavenging and antimutagenic activity

In one of the embodiment fruit rind extract of G. mangostana containing 95% Alpha-mangostin showing free radical scavenging and antimutagenic activity

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

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

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

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

d. Optionally, lyophilizing said supernatant.

e. Analyzing the plant extracts for efficacy and presence of antioxidant, inflammatory and Skin care properties.

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

The choice of selected plant material may be of any type but is preferably the dried fruit rind of the Garcinia mangostana plant.

The solvent extraction process may be selected from direct types such as extraction from plant parts in reflux extractor apparatus in flasks at room temperature or at higher temperature with polar and/or non-polar sohent(s). Typically, the extraction process is as outlined herein. In another embodiment of the invention, the compositions for preventing, treating, or managing antioxidant, antiinflammatory and skin care, comprises of direct composite extract of plant species with a cohol, water and hydroalcohol solvent and successive extract with solvents ftvm 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 Garcinia mangostana 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 testing the antioxidant, anti-inflammatory and skin care property, 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 extracts of xanomax is subjected to HPLC and mass spec analysis.The invention further describes the biotherapeutic potential of various extracts of Garcinia mangostana 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% C02 and 95% air. It is further subcultured when cell become 80% confluent they are subjected to treatment with bioactive under investigation. The incubation is follower by estimating levels of bio-markers for inflammation like high-sensitivity C-Reactive protein (hs-CRP), Tumor Necrosis Factor (TNF-alpha), Interlukins (IL-6), serum amyloid A [SAA], white blood cell [WBC] count and fibrinogen. For antioxidants free radical scavenging, SOD, catalase, and glutathione peroxidase between the bioactive treated ana 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 Garcinia mangostana 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 c: 95%
pH 3-5
Bulk Density > 0.3 g/cc
Heavy Metals < 10 ppm
Arsenic by AAS < 2 ppm
Lead by AAS < 2 ppm
Cadmium by AAS < 0.2 ppm
Assay: Mangostin (HPLC) > 1%
Total Flavonoids (Gravimetric) > 5%

MICROBIOLOGICAL ANALYSES

Specification

Total Plate Count < 1,000 CFU/g
Yeast/Mold < 100 CFU/g
Coliforms < 3g
E.Coli Negative
Salmonella Negative
Staphylococcus Negative
Technical Specifications Xanomax 10% (_ &J'^ ^

DESCRIPTION

Dried extract of Garcinia Composition
mangostana
Common/trade name Mangosteen
Country of Origin India

PHYSICAL PROPERTIES
Appearance Dried reddish-brown powder
Plant Part Fruit rind (pericarp)
Odor/Taste Characteristic
Herb to Extract Ratio 6 : 1
Carrier None
Excipients < 2% Silicone dioxide
Particle Size 95% through 40 mesh
Bulk Density > 0.4 g/ml
Extraction Solvent Hydro Alcohol
Two years under cool and dry Shelf Life
Conditions

CHEMICAL ANALYSES

Specification
Loss On Drying < 5%
Water Soluble Extractives > 40%
60% Alcohol Extractives > 50%
pH 3-5
Arsenic by AAS < 2 ppm
Lead by AAS < 2 ppm
Cadmium by AAS < 0.2 ppm
Heavy Metals < 10 ppm
Assay: Mangostin (HPLC) > 10%
Total Flavonoids (Gravimetric) > 25%

MICROBIOLOGICAL ANALYSES

Specification
Total Plate Count < 1,000 CFU/g
Yeast/Mold < 100 CFU/g
Coliforms < 3g
E.Coli Negative
Salmonella Negative
Staphylococcus Negative
Technical Specifications Xanomax 20%

DESCRIPTION

Dried extract of Garcinia Composition
Mangostana Common/trade name Mangosteen
Country of Origin India

PHYSICAL PROPERTIES

Appearance Dried reddish-brown powder
Plant Part Fruit rind (pericarp)
Odor/Taste Characteristic
Herb to Extract Ratio 12:1
Carrier None
Excipients < 2% Silicone dioxide
Particle Size 95% through 40 mesh
Bulk Density > 0.4 g/ml
Extraction Solvent 80% Ethanol / 20% Water
Two years under cool and dry Shelf Life
Conditions

CHEMICAL ANALYSES
Specification
Loss On Drying < 5%
Water Soluble Extractives > 40%
60% Alcohol Extractives > 50%
pH 3-5
Arsenic by AAS < 2 ppm
Lead by AAS < 2 ppm
Cadmium by AAS < 0.2 ppm
Heavy Metals < 10 ppm
Assay: Mangostin (HPLC) > 20%
Total Flavonoids (Gravimetric) > 40%

MICROBIOLOGICAL ANALYSES

Specification
Total Plate Count < 1,000 CFU/g
Yeast/Mold < 100 CFU/g
Coliforms < 3g
E.Coli Negative
Salmonella Negative
Staphylococcus Negative

Technical Specifications Xanomax 40% L-vjDESCRIPTION
Dried extract of Garcinia Composition
mangostana
Common/trade name Mangosteen
Country of Origin India

PHYSICAL PROPERTIES

Appearance Yellowish brown powder
Plant Part Fruit rind (pericarp)
Odor/Taste Characteristic
Herb to Extract Ratio 30 : 1
Carrier None

Excipients Colloidal Silicon dioxide (< 2%)
Particle Size 95% through 40 mesh
Bulk Density > 0.4 g/ml
Extraction Solvent Hydro Alcohol
Two years under cool and dry
Shelf Life
Conditions

CHEMICAL ANALYSES

Specification
Loss On Drying < 5%
Alcohol Soluble Extractives < 95%
pH 3-6
Heavy Metals < 10 ppm
Arsenic by AAS < 2 ppm
Lead by AAS < 2 ppm
Cadmium by AAS < 0.2 ppm
Assay: Mangostin (HPLC) > 40%
Total Flavonoids (Gravimetric) > 95%

MICROBIOLOGICAL ANALYSES
Specification
Total Plate Count < 1,000 CFU/g
Yeast/Mold < 100 CFU/g
Coliforms < 3g
E.Coli Negative
Salmonella Negative
Staphylococcus Negative
Technical Specifications Xanomax 95% \ DESCRIPTION
Dried extract of Garcinia Composition
mangostana

Common/trade name Mangosteen
Country of Origin India

PHYSICAL PROPERTIES
Appearance Dried yellow powder
Plant Part Dried Fruit Rind (pericarp)
Odor/Taste Characteristic
Particle Size 95% through 40 mesh
Carriers None
Excipients None
Extraction Solvent Hydro Alcohol
Two years under cool and dry Shelf Life
Conditions

CHEMICAL ANALYSES
Specification
Loss On Drying < 5%
Arsenic < 2 ppm
Lead by A AS < 2 ppm
Cadmium by AAS < 0.2 ppm
Heavy Metals < 10 ppm
Assay: Mangostin (HPLC) > 95%

MICROBIOLOGICAL ANALYSES
Specification
Total Plate Count < 1,000 CFU/g
Yeast & Mold < 100 CFU/g
Coliforms < 3/g
Escherichia coli/25g Negative
Salmonella/25g Negative
Staphylococcus aureus/25g Negative

Example 2: HPLC analysis

Metabolic fingerprinting of Xaomax 10% extract was done to establish for 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: 55mins Time constant: 0.64sec Column used: Atlantis dc 18 ,5u.m 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: lml/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: 200|il
Needle stroke: 52mm
Control vial needle stroke: 52mm
Rinsing speed: 35|il/sec
Sampling speed: 15^1/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: lml/min
Samples analyzed: Xanomax_10%
Run conditions
No of runs performed for each sample: 5
Injection volume: 10(il
LC time program used: Xano MD3

Sample preparation

10mg of the extract was weighed out into sterile eppendorf and 100µl of DMSO was added and kept a side for some time (15min) and made it to 1ml 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.

Table 1: HPLC analysis and the peak information of Xanomax 10% extract

The extract was injected at a constant volume of 10(_tl and its metabolite profiling monitored at 254nm. (Fig 6 and Table. 1 ) A comparative profiling was performed between the runs/ extract to check the reproducibility of the extarcts ( Fig. 7)

Example 3: Metagrid profile of XanoMax 10% extracts

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. For analysis, conserved region of the specific wavelength was considered and the standard deviation for batch-to-batch extraction was less than ±10% was maintained through out and any deviated extracts were removed (Fig 8)

DPPH Assay of Xanomax 20%

l,l-Diphenyl-2-picrylhydrazyl (Oxidized form) is a stable free radial with Purple color. In the presence of an antioxidant which can donate an electron to DPPH, the purple color which is typical to free DPPH radical decay and the change in absorbance at 517nm is followed which can be measured spectrophotometrically.

Xanomax 20% exhibited an IC50 value of 25-26 fxg/ml on DPPH activity indicating high antioxidant activity (Fig 9a, 9b)

Cell viability assay of Xanomax 20%

Xanomax 20% did not show any cytotoxicity at lower doses of 0.1-10 ng/ml, howvere significant cytotoxicity was observes at higher doses of 100-500 u.g/ml on 24 hrs of treatment (Fig 10)

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 cnoline, 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.

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,

We Claim:

1. An extract of Garcinia species optimally along with pharmaceutically acceptable additives.

2. The extract as claimed in claim 1, wherein said Garcinia species is Garcinia mangostana.

3. The extract as claimed in claim 1, wherein 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.

4. The extract as claimed in claim 1, wherein said extract is formulated into dosage forms selected from a group comprising tablet, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion in hard or soft gel capsules, syrups and elixirs.

5. A process for obtaining extract from Garcinia species, said process comprising steps of:

(d) powdering plant material of Garcu ia species;

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

(f) concentrating the extract followed by drying to obtain the extract of Garcinia species.

6. The process as claimed in claim 5. wherein said Garcinia species is Garcinia mangostana.

7. The process as claimed in claim 5, wherein said plant materials are selected from a group comprising leaves, seeds, roots, stems, flowers and various combinations thereof preferably the dried fruit rind.

8. The process as claimed in claim 5, wherein said extract is optionally centrifuged followed by lyophilization to obtain the extract of Garcinia species.

9. The process as claimed in claim 5, wherein said solvents are selected from a group comprising water, alcohol and combinations thereof.

10. A method of treating inflammation, said method comprising step of administering pharmaceutically acceptable amount of extract of Garcinia species optionally along with pharmaceutically acceptable additives to the subject.

11. The method as claimed in claim 10, wheiein the subject is animal including human being.

12. An extract of Garcinia species, the process to prepare and the method of treating are substantially as herein described with reference to accompanying examples and drawings.