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 Phyllanthus sp. with specific reference to Phyllanthus emblica. The present invention further relates to extracts which are isolated from different parts of Phyllanthus emblica 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 as a cosmetic application, oxidative stress release, immunomodulation and for antiageing to achieve the best results of body cleansing and beauty. Furthermore, the invention relates to the use of the extracts as a supplement or a medicament useful in the treatment/alleviation or prevention of skin, age and oxidative disorders.

BACKGROUND AND PRIOR ART OF THE INVENTION

The world we live in today poses as a danger to human life due to the many negative effects brought about by the basic elements required for our existence i.e. sunlight and atmospheric air. Skin, the largest organ in our body is the first line of defense separating us from the outside world. The skin employs a host of protective mechanism to defend itself from ravages of the environment. The skin is the most environmentally stressed organ in mammals, particularly in humans. Not only is the skin subjected to germs, toxic chemicals and hostile environments, it is the only organ directly exposed to ultraviolet light (UV). In addition, the vitality of this organ is a consequence of genetic processes, which over time, lead to a decrease in the functionality of the skin. Hence, a variety of dermatological conditions may occur as a result of ongoing intrinsic factors (for example, chronological ageing, disease and allergies) and/or exposure to a number of extrinsic factors (such as infection, trauma, radiation, toxins and steroid use).

A. The need for Cosmetics

Cosmetics are commercially available products that are used to improve the appearance of the skin. The consumer demand for more effective products that more substantively beautify the appearance has resulted in increased basic science research and product development in the cosmetics industry. The result has been more ingredients that may actually improve not just the appearance of the skin, but the health of the skin as well. There is probably no greater focus of interest currently than the incorporation of vitamins and antioxidants in skin care products. There are considerable data to suggest the benefits of such ingredients in cosmetics. This review supports the usefulness of vitamins and antioxidants in cosmetics.

B. Vitamins and Antioxidant activity

The ingestion and absorption of vitamins and antioxidants, most importantly through diet, and secondarily through intake of manufactured supplements, is critical to the health of human beings. The skin is the largest organ; as our primary external barrier, it is on the forefront of the battle with external causes of damaging free radicals. Ultraviolet light and environmental pollutants are known initiators of free radicals. Free radicals are highly reactive molecules with an unpaired electron that result in damage to surrounding molecules and tissues. The most significant damage by free radicals is to biomembranes and to DNA. It is thought that additional, topical use of vitamins and antioxidants in cosmetics can better protect and possibly correct the damage by neutralizing these free radicals. In addition, some vitamins may be beneficial to the skin because of other actions such as effects of suppression of pigmentation and bruising, stimulation of collagen production, refinement of keratinization, or anti-inflammatory effects.

Vitamin C, ascorbate, a water-soluble vitamin present in citrus fruits and vegetables, is important because of its antioxidant actions as well as its function as a cofactor in hydroxylation reactions of collagen production. Humans are unable to synthesize ascorbate, so dietary intake is essential. Interest in vitamin C as a cosmetic ingredient is partly a result of its ability to quench UV induced free radicals directly, and to regenerate vitamin E, another potent antioxidant. Vitamin C is also considered an antiaging ingredient because of its potential to stimulate collagen production. There are three forms commonly available in cosmetics: ascorbyl palmitate, magnesium ascorbyl phosphate, and L-ascorbic acid. Ascorbyl palmitate, a fat-soluble synthetic ester of vitamin C, is stable in cosmetic formulas at neutral pH. Hydrolysis of ascorbyl palmitate yields ascorbic acid and palmitic acid. Topical ascorbyl palmitate in one study was found to be thirty fold more effective than ascorbic acid as an inhibitor of some of the biochemical parameters associated with tumor production in mice skin. Ascorbyl palmitate, applied after UV burning, reduced redness 50% sooner than areas on the same patient that were left untreated. The suspected mechanism is that the ascorbyl palmitate acted as both an antioxidant and anti-inflammatory agent. Other dermatologic conditions that have inflammation as a component of the disease process, such as psoriasis and asteototic eczema, have shown clinical improvement when treated with ascorbyl palmitate. Another vitamin C derivative found in cosmetics is magnesium-L-ascorbyl-2-phosphate, which has been studied for various antioxidant benefits. Magnesium ascorbyl phosphate was found to protect against UVB radiation-induced lipid peroxidation in hairless mice. In vitro evidence was found that magnesium ascorbyl phosphate crossed the epidermis and is converted to ascorbic acid. In an in vitro study of monolayer human fibroblast cultures, magnesium ascorbyl phosphate was found to be equivalent to ascorbic acid in stimulating collagen synthesis and was stable at a neutral pH. Another in vitro study of human skin fibroblast in culture demonstrated enhanced collagen synthesis and cell growth.

Significant also was the extended stability under various conditions and the effectiveness over a wide concentration range. Evidence suggested that cell membrane dephosphorylation converted the magnesium ascorbyl phosphate to ascorbic acid. Subsequent in vitro study of cultured fibroblast suggested that magnesium ascorbyl phosphate regulates type I collagen production. An in vivo study of human skin demonstrated a clinical improvement of melasma and senile freckles when utilizing a 10% magnesium ascorbyl phosphate cream. In situ testing demonstrated absorption into the epidermis. Presence of vitamin C after 48 hr was measurable but minimal (1.6%). The ingredient was stable in nonacid solution, hydrolyzed by phosphatases in the skin to form ascorbic acid, which then inhibited melanin production by reducing o-quinone. Another ascorbate derivative, 2-0- alpha-D-glucopyranosyl-L-ascorbic acid, which is also stable in solution and at wide pH ranges, also stimulated cultured human skin fibroblasts in vitro, resulting in increased collagen synthesis.

L-ascorbic acid is the most bioactive form of vitamin C and has been found to have numerous benefits for the skin. This form of vitamin C is water-soluble but it must be formulated at low pH to be stable. In cultured human skin fibroblast, it increases type I and type III procollagen mRNA. The measured difference was a result of increased synthesis of procollagen polypep polypeptides. The authors speculated that ascorbic acid may have affected gene transcription or mRNA degradation. Another study's results indicated that ascorbic acid stimulated collagen synthesis in collagen matrix. Such 3-D models more closely mimic in vitro conditions. As an electron donor, L-ascorbic acid is able to function as an antioxidant. In porcine studies, it was found to be photoprotective because it reduced sunburn cell formation by both UVB and PUVA exposure. The authors speculate that replenishing the vitamin C that is normally depleted by UV radiation helps protect the skin from free radical damage. Further study is warranted to determine optimal concentration and formulations of ascorbic acid so that it may be a stable formulation perhaps used as an adjunct to sunscreens for more complete photoprotection.

(Mary Lupo, Antioxidants And Vitamins In Cosmetics Clinics In Dermatology 2001;19:467-473.)

C. Nonvitamin Antioxidants

There is great importance for an intact antioxidant defense system for the skin. Directly exposed to a hostile pro-oxidative environment, endogenous skin antioxidants can be easily depleted. In addition to research on antioxidant vitamins, there has also been research into the efficacy of other antioxidants that can be used topically in cosmetics.

Coenzyme Q, or ubiquinone, is a naturally occurring, lipid-soluble substance found in high levels in the epidermis. Its presence in most cells in the body, and its ability to react with reactive oxygen species, has resulted in scrutiny for systemic use for many diseases of aging, such as heart disease and atherosclerosis. Like other naturally occurring skin antioxidants, it is depleted by UV exposure.66 It has also been suggested that it protects vitamin E present in the skin, which can further help the skin fight free radical damage.

These actions have resulted in recent commercial interest in Coenzyme Q-10 as a topical
ingredient in cosmetics. Research has suggested antioxidant effects against UV radiation in cultured skin cells as well as penetration into the epidermis and dermis in vivo. More study must be done to determine the mechanism of its antioxidant actions in human skin after topical application. Flavonoids are a family of compounds that are reported to have health benefits systemically via antioxidant action. Recently, there has been an increase in the use of these polyphenolic compounds in cosmetics (Mary Lupo 2001).

The use of herbs has shown positive effects on the oxidative and immune system. Dry fruits were found to promote and rejuvenate mental and physical health as well as provide a defense against aging and challenging environmental factors.

Amla {Phyllanthus emblica) is a deciduous tree cultivated in SE Asia for its fruit of immense nutritive value. The fruit is richest source of vitamin C and has shown high antioxidant activity and is the subject of ongoing research.

An extract of the fruit has shown free radical scavenging, antimutagenic and antimicrobial properties (Anilakumar et.al.2004; Bhattacharya et.al. 2002) It is used to treat several conditions like scurvy, inflammation, erysipelas, leprosy, anemia, inflammations, burning sensations, urinary discharges, absence of urination painful urination, biliousness, poisoning, and ophthalmia (Kutikar et.al. 1994). Studies show that the antioxidant/free radical scavenging activity of the fruit is primarily due to the presence of Vitamin C; flavonoids and tannins (Scartezzini et.al. 2006; Anila et.al. 2000).

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.

OBJECTIVES OF THE PRESENT INVENTION

The main objective of the present invention is to obtain an extract of Phyllanthus species optionally along with pharmaceutically acceptable additive.

Another main objective of the present invention is to develop a process for obtaining extract from Phytlanthus species.

Yet another main objective of the present invention is to develop a method of preventing and/ or managing oxidative and immunity disorders in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of extract of Phyllanthus species optionally along with pharmaceutically acceptable additives to the subject.

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

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

Yet, another object of the invention is to provide composition comprising active principles of Phyllanthus emblica, and the use of these extracts and constituents for the preparation of nutritional and nutraceutical application.

Still another object of the present invention is to provide Phyllanthus emblica plant extract, which is easily and safely administrable to children and adults.

STATEMENT OF THE INVENTION

Accordingly, the present invention relates to an extract of Phyllanthus species optionally along with pharmaceutically acceptable additive; a process for obtaining extract from Phyllanthus species, said process comprising steps of: a) powdering plant material of Phyllanthus 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 Phyllanthus species and a method of preventing and/ or managing oxidative and immunity disorders in a subject in need thereof, said method comprising step of administering pharmaceutical acceptable amount of extract of Phyllanthus species optionally along with pharmaceutical acceptable additives to the subject.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

Fig. 1: MetaGrid™ analysis of AmlaPure.

Fig. 2: DPPH activity of Amla Pure.

Fig. 3: Effect of Amla on Cell viability on WS-1 cells at 24 hrs of treatment.

Fig. 4: Effect of Amla on Collagen release on WS-1 cells at 24 hrs of treatment.

Fig. 5: Effect of Amla on Elastin release on WS-1 cells at 24 hrs of treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an extract of Phyllanthus species optionally along with pharmaceutically acceptable additive.

In another embodiment of the present invention, said Phyllanthus species is Phyllanthus
emblica.

In yet another embodiment of the present invention, said additive is 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 or a combination thereof.

In still another embodiment of the present invention, said extract is formulated as cosmetics and dosage forms selected from a group comprising tablet, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft gel
capsules, syrups, elixirs.

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

(a) powdering plant material of Phyllanthus 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 Phyllanthus species.

In another embodiment of the present invention, said Phyllanthus species is Phyllanthus emblica.

In yet another embodiment of the present invention, said plant material is selected from a group comprising leaves, seeds, roots, stems, flowers, fruits, or various combinations thereof, preferably fruits.

In still another embodiment of the present invention, said extract is optionally centrifuged followed by Iyophilization to obtain the extract of Phyllanthus 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 oxidative and immunity disorders in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of extract of Phyllanthus species optionally along with pharmaceutically acceptable additives to the subject.

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

Accordingly, the present invention provides Phyllanthus emblica plant extract for its potent antioxidant activity and/ or its support for a healthy immune system in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of standardized Phyllanthus plant composite extract, optionally along with pharmaceutically acceptable additives, to the subject; and a process for enhancing external and internal age related properties of a Phyllanthus plant extract, said process comprising of plant material after pharmacognostic evaluation, then the extractions from batch to batch tested using in-house metabolite fingerprinting QA/QC method based on HPLC equipped with PDA.( Fig1) The extract was subjected to enzymatic assays such as

ORAC Assay, DPPH Assay, and Cell Based Assays such as Viability assay, Collagen release and Elastin release to obtain the plant bioactives which have significant role in reducing oxidative stress and age related disorders. The invention further provides for uses of the extract to manufacture a medicament for multiple therapeutic uses, as well as other healthe benefits.

The present invention is in relation to efficiency of the bioactive component of the plant extract for therapeutic use, wherein said extract from Phyllanthus emblica 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 Phyllanthus emblica as defined herein in the preparation of a comestible (foodstuff) for prophylaxis against the occurrence of oxidative and low immunity diseases. Preferably the mammal is human and the said extract comprises a single extract from a plant part of Phyllanthus emblica or a combination of extracts there from as detailed herein. Thus the present invention further relates to extracts, which may be isolated from fruits of the Phyllanthus emblica plant, the preparation of such extracts, medicaments comprising such extracts, and the use of these extracts and constituents for the preparation of a medicament.

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

In another aspect of the present invention, extracts are isolated from fruits of the Phyllanthus emblica, 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 Phyllanthus emblica 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 Phyllanthus emblica 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 against oxidative and immunity disorders.

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

The choice of selected plant material may be of any type but is preferably the fruits of the Phyllanthus emblica plant.

The solvent extraction process may be selected from direct types such as extraction from plant parts in reflux extractor apparatus or in flasks at room temperature or at higher temperature with polar and/or non-polar solvents). Typically, the extraction process is as outlined herein, In another embodiment of the invention, the compositions for treating oxidative stress and managing skin and beauty aspects and minimizing age related complications as well as immunomodulation, 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 Phyllcmthus emblica plant parts thereby giving each extract an identity of itself.

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

The successive extraction from plant extract will be carried out using soxhlet extractor.

The solvents used, will be based on their sequential polarity starting from non-polar to polar, wherein, various classes of metabolites will be extracted viz. petroleum ether (phytosterols, fixed oils and fats), benzene (fixed oils and fats), chloroform (alkaloids), acetone (phytosterols, phenolics and tannins) ethanol (alkaloids, carbohydrates, glycosides, phytosterols, saponins, phenolics, tannins, proteins and amino acids) and water (alkaloids, carbohydrates, glycosides, saponins, phenolics, tannins, proteins, amino acids, gums and mucilage) at 65°C. These fractions will be lyophilized and stored in amber colored bottles at 4°C.

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

The extracted fractions will be subjected to HPLC using u, bondapak C i8 column (Waters Alliance 2695 Separation Module) to separate the constituent metabolites. The fractions are eluted using a combination (80:20, 60:40, 50:50, 40:60, 20:80) of methanol: water / acetonitrile: water. The gradient run will also be carried out wherever required. 5-lOul of sample is injected with flow rate of 1 ml/min and HPLC run will be performed for 30 minutes. The detection is done on photodiode array and the analysis of the results will be done with the help of Millennium™ software and using MetaGrid the MetaGrid profile for Amla pure ™ was generated. (Figl)

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

The invention further describes the biotherapeutic potential of various extracts of Phyllanthus emblica 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 followed by estimating levels of bio-markers for Oxidative stress and Immunity 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 Phyllanthus emblica 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 the instant invention.

Example 1: Extraction of Phyllanthus emblica:

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

Flow chart for Amla Pure extraction

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.

Table 1. Technical Specifications

1. Enzymatic Assays

ORAC Assay:

The antioxidant capabilities of AmlaPure are evaluated using ORAC assay. The ORAC assay depends on the free radical damage to a fluorescent probe, such as fluorescein, to result in a downward change of fluorescent intensity. The degree of change is indicative of the amount of radical damage. The presence of antioxidants results in an inhibition in the free radical damage to the fluorescent compound. This inhibition is observed as a preservation of the fluorescent signal. AmlaPure is exhibiting a value of 1240 TpDmol/g Sample. This indicates that AmlaPure is a potential antioxidant.

DPPH Assay:

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. (Fig2)

AmlaPure extract exhibited an IC50 value of 10 ug/ml on DPPH activity indicating high antioxidant activity. (Fig.2)

2.Cell Based Assays

Viability assay

AmlaPure extract did not show any cytotoxicity at all the studied doses of 0.1 -500 (ig/ml after 24 hrs of treatment. (Fig.3)

Collagen release

AmlaPure extract demonstrated a statistically significant increase in the collagen release level at 24 hrs of treatment at doses of 10-100 ug/ml with 1,4-1,5 fold increase when compared to sodium ascorbate showing 2.2 fold increase. (Fig. 4) Table: 2.

Table 2: Effect of Amla Pure on collagen release

Elastin release

AmlaPure extract demonstrated a statistically significant increase in the elastin release level at 24 hrs of treatment at doses of 10-100 µg/ml with 1.4 - 2 fold increase when compared to sodium ascorbate showing 2.2 fold increase. Since the DPPH activity showed IC50 value 10 µg/ml, thus it indicates that AmlaPure extract at lower dose of 10

ug/ml exerts its potency and efficacy. (Fig.5). Effect of Amla Pure on Elastin release
shown in table 3.

Table 3: Effect of Amla Pure on Elastin release

Example 2: 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 foodstuff 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 Phyllanthus species optionally along with pharmaceutically acceptable additive.

2. The extract as claimed in claim 1, wherein said Phyllanthus species is Phyllanthus emblica.

3. The extract as claimed in claim 1, wherein said additive is 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 or a combination thereof.

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

5. A process for obtaining extract from Phyllanthus species, said process comprising steps of;

(a) powdering plant material of Phyllanthus 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 Phyllanthus species.

6. The process as claimed in claim 5, wherein said Phyllanthus species is Phyllanthus emblica.

7. The process as claimed in claim 5, wherein said plant material is selected from a group comprising leaves, seeds, roots, stems, flowers, fruits, or various combinations thereof, preferably fruits.

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

9. The process as claimed in claim 1, wherein said solvents are selected from a
group comprising water, alcohol and combinations thereof.

10. A method of preventing and/ or managing oxidative and immunity disorders in a
subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of extract of Phyllanthus species optionally along with pharmaceutically acceptable additives to the subject.

11. The method as claimed in claim 1, wherein the subject is animal including human being.

12. The extract of Phyllanthus species, the process and the method as substantially
described herein with reference to accompanying examples and drawings.