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 Hibiscus sp. with specific reference to Hibiscus sabdariffa. The present invention further relates to extracts which are isolated from different parts of Hibiscus sabdariffa 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, antioxidant, antiobesity to achieve the best results of beauty and weight management. 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 and obesity disorders. The extract of the present invention is also referred as Hibix.

BACKGROUND AND PRIOR ART OF THE INVENTION

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).

Antioxidants may play a significant role in ameliorating or preventing photobiologic damage in skin that could lead to cutaneous disorders such as cancer and premature aging. 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. However, if we flood our bloodstream with an unusually large number of free radicals, oxidative damage can overwhelm the body's repair mechanisms, setting us up for degenerative diseases. Antioxidants protect cells from the damage caused by free radicals, unstable compounds that result from lifestyle factors like environmental stress and strenuous exercise, as well as natural processes like aging.

Enzymatic and Non-enzymatic Antioxidants

Antioxidants are commonly found in two forms: enzymatic and non-enzymatic. The enzymatic antioxidants include superoxide dismutase (SOD), which catalyses the conversion of Oxygen radical to Hydrogen peroxide and water; catalase, which then converts Hydrogen peroxide to water and oxygen; and glutathione peroxidase, which reduces Hydrogen peroxide to water. 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. 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.

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.

(Tamer Fouad "Antioxidants Nature and Chemistry", www.doctors lounge.com)

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 recommended 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.

Obesity
Obesity has reached epidemic proportions globally, with more than 1 billion adults overweight -at least 300 million of them clinically obese - and is a major contributor to the global burden of chronic disease and disability. Often coexisting in developing countries with under-nutrition, obesity is a complex condition, with serious social and psychological dimensions, affecting virtually all ages and socioeconomic groups. The prevalence of overweight and obesity is commonly assessed by using body mass index (BMI), defined as the weight in kilograms divided by the square of the height in metres (kg/m2). These markers provide common benchmarks for assessment, but the risks of disease in all populations can increase progressively from lower BMI levels.

Obesity and overweight pose a major risk for serious diet-related chronic diseases, including type 2 diabetes, cardiovascular disease, hypertension and stroke, and certain forms of cancer. The health consequences range from increased risk of premature death, to serious chronic conditions that reduce the overall quality of life. Of especial concern is the increasing incidence of child obesity.

Overweight and obesity lead to adverse metabolic effects on blood pressure, cholesterol, triglycerides and insulin resistance. The non-fatal, but debilitating health problems associated with obesity include respiratory difficulties, chronic musculoskeletal problems, skin problems and infertility. The more life-threatening problems fall into four main areas: CVD problems; conditions associated with insulin resistance such as type 2 diabetes; certain types of cancers, especially the hormonally related and large-bowel cancers; and gallbladder disease.

Raised BMI also increases the risks of cancer of the breast, colon, prostrate, endometroium, kidney and gallbladder. Chronic overweight and obesity contribute significantly to osteoarthritis, a major cause of disability in adults. Although obesity should be considered a disease in its own right, it is also one of the key risk factors for other chronic diseases together with smoking, high blood pressure and high blood cholesterol. In the analyses carried out for World Health Report 2002, approximately 58% of diabetes and 21% of ischaemic heart disease and 8-42% of certain cancers globally were attributable to a BMI above 21 kg/m2.

Effective weight management

Effective weight management for individuals and groups at risk of developing obesity involves a range of long-term strategies. These include prevention, weight maintenance, management of comorbidities and weight loss. (Dr. P. Puska, Dr. C. Nishida & D. Porter, 2003, World Health Organization, Global Strategy On Diet, Physical Activity And Health., Obesity And Overweight. http://www.who.int/dietphysicalactivity/media/en/gsfs_obesity.pdf)

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 the skin particularly due to its antioxidative property and weight management. Dry plant parts were found to promote and rejuvenate mental and physical health as well as provide a defense against challenging environmental factors.

"Roselle" flower (Hibiscus sabdariffa L. var. ruber ; family Malvaceae) is a tropical, erect, annual, herbaceous shrub. Roselle is widely cultivated in the tropics and is found in many different countries, including China, India, Thailand, Malaysia, the Philippines, Australia, Africa, Central America, Mexico, the West Indies, and Florida (Morton, 1974; Wichtl, 2004). Major commercial sources of the dried flowers are China, Thailand, Sudan, Egypt, and Mexico (Wichtl, 2004; Leung and Foster, 1996).

Apart from the German "roselle", the plant is known by numerous common names, including the French "karkade" (red tea) (Wichtl, 2004), the Thai "krachiap daeng" (Hirunpanich et al., 2006), the Persian "sour tea" (Haji Faraji and Haji Tarkhani, 1999), the Chinese "Lo Shen" (Duh and Yen, 1997), and the English "Roselle of Rama" (Farnsworth and Bunyaprephatsara, 1992), "Jamaica sorrel", "Indian sorrel", "jelly okra", "sour-sour Queensland jelly plant", and "Florida cranberry" (Morton, 1974).

The bracts and the thick, fleshy, red calyces (enveloping sepals) that form the flower are commonly referred to simply as "roselle flower" and are usually dried before use. As early as the 1930s, an infusion of the calyces was noted as a traditional African treatment for the relief of cough and bronchitis (Hutchinson and Dalziel, 1937), as well as hypertension (Morton, 1974; Sharaf, 1962); a use that may be even older in Iran (Haji Faraji and Haji Tarkhani, 1999). In Thailand, the calyces of "krachiap" are used in the treatment of high cholesterol levels, urinary bladder stones, kidney stones, and as an expectorant (Farnsworth and Bunyaprephatsara, 1992). In Europe, the calyces have been used in herbal medicine for treating high blood pressure, heart diseases, nerve diseases, calcified arteries (El-Saadany et al., 1991), arthritis (Stirn, 1978), arteriosclerosis (Perry and Metzger, 1980), circulatory disorders, poor appetite, colds, catarrhs of the stomach and upper respiratory tract, for dissolving phlegm, and as a diuretic and mild laxative.

Antioxidant Activity
An ethanol extract of the dried flowers of roselle from Nigeria was further extracted to obtain a chloroform-soluble fraction (HSCF) and an ethyl acetate-soluble fraction (HSEA). In vitro scavenging of hydroxyl radical, hydrogen peroxide, and superoxide anion radical production was found with greater potency from HSEA compared to HSCF. When compared for total antioxidant activity in a peroxidase system for equivalent activity to that of vitamin C, HSEA was significantly more potent than HSCF and held a significantly greater content of total phenolics (Farombi and Fakoya, 2005).

Using the dried calyces obtained from Egypt, a decoction and a series of cold infusions (2 g dried petals in 100 mL water) were tested for antioxidant activity using Briggs-Rauscher oscillating reaction, a method developed for wine. The reaction generates hydroperoxyl radicals in a medium to simulate pro-oxidant conditions. According to the Briggs-Rauscher Antioxidant Index (BRAI), overall antioxidant potential was less than that of red wine and greatest from the 3-minute (100°C) decoction measured at 37°C (BRAI = 665) and the 30- and 180-minute cold infusions measured at 37°C (BRAI = 571 and 522, respectively). Since the quantity of gallic acid equivalents corresponded to the degree of BRAI, it is reasonable to suppose that that polyphenol content corresponded to the antioxidants activity. In terms of acidity, the extraction methods showed no significant differences, likely because the acids are more readily extractable in water. (Prenesti et al., 2007).

A water extract of the sun-dried calyces exhibited dose-dependent scavenging of the DPPH radical (l,l-diphenyl-2-picrylhydrazyl) with concentrations of 5, 15, and 25 mg/mL inhibiting radical formation by 49.9%, 771.%, and 88.6%, respectively (Duh and Yen, 1997). From an ethanol extract of the dried flowers obtained in an herbal pharmacy in Taiwan, an ethyl-acetate-soluble fraction showed greater scavenging of the DPPH radical (EC50 = 0.017 mg/mL) than either a chloroform-soluble fraction or an insoluble ethyl acetate fraction. However, in the xanthine oxidase (XO) assay in which XO "catalyses the oxidation of hypoxanthine to xanthine and of xanthine to uric acid", the chloroform-soluble fraction was the most potent inhibitor of XO activity (EC50 = 0.742 mg/mL) (Tseng et al., 1997).

The total anthocyanin fraction of the dried calyces (standardized to cyaniding and delphinidin) dose-dependently scavenged the DPPH radical by greater than 50% at concentrations of 0.05 mg/mL and more. (Chang et al., 2006). However, others reported that the total anthocyanins of the dried flowers at a concentration of 0.20 mg/mL quenched the DPPH radical by about 50% (Wang et al, 2000).

The petals of a cultivar of roselle (F141) grown in Taiwan were dried (50°C for 36 hours) before preparing a hot water extract made by boiling them in water for 3 minutes (1 g/100 mL). The antioxidant activity of the extract in the ferric reducing ability of plasma (FRAP) assay was shown to correlate with the results obtained in the ORAC (oxygen radical absorbance capacity) assay and total antioxidant status (TAS) method. The results clearly suggest that either of these methods would accurately measure the antioxidant activity of roselle. Preliminary tests showed that extracts of the petals prepared at 3-5 minutes of boiling held the maximum antioxidant activity.

Using the FRAP assay, the antioxidant activity of the extract was compared to those of coffee and tea also prepared by boiling in water for 3 minutes (1 g/300 mL). Samples of green and black teas, bottled orange juice, and red and white wines obtained from commerce were used for further comparisons. Red wine showed a value of about 20 which was by far the greatest of all the substances tested. Among the teas, green tea held the greatest antioxidant activity with a value of about 8, which was comparable to that of rose wine and orange juice. The value of the roselle extract was about 16%-25% that of green tea or about 1.28-2 (Tsai et al., 2002).

When the anthocyanin content of the roselle extract correlated with antioxidant capacity in the FRAP assay, efforts were made to fractionate the extract to characterize the component anthocyanins responsible. Delphinidin 3-sambubioside was responsible for the red pigmentation of the extract and constituted 85% of the anthocyanins while cyanidin 3-sambubioside (provisionally identified) appeared to account for the pink pigmentation. Respectively, they accounted for 48% and 3% of the antioxidant activity obtained in the FRAP assay, while the remaining 24% of activity was attributed to brown pigments composed of phenolics. The authors concluded that roselle petals represent "a good source of antioxidants" and that although relatively stable during storage, the antioxidant activity "will ultimately depend on the bioavailability of the anthocyanins" (Tsai et al., 2002) (see Pharmacokinetics).

DERMATOLOGICAL ACTIVITY

Roselle is reported to inhibit the in vitro growth of Staphylococcus epidermis (minimum inhibitory concentration = 0.625 mg/mL; minimum bactericidal concentration = 5 mg/mL), a microorganism involved in superficial infections of the skin. Weaker activity was found against the growth of Propionibacterium acnes (MIC = 2.5 mg/mL; MBC = 5 mg/mL), an organism associated with the development of inflammatory acne (Chomnawang et al., 2005). The total crude polysaccharide fraction and 4 subtractions were isolated from the flowers of roselle and tested for proliferative and other effects on human keratinocytes (HaCaT cells). The crude polysaccharide fraction (0.1- 10 mg/mL) significantly increased the rate of proliferation by 10% to 20%. Whereas a neutral subtraction showed no activity, 3 acidic subfractions were significantly active, stimulating proliferation by up to 30%. Concentrationdependent activity was absent, however; likely due to the activation of membrane-associated receptors and the subsequent involvement of transduction cascades. Further tests using natural human keratinocytes found significant stimulation of differentiation from the crude polysaccharide fraction (10 mg/mL). Tests using the MTT assay revealed that the enhanced proliferation of HaCaT cells by the polysaccharides did not involve increased energy metabolism and that they were devoid of cytotoxicity. The authors concluded that while extracts of roselle rich in highly acidic polysaccharides have some rationale for use in cosmetic or dermatological applications, bioavailability and relevant concentrations remain to be determined (Brunold et al., 2004).

Anti-obesity effect

Francisco et.al. 2007 determined that Hibiscus sabdariffa calyces aqueous extract had an effect on body weight in an obese animal model induced by the administration of monosodium glutamate. It significantly reduced body weight gain in obese mice and increased liquid intake in healthy and obese mice. ALT levels were significantly increased but AST levels did not show significant changes. Triglycerides and cholesterol levels showed non-significant reductions in treated animals confirming the anti-obesity effect of Hibiscus sabdariffa reported by the Mexican population.

Jin-Kyung Kim etal. 2007 investigated the inhibitory mechanisms of hibiscus extract on adipocyte differentiation in 3T3-L1 preadipocytes. They examined whether hibiscus extract affected the adipogenesis. To differentiate preadipocyte in adipocyte, confluent 3T3-L1 preadipocytes were treated with the hormone mixture including isobutylmethylxanthine, dexamethasone, and insulin (MDI). Hibiscus extract inhibited significantly the lipid droplet accumulation by MDI in a dose-dependent manner and attenuated dramatically the protein and mRNA expressions of adipogenic transcriptional factors, C/EBPgreek small letter alpha and PPARy, during adipogenesis. The increase of phosphorylation and expression of PI3-K/Akt during adipocytic differentiation was markedly inhibited by treatment with hibiscus extract or PI3-K inhibitors. Furthermore, the phosphorylation and expression of MEK-1/ERK known to regulate the early phase of adipogenesis were clearly decreased with the addition of hibiscus extract. Taken together, this suggests that hibiscus extract inhibits the adipocyte differentiation through the modulation of PI3-K/Akt and ERK pathway that play pivotal roles during adipogenesis.

Pau-Ling Tee et.al. 2002 studied the benefits of roselle in vivo using the model of male Sprague Dawley rats fed with normal diets (C), 2.5 percent roselle (R), 2.5 percent roselle with 15 percent soybean oil (RO) and 15 percent soybean oil (O) for 25 weeks. The results demonstrated that supplementation of roselle in the diets significantly reduced the gross body weight and increased the high-density lipoproteins cholesterol (HDL-C) compared to the rats fed with normal diets and oil diets, and reduced the level of triglycerides in the serum. Addition of roselle in oil diet significantly decreased the malondialdehyde formation at week 20 and 25 but there was no significant effect of roselle on the catalase activity among the treatments at all time points. This suggests that roselle might play a role in the prevention of atherosclerosis and obesity.

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

Another object of the present invention is to provide an active extract and bioactive fraction obtained from different parts of Hibiscus sabdariffa plant.

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

Still another object of the invention is to provide composition comprising active principles of Hibiscus sabdariffa, 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 Hibiscus sabdariffa plant extract, which is easily and safely administrable to children and adults.

STATEMENT OF THE INVENTION

Accordingly, the present invention provides an extract of Hibiscus species optionally along with pharmaceutically acceptable additives; A process for obtaining extract from Hibiscus species, said process comprising steps of: powdering plant material of Hibiscus species; extracting and refluxing the powder with solvents to obtain extract; concentrating the extract followed by drying to obtain the extract of Hibiscus species; A method of preventing and/ or managing oxidative stress and obesity in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of extract of Hibiscus species optionally along with pharmaceutically acceptable additives to the subject.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

Figure 1: Process flow diagram for Hibix 45% extract

Figure 2 : HPLC chromatogram of Hibix 45% extract at 254 nm

Figure 3: Comparative overlay HPLC chromatogram of three replicate runs of Hibix 45% extract at 254 nm

Figure 4: Metagrid analysis of Hibix 45% extract

Figure 5: DPPH activity of Hibix extract of Hibix 45% extract

Figure 6: Effect of Hibix extract on cell viability of Hibix 45% extract

Figure 7: Effect of Hibix extract on cell viability in WS-1 cells at 24 hours of Hibix 45% extract

Figure 8: Effect of Hibix extract on adipolysis of Hibix 45% extract

Figure 9: Effect of Hibix extract on adipogenesis of Hibix 45% extract

Figure 10: (a) Effect of Hibix extract on Collagen release of Hibix 45% extract; (b)

Effect of Hibix extract on Elastin release of Hibix 45% extract

DETAILED DESCRIPTION OF THE INVENTION

The present invention is in relation to an extract of Hibiscus species optionally along with pharmaceutically acceptable additives.

In another embodiment of the present invention said Hibiscus species is Hibiscus sabdariffa.

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 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 is in relation to a process for obtaining extract from Hibiscus species, said process comprising steps of: powdering plant material of Hibiscus species; extracting and refluxing the powder with solvents to obtain extract; and concentrating the extract followed by drying to obtain the extract of Hibiscus species.

In another embodiment of the present invention said Hibiscus species is Hibiscus sabdariffa.

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

In still another embodiment of the present invention said extract is optionally centrifuged followed by lyophilization to obtain the extract of Hibiscus 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 is in relation to a method of preventing and/ or managing oxidative stress and obesity in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of extract of Hibiscus species optionally along with pharmaceutically acceptable additives to the subject.

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

The present invention provides Hibiscus sabdariffa plant extract for its potent antioxidant activity and/ or its support for a healthy skin and weight management in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of standardized Hibiscus plant composite extract, optionally along with pharmaceutically acceptable additives, to the subject; and a process for enhancing external and internal skin properties of a Hibiscus plant extract, said process comprising of plant material after pharmacognostic evaluation, followed by the extractions from batch to batch tested using in-house metabolite fingerprinting QA/QC method based on HPLC equipped with PDA ( Figure 1). The extract was subjected to enzymatic assays such as ORAC Assay, DPPH Assay, and Cell Based Assays such as Viability assay, Efficacy studies, Collagen release and Elastin release to obtain the plant bioactives, which have significant role in improving skin appearance and weight management. 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 Hibiscus sabdariffa 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 Hibiscus sabdariffa as defined herein in the preparation of a comestible (foodstuff) for prophylaxis against the occurrence of skin and obesity related diseases. Preferably the mammal is human and the said extract comprises a single extract from a plant part of Hibiscus sabdariffa or a combination of extracts there from as detailed herein. Thus the present invention further relates to extracts, which may be isolated from corolla of the Hibiscus sabdariffa 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 corolla of the Hibiscus sabdariffa, 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 Hibiscus sabdariffa 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 Hibiscus sabdariffa 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 skin and obesity 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 corolla of the Hibiscus sabdariffa plant.

The solvent extraction process is 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 weight management, 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 describes the HPLC profiles of direct and successive solvent extracts of Hibiscus sabdariffa 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 obesity 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 invention further describes the biotherapeutic potential of various extracts of Hibiscus sabdariffa 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 Hibiscus sabdariffa 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 technology of the instant Application is further elaborated with the help of following examples. However, the examples should not be construed to limit the scope of the invention.

Example 1:

Extraction of Hibiscus sabdariffa:

Extraction of Hibiscus sabdariffa 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 Hibiscus sabdariffa 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

Example: 2

Room Temperature Extraction

Powdered Hibiscus sabdariffa 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. The process of instant invention is provided in flow chart as Figure 1.

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 as Hibix 45% Example: 3

Technical Specifications Description

Physical Properties

Chemical Analysis

Microbial Analysis

Example: 4 HPLC analysis
Metabolic fingerprinting analysis of Hibix45% by using Shimdzu HPLC was done to o to establish its authenticity by providing reproducibility

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: 75 mins Acquisition time (PDA) Sampling: 3.125 Hz Start time: Omins End time: 65mins Time constant: 0.64sec
Column used: Xterra® RP 18.3 5um 4.6x150mm.

Mobile phase
Mobile phase A: 0.1% TFA acidified water Mobile phase B: 0.1% TFA acidified acetonitrile

LC time program ACN Gradient 14_d)
Pump

Mode: Low pressure gradient
Total pump A flow: lml/min
Solvent B Cone: 0
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: 200ul
Needle stroke: 52mm
Control vial needle stroke: 52mm

Rinsing speed: 35pJ/sec Sampling speed: 15 pi/sec Purge time: 25 min

Rinse mode: Before and after aspiration Rinse dip time: l0Sec 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 :HIBIX 45%
Run conditions
No of runs performed for each sample: 4
Injection volume: 20ul
LC time program used: ACN Gradient 14_d
Sample preparation

20mg of the lyophilized extract was weighed out into sterile eppendorf and 2ml of 50% methanol is added to it and sonicated for 15 minutes to ensure that the extract dissolves completely. The extract was centrifuged at 12,000rpm for 20 minutes at room temperature and the supernatant was filtered through 0.2(x filters and collected into sterile recovery vials before analysis was performed.

Table 1 : HPLC anlaysis and thepeak information of Hibix 45 % at 254nm.




The extract was injected at a constant volume of 20^1 and its metabolite profiling monitored at 254nm (Figure 2). A comparative profiling was performed between the runs/ extract to check the reproducibility of the extracts at 254nm (Figure 3).

Example: 5

1. Metagrid Profile of Hibix 45% 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. Figure 4.

2. Enzymatic Assays ORAC Assay

The antioxidant capabilities of Hibix 45% 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 and indicates that Hibix 45% 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. (Figure 5)

Hibix extract exhibited an IC50 value of 10 ug/ml on DPPH activity indicating high antioxidant activity. (Figure 1)

3. Cell Based Assays
Adipolysis and Adipogenesis Activities:
Viability assay: Various concentrations viz., 0.1 ug/ml, 1 ug/ml, 10 ug/ml and 100 ug/ml of Hibix 45% treatment on adipocytes retained its viability. (Figure 6 and Figure 7)

Efficacy studies

Lipolysis is an important process determining fuel metabolism, and insulin regulates this process in adipose tissue. Our lipolysis assay measures the ability of a compound to stimulate lipolysis in human adipocytes. Compounds that can stimulate lipolysis have potential use as an anti obesity drug. Assay measures glycerol release. Triglyceride measurements are used in the diagnosis and treatment of various diseases involving lipid metabolism.

Hibix 45% treatment on the adipocytes for 24 hours, showed significant lipolysis at concentrations of l0ug/ml, lug/ml and 0.1 ug/ml as determined by estimation of glycerol concentrations. At the concentration of l00ug/ml Hibix 45% was found to be two fold more efficacious compared to the positive control arachidonic acid. (Figure 8)

Hibix 45% treatment on the preadipocytes along with the differentiation mix, dexamethasone, IBMX, insulin, showed significant reduction in the extent of adipogenesis, compared to control. L0ug/ml, lug/ml, 0.1 ug/ml showed an inhibition of 53% 48%, 45% respectively. The extract did not have any effect at a higher concentration of 100 ug/ml. This analysis showed the potential of the bioactive as an anti obesity drug by prevention of adipogenesis. (Figure 9)

Collagen and Elastin release activity studies:

Hibix 45% extract did not show any cytotoxicity at all the studied doses of 0.1-500 ug/ml on 24 hrs of treatment.

Hibix 45% extract demonstrated a statistically significant increase in the collagen release level at 24 hrs of treatment at doses of 100 ug/ml with 1.5 fold increase when compared to sodium ascorbate showing 1.5 fold increase. (Figured and Table 2)

Table 2 Effect of Hibix extract on Collagen release

Hibix 45% extract demonstrated a statistically significant increase in the elastin release level at 24 hrs of treatment at doses of 100-500 ug/ml with 1.2-1.4 fold increase when compared to sodium ascorbate showing 1.4 fold increase. (Figure. 9 and Table 3)

Table3 Effect of Hibix extract on Elastin release

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 antioxidant 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.

WE CLAIM:

1. An extract of Hibiscus species optionally along with pharmaceutically acceptable additives.

2. The extract as claimed in claim 1, wherein said Hibiscus species is Hibiscus sabdariffa.

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 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.

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

(a) powdering plant material of Hibiscus 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 Hibiscus species.

6. The process as claimed in claim 5, wherein said Hibiscus species is Hibiscus sabdariffa.

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 corolla.

8. The process as claimed in claim 5, wherein said extract is optionally centrifuged followed by lyophilization to obtain the extract of Hibiscus 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 preventing and/ or managing oxidative stress and obesity in a subject in need thereof, said method comprising step of administering pharmaceutically acceptable amount of extract of Hibiscus species optionally along with pharmaceutically acceptable additives to the subject.

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

12. An extract of Hibiscus species, the process to prepare and the method of preventing and/ or managing are substantially as herein described with reference to accompanying examples and drawings.