Description:
TECHNICAL FIELD
The present invention relates to antiobestic clothing coated with cyanidin 3-glucoside, polyphenols as a bioactive ingredient. This medicinal extract is utilized for its anti-antiobestic properties. The pharmacological action of antiobestic clothing is based on the kinetics and absorption of bioactive material Cyanidin 3-glucoside from plant Ricinus communis L. In this present invention the dermal bioavailability of cyanidin 3-glucoside. poKphenols was investigated. Viable human skin, adapted on continuously perfused Franz cells, was applied with 3 % (w/v) cyanidin 3-glucoside and polyphenols solution. Samples were taken at 0.5, 1, 2, 4, 6, 8, 10, and 12-hour intervals and analyzed for detection of cyanidin 3-glucoside by high performance liquid chromatography (HPLC) (reversed phase column, isocratic conditions) coupled with an electrochemical detector (EC). These findings indicate that cyanidin 3-glucoside from the bark extract of plant Ricinus communis L. is readily absorbed by human skin and can be used for other topical application.
BACKGROUND ART
Recently, as western diet becomes more prevailing, obesity keeps on increasing. The obesity is condition of adipose tissue increasing beyond its normal proportion in a body because of excessive intake of sugars (carbohydrates) and fats. Obesity is a medical condition in which excess body fat has accumulated to the extent that it may have an adverse effect on health, leading to reduced life expectancy.
The primary treatment for obesity is dieting and physical exercise. To supplement this, or in case of failure, many anti-obesity drugs may be taken to reduce appetite or inhibit fat absorption. In severe cases, surgery is performed or an intragastric balloon is placed to reduce stomach volume and/or bowel length, leading to earlier satiation and reduced ability to absorb nutrients from food.
Normally, the sugars and fats, after being decomposed by digestive enzymes, are used as an energy source or the like. However, if taken excessively, an excess of sugars is changed into fatty acids via acetyl CoA and stored in tissue together with fats, resulting in obesity. Adipose cells storing fats secret free fatty acids and a tumor necrosis factor (TNF) and inhibit the action of insulin which regulates metabolism of sugar including blood glucose, fats and amino acids. Therefore, if adipose cells increase owing to obesity, insulin does not act well and blood glucose increases to cause diabetes. It has been pointed out that the illness, if it hangs on for a long time, leads to peripheral vessel disorder and arterial sclerosis. Anthocyanin pigments having a wide distribution in the plant kingdom are used as colorants, but are also known to have an decreasing effect on cholesterol in blood, for example, as bioacitivity ( Agric. Biol. Chem., 54(1), 171-175. 1990).
No prior art discloses the treatment and prevention of obesity by regular wearing of garment like shirt pant underwear and west etc.
The invention is aimed to develop the natural fiber colored and coated with the bioactive plant material designed for enhancing a wearer's as colored cloth, while giving the wearer's a very natural appearance with therapeutic property with many other benefits used in colour therapy .
DISCLOSURE OF INVENTION
The present invention has objects to provide a highly bioactive material from plant Ricimis communis L.. which is rich in Cyanidin 3-glucosides , readily handleable. scarcely hygroscopic, and relatively high in storage stability; and its preparation
and uses.
To solve the above objects, the present inventors energetically studied the preparation method of such an extract powder of Ricinus communis L plant. As a result, they found that the desired powder, which is rich in Ricinus communis L extract and high in storage stability, can be easily produced by incorporating a partial starch hydrolyzate with a dextrose equivalent (abbreviated as "DE", hereinafter) of 10.2 or lower in an amount of 0.25 part by weight or higher but not higher than 5 parts by weight, d.s.b., to one part by weight, d.s.b., of a solid of liquid extract of Ricinus communis L dissolved and/or suspended in any of the later described aqueous solvents, drying the resulting mixture, and optionally pulverizing the dried product. Further, the present inventors found that external dermal agents such as cosmetics, quasi-drugs, and pharmaceuticals; orally ingestible compositions such as pharmaceuticals, and quasi-drugs and miscellaneous goods can be easily prepared by using the extract powder of Ricinus communis L of the present invention. Thus, they accomplished this invention. The present invention solves the above objects by providing a preparation method comprising the steps of incorporating a partial starch hydrolysate with a DE of 10.2 or lower in an amount of 0.25 to 5 parts by weight, d.s.b., to one part by weight, d.s.b.. of the solid of a liquid extract of Ricinus communis L, and drying the resulting mixture; an extract powder of Ricinus communis L produced by the method; and a composition incorporated with the extract powder.
The extract powder of Ricinus communis L of the present invention has a relatively high safeness, insubstantial hygroscopicity and browning, and improved storage stability. Since the extract powder of Ricinus communis L has a melanin-formation inhibitory activity, etc., it exhibits a satisfactory skin-whitening effect of lightening the skin color of post sunburn pigmentation, spots, freckles, and chloasmata, when applied extradermally or ingested orally. Also, the extract powder of Ricinus communis L has an elastase inhibitory activity, turnover-accelerating activity of skin cells, etc., and this inhibits the formation of wrinkles and line wrinkles and recovers/maintains the tension and elasticity of the skin, resulting in exertion of satisfactory effects of the prevention of skin ageing and the maintenance of youthful skin conditions. The extract powder of Ricinus communis L has various physiological actions such as moisture-retaining action to the skin, lipase- activity-inhibitory action, action of inhibiting secretion of sebums from sebaceous glands, action of promoting decomposition of body fats, antibacterial action, antioxidant action, anti-inflammatory action, anti-periodontal action, action of improving lipid metabolism, action of lowering body fats, etc. By preparing external dermal agents and orally ingestible compositions using the extract powder of Ricinus communis L of the present invention, the physiological activities of the indigo plant can be imparted thereto.
The first principle for anti obesity activity of the novel clothing is the decomposition of the sugars and fats by transmitting rage and tarns dermal delivery of bioactive plant material and stimulating effect on leptin. Our in -vivo experimental study confirms that medicinal plant pigment coating on cloths stimulate the signaling factors of leptin and plays an important role in sebaceous gland physiology and helps in treating and preventing the Obesity.
The Second principle for anti obesity activity is that the layers of the highly bioactive coating material onto of the novel clothing selectively transmits of Far Infrared light and allow the Far Infrared light to reach upto the skin and other pilosebaceous unit and refract the of the other harmful rays.
In the present invention textiles was formed by weaving, knitting, crocheting, knotting of Ricinus communis L.plant fibers pressing together.
Before the spinning step, the wood pulp was mixed with cotton fibers. A preferred mixture is one in which the different fibers exist in a proportionate ratio of 20%-25% Ricinus communis Lfibers and 80% cotton fibers.
The fibers produced by the present invention have high strength, especially when wet. and good dimensional stabillity and firmness. In addition, the fibers produced by the process of the invention are highly absorbent. The fibers which result from the process of the present invention will be used in the manufacture of clothing, as medical fabrics.
Currently existing garments products, such as shirt, trousers, skirt, sari etc, are made from non coated fibers and do not include any bioactive coating agent applied topically to the fiber which capture and kill pathogenic microbes and have therapeutic effect by controlled release processes by the cloth media surface

during normal use through the skin .However, till the date none of garments individually demonstrate a therapeutic effect by skin absorption on prevention and treatment of obesity .
(Figure Removed)
The fibers produced by the present invention is composed of the plant material and coated with the natural bioactive component selectively allow the Far Infrared light and inhibits the other harmful rays. The effect of the coating onto the invented fabric will transmit solar radiation of wavelengths between 280 nm and 2500 nm, while absorbing infrared in the 5000 nm to 35000 nm region.
Although the wavelengths of Far Infrared light are too long to perceive by the naked eyes, we can experience its energy as gentle, radiant heat, which can penetrate up to [3.5] inches beneath the skin. Among FIR's healing benefits is its
ability to stimulate inflammation, which is necessary for a period of time to heal injuries (such as a pulled muscle). Far Infrared light is also capable of enhancing white blood cell function, thereby increasing immune response and eliminating foreign pathogens and cellular waste products in the body. Additional benefits of FIR include the ability to stimulate the hypothalamus, which controls the production of neurochemicals, which is involved in such biological processes as sleep, mood, pain sensations, and blood pressure; enhancing the delivery of oxygen and nutrients to the body's soft tissue areas; and the removing accumulated toxins by improving lymph circulation.
Example: - Skin is the integument and composed of numerous layers of cells there are two main layers: Epidermis, or outer layer
The Dermis is the thicker inner portion of the skin. It gives rise to the epidermis. The dermis has connective tissue which has elastic properties satisfying the need for skin to change shape as the body does. Blood vessels, nerve endings, and various glands reticulate throughout the dermis supplying its metabolic needs. The skin contains blood vessels, sweat glands, fat deposits, and hair follicles which can be controlled and variously adjusted to regulate core body temperature.
Blood vessels open or constrict to regulate the passage of blood warmed by metabolic processes to be cooled under the skin by the evaporation of sweat. Sweat can also function to excrete water, salts, and other organic wastes as needed and this is a primary route for the lymphatic system to remove toxins from the body
We have undertaken this study to demonstrate that cross exchange between medicinal ingredient and natural fiber of cloth with skin receptors and sweat glands is possible to the humans. Edmond Locard gave a law which is related with crime research & famous as a Principle of Exchange. This law is playing a very important role in Forensic researches. According to Edmond Locard when two things come into contact they mutually transfer the materials. Our study is based on the scientific principle.
Thus there is a need for novel textiles which is organic, colored and coated by accurate and specified medicinal plants describes in Ayurvedic text for the prevention and treatment of obesity .
SUMMARY OF THE INVENTION
In our study. Natural dyes from various medicinal plants has applied to 100% organic cotton fabric . 100% silk and wool to develop a fabric, which is specific for the prevention and treatment of obesity . Specific solvents have been used for extraction of natural dyes from various plants according to the chemical property of the natural dye. The finished fabric samples have been tested for the specific activity related with the disease by in-vitro trials. The natural dye treated fabrics was exhibited significant prevention of diabetes. The wash durability of the treated sample was found good even after 50 wash. The beneficial aspects of the medicinal plant dyes in comparison to any of the synthetic dyes are high. Chemically created colors are terrible poisons.
The present invention aims at developing an eco friendly textiles which is organic and 100 % natural, which colored and coated by accurate and specified medicinal plants describes in Ayurvedic text for the prevention of Diabetes. Some selective species of medicinal plants were identified and screened for their activity and the extracts were applied to cotton fabrics.
The principle for therapeutic value of textile is the rubbing fastness of medicinal plant active ingredients and trans dermal application it absorbed into the tissue and then move through the capillaries into the blood stream.
Detail Description of Invention for natural colors
The most characteristic compounds present in fruits are colorful polyphenols belonging to the classes of anthocyanins and of proantho cyanidins. The pharmacological properties of these substances seem nowadays well ascertained; they are mainly linked to a strong antioxidant capacity, although associated with many other biological activities, such as anti-inflammatory, vasoactive, hypolipemic. hypoglycemic, cell-regenerating, antimicrobial, chemopreventive, etc.
All of these properties have been intensively studied, both in vitro and in vivo. In humans, the majority of the studies carried out up to now have been limited to few groups of pathologies, namely to vasculopathies of different nature, retinal diseases and ulcerations of various ethiology in the digestive tract. The results have confirmed the efficacy of both whole extracts and purified anthocyanosides.
Antioxidant activity:
Bilberry anthocyanins and some related aglycones are reported to be potent scavengers of free radicals, as when tested in vitro in the superoxide anion generating system hypoxanthine/xanthine oxidase (Salvayre et al. 1981 and Acquaviva et al. 2003). Anthocyanin extracts of Vaccinium myrtillus fruits have been shown to act both as scavengers against superoxide anion and as inhibitors of lipid peroxidation in rat liver microsomes (Meunier et al. (1989). Martin et al. (1997. 1998) and Martin - Aragon et al. (1999).
Cyanidin and delphinidin chlorides proved to be potent scavengers, interacting with 1, 1-diphenyl-l-picrylhydrazyl (DPPH) free radical: the IC50 values were 2.5 and 4.0 µM, respectively, comparable to that of quercetin.
Cyanidin chloride is the most active compound on CC14 induced lipoperoxidation
(Morazzoni et al. 1988 and Laplaud et al. (1997) reported that an aqueous extract
of V. myrtillus berries protected low-density lipoproteins (LDL) from copper-
mediated oxidation.
As reported by Rasetti et al. (1996/97) a proprietary Bilberry extract was capable
of protecting apolipoprotein B from UV-induced oxidative fragmentation.
Ichiyanagi et al. (2003) studied the activity of 11 major bilberry anthocyanins
against hydroxyl radicals (OH°), superoxide anion, and singlet oxygen by using
capillary zone electrophoresis. The reactivity of anthocyanins towards OHo was
comparable to that of (+)-catechin used as reference substance, and was neither
significantly affected by the aglycon structure nor by the conjugated sugar type.
On the contrary, the reactivity towards superoxide anion and singlet oxygen was determined by the aglycon structure.
Prior et al. (1998) comparing the antioxidant capacity (oxygen radical absorbance
capacity. ORAC) of different variety of four Vaccinium species found that V.
myrtillus and V. angustifoliam (low bush) exhibited potent ORAC activity
(44.6±2.3 and 45.9±2.2, respectively).
A linear relationship existed between ORAC and anthocyanin (rxy = 0.77) or total phenolic content (rxy = 0.92).
Anthocyanins can also prevent the oxidation of ascorbic acid caused by metal ions by chelating the metals ions and forming an ascorbic acid (copigment)-metal-anthocyanin complex. Sarma et at. (1997). In addition the anthocyanin extract is
reported to inhibit the K+ loss induced by free radicals in human erythrocytes as well as the cellular reactions induced by the oxidative compounds daunomycin and paraquat. (Maridonneau et al 1982 and Mavelli et al. (1983).
Most recent studies by Bao et al. (2008) indicated that Mirtoselect ® is active in protecting the kidneys form damage induced by potassium bromate in mice. Potassium bromate is an environmental pollutant, which can be formed as a by-product in the process of ozone purification of drinking water. It may form free radicals triggering harmful modifications in the kidney tissue.
The protective properties of bilberry extract are due to the improved antioxidant capacity of the kidney tissue promoted by bilberry anthicyanins: the reduction of NO production and the improved ability to absorb oxygen radical (ORAC) Li Bao et al. (2008) showed that Mirtoselect® is able to alleviate stress-induced liver demage in mice by both scavenging free radicals activity and lipid peroxidation inhibitor effect.
Inhibition of cyclic nucleotide phosphodiesterases:
The anthocyanins cyanidin, delphinidin and malvidin 3-O-glucosides and their
aglyeones are reported to inhibit phosphodiesterase (PDE) isoforms from different
sources as retina, choroid, large vessels and platelets. The compounds were more
active on retinal than on platelet PDEs and in particular on the retinal calmodulin
stimulated enzymes. IC50 of malvidin and delphinidin 3-O-glucosides on
calmodulin stimulated enzymes ranged from 5.4 to 35.6 µM. Anthocyanins
appeared more active than isobutylmethylxanthine used as reference( Ferretti et al.
1988. Ferretti et al. 1990 and Pifferi et al. 1992)
Antiplatelet activity:

Morazzoni and Magistretti et al 1990 studied the antiplatelet activity of bilberry
36% extract against aggregation induced by ADP, collagen and sodium
arachidonate on rabbit platelet-rich plasma (PRP). Bilberry extract was a strong
inhibitor of platelet aggregation with IC50 values ranging from 0.36 to 0.81
mg/mL PRP, comparable to those obtained with dipyridamole. Moreover, the
Bilberry extract exerted an inhibitory effect on ADP-induced platelet aggregation
in rats maintained on extracorporeal circulation.
The Bilberry extract when orally administered to rats at doses up to 400 mg/kg, prolonged bleeding time for 24 h, without affecting blood coagulation pathways; administration of 400 mg/kg by oral route to mice, reduced the adhesiveness of platelets to glass micropellets. An anthocyanin extract of V. myrtillus fruits was reported to inhibit platelet aggregation in vitro when induced by ADP or adrenalin on human plasma. Serranillos et al 1983 The inhibitory effect on platelet aggregation, demonstrated in vitro, was confirmed ex vivo on ADP- and collagen-induced aggregation of platelets obtained from the blood of 30 healthy subjects treated by oral route (480 mg/day for 30-60 days). Pulliero et al 1989.
Interaction with collagen, phospholipids and proteoglycans:
In vitro anthocyanin extracts of V. myrtillus fruits are able to inhibit proteolytic enzymes like elastase, which are involved in the degradation of collagen and other components of the extravascular matrix in certain pathological conditions such as atherosclerosis, pulmonary emphysema, rheumatoid arthritis (Jonadet et al. 1983). Anthocyanin extracts may interact with collagen metabolism, by cross-linking collagen fibres and making them more resistant to collagenase action (Robert et al. 1979). A reduction in biosynthesis of polymeric collagen and structural glycoproteins, responsible for thickening of capillary in diabetics, has also been described (Boniface et al. 1986). Hystochemical and biochemical studies showed that anthocyanins from V. myrtillus interact with phospholipidic constituents of

plasma membranes from the rat brain, potentially modifying their physical chemical properties and enhancing their resistance to lesive stimuli (Curri et al. 1976). Salmona et al. (1990) studied the influence of bilberry 36% extract on membrane viscosity of platelets and confirmed that anthocyanins were able to modify the membrane fluidity due to their high affinity for membrane phospholipids.
A local stimulating effect of the anthocyanins from V. myrtillus on the biosynthesis of mucopolysaccharides in granuloma induced by foreign bodies was reported b} Mian et al. (1977) Mucopolysaccharides are recognised to play an important role in maintaining the integrity of both perivascular tissue and the basal membrane. In an in vitro study, using endothelial cells from human umbilical cord. Piovella et al. (1979)and Piovella et al. (1981) reported that anthocyanins induced active phagocytosis of pigment material and intense cell regeneration. A growth promoting activity on fibroblasts and on smooth muscle cells was also reported in the same study.
Anthocyanins may facilitate the regeneration both of the cellular component of the vessel wall and of the perivascular tissues, due to their stimulating effect on mucopolysaccharides.
Effect on arteriolar vasomotion:
The arteriolar vasomotion, a rhythmic variation of diameter of arterioles in microvascular network, influences the microvascular mechanism which regulates the formation of interstitial fluid. Colantuoni et al. (1991) studied the effects of bilberry 36% extract on arteriolar vasomotion in two experimental models: the cheek pouch of anaesthetised hamster and the skin fold window preparation (muscular type) of unanaesthetised hamster. Bilberry 36% extract (5-10 mg/kg i.v.) induced vasomotion suppressed by anaesthetic in cheek pouch arterioles and

terminal arterioles, and increased vasomotion frequency in the skeletal muscle arteriolar network. These findings indicate that the Bilberry extract may prevent or control interstitial fluid formation and contribute to control the blood flow redistribution in the microvascular network.
Robert Byamukama et al. (2007) reported the two new anthocyanins which were isolated from the stem bark of the castor plant, Ricinns communis L. by chromatographic techniques. Thy elucidated the structure by nuclear magnetic resonance spectroscopy and high-resolution electrospray mass spectrometry to be cyanidin 3-O-ß-xylopyranoside-5-O-ß-glucopyranoside, 1 (21 %) and cyanidin 3-O-ß-xylopyranoside-5-O-(6"'-O-malonyl-ß- glucopyranoside), 2 (79 %). In addition, they identified cyanidin 3-O-ß-xylopyranoside-5-O-(6'"-O methylmalonate- ß glucopyranoside) (3) which were formed by methyl esterification of the malonyl unit of 2 during isolation and storage.They concluded that three anthocyanins are among the few anthocyanins having a xylosyl moiety linked directly to the anthocyanidin.
Linda et al. (2007) studied on polar anthocyanin pigments that are present in water fractions after partitioning methanolic extracts with hexane and ethyl acetate (EtOAc). Anthocyanins are almost exclusively responsible for the red, blue and purple colours in fruits. Cyanidin is the most common anthocyanidin. and the 3-glucoside is the most active antioxidant anthocyanin (Wanget et al. 1997). The polarity and complexity of water extracts can make it difficult to isolate pure components (Degenhardt, Knapp, & Winterhalter, 2000). Yet the aqueous extracts often contain potent polyphenolic antioxidants, such as anthocyanins and tannins ( Wanget et al. 1997).
Material and Methods extraction of pigment from medicinal plants for the prevention and treatment of obesity.
Bark and leafs of plant Ricinus communis (Linn.) were obtained from Divya Nursury. Haridwar and about 200 g of each were used from same plant for present study.
Anthocyanins extraction
Anthocyanins were normally extracted with methanol containing trifluoroacetic acid but the direct extraction of anthocyanins in methanol produces very poor yield. In the present investigation 200g bark of plant Ricinus communis (Linn.) was pre-soaked in water containing 0.6% TFA. Extraction of homogenized material was performed with 850 ml of methanol containing 0.5% trifluoroacetic acid upto 5 hours. After extraction the extract was filtered, and the methanol was removed by evaporation under reduced pressure at relatively low temperatures (20°C). to avoid hydrolysis of potential acyl groups in the anthocyanin structure, and degradation. The filtered extract was concentrated under reduced pressure at 30°C. purified by partition (several times) against ethyl acetate and applied to a column packed with styrene ion exchange resin. The anthocyanins were adsorbed to the column, and then the column were washed with water, and eluted with methanol containing 1.5 % TFA. Again the concentrated elute of anthocyanin was purified by passing it through the bead-formed by dextran medium by using 50% aqueous methanol containingl.5% TFA as eluting agent.
HPTLC Analysis
Sample solution: 1 g of concentrated elute of anthocyanin was dissolved in 3 ml methanol and 2 µl was spotted on coated silica gel 60 F254 TLC plate by CAM AG LINOMATE-5.
Development system: For the separation of anthocyanins the solvent mixtures employed were: 1-butanol: acetic acid: water (4:1:5, v/v/v). Stationery Phase: Pre coated silica gel 60 F254 TLC plate of 0.2mm thickness Silica (20 x 20 cm, Merck).
Liquid-liquid partition: - The combined aqueous concentrates after evaporation were purified by partition against ethyl acetate to remove chlorophylls and. less polar flavonoids and other non polar compounds from the mixture. The aqueous extracts obtained after the liquid-liquid partition step will also contain other water soluble compounds than anthocyanins, like free sugars and aliphatic acids. These non-aromatic compounds were removed with the use of styrene ion exchange resin for column chromatography.Ion exchange resin adsorbs the aromatic compounds including anthocyanins and other flavonoids in aqueous solutions, whereas free sugars and other polar non-aromatic compounds were removed by washing with distilled water until the eluted water has a neutral pH. Then the adsorbed anthocyanins and other flavonoids were eluted using methanol containing 0.5% TFA (v/v) as mobile phase (Andersen, 1988).
HPLC Analysis: - The individual anthocyanins were further separated by using preparative HPLC (Konic 550 A Pump equipped with a UV detector) equipped with an ODS Hypersilcolumn (25 x 2.2 cm; i.d.; 5 urn) for the confirmation of sample fractionation and isolation of pure pigments First solvent used for elution was: HC02H-H20(1:9; v/v) and second was HC02H-H20-MeOH; (1:3.9:5;
v/v).The elution profile consisted of a linear gradient from 10 % to 100 % B for 30 min. isocratic elution (100 % B) for the next 10 min, followed by a linear gradient from 100 % to 10 % B for 1 min. The flow rate was 14 ml/min for 41 min, and aliquots of 250 µl were injected.
Preparative HPLC: - Preparative HPLC have been used in fractionation and isolation of pure pigments. The instruments used were a Konic 550 A,( Different flow cell) equipped with C18 reversed-phase column (ODS-Hypersil column (25 x 2.2 cm. 5 urn)) coupled with UV/Vis spectra and peak purities was obtained. The polar mobile phase used was a gradient consisting of variable proportions of H2O-HCOOH (9:1, v/v) and H2O HCOOH- CH3OH (4:1:5, v/v).Prior to injection the solutions were filtered through a 0.45 urn Millipore membrane filter and 15 µl of the extracts was injected on the HPLC. The quantitative amounts were determined from a HPLC calibration curve of pure cyanidin 3-O-ß-galactoside.
NMR Analysis:-Two dimensional nuclear magnetic resonance (NMR) spectroscopy, in which NMR spectrum is obtained as a function of two frequency variables, had a profound effective on the study of bio-molecules and other systems which yield complex NMR spectra. 1D 1HNMR:-
The 1D proton spectra of anthocyanins provide quantitative information about proton chemical shifts and their coupling constants (JHH) and give quantitative information by integrating baseline-separated signals or selected spectral regions. Information about the nature of the aglycone. type and number of sugar and acyl substiluents can also be provided.The chemical shift values also indicate linkage positions between different sub-units of the anthocyanin.
Results: -HPTLC Analysis
The anthocyanins extracted from the bark and leaf were separated by(Fig-24) HPTLC (Camag).Leaf track -1 represent the one major band(Deep Pink) where as track -2 exhibited three bands of anthocyanins (Deep Pink to light Pink). Track -3 also exhibits the three major bands(Deep Pink to Hot Pink) .Tack -4 exhibit only one band, which was isolated by preparative HPTLC.The major three bands was exhibited by using the 1-butanol: acetic acid: water (4:1:5, v/v/v) as mobile phase. Whereas H2O-MeOH-TFA (89.5:10:0.5) as mobile phase, reported earlier was not exhibited the clear bands of anthocyanins. First band contained pigment 1. second band contained pigment 3 and third band pigments 3 and 2. The HPLC chromatogram of the fresh acidified methanolic extract of the stem bark of Ricinus communis L. detected in the visible spectral region revealed two anthocyanins (1 and 2).After storage in the extraction solvent, the HPLC chromatogram showed three anthocyanins (1, 2, 3).The relatively amounts of 1, 2 and 3 in the fresh extract were 19, 79 and 2 %, respectively.
(Figure Removed)
Fig.-24 TLC chromatogram of anthocyanins extracted from the bark and leaf
Trak-1 Leaf treated passed cation resin
Trak-2 Leaf extract
Trak-3 Bark
Trak-4 Isolated Spot of pigment -1
Interpretation:- Leaf treated passed cation resin in track-1 exhibits only two bands anthocyanins, non treated leaf extract in Track-2 exhibited three clear bands of anthocyanins , Bark in Track-3 also exhibited the three clear bands (1, 2 and 3)of anthocyanins. Track -4, isolated spot was confirm the presence of single anthocyanins from plant Ricinus communis Linn.
(Figure Removed)
Rt [min] Area [mV.s] Height [mV] Area [%] Height
[%] W05 [min]
2.460 347.353 15.479 100.0 100.0
0.32
Fig-25 HPLC chromatogram of isolated pigment -1.
Interpretation:-Isolated spot from the bark extract was exhibits the single peak at Rt 2.460 with 100.0% area in HPLC, which showed that the isolation and purification of each anthocyanins from bark extract was clean for NMR study.
HPLC Analysis: - The HPLC equipped with a UV detector, analyses was performed using a C18 reverse phase column (250 x 4.6 mm, 5 µm particles). The elution system was binary, with an aqueous acidified solvent (A) and a less polar acidified acetonitrile (B) solvent. Elution retention was in the order; cyanidin < delphinidin < petunidin < peonidin < malvidin. Anthocyanin glycosides elute in the following order: 3, 7-diglucosides < 3, 5-diglucosides < 3-sophorosides < 3 galactosides< 3-lathyrosides < 3-sambubioscides < 3-glucosides < 3-arabinosides < 3-rutinosides < 3-rahmnosides.
NMR Analysis:
NMR instrument was used for the characterization and structure determination of individual pigments conclusive structure identification of anthocyanins 1, 2 and 3 from bark of Ricinus communis linn. Structure elucidation of anthocyanins comprises 1) aglycone (non-sugar unit), 2) sugar units, as well as 3) determination of linkage positions between the different sub-groups.
Examples:-
For characterization and structure determination of individual pigments different chromatographic and spectroscopic techniques have been used in present investigation such as thin layer chromatography (TLC), analytical HPLC, UV-Vis spectroscopy and Nuclear Magnetic Resonance spectroscopy (NMR) .TLC, HPLC and UV-Vis spectroscopy would give the lot of characteristic information about the type of anthocyanins.The presence of the anthocyanins in the leaf of the castor plant has previously not been reported.
The anthocyanins extracted from the bark were separated by HPTLC (Camag).They exhibited the three bands by using the 1-butanol: acetic acid: water (4:1:5, v/v/v) as mobile phase. First band contained pigment 1, second band contained pigment 3 and third band pigments 3 and 2. The HPLC chromatogram of the fresh acidified methanolic extract of the stem bark of Ricinus communis L. detected in the visible spectral region revealed two anthocyanins (1 and 2).After storage in the extraction solvent, the HPLC chromatogram showed three anthocyanins (1,2, 3).The relatively amounts of 1 and 2 in the initial extract were 19 . 79 and 2 %, respectively. TLC is considered to be one of the simplest of the chromatographic techniques. TLC facilitates short acquired time, and is a relatively inexpensive procedure. In this solvent system anthocyanins with similar structure regarding sugars and acyl units will be separated with respect to the number of hydroxyl and methoxyl groups on the bring of the aglycone.
Increasing hydroxylation and methoxylation will result in a decreasing retention factor (Rf). The hydroxyl groups have a greater impact than the methoxyl groups.The retention time was increase with increasing number of sugars and acyl groups in the anthocyanin structure (Andersen. 1988b.). Stintzing Florian C et al 2002 discussed about the antioxidant efficacy with the colours of anthocyanins.
They examined individual isolated compounds purified total pigment isolates from blackberry, elderberry, black carrot, red cabbage, and sweet potato. Acylation with cinnamic acids shifted color tonality (hue angle) to purple and markedly increased pKH and antioxidant activity, but lowered the visual detection threshold. Glycosidic substitution at the 5 position moved tonalities toward purple and decreased pKH. and tended to lower the ORAC value, but raised the visual detection threshold. Increasing the number of sugar substituents at the 3 position also affected all of these parameters; however, the extent was not predictable.
HPLC was the method of choice for the accurate determination of both the composition and the concentration of anthocyanins in leafs and bark of the Ricimis communis linn. The main chromatographical separation principle involved in reversed-phase HPLC is the partition of solutes between the polar mobile phase and the non-polar stationary phase. The overall polarity and the stereochemistry of the anthocyanins are the key factors for separation.
The elution of anthocyanins in reversed-phase HPLC columns depends on the pattern of hydroxylation/methoxylation of the aglycone, the degree of glycoslyation and acyl substitution, as well as on the mobile phase composition and solvent gradient steepness. The presence of aromatic or aliphatic acylation increases retention times compared to the corresponding non-acylated derivatives. Structural elucidation of individual anthocyanins by NMR
Structural elucidation of pigments (J, 2 and 3)
The downfield part of the 1D 1H NMR spectrum of 1 showed a singlet at 9.04 ppm (H-4). a3H AMX system at 8.42 ppm (dd, 8.8 Hz, 2.3 Hz; H-6')- 8.15 ppm (d. 2.3 Hz; H-2') and 7.12 ppm (d, 8.8 Hz; H-5') and an unresolved 2H AB system at
7.17 ppm (H-8) and 7.13 ppm (H-6), respectively, in accordance with the anthocyanin, cyanidin. The sugar region of the 1D 1HNMR of 1 showed the presence of two sugar units revealed by two anomeric protons with a ß-configuration (H-1": 3JHH = 7.0 Hz, H-1"': 3JHH = 7.9 Hz). The COSY and TOCSY spectrawere in accordance with 13 sugar protons, which indicated that one of the sugar units, was apentose, and the other a hexose. Starting from H-1" at § 5.49 {J =7.0 Hz), the observed cross peak at 5.49/3.81 ppm in the COSY spectrum supported by corresponding cross peak in the HSQC spectrum, permitted the assignments of H-2", H-3", H-4", H-5A" and H-5B".
The chemical shifts and the coupling constants of this glycosyl unit were in accordance with a ß-xylopyranosyl. A crosspeak at 8 5.49/145.26 in the HMBC spectrum between H-1" and C-3 of the aglycone confirmed the connection point of the xylosyl unit to the 3-position of the aglycone. By using the doublet at 5 5.28 (J = 7.9 Hz) as the starting point in the COSY and TOCSY spectra, it was likewise possible to assign all the chemical shifts for the second monosaccharide moiety, ß-glucopyranosyl. A cross peak at 8 5.28/156.95 in the HMBC spectrum confirmed the connection point of this unit to be in the 5-position of the aglycone. The molecular mass (m/z 581.1495) in the ES1+ high resolution mass spectrum of 1 corresponding to C26H29015 +, confirmed the structure to be cyanidin 3-O-ß-xylopyranoside-5-O-ß-glucopyranoside, which is a new anthocyanin in plant.
(Figure Removed)
Fig. 25 Structure of pigments 1 (cy 3-xyl-5glc).
The NMR resonances of pigment 2 shared many similarities with the corresponding resonances of 1, in accordance with a cyanidin 3-O-ß-xylopyranoside-5-O-ß- glucopyranoside derivative. However, the chemical shift values of H-6A'" {3 4.62), H-6B'" (3 4.42), H-5"' (3 3.89) and C-6"' {3 65.3), indicated the presence of acylation at the 6"'-hydroxyl.
The cross peaks at 3 4.62/168.7 (H-6A"7MI) and 4.42/168.7 (H-6B'"/MI) in the HMBC spectrum confirmed that an acyl moiety was linked to this hydroxy 1 group. The molecular mass (m/z 667.1478) in the ES1+ high resolution mass spectrum of 2 corresponding to C29H31018+ , was in accordance with cyanidin 3-xylopyranoside-5-glucopyranoside with an additional malonyl unit. Thus, the identity of 2 was determined to be the new anthocyanincyanidin3-O-ß-xylopyranoside-5-O-ß-(6'" malonyl glucopyranoside).

(Structure Removed)
Fig. 26 Structure of pigments 2(cy 3-xyl-5-[6(mal)glc]) .
Pigment 3 was identified as the esterified form of pigment 2 Methyl esterification of the terminal carboxyl group of malonyl units occurs in the acidified methanolic solvents normally used for extraction and isolation.
The molecular ion at mlz 681.1478 in the positive ion ESI was in accordance with cyanidin 3-O-ß-xylopyranoside-5-O-ß-(6'"-malonylglucopyranoside) with an additional mass of 14 amu. The cross peak at 5 3.76/168.6 (H-MIV/C-MIII), and the cross peaks at S 4.66/167.9(H-6A"7C-MI) and 4.42/167.9 (H-6B"7C-MI) in the HMBC (Figure-25 ) confirmed the identity of pigment 3 to be cyanidin 3-O-ß-xylopyranoside-5-O-ß-(6"'-methylmalonateglucopyranoside).
(Figure Removed)
Fig. 28 Expanded region of the HMBC spectrum of pigment 3 in CF3C02D-CD30D (5:95, v/v) recorded at 25°C.
Table 1.8 C-l Table C-l 1H NMR spectra data for pigment 1, 2 & 3 dissolved in CD30D-CF3COOD (95:5, v/v) recorded at 25 OC
C 1H NMR data reported in the thesis (Chemical shifts are given in ppm and coupling constants are given in Hz

(Table Removed)
*overlap; s. Singlet; d, Doublet, t, triplet; m, multiplets;
#. not detected adequately (For structure see Fig B1 and B-3)
Table 1.9 D-1 13C NMR spectra data for pigment 1, 2 & 3 dissolved in CD30D-CF3COOD (95:5, v/v) recorded at 25 OC

(Table Removed)
13C NMR data reported in the thesis (Chemical shifts are given in ppm )
#. not detected adequately (For structure see Fig B1 and B-3)

What it claimed is:
1. Therapeutic clothing comprising of natural fabric that's coated and dyed with the highly bioactive extract which have a therapeutic property for the prevention and treatment of Obesity.
2. The therapeutic clothing claim 1 where the fabric is selected from the clothing that is made from materials that was grown without the use of synthetic chemicals such as pesticides - herbicides or other chemicals.
3. The therapeutic clothing of claim 2 where the fabric is cotton, silk and wool.
4. The therapeutic clothing claim 1 where the first therapeutic agent for the prevention and treatment of Obesity is the natural bioactive material obtained from the Fruits, Leaves, bark and root of the different species of Euphorbiaceae, Combretaceae Zingiberace and Solanaceae family, which includes Ricinus cumminis (Erand), Jatropha spp. (Jamalgota) Terminalia chebula (Harad), Zingiber officinale (Saunth) and Withania somnifera (Ashwagandha).
5. The product of claim 1 where the entire ingredient claimed as therapeutic agent for the dyeing of organic fiber and cloths was tested for their efficacy by various in-vivo and in-vitro clinical trail the results was very significant on prevention and treatment of Obesity.
6. A method of making an therapeutic product comprising contacting a fabric dyed and coated with an natural colors and other active ingredients from the various parts of the plant Ricinus cumminis (Erand), Jatropha spp. (Jamalgota) Terminalia chebula (Harad), Zingiber officinale (Saunth) and Withania somnifera (Ashwagandha).