DESC:FIELD OF THE INVENTION
The present invention relates to novel complexes of ß-glucan with a -arbutin and its process for preparation.

BACKGROUND OF THE INVENTION
ß-glucan is a polysaccharide or a mixture of oligosaccharides of varying lengths with a basic skeleton of ß-(1?3)-glycosidic bonds which are found in the cell walls of yeast, bacteria, algae, fungi, lichens, oats and barley. They can be classified based on the source, cereal or non-cereal which is largely based on structure. ß-glucans from barely also differ structurally from those of oats origin. A similar diversity exists within ß-glucans from non-cereal sources. In general, all the ß-glucans are homo-polysaccharides and essentially, composed of glucose units linked together with a characteristic 1?3 linked backbones. The structural difference occurs at branching off this backbone, which is dictated by source. ß-glucans can be either branched or unbranched. Branching can usually occur at either the 1?4 or 1?6 position. These molecular and structural characteristics are fundamental to their activity which determine defined structure–activity relationship.

Cereal or grain derived ß-glucans usually have 1?3, 1?4 glycosidic linkages without any 1?6 bonds or branching. Non-cereal sources usually have 1?6 linked branches off the main side chain. Other glucans such as Curdlan, a glucan isolated from Agrobacterium does not contain side branching, but just has a ß-glucan backbone. Some exceptions such as Sorghum arundinaceum, an ancient cereal grain, was found to contain ß-glucans with alpha 1?4 linked D-glucopyranose residues with 1?3, 1?6 branching points. Moreover, different species of Sorghum have different structures; Sorghum bicolor contains 1?3 with 1?4 linkages.

ß-glucans can be recognized as antigens by macrophages via pathogen-associated molecular patterns, and possess the ability to activate peripheral immune cells and hence known to possess immunity-enhancing properties. Several studies, indicate that ß-glucans are effective substances that help the body in the key defense mechanisms of immunomodulation. This effect occurs through numerous mechanisms, including the stimulation of cellular and humoral immunity, control of metabolic diseases, such as diabetes, stimulation of regenerative systems, such as wound healing, attenuation of chronic fatigue and stress, cancer inhibitory stimuli, and lowering cholesterol to name a few. Oral supplementation with ß-glucan is the most used and widely studied and known to be safe in the doses 35 mg to 500 mg a day. ß-Glucan from oats is acknowledged by the U.S. Food and Drug Administration as being safe and is listed as a GRAS (Generally Recognized as Safe) ingredient. It is an irreplaceable supplement for diabetics as it regulates the level of glucose in the blood by slowing down its absorption after eating. The beneficial effects also include reduction of serum cholesterol and glucose immunomodulation, antitumor activity and obesity prevention.

Topical application of ß-glucans in dermatology is another interesting segment where their pluripotent mechanisms of actions such as antioxidant, anti-inflammatory, regeneration effects, immunomodulation, radioprotection, moisturization, rejuvenation could help as complementary therapy in the management of various skin diseases. In clinical medicine, topical application of ß-glucans was successfully studied in the treatment of various skin diseases and conditions such as radiation dermatitis, venous ulcers, wound healing, solar keratosis, HPV-associated vulvar lesions and contact dermatitis.

Arbutin, also known as 4-hydroxyphenyl ß-D-glucopyranoside, is a naturally occurring glucoside of hydroquinone found in various plants such as Asteraceae, Ericaceae, Proteaceae, and Rosaceae families. Arbutin is found in a number of edible berry-producing plants such as blueberry and cranberry, marjoram, and most pear species. Chinchircoma (Muticia acuminatai) that contains arbutin, has been traditionally used by South American populations internally. The fresh juice is used for gastric ulcers and internal tumors; the water of boiled leaves and flowers for illness of the respiratory tract; for hearth disorders or pain. According to pharmacological results in vitro, liver protective effects as well as anti-inflammatory activity were proven. It can also be beneficial for asthma and other anaphylactic reactions. To date, the substance has been found in at least 45 other plant families.

Applied topically, it inhibits tyrosinase and thus prevents the formation of melanin. Arbutin is therefore used as a skin-lightening agent. In vitro studies of human melanocytes exposed to arbutin at concentrations below 300 µg/mL reported decreased tyrosinase activity and melanin content with little evidence of cytotoxicity.

a-Arbutin (4-hydroxyphenyl a-D-glucopyranoside), is an isomer of arbutin with the a-anomeric form of the glycoside bond. a-Arbutin is synthetic compound and can also be produced by microbes or microbial enzymes, which include sucrose phosphorylase, a-glucosidase, cyclodextrin glycosyltransferase, dextransucrase, amylosucrase, and sucrose isomerase.

a-Arbutin has been found wide applications in medical, pharmaceutical and cosmetics industries due to broad spectrum of biological activities like wound healing, anti-inflammatory, anti-cancer, anti-diabetic, anti-osteoporosis, anti-ulcerogenic, anti-aging, anti-pigmentation and antioxidant activity. It is also used to treat acute lung and optic nerve injury, Alzheimer’s disease.

Predominant application of a-arbutin in cosmetic industry for skin pigmentation. a-Arbutin is a safe substitute for a skin lightening agent hydroquinone and other harmful ingredients used for skin lightening application. a-Arbutin can inhibit the activity of tyrosinase enzyme and reduce the formation of melanin while not affecting cell growth or inducing cell toxicity (Sugimoto et al., 2004; Huang et al., 2012). The inhibition of tyrosinase activity was found to be up to 10 times greater for a-arbutin than for ß-arbutin (Seo et al., 2012; Liu et al., 2013). a-Arbutin has been widely approved as an additive in cosmetics applications for enhancing the skin-whitening effect.

Despite promising tyrosinase inhibiting properties of a-arbutin, its high hydrophilicity (Iog P value, - 1.49) and low penetration through the stratum corneum limit its use in topical preparations. Therefore, traditional skin formulations such as lotions and creams are unsuccessful in achieving adequate skin deposition.

To improve the bioavailability of a-arbutin, techniques such as encapsulation, dissolving microneedles using Gantrez™ S-97, hydroxypropyl methylcellulose (HPMC), polyvinylpyrrolidone K-90 (PVP), chitosan, and their combinations were explored.

Skin penetration studies performed on human abdominal skin using beta-glucan solution indicated that beta-glucan, despite its large molecular size, deeply penetrated the skin into the epidermis and dermis. With availability of data on excellent beneficial properties of ß-glucan and a -arbutin, tapping unexplored potential of synergistic properties of the combination of ß-glucan with a -arbutin holds great promise in Cosmeceutical, Pharmaceutical and Nutraceutical industries. It is desirable to develop an efficient and robust process for preparation of stable complexes of ß-glucan and a-arbutin in high purities and yields, such that the bioavailability both the active components is enhanced.

SUMMARY OF THE INVENTION
In one aspect, the present invention provides process for the preparation of novel complexes of ß-glucan with a -arbutin which comprises:
i) dissolving ß-glucan with a -arbutin in distilled water and stirring at room temperature;
ii) the above obtained solution was lyophilized using freeze dry system to obtain the complex.

In another aspect, the present invention provides process for the preparation of novel complexes of ß-glucan with a -arbutinwhich comprises:
i) dissolving ß-glucan and a -arbutin in distilled water and stirring at room temperature;
ii) evaporated the solvent under vacuum to obtain the complex.

In another aspect, the present invention provides process for the preparation of novel complexes of ß-glucan and a -arbutin, which comprises:
i) ß-glucan with a -arbutin were taken in a mortar and add water dropwise;
ii) the mixture obtained in step-a is grounded with pestle to obtain the complex.

FIGURES
Figure 1: IR of ß-glucan
Figure 2: IR of a-arbutin
Figure 3: IR of Lyophilization complex of ß-glucan and a-arbutin

DETAILED DESCRIPTION OF THE INVENTION
ß-glucan obtained from various sources like microbial, fungal, mushroom, yeast and plant sources were used.

Naturally occurring a -arbutin was used in the formation of the complex.

In one embodiment, the present invention provides a process for the preparation of novel complexes of ß-glucan with a -arbutin by the freeze drying method.

In step-a, ß-glucan and the a -arbutin were dissolved in double distilled water and stirred.

The reaction is carried out at a temperature range of 20-50°C for the duration of 2-14 hours. Preferably at a temperature in the range from 25-35oC for the duration of 10-12 hours.

In step-b, the resultant solution was subsequently lyophilized using freeze dry system to obtain the complex.

In another embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan with a -arbutin.

In step-a, ß-glucan with arbutin derivatives were dissolved in distilled water and stirred.

The reaction is carried out at a temperature range of 20-50°C for the duration of 2-14 hours. Preferably at a temperature in the range from 25-35oC for the duration of 10-12 hours.

In step-b, the mixture obtained in step-a is taken and evaporated the solvent under vacuum to obtain the complex.

In another embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan with a -arbutin by grinding method.

In step-a, ß-glucan with a -arbutin were taken in a mortar and add water dropwise, preferably 3-10 drops.

In step-b, the mixture obtained in step-a is grounded with pestle to obtain the complex.

In another embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan and a- arbutin by extrusion method.

Appropriate stoichiometric blends of ß-glucan with arbutin derivatives were prepared and used in extrusion experiments. Extrusion experiments were conducted by passing the above blends through a co-rotating twin-screw extruder. Twin Screw Extrusion (TSE) parameters such as screw design, temperature, and residence time were studied in a series of experiments to evaluate conditions required for formation of respective complexes.

EXPERIMENTAL SECTION
The details of the invention are given in the examples provided below, which are given to illustrate the invention only and therefore should not be construed to limit the scope of the invention.

Example-1: Process for preparation of novel complex of ß-glucan and a-arbutin by freeze drying method
Step-a:
A 2.0 grams of ß-glucan and 2.0 grams of a- arbutin was dissolved in 50 mL of double distilled water and stirred at room temperature for 3 hour.
Step-b:
The resultant solution obtained in Step-a was subsequently lyophilized using freeze dry system to obtain the complex.

Example-2: Process for preparation of novel complex of ß-glucan and a-arbutin by solvent assisted grinding method
Step-a:
2.0 grams of ß-glucan and 2.0 grams of a-arbutin were taken in a mortar and added 3 drops of water then ground for 1 h using mortar & pestle.

Example-3: Process for preparation of novel complex of ß-glucan and a-arbutin by solvent evaporation method
Step-a:
A 2.0 grams of ß-glucan and 2.0 grams of a-arbutin was dissolved in 12 mL of double distilled water and stirred at room temperature for 3 hour.
Step-b:
The resultant solution obtained in Step-a was evaporated by rota to obtain the complex.

Example-4: Evaluation of Mushroom Tyrosinase inhibitory activity by Mushroom Tyrosinase Inhibition assay
Mushroom Tyrosinase Assay Tyrosinase inhibitory activity was measured spectrophotometrically according to the method of Masamoto et al.17. First, 10µL (1000-62.5 µg/mL) in 1% DMSO was added to a 96-well microplate and mixed with 60 µL phosphate buffer (pH 6.8). Then, 20 µL 0.9mg/ML L-DOPA in phosphate buffer was added. Finally, 10 µL Mushroom tyrosinase (500 U/mL in phosphate buffer) was added and then this assay mixture was then incubated microplate reader. 8. RESULTS at 270C for 10 min. Following incubation, the amount of dopachrome production in the reaction mixture was determined spectrophotometrically at 450nm (OD 450nm) in a Kojic acid (1000- 62.5 µmol/L) was then dissolved in 50 mmol/L phosphate buffer which was used as a positive control. The concentration for 50% inhibition (IC50) was determined. Each measurement was performed in duplicates.

Beta glucan and Alpha Arbutin complex & Alpha Arbutin samples were evaluated to determine the inhibition potential on Mushroom Tyrosinase enzyme using Mushroom Tyrosinase Inhibition assay by fixing the test conc. of 1000 to 62.5 ?g/mL. As shown in the Table 1, the test samples Beta glucan and Alpha Arbutin complex indicated relatively superior Mushroom Tyrosinase inhibition activity with IC50 (470.81 ± 3.81 µg /mL) compared to Alpha Arbutin (>1000 µg/mL). Assays were carried out against Kojic acid as reference standard.

Table 1: Mushroom tyrosinase inhibitory activity assay results
S. No Name of test sample IC50 (µg/mL) Parameter
1 Beta glucan and Alpha Arbutin complex 470.81±3.81 Mushroom tyrosinase inhibitory activity assay

,CLAIMS:1. Novel complexes of ß-glucan and a-Arbutin and its process for preparation.

2. The process of preparation of novel complex claimed in claim 1, which comprises of
i) dissolving ß-glucan with a -arbutin in distilled water and stirring at room temperature;
ii) the above obtained solution was lyophilized using freeze dry system to obtain the complex.

3. The process of preparation of novel complex claimed in claim 1, which comprises of
i) dissolving ß-glucan and a -arbutin in distilled water and stirring at room temperature;
ii) evaporated the solvent under vacuum to obtain the complex.

4. The process of preparation of novel complex claimed in claim 1, which comprises of
i) ß-glucan with a -arbutin were taken in a mortar and add water dropwise;
ii) the mixture obtained in step-a is grounded with pestle to obtain the complex.

5. The novel complex of ß-glucan and a-Arbutin claimed in claim 1 is used for the treatment of Anti-inflammatory diseases.

6. The novel complex of ß-glucan and a-Arbutin claimed in claim 1 is showed relatively superior Mushroom Tyrosinase inhibition activity with IC50 (470.81 ± 3.81 µg /mL) compared to Alpha Arbutin (>1000 µg/mL).

7. The novel complex of ß-glucan and a-Arbutin claimed in claim 1 is showed good anti-oxidant activity.