DESC:FIELD OF THE INVENTION
The present invention relates to novel complexes of ß-glucan with Niacinamide 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.

Niacinamide is a form of vitamin B3 found in food and used as a dietary supplement and medication. nicotinamide consists of a pyridine ring to which a primary amide group is attached in the meta position. It is an amide of Nicotinic acid. As a supplement, it is used by mouth to prevent and treat pellagra. It has anti-inflammatory actions, which may benefit people with inflammatory skin conditions. Niacinamide increases the biosynthesis of ceramides in human keratinocytes in vitro and improves the epidermal permeability barrier in vivo.

Niacinamide has been shown to prevent Cutibacterium acnes-induced activation of toll-like receptor 2, which ultimately results in the down-regulation of pro-inflammatory interleukin-8 production.

Nicotinamide, as a part of the cofactor nicotinamide adenine dinucleotide (NADH / NAD+) is crucial to life. In cells, nicotinamide is incorporated into NAD+ and nicotinamide adenine dinucleotide phosphate (NADP+). NAD+ and NADP+ are cofactors in a wide variety of enzymatic oxidation-reduction reactions, most notably glycolysis, the citric acid cycle, and the electron transport chain.
Niacinamide is found to show reduce in the rate of new nonmelanoma skin cancers and actinic keratoses in a group of people at high risk for the conditions.
Niacinamide has been investigated for many additional disorders, including treatment of bullous pemphigoid nonmelanoma skin cancers. Niacinamide may be beneficial in treating psoriasis.

There is tentative evidence for a potential role of niacinamide in treating acne, rosacea, autoimmune blistering disorders, ageing skin, and atopic dermatitis. Niacinamide also inhibits poly(ADP-ribose) polymerases (PARP-1), enzymes involved in the rejoining of DNA strand breaks induced by radiation or chemotherapy. ARCON (accelerated radiotherapy plus carbogen inhalation and nicotinamide) has been studied in cancer. Research has suggested niacinamide may play a role in the treatment of HIV.

Niacinamide is also found in many foods, including meat, fish, milk, eggs, vegetables, cereals, nuts, and mushrooms, as well as to a lesser extent in some vegetables. It is commonly added to cereals and other foods. Many multivitamins contain 20–30 mg of vitamin B3 and it is also available in higher dose.

With availability of data on excellent beneficial properties of ß-glucan and Niacinamide, tapping unexplored potential of synergistic properties of the combination of ß-glucan with Niacinamide and its derivatives holds great promise in Cosmeceutical, Pharmaceutical and Nutraceutical industries. It is desirable to develop an efficient and robust process for preparation of stable complex of ß-glucan and Niacinamide in high purities and yields, such that the bioavailability both the active components is enhanced.

SUMMARY OF THE INVENTION
The present invention provides novel complexes of ß-glucan with Niacinamide and their process for preparation.

In one embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan and Niacinamide which comprises:
a) dissolving ß-glucan and Niacinamide in distilled water and stirring at room temperature;
b) the above obtained solution was lyophilized using the freeze dry system to obtain complex.

In another embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan and Niacinamide, which comprises:
a) dissolving the ß-glucan and Niacinamide in distilled water and stirring at room temperature;
b) the above obtained solution is evaporated under vacuum to obtain complex.

In another embodiment, the present invention provides a process for the preparation of novel complexes of ß-glucan and Niacinamide, which comprises; Grinding mixtures of ß-glucan and Niacinamide to obtained complex.

FIGURES
Figure 1: PXRD of ß-glucan
Figure 2: PXRD of Niacinamide
Figure 3: PXRD of Lyophilization complex of ß-glucan and Niacinamide
DETAILED DESCRIPTION OF THE INVENTION
ß-glucan obtained from various sources like microbial, fungal, mushroom, yeast and plant sources was used.

Naturally occurring Niacinamide extracts, synthetic forms Niacinamide were used in the formation of the complexes. A general terminology “Niacinamide” is used in the text which should be considered as any of the natural extracts containing Niacinamide or synthetic Niacinamide.

Accordingly, the present invention provides novel complexes and various processes in different ratios for the preparation of novel complexes of ß-glucan with Niacinamide.

In one embodiment, the present invention provides a process for the preparation of novel complexes of ß-glucan and Niacinamide by the freeze drying method.
In step-a, ß-glucan and Niacinamide were dissolved in 100 mL of 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 25-35 °C 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 and Niacinamide by evaporation method.

In step-a, ß-glucan and Niacinamide was 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 25-35 °C 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 and Niacinamide by grinding method.

In step-a, ß-glucan and Niacinamide was taken in a mortar and add water , preferably 3 to 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 Niacinamide derivatives by extrusion method.
Appropriate stoichiometric blends of ß-glucan with Niacinamide 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 Niacinamide by freeze drying method
Step-a:
Charged water 500 mL into RBF, charged ß-glucan (50 grams) and stirred for 10 minutes, then added Niacinamide (250 grams) and added water 500 mL Stirred for 1 hour at room temperature.
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 Niacinamide by solvent evaporation method
Step-a:
A 2.0 grams of ß-glucan and 2.0 grams of Niacinamide was dissolved in 12 mL of double distilled water and stirred at room temperature for 3 hours.
Step-b:
The resultant solution obtained in Step-a was evaporated by rota to obtain the complex.

Example-3: Process for preparation of novel complex of ß-glucan and Niacinamide by grinding method
6.0 grams of ß-glucan and 0.3 grams of Niacinamide extract powder were taken in a mortar and ground for 10 minutes using mortar & pestile.

Example-4:
Evaluation on pore tightening activity of beta glucan and niacinamide complex through MAGP-1 gene expression in human keratinocytes:
The in vitro MTT assay was performed for the test samples on Human keratinocyte (HaCaT) cell line to determine the level of cytotoxicity. The effect of on beta glucan and niacinamide complex modulation of MAGP-1 was estimated by gene expression method, where the level of the expression of MAGP-1 in Human keratinocyte (HaCaT) cell line was determined with respect to untreated HaCaT cells.
Table 1: In vitro cytotoxicity of test samples in terms of percentage cell viability against Human Keratinocytes (HaCaT) cell line by MTT assay.
Test sample concentration (µg/mL) Percentage of cell viability after treatment (Mean ± SD)
Niacinamide beta glucan and Niacinamide complex
1000 60.16 ? 0.47 62.81 ± 0.52
500 73.23 ? 1.25 80.31 ± 0.10
250 83.33 ± 1.56 83.65 ±1.04
125 87.40 ? 1.35 89.32 ± 1.61
62.5 91.15 ? 0.83 93.80 ± 0.47
31.25 93.75? 0.73 95.73 ± 0.52
15.62 96.98? 0.31 97.34 ± 1.41
7.81 99.06? 0.42 98.96 ± 0.21

In vitro cytotoxicity of test sample in terms of percentage cell viability against Human Keratinocytes (HaCaT) cell line by MTT assay.
In vitro cytotoxicity studies for the samples were tested on Human Keratinocytes (HaCaT) cell line by MTT assay. The CTC50 value of the test substances on HaCaT cell line was above 1000µg/mL. The cytotoxicity of niacinamide & beta glucan and niacinamide complex was determined in terms of percentage cell viability and it was found to be 60.16±0.47 % and 62.81±0.52 % at higher concentration (1000µg/mL) on HaCaT cell line respectively indicating that the complex is safe for usage.
Table 2: The quantitative expression level of MAGP-1 gene in test samples treated cells
Sl. No Test sample concentration MAPG - 1 Expression Fold
Niacinamide Beta glucan and Niacinamide complex
1 250 µg/ml 2.41 1.72
2 125 µg/ml 1.35 0.68
3 Standard- Hyaluronic acid
250 µg/ml 1.82 1.82
4 Standard- Hyaluronic acid
125 µg/ml 0.98 0.98
5 Cell control 0.14 0.14

In the present study, the mean level of MAGP-1 expression in the test samples treated cells with control cells were compared and relative gene expression was reported. Results showed that the up regulation of MAGP-1 gene expression in cells treated with test samples Niacinamide & Beta glucan and Niacinamide complex were found to be 2.41 & 1.72 fold respectively, when compared with the control cells (0.14 fold), at 250 µg/mL concentration. This result indicates the MAGP-1 gene modulation efficacy of both samples and thereby it upsurges the ability to assemble elastin, microfibrils, and proteoglycans together, and helps organize them into a complex dermal structure within the epidermis.

Summary: The result concluded that the test samples Niacinamide & Beta glucan and Niacinamide complex exhibited with skin pore tightening property by modulating the MAGP-1 gene expression in Human Keratinocyte cells.
,CLAIMS:
1. Novel complexes of ß-glucan and Niacinamide and its process for preparation.

2. The process of preparation of novel complex claimed in claim 1, which comprises of
i) dissolving ß-glucan and Niacinamide 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 Niacinamide 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 Niacinamide 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 Niacinamide as claimed in claim 1 for the treatment pore tightening activity through MAGP-1 gene expression in human keratinocytes.