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

Sodium hyaluronate is a derivative of hyaluronic acid, a naturally occurring polysaccharide found in connective tissues such as cartilage, extracellular matrix of mammalian connective, epithelial, and neural tissues, as well as the corneal endothelium. It has many uses, including the treatment of arthritis, dry eyes, ulcers, vaginal dryness and wounds. It is also present in skin care products and cosmetics.
Sodium hyaluronate is a humectant, which means that it attracts moisture. It can be applied topically in creams and serums to hydrate the skin and also as use as a dermal filler to the skin to reduce the appearance of fine lines and wrinkles.
Sodium hyaluronate comes from hyaluronic acid, a substance that occurs naturally in the synovial fluid of the joints, eyes, osteoarthritis and skin. The “sodium” component reflects that sodium hyaluronate is a salt. Sodium hyaluronate is also more stable than hyaluronic acid and less prone to oxidation, which is why it often appears in skin care products.

There are two main sources of Hyaluronic Acid: plant origins and animal origins. Plant-based Hyaluronic Acid is extracted from microbial fermentation. This is a fancy way of saying that a bacterial strain naturally contains Hyaluronic Acid and is then fermented to yield the desired molecular weights ideal for skin care purposes.
Some plant-based Hyaluronic Acid may contain wheat. Animal-based Hyaluronic Acid utilizes the combs of roosters—this is the red flesh at the top of a rooster’s head. Like humans, other animals also produce Hyaluronic Acid in their bodies, and the rooster’s comb is considered one of the best animal sources. Microbial fermentation is the most common way to source Hyaluronic Acid. Not only is it more cost-effective for manufacturers, and therefore consumers, but it’s also more eco-friendly since it produces less environmental pollution. Most natural, organic, and vegan skin care lines utilize plant-based Hyaluronic Acid in their formulations, but animal-based Hyaluronic Acid is still commonly used in Hyaluronic Acid supplements and injections

With availability of data on excellent beneficial properties of ß-glucan and Sodium hyaluronate, tapping unexplored potential of synergistic properties of the combination of ß-glucan with Sodium hyaluronate 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 complexes of ß-glucan and Sodium hyaluronate 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 Sodium hyaluronate which comprises:
a) dissolving ß-glucan and Sodium hyaluronate 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 Sodium hyaluronate, which comprises:
a) dissolving the ß-glucan and Sodium hyaluronate 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 Sodium hyaluronate, which comprises; Grinding mixtures of ß-glucan and Sodium hyaluronate to obtained complex.

FIGURES
Figure 1: PXRD of ß-glucan
Figure 2: PXRD of Sodium hyaluronate
Figure 3: PXRD of Lyophilization complex of ß-glucan and Sodium hyaluronate

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

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

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

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

In step-a, ß-glucan and Sodium hyaluronate 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 Sodium hyaluronate by evaporation method.

In step-a, ß-glucan and Sodium hyaluronate 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 Sodium hyaluronate by grinding method.

In step-a, ß-glucan and Sodium hyaluronate 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 Sodium hyaluronate derivatives by extrusion method.

Appropriate stoichiometric blends of ß-glucan with Sodium hyaluronate 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 Sodium hyaluronate by freeze drying method
Step-a:
Charged water 2000 mL into RBF, charged ß-glucan (300 grams) and stirred for 10 minutes, then added Sodium hyaluronate (150 grams), water 2000 mL and Stirred for 15 minutes, clear solution not observed so added water 2000 mL and stirred for 10 minutes, transparent jelly type reaction mixture observed and further diluted with water 3000 mL to get the clear solution. Reaction mixture 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 Sodium hyaluronate by solvent evaporation method
Step-a:
A 2.0 grams of ß-glucan and 2.0 grams of Sodium hyaluronate are 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 Sodium hyaluronate by grinding method
5 grams of ß-glucan and 0.3 grams of Sodium hyaluronate extract powder are taken in a mortar and ground for 10 minutes using mortar & pestle.
Exsmple-4:
Evaluation of moisturizing effect of beta glucan and hyaluronic acid complex by AQP-3 gene expression modulation and Hyaluronidase inhibition in Human Keratinocytes
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 test samples beta glucan & beta glucan and hyaluronic acid complex was determined in terms of percentage cell viability and it was found to be 65.57±3.6 % & 64.55±2.72% at higher concentration (1000µg/mL) on HaCaT cell line respectively indicating that the complex is safe for usage.

Table 1. In vitro cytotoxicity of test samples in terms of percentage cell viability on Mouse Skin Melanoma (B16-F10) cell line by MTT assay.
Test sample concentration (µg/mL) Percentage of cell viability after treatment (Mean ± SD)
beta glucan beta glucan and hyaluronic acid complex
1000 65.57 ? 3.6 64.55 ? 2.72
500 81.67 ? 1.13 80.30 ? 3.74
250 84.94 ± 3.61 85.00 ? 146
125 88.12 ? 0.84 87.42 ? 1.26
62.5 91.84 ? 1.25 91.11 ? 0.91
31.25 95.11? 0.83 94.09 ? 1.57
15.62 97.09? 1.22 96.31 ? 1.16
7.81 99.18? 0.33 98.13 ? 1.06

Table 2: The quantitative expression level of AQP-3 gene in test samples treated cells
Sl. No Test sample concentration AQP-3 Expression Fold
Beta glucan Beta glucan and Hyaluronic acid complex
1 500µg/ml 0.93 1.12
2 250µg/ml 0.41 0.61
3 Standard- Hyaluronic acid
500µg/ml 1.56 1.56
4 Standard- Hyaluronic acid
250µg/ml 0.97 0.97
5 Cell control 0.32 0.32

Reverse Transcriptase-PCR experiment was performed by using gene specific primers. Quantitative RT-PCR analysis revealed the modulatory effect of test product on the mRNA expression of AQP3 gene over cell control. The gene AQP3 encodes the water channel protein aquaporin 3, water-transporting proteins, which play key role in providing proper skin hydration and maintaining water balance in the cell layers. The activity level of AQP3 in the epidermis showed to be associated with the degree of skin hydration moisturizing property.

The mean level of AQP3 expression in the test samples treated cells with control cells was compared and relative gene expression was reported. Results showed that the test samples Beta glucan & Beta glucan-Hyaluronic acid complex were found to exhibit 0.93 & 1.12 fold up regulation of AQP3 gene expression in cells, when compared with the control cells (0.32 fold), at 500 µg/mL concentration. Beta glucan-Hyaluronic acid complex showed relatively better upregulation than only Beta glucan at 250 µg/ml concentration. The results indicated the superior AQP-3 gene modulation efficacy of both the test samples and thereby indicating the ability of the complex to upsurge and transport water and glycerol within the epidermis. This transported water is known to play a role in epidermal hydration and hydrostatic pressure which maintain the skin texture by moisturizing effect.
Table 3: Dose-dependent percentage inhibition of Hyaluronidase enzyme by test product, and standard.
Concentration (µg/ml) Ascorbic acid (Standard) Percentage Hyaluronidase inhibition by test product and standard (Mean ± SEM)
Beta glucan Beta glucan and Hyaluronic acid complex
1000 39.20± 0.4 26.38 ± 4.1 31.28 ± 3.9
500 21.2 ± 4.5 12.51 ± 4.1 22.56 ± 2.3
250 14.58±2.3 7.77 ± 3.2 11.25 ± 3.8
125 9.78 ± 1.2 2.12 ± 0.8 9.27 ± 2.5
62.5 4.5± 2.3 0.32 ± 0.1 2.32± 0.9

In-vitro estimation for Hyaluronidase inhibition of the test samples with standard compound (Ascorbic acid) was carried out using enzyme assay method at different concentrations ranging from 62.5 µg/mL to 1000 µg/mL (Table 3). From the perceived values, it was observed that beta glucan and hyaluronic acid complex exhibited a superior Hyaluronidase inhibitory effect than only Beta glucan and exhibited similar effect as the standard ascorbic acid (at 125 µg/mL) respectively.

Summary: The result also indicated that beta glucan & beta glucan and hyaluronic acid complex exhibited skin hydration/moisturizing property by modulating the AQP-3 gene expression as well as by inhibiting the Hyaluronidase enzyme in Human Keratinocyte.

Conclusion: The beta-glucan-hyaluronic acid complex demonstrates enhanced efficacy compared to hyaluronic acid alone in promoting skin hydration. It showed higher AQP-3 expression levels at both 500 µg/mL (1.12 vs. 0.93) and 250 µg/mL concentrations (0.61 vs. 0.41). Additionally, the complex exhibited superior Hyaluronidase inhibition across all tested concentrations, notably achieving 9.27% inhibition at 125 µg/mL compared to only 2.12% for beta-glucan. The beta-glucan-hyaluronic acid complex at 500 µg/ml, which is equivalent to 166 µg/ml of hyaluronic acid showed 1.12 folds AQP-3 expression, which is better than hyaluronic acid at 250 µg/ml concentration (Tabel 2). These findings indicate that the beta-glucan-hyaluronic acid complex not only improves hydration but also better preserves hyaluronic acid in the skin. This data supports its use in skincare formulations aimed at optimizing moisture retention more effectively than hyaluronic acid alone.

,CLAIMS:1. Novel complexes of ß-glucan and Sodium hyaluronate 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 Sodium hyaluronate 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 Sodium hyaluronate in distilled water and stirring at room temperature;
ii) the above obtained solution is evaporated 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 Sodium hyaluronate 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 Sodium hyaluronate as claimed in claim 1 is used as moisturizing applications in cosmetics.

6. The novel complex of ß-glucan and Sodium hyaluronate as claimed in claim 1 exhibited moisturizing effect by AQP-3 gene expression modulation and Hyaluronidase inhibition in Human Keratinocytes.