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

Adenosine is an organic compound that occurs widely in nature in the form of diverse derivatives. Adenosine is an endogenous purine nucleoside that modulates many physiological processes. Cellular signaling by adenosine occurs through four known adenosine receptor subtypes (A1, A2A, A2B, and A3). The molecule consists of an adenine attached to a ribose via a ß-N9-glycosidic bond. Adenosine is one of the four nucleoside building blocks of RNA (and its derivative deoxyadenosine is a building block of DNA), which are essential for all life on earth. Adenosine is used as an intravenous medication for cardiac arrhythmias. In individuals with supraventricular tachycardia (SVT), adenosine is used to help identify and convert the rhythm. Because of the effects of adenosine on AV node-dependent SVTs, adenosine is considered a class V antiarrhythmic agent. When adenosine is used to cardiovert an abnormal rhythm, it is normal for the heart to enter ventricular asystole for a few seconds. This can be disconcerting to a normally conscious patient, and is associated with angina-like sensations in the chest. Adenosine used as a second messenger can be the result of de novo purine biosynthesis via adenosine monophosphate (AMP), though it is possible other pathways exist.

When adenosine enters the circulation, it is broken down by adenosine deaminase, which is present in red blood cells and the vessel wall.
Adenosine deaminase deficiency is a known cause of immunodeficiency. Adenosine is believed to be an anti-inflammatory agent at the A2A receptor. Topical treatment of adenosine to foot wounds in diabetes mellitus has been shown in lab animals to drastically increase tissue repair and reconstruction. Topical administration of adenosine for use in wound-healing deficiencies and diabetes mellitus in humans is currently under clinical investigation.

In general, adenosine has an inhibitory effect in the central nervous system (CNS). Caffeine's stimulatory effects are credited primarily (although not entirely) to its capacity to block adenosine receptors, thereby reducing the inhibitory tonus of adenosine in the CNS. This reduction in adenosine activity leads to increased activity of the neurotransmitters dopamine and glutamate. Experimental evidence suggests that adenosine and adenosine agonists can activate Trk receptor phosphorylation through a mechanism that requires the adenosine A2A receptor. Adenosine has been shown to promote thickening of hair on people with thinning hair. A study compared topical adenosine with minoxidil in male androgenetic alopecia, finding it was as potent as minoxidil (in overall treatment outcomes) but with higher satisfaction rate with patients due to “faster prevention of hair loss and appearance of the newly grown hairs”.

Adenosine is a key factor in regulating the body's sleep-wake cycle. Adenosine levels rise during periods of wakefulness and lowers during sleep. Higher adenosine levels correlate with a stronger feeling of sleepiness, also known as sleep drive or sleep pressure. Cognitive behavioural therapy for insomnia (CBT-I), which is considered one of the most effective treatments for insomnia, utilizes short-term sleep deprivation to raise and regulate adenosine levels in the body, for the intended promotion of consistent and sustained sleep in the long term. It also plays a role in regulation of blood flow to various organs through vasodilation.

Adenosine serves as a building block for adenosine triphosphate (ATP). When ATP is metabolized for energy production due to increased energy demand, adenosine levels increase. Adenosine is one of the four building blocks of DNA, and RNA, these are essential for all life. Foods that provide adenosine are Meat that is fed on grass, well-pastured poultry, and organ meats, such as liver or kidneys, Fish and seafood, such as salmon, sardines, halibut, orange roughy, tuna, ling, pike, cod, cusk, sunfish, haddock, and whitefish, eggs, seeds and nuts, whole grains and legumes after soaking them, vegetables and fruits, including sea vegetables like algae and spirulina.

With availability of data on excellent beneficial properties of ß-glucan and Adenosine, tapping unexplored potential of synergistic properties of the combination of ß-glucan with Adenosine 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 Adenosine 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 and Adenosine and their process for preparation.

In one embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan and Adenosine, which comprises:
a) dissolving ß-glucan and Adenosine 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 Adenosine, which comprises:
a)dissolving the ß-glucan and Adenosine 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 Adenosine, which comprises; Grinding mixtures of ß-glucan and Adenosine in presence of water to obtained complex.

FIGURES
Figure 1: PXRD of ß-glucan
Figure 2: PXRD of Adenosine
Figure 3: PXRD of Lyophylization complex of ß-glucan and Adenosine

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

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

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

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

In step-a, ß-glucan and Adenosine 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 25-35 °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 Adenosine by evaporation method.

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

In step-a, ß-glucan and Adenosine are 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 with Adenosine derivatives by extrusion method.

Appropriate stoichiometric blends of ß-glucan with Adenosine 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 Adenosine by freeze drying method
Step-a:
Charged 20% Acetonitrile in Water (700 mL) into RBF, charged ß-glucan (60 grams) and Adenosine (5 grams) and stirred for 3 hours 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 Adenosine by solvent evaporation method
Step-a:
A 4.0 grams of ß-glucan and 4.0 grams of Adenosine 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 Adenosine by grinding method
3 grams of ß-glucan and 0.3 grams of Adenosine extract powder are taken in a mortar and ground for 10 minutes using mortar & pestle.

Example-4:
ANTIOXIDANT ACTIVITY OF BETA GLUCAN AND ADENOSINE COMPLEX BY ABTS ASSAY AND HYDROGEN PEROXIDE RADICAL SCAVENGING ASSAY
Beta glucan and Adenosine complex was evaluated for its antioxidant activity by ABTS Radical Scavenging assay by fixing the test conc. of 1000 to 62.5 ?g/mL (Table 1). Beta glucan and Adenosine complex was evaluated for its antioxidant activity by ABTS have exhibited high antioxidant activity with IC50 value of 305.17 ± 3.68µg /mL.

Table 1: The antioxidant activity of Beta glucan and Adenosine complex for ABTS assay.
S. No Sample Name IC50 (µg/mL) Parameter
1. Beta glucan and Adenosine complex 305.17 ± 3.68 ABTS Inhibition Assay.
2. Ascorbic acid (Standard) 64.97 ± 0.06

Example-5:
Evaluation of the Anti-aging property of beta glucan and adenosine complex against UV induced stress in Human Keratinocytes (HaCaT) cell line
Determination of cell cytotoxicity by MTT Assay:
Beta glucan and Adenosine complex was tested in vitro cytotoxicity studies on the Human keratinocyte (HaCaT) cell line by MTT assay. The CTC50 value of the test product on the keratinocyte cells was above 1000 µg/mL. The cytotoxicity of the test product was determined in terms of percentage cell viability, and it was found to be 67.29 ± 2.15 % at higher concentration (1000µg/mL) on Human keratinocyte (HaCaT) cell line (Table 2).
Table 2: In vitro cytotoxicity of Beta glucan and Adenosine complex in terms of percentage cell viability against Human Keratinocyte (HaCaT) cell line by MTT assay
Test Product Concentration (µg/mL) Percentage of cell viability after treatment
Beta glucan and Adenosine complex 1000 67.29 ± 2.15
500 75.86 ± 3.64
250 80.26 ± 3.23
125 86.78 ± 1.28
62.5 92.60 ± 2.00
31.25 94.73 ± 0.86
15.625 97.03 ± 0.87
7.8 99.12 ± 0.54

Table 3: Anti-oxidant activity of Beta glucan and Adenosine complex in Human Keratinocyte (HaCaT) cells against UV induced stress by estimation of reduced glutathione (GSH) and Superoxide dismutase (SOD) levels.
S. No Sample Name GSH Level (U/mgprot) SOD Level (U/mgprot)
1. Cell control- Irr- 0.178 ± 0.002 15.63 ± 1.2
2. Cell control- Irr+ 0.008 ± 0.001 4.4 ± 0.9
3. Beta glucan and Adenosine complex - 500 µg/ml 0.203 ± 0.001 30.83 ± 1.2
4. Beta glucan and Adenosine complex - 250 µg/ml 0.174 ± 0.001 19.53 ± 1.1
5. Standard- Quercetin 500 µg/ml 0.250 ± 0.002 29.66 ± 2.8
6. Standard- Quercetin 250 µg/ml 0.171 ± 0.002 13.25 ± 1.8
*Irr+ UV Irradiated; Irr-UV nonirradiated

The UV induced cell damage is associated with free radical injury and oxidative stress. Oxidative stress is characterized by increased lipid peroxidation and/or altered non-enzymatic and enzymatic antioxidant systems. In this study it is found that chronic UV treatment caused significant decreases in reduced glutathione (GSH) and Superoxide dismutase (SOD) activities in keratinocyte cells. Human skin exposed to solar UV radiation dramatically increases ROS production /oxidative stress. These free radicle formation and imbalance of antioxidants results in aging. In the present study, Beta glucan and Adenosine complex significantly increase the GSH and SOD activities in UV treated groups when compared with the cell control (Table 3). This result indicates the effective modulatory properties of Beta glucan and Adenosine complex on oxidative free radical scavenging enzymes activity against UV induced oxidative stress states in keratinocyte cells.

Table 4: In vitro cell - proliferative activity of the test product in Human Keratinocytes (HaCaT) cell line against UV induced stress.
Sample Name Concentration (µg/ml) Percentage viability in 24 hr (%) Percentage viability in 72 hr (%) Percentage protection over control (%)
Beta glucan and Adenosine complex 500 67.29 ± 2.15 46.58 ± 1.25 29.91
250 75.86 ± 3.64 45.12 ± 1.47 19.88
Cell Control 100 49.38 -

In order to ascertain the percentage of protection provided by the test product on maintaining cell viability following UV exposure, the test product was evaluated for its capacity to increase both the quantity and function of cells. A predetermined number of cells (5x104 cells) were seeded into 24 well plates for the proliferation investigation, and the cells were monitored for proliferation after 24 and 72 hours. The MTT test was used to determine the proportion of viable cells in each well. When compared to the 67.29 % & 75.86 % viability at 24 hours of exposure, Beta glucan and Adenosine complex treated cells, revealed 46.58 % and 45.12 % viability after 72 hours of UV exposure at 500 and 250 µg/ml concentrations. It shows that, in contrast to the 50% reduction in cell control, less than 30% of the cells in the treated groups were reduced. This result indicated 29.91% and 19.88% relatively superior protection activity of test product at 500 and 250 µg/ml over the control (Table 5).

Summary: The test result concluded that the test product Beta glucan and Adenosine complex exhibited Anti-aging efficacy by protecting the cells from oxidative damage.
,CLAIMS:1. Novel complexes of ß-glucan and Adenosine 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 Adenosine 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 Adenosine 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 Adenosine 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 Adenosine as claimed in claim 1 is used as anti-oxidant.

6. The novel complex of ß-glucan and Adenosine as claimed in claim 1 is used as anti-aging agent by protecting the cells from oxidative damage.