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

Cordyceps, one of the large genuses in fungal family belongs to cordycipitaceae family, subfamily Hypocreales, Ascomycota genus and it is an entomopathogenic fungi having more than 600 species in mycobank. Cordyceps is a parasitic fungus, mostly infect lepidopteron insects. The word cordyceps was derived from Greek word “kordyle” means “club” and “ceps” means “head” in Latin. Cordyceps are mostly used as traditional medicine widely in Asia. It is rich in a variety of bioactive compounds with broad applications in pharmacological, Therapeutic and Cosmetics.

Many bioactive molecules present in cordyceps militaries like cordycepin, pentostatin, ergosterol, d- mannitol, trehalose, and several polysaccharides’ nucleosides and amino acid, which can satisfy the industrial applications and market value.

Cordyceps related products are popular in western countries as nutraceuticals consumed in different forms like extracts, fermented powder, tinctures, dried fruit bodies etc. cordycepin tonics/ other form of products clamming beneficial in preventing & slowdown progress of chronic kidney, respiratory, heart, liver and lung diseases along with anti-aging, hypoglycemic, hypolipidemic, anti-inflammatory, anti-tumor, antibacterial. Antiviral, antimalarial, immunomodulatory, prosexual, neuroprotective, antioxidant, and immune-protective activities. Among the bio actives present in cordyceps militaris, cordycepin is a structural analog of adenosine and imidazole and is considered as most economically valuable bioactive molecule mostly produced by cordyceps militaris and sinensis.

Cordycepin was reported to exhibit antioxidant properties by hydroxyl radicals scavenging activity in cosmetics. It is also used for cosmetic applications like decreasing the melanin production by suppressing cellular tyrosinase activity. It can also be used as functional food for nutritional enhancement and fortified foods like cookies which are having high protein, antioxidant and phenolic content which can be a healthy alternative for consumers.

As a natural antibiotic, Cordycepin has many strong pharmacological activities, but its rapid deamination metabolism by ADA in vivo leads to its short half-life, poor bioavailability and unsatisfactory efficacy, which limits its applications in disease prevention and treatment. There are currently three main methods, co-administration of an ADA inhibitor and Cordycepin, structural modifications to produce Cordycepin derivatives and new drug delivery systems for delivering Cordycepin, can be applied

Recent literature shows promising use of combination of polysaccharides and cordyceps militaris to modulate gut microbacteria and improve metabolic disorders in mice (J Sci Food Agric. 2023 Mar 15;103(4):1885-1894).

Literature related to dietary supplement compositions, cosmetic formulations and drug combinations containing ß-glucan, and Cordyceps militaris extracts or Cordyceps sinensis extracts or cordycepin is available. However, to date there are no reports of the molecular complexes of ß-glucan with cordycepin or Cordyceps mushroom extracts.

In spite of the knowledge of the beneficial properties of ß-glucan and Cordycepin there still remains an unrealized scope to explore the synergistic properties of this combination. In view of the above enormous potential combinations of ß-glucan and Cordycepin in Cosmeceutical, Pharmaceutical and Nutraceutical industries, it is desirable to develop an efficient and robust process for preparation of their stable complexes, such that the bioavailability of the active ingredient Cordycepin is increased.

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

In one embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan and Cordyceps which comprises:
a) dissolving ß-glucan and Cordyceps in distilled water and stirring at room temperature or heating up to 50 °C;
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 Cordyceps, which comprises:
a) dissolving the ß-glucan and Cordyceps in distilled water and stirring at room temperature or heating upto 50 °C.
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 Cordyceps, which comprises; Grinding mixtures of ß-glucan and Cordyceps to obtained respective complex.

In another embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan and Cordyceps, which comprises:
Extrusion of mixtures of ß-glucan and Cordyceps to obtain complex.

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

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

Cordyceps means both Cordyceps militaris and Cordyceps sinensis. Extracts of Cordyceps militaris and Cordyceps sinensis were obtained and per the reported experimental procedures. Cordycepin was directly purchased and used as is.

Accordingly, the present invention provides novel complexes of ß-glucan and Cordyceps as shown below.

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

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

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

In step-a, ß-glucan and Cordyceps 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 Cordyceps by extrusion method.

Appropriate stoichiometric blends of ?-glucan and Cordyceps 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 ?-Glucan and Cordyceps complex. For the ?-Glucan and Cordyceps complexes, extruder temperature was performed at room temperature to 50 ? throughout the screw zones, while the volumetric feed rate was set at 5%, and screw speed was fixed at 100 rpm.

EXPERIMENTAL PORTION
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 Cordyceps by freeze drying method
Step-a:
A 140 grams of ß-glucan and 7.0 grams of Cordyceps powder was dissolved in 200 mL of double distilled water and stirred at room temperature for 1 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 Cordyceps by grinding method
Step-a:
6.0 grams of ß-glucan and 0.3 grams of Cordyceps extract powder were taken in a mortar and ground for 10 minutes using mortar & pestle.

Example-3: Evaluation of the Anti-aging property of beta glucan and cordyceps militaris complex against UV induced stress in Human Keratinocytes (HaCaT) and hepatocellular Carcinoma cells
Determination of cell cytotoxicity by MTT Assay:
Beta glucan and Cordyceps militaris 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 51.21 ± 0.10 % at higher concentration (1000µg/mL) on Human keratinocyte (HaCaT) cell line (Table 1).
Table 1: In vitro cytotoxicity of Beta glucan and Cordyceps militaris 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 Cordyceps militaris complex 1000 51.21 ± 0.10
500 72.83 ± 3.84
250 82.07 ±1.46
125 87.12 ± 1.06
62.5 91.66 ± 0.05
31.25 93.08 ± 0.45
15.625 95.51 ± 0.66
7.8 98.23 ± 0.96

Table 2: Anti-oxidant activity of beta glucan and cordyceps militaris complex in Human hepatocellular carcinoma (HepG2) cells against LPS 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 0.34 ± 0.003 74.58 ± 2.3
2 LPS – 1 µg/ml 0.22 ± 0.001 58.36 ± 1.5
3 Beta glucan and Cordyceps militaris complex - 500 µg/ml 0.52 ± 0.002 76.82 ± 1.4
4 Beta glucan and Cordyceps militaris complex - 250 µg/ml 0.34 ± 0.002 62.13 ± 1.7
5 Standard- Quercetin 500 µg/ml 0.47 ± 0.004 77.58 ± 2.1
6 Standard- Quercetin 250 µg/ml 0.31 ± 0.002 65.22 ± 2.1

Table 3: Anti-oxidant activity of Beta glucan and Cordyceps militaris 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 0.178 ± 0.001 15.63 ± 1.2
2 LPS – 1 µg/ml 0.114 ± 0.001 10.44 ± 0.9
3 Beta glucan and Cordyceps militaris complex - 500 µg/ml 0.165 ± 0.003 23.47 ± 1.2
4 Beta glucan and Cordyceps militaris complex - 250 µg/ml 0.131 ± 0.002 18.75 ± 1.6
5 Standard- Quercetin 500 µg/ml 0.161 ± 0.002 23.19 ± 1.9
6 Standard- Quercetin 250 µg/ml 0.141 ± 0.001 15.21 ± 1.1

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 LPS induction in cells caused significant decreases in reduced glutathione (GSH) and Superoxide dismutase (SOD) activities in both keratinocyte and hepatic cells. In the present study, beta glucan and cordyceps militaris complex significantly increase the antioxidant indicators Glutathione (GSH) and Super oxide dismutase (SOD) levels in LPS treated groups when compared with the cell control (Table 2 & Table 3). This result indicates the modulatory effect of beta glucan and cordyceps militaris complex on oxidative free radical scavenging enzymes activity against LPS induced oxidative stress states in human hepatic and keratinocyte cells.
Table 4: In vitro cell - proliferative activity of beta glucan and cordyceps militaris complex 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 Cordyceps militaris complex 500 72.83 ± 3.84 45.58 ± 1.56 23.37
250 82.07 ± 1.46 41.25 ± 1.54 9.8
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 72.83 % & 82.07 % viability at 24 hours of exposure, Beta glucan and Adenosine complex treated cells, revealed 45.58 % and 41.25 % 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 23.37% and 9.8% relatively superior protection activity of beta glucan and cordyceps militaris complex at 500 and 250 µg/ml over the control (Table 4).

Summary: The test result concluded that the test product Beta glucan and Cordyceps militaris complex exhibited Anti-aging efficacy by protecting the cells from oxidative damage.
,CLAIMS:1. Novel complexes of ß-glucan and Cordyceps 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 Cordyceps 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 Cordyceps 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 Cordyceps 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 Cordyceps as claimed in claim 1 for the treatment of Anti-aging property against UV induced stress.

6. The novel complex of ß-glucan and Cordyceps as claimed in claim 1 as Antioxidant.

7. The novel complex of ß-glucan and Cordyceps as claimed in claim 1 for the treatment of protective effect in human keratinocyte cells against UV induced stress.