Description:TECHNICAL FIELD OF INVENTION

Present embodiment is a kind of unique solution to extract the broad-spectrum high saturated metabolome and metabolites of Cordyceps militaris with cheap, chemical free, rapid and versatile in various food and health application.

BACKGROUND OF THE INVENTION

The background information herein below relates to the present disclosure but is not necessarily prior art.

Cordyceps militaris is a rare folk Chinese medicine with high health care and medical value and is enriched with the many kinds of human body needs. It contains all unique nutrient component like adenosine, cordycepic acid, cordycepin, amino acid, sterol, mannitol, alkaloid, vitamin B1, B2, polysaccharide and mineral matter as well as various rare compounds and metabolites, that can majorly require to any animals and plants. It has the property of medicine leniency and hence edible throughout the year to any human age group or gender with any type of deficiency or disease. It has variety of biological activities, including immunomodulation, antioxidant, anti-tumor, and anti-aging activities, among other and hence It is commonly used to treat fatigue, renal and pulmonary dysfunction, hyperglycemia, hyperlipidemia, and heart disease, such as arrhythmia.

C. militarisfruiting bodies contains the wide range of active metabolites but mainly reported are cordycepin, adenosine, Beta glucan and polysaccharides. The cordycepin (3'-deoxyadenosine), an indexed components of C. militaris, is a nucleoside analogue and is considered as anucleic acid antibiotic that might impede thecells canceration. The wide range of reported pharmacological effects of Cordycepin, such as anti-inflammatory, antiviral, anticancer, anti-leukemia, antidiabetic, antitumor, anti-obesity effects, immunomodulation etc. have been revealed the possibility of Cordycepin as a preventive material for mankind uses.

Adenosine, an endogenous purine nucleoside, is another major component of C. militaris fruiting bodies. It acts as an extracellular signalling molecule and proven as potent negative inotropic as well as coronary vasodilator agent. Also, act as raw material for ATP molecule synthesis.Further the Ministry of Food safety, 2017 (http://www.law.go.kr/admRulInfoP.do?.admRulSeq=2100000107137) has recognized the fungal ß-glucan as functional ingredients that can help improve immune function.

To extract these potential metabolites from C. militaris, decoction, reflux distillation, probe assisted ultrasonic extraction, high pressure, microwave extraction, enzyme assisted cell disruption etc. Also, these methods need to maintain various factors such as temperature, pressure, precise material-solvent. Though, these methods have adverse effect on the various low molecular weight active metabolites and hence the quality yield of extract ismoderately low. Moreover, these techniques are fairly dependents on the chemical solvents as ethylAlcohol n-butyl alcohol, Benzene, Hexane, Petroleum ether, methanol, acetonitrile, acetone, chloroform, ethyl acetate as well as other non-food-grade chemical reagents which traces can be available as a toxic impurity with the active ingredients.Hence, such methodologies create a hidden hazards to mankind.

With the above background, it is clear that a chemical free extraction method is in higher requirements to increase the production of quality extract, a hidden treasure of C. militaris, with cost effective, rapid and chemical free manner. Here some of the potential embodiment of C. militaris extraction is given below:

There exists in the art examples in the patent 201410199236 has described the procedure forextraction of cordycepin from medicinal fungi Cordyceps militaris via alcohol solvent methodology. It contains the steps of cordyceps in extraction, then dewaxing of concentrate form of extract, their purification, membrane-based extraction and finally refining and its drying. The overall embodiment is multistep process and based on the organic solvent.

Another example of extractive-rich cordyceps militaris extract under the patent CN103766545A has claimed the availability of polysaccharides within the range of 32.30-60.32mg/g. Extraction procedure was based on ethanol concentration ranges 20%-95%.

Similarly, an ethanol-based polysaccharides precipitation method from Cordyceps fruiting bodies was described in the patent CN107501428. This method leads by the decoction, Soxhlet method, microwave radiation as well as ultrasonic waves. Overall process is done with the alcohol or alcohol and water. Moreover, the ultrasonication is done with the double-aqueous phase system of Ammonium Sulfate and Polyethylene Glycol 6000 (PEG 6000) Liquid-solid extraction.

Patent AU2020102126A4 has revealed the preparation method of Cordyceps militaris Extract and described their potentiality as a functional food. This embodiment is based on the leaching of Cordyceps militaris and further the filtration of aqueous portion via microporous resin. Repetition of process done to enhance the recovery which is based on the Executing liquid chromatography. Further, the total filtrate was treated with alcohol to precipitate the targeted metabolites.

Patent CN103919712A disclosed the warm extraction process for the preparation of C. militaris extract and indicated its uses in the cosmetics. The process comprises the repeated treatment under the temperature range 35-80 ? for 6-24h then filtration and lyophilization. Further, the invention described its uses as raw material in the beauty cream, mask, skin whitener, frost or essence etc.

Patent 201410041854.4 displayed the techniques of extraction from C. militaris by freeze pulverization and then extraction of crude by acetone treatment. Elution of active compounds were performed by the liquid chromatography with the elution agent n-hexane (100%), ethyl acetate-n-hexane (25%), and ethyl acetate (100%). Though, the uses of these elution agent can be problematic in impurities removal.

The patent application 20190374590 has disclosed the extraction of C. militaris with ethanol as solvent and its application in the treatment of renal disease. The embodiment was validated by the in-vitroeffect of C. militaris extract containing cordycepin has profound effect on nephrin protein level, smooth muscle actin protein level in AB8/13 human podocytes and renal interstitial fibrosis, renal extracellular matrix accumulation and glomerular fibrosis level in mice.

Another patent US20140255385 revealed the treatment of glioblastoma by ingredient (0.5 mg/mL or 1.5-2.5 mg/mL) of cultivated C. militaris.

OBJECTIVE OF THE INVENTION

The main objective of the invention is to provide a method of extract preparation of cordyceps militaris.

Further object of the present invention is to provide a natural, chemical free, room temperature based, large shelf life, concentrate, enriched with high concentration of cordycepin, adenosine and beta-glucan having low-cost based C. militaris extract which is safe for human use.

Another objective of the present invention is to provide a process of preparation of said C. militaris extract which shows versatility in the further application as food.

Yet another objective of the present invention is to provide a method of preparation of said embodiment is in the form of semi-solid, 100% water soluble extract.
A further objective of the present invention is to provide a method of preparation of said C. militaris extract without any chemical/ alcohol base.

Added objective of the present invention is to revealed the efficient solution for the treatment of obesity and indicate its potential application for the treatment of vitality, enhancement of endurance and several other in human.

Further, the abundance of wide range of metabolites revealed its probable application in comestology.

This and other objects and characteristics of the present invention will become apparent from the further disclosure to be made in the detailed description given below.

SUMMARY OF THE INVENTION

Accordingly following invention providesa method of extract preparation of cordyceps militaris. The principle of present embodiment is to create the conditions at room temperature to increase the exposure to low osmotic pressure that cause water to quickly enter in to the cells and rapture it by the ultrasonic sound waves. Further, the water-soluble,low size metabolites will be taken out by high-speed centrifugation and rest pellets will be treated again with a quantified sonication wavewith a desired quantity of water. Further, the big size metabolites and interphase shall be harvested by different sets of centrifugations. At the end all the harvest diluents will be collected in the large surface area vessel to remove the maximum water part.

BRIEF DESCRIPTION OF DRAWING

This invention is described by way of example with reference to the following drawing where,

Figure 1 of sheet 1 shows HPLC data of cordycepin and adenosine concentration in the C. militaris extract CXT-91.

Figure 2 of sheet 1 shows cell toxicity assay performed on kidney cell line.

Figure 3 of sheet 2 shows A: Staining of lipid droplets by Oil Red -O-stain after conversion of preadipocyte into mature adipocyte without extract. B: Staining of lipid droplets by Oil Red -O-stain after conversion of preadipocyte into mature adipocyte with extract (100 µl/ml).

In order that the manner in which the above-cited and other advantages and objects of the invention are obtained, a more particular description of the invention briefly described above will be referred, which are illustrated in the appended drawing. Understanding that these drawing depict only typical embodiment of the invention and therefore not to be considered limiting on its scope, the invention will be described with additional specificity and details through the use of the accompanying drawing.

DETAILED DESCRIPTION OF THE INVENTION:

The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. If the specification states a component or feature “may”, “can”, “could”, or “might” be included or have a characteristic, that particular component or feature is not required to be included or have the characteristic.

As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

The present discloser relates, in general toa method of extract preparation of cordyceps militaris.

The 45-65 days old C. militaris fruiting bodies (Fresh or dried) have been taken and pulverized in to the paste or coarse powder form with mesh size range 20-100.

Further, the processed material was soaked and mixed well with the40%-90% volume of distilled water or pure water, or both, in the automatic blender or in the beaker and hold it for 2H-24H at room temperature. Then, mixture was sonicated in the sonication tank at 12 kHz to 400 kHzfor 5 min-2h, then hold for 5min-2h and again repeated the same process.

Further, the sonicated material was centrifuged at 6000RPM-14000RPM for 3 min-15 min to sediments the insoluble particles. The aqueous part was taken carefully in the cleaned or sterilized pot. Sedimented part was collected again and re-saturated with the distilled wateror pure wateror both,with the volume of 25%-200% and blended or mixed for 5-15 min. Mixed material was hold for 1h-15H at room temperature and was sonicated in the sonication tank at 40 kHz to 800 kHzfor 5 min-2h, then harvested in the centrifuge for 5-12 min at 4000RPM-12000 RPM for 2-12min to separate the insoluble material.

Further, the aqueous as well as interphase layer was collected very carefully to avoid any type of sedimented materials. The harvested solution was further filtered with the 10micron to 100micron size filtration unit. Then all the liquid solution were kept in the cleaned or sterilized pot. Further, the maximum water portion was remover by vacuum drying or with airdrying with mild temperature range 20 -37.

The obtained gel type material or semisolid concentrate material has further stored in the cleaned or sterilized or both, in vessel or container made from wood, glass, steel or foodgrade plastic. The storage temperature of C. militaris extract concentrate is -20 to 28.

Quantification of cordycepin and adenosine contents in present invention was estimated by National Accreditation Board for Testing and Calibration Laboratories (NABL) accredited laboratory,Farelabs, Gurugram, India (Reference number FL/OT/SL/05052022-006). The in-house protocol (data not shown) was followed with the injection volume of 20µl for 10min with process channel 2998_chl_259nm@1.2nm and plotted against the standard cordycepin and adenosine (Fig. 1). The results revealed the highest concentration of cordycepin 16.83mg/gram as well adenosine 9.28mg/gram.

The Beta-glucan in the C. militaris extract was evaluated as 41.15% (data not shown) by the methodology of National Accreditation Board for Testing and Calibration Laboratories (NABL) accredited laboratory,Farelabs, Gurugram, India (Reference number FL/SOP/FC-185).

Kidney cell linecytotoxicity assay:

Human embryonic kidney cells (HEK293) were used for evaluation of cytotoxicity of Cordyceps militaris extract at different concentration.The assessment was carried out by the MTT assay (calorimetric assay). The principle involved in the procedure is the capability of reducing MTT into colored formazan by the viable cells,and the amount of formazan produced was a marker for cell viability.

HEK cell lines were cultured in DMEM supplemented with 10% fetal calf serum and 1% antimycotica-antibiotic mixture culture plates at 37°C and 5% CO2-humidified incubator. Cells seeded at a density of 1?×?105cells/well in 96-well plate for 24h; then, cells were washed and incubated in fresh medium. C. militaris extracts (CXT-91A, CXT-9100A) were added at equivalent concentration of 500 µg and 1000 µg to triplicate wells and kept for 24h, after which cells were washed three times with PBS. After washing, 20µL of MTT solution (5-mg/mLstock solution) was added to each well, and cells were then incubated for additional 4h.

The un-reacted MTT dye and medium were aspirated off,and 100µL of DMSO was added to each well to ensure solubilization of formazan crystals. The contents of the plates were mixed for 15min to achieve complete solubilization of the formazan crystals, and the measurement of optical density was carried out at 570nm with a micro plate spectrophotometer (MRX Micro plate Reader, Dynatech Laboratories Inc., Chantilly, VA, US) at 570nm. Data comparison with control was displayed in the fig. 2.

Application of C. militaris extract CXT-91 as an anti-obesity ingredients, the mouse 3T3-L1preadipocyte cell line was used to displayed the inhibitory effect of a CXT-91 extract on adipocyte differentiation in 3T3-L1 cells, in the present embodiment. It is because of the clear consideration of the effect of adipogenesis has been considered a vitalfactor for the development of anti-obesity agents. The Adipogenesis subsidizes to surplus fat accumulation in adipocytes through the differentiation of preadipocytes.

In this invention, the 3T3-L1 preadipocyte cells were induced by adding of 0.5 mM 3-isobutyl-1 mM dexamethasone, 1-methylxanthine, and 1 µg/mL insulin. Lipid-rich vacuoles seeming in the differentiated adipocytes were histologically stained with Oil Red O because it is a reliable indicator of adipogenesis and is used in the qualitative and quantitative valuation of adipocyte differentiation in 3T3-L1 preadipocyte cells. Treatment of3T3-L1 preadipocyte cells with CXT-91 extracts ofconcentration of 100µl/ml displayed the significanteffect in the cell proliferation during the differentiation period of 8 days in 3T3-L1 preadipocytes (Fig. 3 a,b). The number of Oil Red O staining was increased by inducing adipocyte differentiation but was reduced upon CXT-91 treatment (Fig 3 a, b). In the control, the mean value was 0.9760±0.0160 but in the CXT treated cell line the mean value was 0.7150±0.0250. Which revealed the >30% adipogenesis property of the present invention. These results suggested that CXT-91 treatment prevented adipocyte differentiation in 3T3-L1 preadipocytes.
, Claims:1. A method of extract preparation of Cordyceps Militaris; consisting of following steps;

a) Pulverizing 45-65 days old Cordyceps Militaris fruiting bodies into the paste or coarse powder form with mesh size range 20-100;

b) Soaking and mixing the processed material with the 40% - 90% volume of distilled water, in the automatic blender and holding it for 2H-24 H at room temperature;

c) Sonicating the mixture in the sonication tank at 12 kHz to 400 kHzfor 5 min-2hour, then holding for 5min-2 h and again repeating the same process;

d) Further, the sonicated material centrifuging at 6000RPM-14000RPM for 3 min-15 min to sediments the insoluble particles, the aqueous part separated in the cleaned or sterilized pot;

e) Collecting the sedimented part and re-saturating with the distilled water with the volume of 25%-200% and blending for 5- 15 min;

f) Holding mixed material for 1 H – 15 H at room temperature and sonicating in the sonication tank at 40 kHz to 800 kHzfor 5 min-2h, then harvesting in the centrifuge for 5-12 min at 4000RPM-12000 RPM for 2-12 min to separate the insoluble material;

g) Further, the aqueous as well as interphase layer collecting to avoid any type of sedimented materials and the harvested solution filtering with the 10micron to 100 micron size filtration unit;

h) Then keeping all the liquid solution in the cleaned pot and the maximum water portion remove by vacuum drying with mild temperature range 20 -37; and

i) The obtained gel type material or semisolid concentrates material storing in the cleaned in vessel made from wood, glass, steel or foodgrade plastic at the storage temperature of -20? to 28 ?.