KR20180015873A - composition for improving wrinkle or elasticity comprising the extract of beluga lentil - Google Patents

Abstract

The present invention relates to a wrinkle or elasticity recovering composition containing a beluga lentil extract as an active ingredient, and more particularly, to a composition containing a beluga lentil extract as an active ingredient which increases expression of adiponectin, SPEBP1 and PPAR-γ to promote differentiation of adipocytes, thereby being confirmed to have an excellent effect of recovering wrinkles or elasticity. In addition, the composition containing the beluga lentil extract as an active ingredient of the present invention is useful as a pharmaceutical composition for preventing or treating wrinkles, a health food composition for recovering wrinkles or elasticity, a composition for would healing and plastic surgery, and a composition for treating hair loss or promoting hair growth.

Description

TECHNICAL FIELD The present invention relates to a composition for improving wrinkles or elasticity comprising a belluga lentil extract as an active ingredient, a composition for improving wrinkle or elasticity comprising the extract of beluga lentil,

The present invention relates to a wrinkle or elasticity improving composition containing bellucca lentil extract as an active ingredient, and more particularly to a composition for improving wrinkle or elasticity by promoting differentiation of adipocytes containing belluga lentil extract as an active ingredient ≪ / RTI >

The skin, which is the largest organ of the human body, occupies about 16% of the whole human body volume. The skin is composed of epidermal layer, dermal layer and subcutaneous fat layer and is in direct contact with the external environment and can be used for a variety of applications including temperature and humidity change, ultraviolet rays, harmful microorganisms, It is an organ that plays a primary protective role in protecting the internal organs from environmental stimuli and at the same time is responsible for the biochemical functions necessary for metabolism of the whole body. Particularly, the subcutaneous fat layer existing under the dermis is composed of adipocyte lobules, and serves as a cushion for protecting internal organs of the skin by absorbing shocks from external stimuli, preventing heat damage, (triacylglycerol) and stored in the form of a nutrient storage that functions as a source of energy when needed. However, as the skin ages with aging, natural factors and various external stimuli damage the skin. As a result, the skin cells do not function properly, and wrinkle formation, elastic loss and skin atrophy A phenomenon occurs.

The basic structure of the skin is maintained by subcutaneous fat tissue, and this subcutaneous fat tissue plays a role in the volume and intensity of the skin. Therefore, it is better to increase the volume of adipose tissue than to give elasticity to the dermis or epidermis in the subcutaneous layer. Adipocytes secrete a variety of cytokines, particularly adiponectin and leptin, which increase the synthesis of hyaluronic acid and collagen from fibroblasts in skin dermal fibroblasts, It was reported to promote healing. Since the preadipocyte is differentiated into adipocytes by differentiation inducers such as insulin, it is known that the pre-adipocyte activity is different from that of the adipocyte Recently, it has been widely used in researches for developing substances promoting the differentiation of adipocytes for the purpose of promoting volume and elasticity of facial skin, skin regeneration and wound healing.

In addition, the fact that adipocytes in the subcutaneous tissues of the scalp plays a key role in hair growth has recently become a new method of treating hair loss. It is known that during the period of new hair generation during hair cycle, new adipocytes are created in the subcutaneous tissues to send a hair-generating signal to hair follicle cells. In animal experiments, mice that are deficient in adipogenesis function, Studies have shown that hair follicles are not regenerated. About 75% of the total hair loss population is arthropod alopecia because hair scar is not formed and hair follicle is maintained. Therefore, hair regeneration is possible by promoting differentiation of adipocytes in subcutaneous tissue after symptom improvement. Thus, promotion of adipocyte differentiation in the subcutaneous tissues induces hair regeneration by promoting hair regeneration and hair follicle cell growth, and further activates fibroblast by cytokines secreted by adipocytes and improves scalp by increasing collagen synthesis, thereby preventing hair loss Or promoting hair growth.

Lens culinaris , recently known as superfood, is a plantaceous herbaceous plant ( Lens culinaris or L. esculenta ) that grows well in low precipitation and dry climates, and is one of the foods consumed mainly in Africa, the Middle East and South Asia. The color of the seed and the color of the flower vary according to the variety of the lentil (lentil), and the folate, thiamine, phosphorus, iron, carbohydrate, dietary fiber ) And vitamins (vitamins) are evenly contained. It is also known that the content of protein and fiber is high, there is little fat, and the starch content is about 50%.

Lentil has been reported to not only help maintain blood sugar due to high dietary fiber content but also regulate cardiovascular function due to blood pressure control. In addition, flavanol, flavonol, soybean saponins, Phytic acid, and condensed tannin, which are known to be useful in the art. The results of a study on the possibility of cancer prevention of lentil showed that consumption of boiled lentil was effective on azooxymethane-induced early colorectal cancer and polyphenol composition and antioxidant activity It has been reported that it can be regarded as a functional food for helping chronic diseases.

In addition, Lentil beans are based on color, green lentil, masoor lentil, french lentil and beluga lentil. Green lentil beans contain shells, Mossur Lentil beans are brown in color and orange in color. French lentil beans are brown in color and yellow in color. Beluga lentil beans are black in color and yellow in color. Among them, belluga lentil bean shows differentiation promoting activity of adipocytes, and it shows the activity of promoting the differentiation of adipocytes. It shows a cosmetic composition for improving skin wrinkles and elasticity, a pharmaceutical composition for preventing or treating wrinkles, a wound healing and molding composition, a health food composition for improving wrinkles or elasticity, A composition for promoting hair growth and a composition for treating cancer.

Japanese Patent Application Laid-Open No. 2003-514001 (published on April 15, 2003)

The present invention relates to a composition for improving wrinkle or elasticity by promoting differentiation of adipocytes containing an extract of beluga lentil as an active ingredient, and a composition comprising the belluga lentil extract as an active ingredient is adiponectin, SPEBP1 (SRE-binding protein 1) and PPAR-γ (peroxisome proliferator-activated receptor-γ), thereby promoting differentiation of adipocytes.

Also, the composition containing the bellucca lentil extract of the present invention as an active ingredient is useful as a pharmaceutical composition for preventing or treating wrinkles, a health food composition for improving wrinkles or elasticity, a composition for wound healing and molding, and a composition for improving hair loss or promoting hair growth .

In order to achieve the above object, the present invention provides a cosmetic composition for improving wrinkles or elasticity, a pharmaceutical composition for preventing or treating wrinkles, a health food composition for improving wrinkle or elasticity, a wound healing composition containing a beluga lentil extract as an active ingredient, And a composition for molding and a composition for promoting hair loss improvement or hair growth.

The present invention also relates to a process for the preparation of a composition comprising i) washing, drying and pulverizing belluccales; ii) culturing the washed, dried and pulverized bovalgantil with an aqueous methanol solution and shaking it; iii) extracting the extract by repeating the step ii) twice to five times; iv) filtering the extract on a filter paper; V) Concentrating the filtered extract under reduced pressure; And vi) lyophilizing the reduced-pressure concentrated extract.

The present invention relates to a composition for improving wrinkles or elasticity, comprising a belluga lentil extract as an active ingredient, and more particularly, to a composition for improving wrinkle or elasticity comprising a bellucca lentil extract as an active ingredient, To promote adipocyte differentiation, thereby confirming that the wrinkle or elasticity improving effect is excellent.

Also, the composition containing the bellucca lentil extract of the present invention as an active ingredient is useful as a pharmaceutical composition for preventing or treating wrinkles, a health food composition for improving wrinkles or elasticity, a composition for wound healing and molding, and a composition for improving hair loss or promoting hair growth .

FIG. 1 shows the results of confirming the promotion of adipocyte differentiation after treatment of differentiation-inducing factors in pre-adipocytes and treatment of lentil soybean extracts by type and concentration.
FIG. 2 shows the results of pretreatment of precursor adipocytes with differentiation-inducing factors and treatment of bellulandil soybean extract with different concentrations, followed by oil-red-O staining for promotion of adipocyte differentiation.
FIG. 3 is a result of western blotting to measure the expression of adiponectin release and differentiation-related proteins secreted from adipocytes treated with lentil soybean extract.

The inventors of the present invention produced bellugalante extract by washing, drying and pulverizing bellugal, then repeatedly shaking culture for 24 hours with 80% (w / v) methanol aqueous solution 10 times, By analyzing the functional components and confirming the promotion effect of adipocyte differentiation in human preadipocyte, the present inventors confirmed the function of improving wrinkles or elasticity and completed the present invention.

As used herein, the term " adipocyte differentiation " refers to the differentiation of mammalian adipose precursor cells or mesenchymal stem cells, most preferably mesenchymal stem cells derived from human bone marrow, into adipocytes will be.

The term "extract" used in the present invention refers to an extract obtained by extracting a plant, a diluted solution or concentrate of the extract, a dried product obtained by drying the extract, a controlled preparation or a purified product of the extracted solution, Extracts themselves and extracts of all formulations which can be formed using extracts. The extract of the present invention can be preferably prepared in the form of a dry powder after extraction.

The present invention provides a cosmetic composition for improving wrinkle or elasticity, which comprises a beluga lentil extract as an active ingredient. Specifically, the belluga lentil extract can be extracted with water, a C1 to C4 alcohol or an aqueous solution thereof, and can promote adipocyte differentiation to improve wrinkles or elasticity.

When the composition of the present invention is a cosmetic composition, the cosmetic composition may contain conventional additives such as stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers in addition to the active ingredients. In addition, the cosmetic composition may further include a skin absorption promoter to promote its effect.

The formulations of the cosmetic composition may be in any form conventionally prepared in the art and may be in the form of lotions, milks, skins, toners, emulsions, lotions, essences, sunscreens, creams, makeup bases, Hair conditioner, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nutrition cream, hair conditioner, hair conditioner, powder, pack, gel, shampoo, rinse, spray, make-up remover, Hair Moisture Cream, Hair Massage Cream, Hair Wax, Hair Aerosol, Hair Pack, Hair Nutrition Pack, Hair Soap, Hair Cleansing Foam, Hair Oil, Hair Drying Agent, Hair Preservative, Hair Dye, Hair Dye, Hair Decolorant, Hair Gel, Hair Such as, but not limited to, glaze, hair dresser, hair lacquer, hair moisturizer, hair mousse, and hair spray, Specify.

When the formulation is a paste, cream or gel, an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component .

When the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, chlorofluorohydrocarbons, propane / Or propellants such as dimethyl ether.

When the formulation is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, - butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, a microcrystalline cellulose , Aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

The present invention also provides a pharmaceutical composition for prevention or treatment of wrinkles containing a beluga lentil extract as an active ingredient.

When the composition of the present invention is a pharmaceutical composition, it may contain a pharmaceutically acceptable carrier in addition to the above-mentioned active ingredients. Such pharmaceutically acceptable carriers are those conventionally used in pharmaceutical preparations, and include lactose, dextrose, The present invention relates to a process for the preparation of a medicament for the treatment and / or prophylaxis of cancer, comprising administering a therapeutically effective amount of a compound selected from the group consisting of cross, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, Carboxymethylcellulose, carboxymethylcellulose, hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. The pharmaceutical composition may further contain a filler, a weight agent, a binder, a lubricant, a wetting agent, a disintegrant, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a fragrance, a preservative and the like as an additive and an auxiliary agent.

The pharmaceutical composition may be formulated into tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, nonaqueous solutions, emulsions, lyophilized preparations, suppositories and sterile injectable solutions. The pharmaceutical composition of the present invention may be administered orally or parenterally such as intravenously, intraarterally, intraperitoneally, intramuscularly, intrasternally, subcutaneously, intradermally, intranasally, intrapulmonary, rectally, topically, RTI ID = 0.0 >

The method of administration of the pharmaceutical composition is determined depending on the severity of symptoms, and local administration is generally preferred. The effective amount of the above-mentioned effective ingredient of the above pharmaceutical composition means an amount required for prevention or treatment of the disease. Accordingly, the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, But are not limited to, various factors, including, for example, the rate of administration, the rate of administration, the duration of the treatment, the drugs used concurrently.

The present invention also provides a health food composition for wrinkle or elasticity improvement comprising a beluga lentil extract as an active ingredient.

The health food composition may be provided in the form of powder, granules, tablets, capsules, syrups or beverages. The health food composition may be used in combination with other food or food additives other than the active ingredient, . The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.

The effective dose of the active ingredient contained in the health food composition may be used in accordance with the effective dose of the pharmaceutical composition. However, in the case of long-term consumption intended for health and hygiene purposes or health control purposes, And it is clear that the active ingredient can be used in an amount exceeding the above range since there is no problem in terms of safety.

There is no particular limitation on the type of the health food, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.

The present invention also provides a wound healing and molding composition comprising a beluga lentil extract as an active ingredient.

The present invention also provides a composition for improving hair loss or promoting hair growth, comprising a beluga lentil extract as an active ingredient.

The composition for wound healing and molding, and the composition for improving hair loss or promoting hair growth can be used as a cosmetic composition, a pharmaceutical composition, and a health food composition.

The present invention also relates to a process for the preparation of a composition comprising i) washing, drying and pulverizing belluccales; ii) culturing the washed, dried and pulverized bovalgantil with an aqueous methanol solution and shaking it; iii) extracting the extract by repeating the step ii) 2 to 5 times; iv) filtering the extract on a filter paper; V) Concentrating the filtered extract under reduced pressure; And vi) lyophilizing the reduced-pressure concentrated extract.

Preferably, the aqueous methanol solution in step ii) may be, but is not limited to, an aqueous 70 to 90% (w / v) methanol solution. If the methanol aqueous solution is out of the above range, the production yield of bellucca lentil extract may be lowered, which may be undesirable.

Preferably, in step ii), the weight ratio of the bergulanate extract to the methanol aqueous solution may be 1: 5 to 1:15, but not limited thereto. When the mass ratio of the belluga lentil extract to the aqueous methanol solution is less than 1: 5, the amount of the aqueous methanol solution required is not sufficient. It may be undesirable from the viewpoint of cost.

Preferably, step ii) may be performed at 15 to 60 < 0 > C for 20 to 30 hours, but is not limited thereto. If the reaction temperature and time are out of the above range, the reaction rate may be significantly lowered and the yield of the bergalgantile extract may be lowered, which may be undesirable.

Preferably, step (iii) may be repeated two to five times, but is not limited thereto.

Preferably, step (v) may be, but is not limited to, treatment at 45 to 65 ° C for 40 to 60 hours. If the reaction temperature and time are out of the above ranges, there may be a problem in that the concentration of reduced pressure of the belluccalente extract may be lowered, which may be undesirable.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  One : Lentil beans  Preparation of extract

In order to evaluate the activity of promoting adipocyte differentiation of Bellulase lentil, we used belluga lentil from the right herb and used it in the experiment. The benthic lentil was washed, dried and pulverized, and then an 80% (w / v) aqueous methanol solution was added 10 times, followed by shaking culture for 24 hours for 3 times to extract the lentil bean extract. The extract was filtered using a filter paper (Whatman No. 3, England) and concentrated under reduced pressure at 55 ° C using a rotary vacuum evaporator (UNI TRAP UT-1000, Eyela, Tokyo, Japan) Lt; / RTI > The extracts were stored in powder form at -20 ° C.

Example 2: 3T3 -L1 Adipose precursor cell  Differentiation induction and sample treatment

The 3T3-L1 cells used in the experiments were purchased from ATCC (Manassas, VA, USA). 3T3-L1 cells were differentiated into adipocytes by treatment with differentiation inducers. DMEM medium (Dulbecco's modified eagle's medium high glucose, DMEM; Invitrogen, Carlsbad, Calif., USA ) Were supplemented with 10% bovine calf serum (BSA) and antibiotics and incubated in a 5% CO ₂ incubator at 37 ° C. When the cells become confluent, trypsin / ethylenediaminetetraacetic acid (EDTA) is applied, and the cells are collected and centrifuged (L-70, Bekman Coulter, Fullerton, CA, USA) . After that, the cells were divided into 6 wells at a density of 3 × 10 ⁵ cells / well. After 100 hours of saturation, the cells were allowed to stand for 48 hours. DMEM was supplemented with 10% elegance serum and a differentiation inducer, 23 mg / ml 3-isobutyl- 5 mg / ml insulin (Sigma-Aldrich, Chem. Co.), and 5 mg / ml insulin were added to a solution of 10 mg / The medium (MDI) supplemented with 1 mM dexamethasone (Sigma-Aldrich, Chem. Co.) was treated to induce differentiation for 48 hours. Subsequently, medium supplemented with 5 mg / ml of insulin in DMEM medium supplemented with 10% fetal bovine serum (FBS) was changed every two days. The treatment of the sample was carried out from the time of addition of the differentiation inducing medium.

Example  3: Oil-red-O staining

3T3-L1 precursor adipocytes were differentiated for 7 days, and the medium was removed, washed with phosphate buffered saline (PBS), and fixed with 10% formaldehyde solution. After washing with phosphate-buffered saline, the oil-red-o solution was added and stained for 30 minutes at room temperature. The oil-red-o solution was removed, washed with distilled water and dried. Thereafter, observation was made using a phase contrast microscope, and lipids stained with 100% isopropyl alcohol were dissolved and the absorbance was measured at 510 nm.

Example  4 : Adiponectin protein  Expression measurement

Western bolt was performed to measure adiponectin release amount and differentiation-related protein expression of adipocyte by Veluga lentil treatment. The supernatant and cells of the sample-treated cells (7th day of differentiation) were taken to quantitate the proteins. SDS-polyacrylamide gel electrophoresis was performed at 125V using 10% running gel and 4.5% SE260, Amersham Biosciences, Uppsala, Sweden). The protein separated by electrophoresis was immobilized at 350 mA for 120 minutes using transfer buffer (20% methanol, 25 mM Tris-HCl, 192 mM glycine) to immobilon-P transfer membrane ; PVDF transfer membrane, Port Washington, NY, USA). The membrane was blocked with 5% non-fat dry milk (skim milk solution). To investigate the extent of primary antibody expression at 4 ° C, the cells were diluted 1: 1000 in TBST (tris-buffered saline and tween 20) solution, reacted for 24 hours, and washed three times with TBST. Subsequently, the secondary antibody was diluted 1: 2000 in TBST solution for 2 hours, reacted and washed three times with TBST. After washing once more with distilled water, the mixture was mixed with 1: 1 mixture of coloring reagents I and II of ECL detection kit (WSE-7120s, ATTO Co., Tokyo, Japan) X-ray film; CP-G plus, Agfa Healthcare Ltd, New Orleans, LA, USA) and then observed the degree of protein expression on the film.

Experimental Example  One : Beluga Lentil  Adipocyte differentiation inducing activity

The effect of differentiation inducers such as insulin, dexamethasone, isobutylmethylxanthine and four kinds of lentil extracts (beluga, french, green, and masoor lentil) at 10 and 20 μg / As shown in FIG. 1, when the lentil was treated with different concentrations of lentil, the differentiation promoting effects were 136, 127, 116, and 119% at a concentration of 10 μg / ml. However, when treated at a concentration of 20 μg / ml, the lentil extracts except bellugalantol decreased the differentiation, and the bellugalantil extract showed a high promoting effect of 164% on the differentiation. As a result of treatment with pregelatinized bellucca lentil extracts at 5, 10 and 20 μg / ml, the differentiation promoting effect was 128, 136 and 160%, respectively. And the highest adipocyte differentiation activity of lentil was confirmed.

Experimental Example  2 : Beluga Lentil Adiponectin  And adipocyte transcription factor

Adiponectin is a typical protein hormone secreted by adipocytes. It has been reported that the expression level of adiponectin increases with the differentiation of adipocytes. Thus, the amount of adiponectin, which is an index of promotion of adipocyte differentiation, was secreted into a cell culture medium. As shown in FIG. 2, when adiponectin secreted into cell culture medium was observed when adipogenic factors and bellulagen were administered to precursor adipocytes, the concentration of adiponectin when treated with belugalantil was higher than that of the differentiated control Adiponectin expression was also increased.

In addition, differentiation of adipocytes is expressed in the process of differentiation of adipocytes into transcription factors acting on the regulatory regions of genes that are specifically expressed in adipose tissue cells. As a result, the expression of SREBP1, PPAR-γ and C / EBP alpha was increased, and SREBP1 and PPAR-γ The results showed that the lipolytic activity of bellugal was enhanced.

Therefore, the belluga lentil extract is useful as a cosmetic composition for improving skin wrinkle or elasticity through promotion of adipocyte differentiation, a pharmaceutical composition for preventing or treating wrinkle, a wound healing and molding composition, a health food composition for improving wrinkle or elasticity, And a cancer treatment composition, and confirmed the possibility as a main active material for the development of the above-mentioned composition for various uses.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (12)

A cosmetic composition for improving the wrinkle or elasticity comprising an extract of beluga lentil as an active ingredient. The cosmetic composition according to claim 1, wherein the bellugenta extract is extracted with water, a C1 to C4 alcohol or an aqueous solution thereof. The cosmetic composition for improving wrinkles or elasticity according to claim 1, wherein the bellugenta lentil extract promotes adipocyte differentiation and promotes skin elasticity. A pharmaceutical composition for the prevention or treatment of wrinkles containing belluga lentil extract as an active ingredient. A health food composition for improving wrinkle or elasticity comprising an extract of beluga lentil as an active ingredient.  A composition for wound healing and molding comprising a beluga lentil extract as an active ingredient. A composition for enhancing hair loss or promoting hair growth comprising an extract of beluga lentil as an active ingredient. i) washing, drying and crushing the beluga lint;
ii) culturing the washed, dried and pulverized bovalgantil with an aqueous methanol solution and shaking it;
iii) extracting the extract by repeating the step ii) 2 to 5 times;
iv) filtering the extract on a filter paper;
V) Concentrating the filtered extract under reduced pressure; And
vi) lyophilization of the reduced-pressure concentrated extract. The method according to claim 8, wherein the methanol aqueous solution in step ii) is 70 to 90% (w / v) methanol aqueous solution. [Claim 9] The method according to claim 8, wherein the weight ratio of the bellucose lentil extract to the aqueous methanol solution is 1: 5 to 1:15. 9. The method according to claim 8, wherein the step ii) is carried out at 15 to 60 DEG C for 20 to 30 hours. 9. The method according to claim 8, wherein the step v) is carried out at 45 to 65 DEG C for 40 to 60 hours.

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