Cordyceps militaris fermentation product with anti-inflammatory effect as well as preparation method and application thereof

Abstract

The invention discloses a cordyceps militaris fermentation product with an anti-inflammatory effect as well as a preparation method and application thereof. Cordyceps militaris is fermented to obtain cordyceps militaris fermentation liquor, the cordyceps militaris fermentation liquor is separated to obtain mycelia and supernate, the mycelia are smashed and then extracted with water and ethanol respectively, mycelium water extract and mycelium alcohol extract are obtained, and the cordyceps militaris fermentation product, the mycelium water extract and the mycelium alcohol extract are the cordyceps militaris fermentation product. The invention finds that the fermentation product obtained after optimization of the culture condition has excellent performance in anti-inflammatory efficacy, and can significantly reduce the generation of inflammatory factor NO, so that the liquid fermentation of cordyceps militaris can be applied to the industry of research, development and production of soothing cosmetics to enhance the efficacy of the product.

Description

Technical Field:

[0001] The present invention belongs to the technical field of daily-use cosmetics, and particularly relates to a Cordyceps militaris fermentation product with anti-inflammatory effects, its preparation method and application. Background Art:

[0002] Cordyceps militaris, also known as Cordyceps sinensis var. militaris, Golden Grass, and Cordyceps flower, is a member of the Cordyceps family. This precious fungus parasitizes in the larvae in winter and grows by absorbing the nutrients of the larvae, resulting in the death of the larvae. In summer, the mycelium grows stroma from the corpse of the larvae, resembling a herbaceous plant, and is usually collected around the summer solstice. Cordyceps militaris is known as the "treasure among treasures" because of its rich nutritional and medicinal values and is widely used in traditional Chinese medicine. According to the "Outline of New Chinese Materia Medica", Cordyceps militaris "has a sweet taste and a neutral nature" and has the effects of "benefiting the lungs and kidneys, tonifying the essence, stopping bleeding and resolving phlegm".

[0003] Modern medical research also shows that Cordyceps militaris contains various active ingredients such as cordycepin, polysaccharides and adenosine. These ingredients exhibit significant biological activities in aspects such as anti-inflammatory, immunomodulatory and anti-tumor, and have multiple benefits for human health. The above-mentioned active substances can inhibit the production of inflammatory mediators such as NO, IL-1β, TNF-α and IL-6, thereby reducing the inflammatory response, can activate macrophages, stimulate the secretion of cytokines, and enhance the immune system's ability to regulate inflammation. The anti-inflammatory effects of Cordyceps militaris and its cells have important application values in the fields of medicine and cosmetics. Through liquid fermentation technology, the active ingredients of Cordyceps militaris can be efficiently obtained, providing strong support for the development of related products. Summary of the Invention:

[0004] The purpose of the present invention is to provide a Cordyceps militaris fermentation product with anti-inflammatory effects, its preparation method and application.

[0005] The first purpose of the present invention is to provide a preparation method of a Cordyceps militaris fermentation product, which is characterized in that: Cordyceps militaris is fermented to obtain a Cordyceps militaris fermentation broth, the Cordyceps militaris fermentation broth is separated to obtain mycelium and supernatant, the mycelium is pulverized and then extracted with water and ethanol respectively to obtain a mycelium water extract and a mycelium ethanol extract, and the Cordyceps militaris fermentation product, the mycelium water extract and the mycelium ethanol extract are the Cordyceps militaris fermentation product.

[0006] Preferably, the Cordyceps militaris fermentation broth is centrifuged and separated to obtain the supernatant and the precipitate; the supernatant is filtered through a filter membrane to obtain a Cordyceps militaris supernatant; the precipitate is dried to constant weight at 60°C, pulverized using a pulverizer and then ground with liquid nitrogen added, and the process of adding liquid nitrogen and grinding is repeated 3 - 4 times to obtain mycelium powder; the mycelium powder is weighed, pure water is added, and it is extracted at room temperature and then heated in a water bath for h, concentrated and filtered through a filter membrane to obtain a mycelium water extract; the mycelium powder is weighed, 70% ethanol is added, it is extracted at room temperature, and then ultrasonically extracted, and filtered to obtain a mycelium ethanol extract.

[0007] Preferably, the Cordyceps militaris fermentation broth is centrifuged to obtain the supernatant and the precipitate; the supernatant is filtered through a 0.22 μm filter membrane to obtain the Cordyceps militaris fermentation broth; the precipitate is taken, dried to constant weight at 60 °C, pulverized with a pulverizer and then ground with liquid nitrogen added, and the addition of liquid nitrogen for grinding is repeated 3 to 4 times to obtain the mycelium powder; the mycelium powder is weighed, pure water is added, and after extraction at room temperature for 30 min, it is placed in a water bath at 90 °C for heat extraction for 3 h. After centrifugation, the precipitate is extracted once again, and the filtrates obtained twice are combined. After concentration to 50% of the original volume at 80 °C, filtration through a 0.22 μm filter membrane gives the aqueous extract of the mycelium; the mycelium powder is weighed, 70% ethanol is added, and after extraction at room temperature for 60 min, it is placed in an ultrasonic cleaner and kept for 30 min, with an ultrasonic power of 240 W and a frequency of 40 kHz. After centrifugation at 10000 rpm for 10 min, filtration gives the ethanol extract of the mycelium.

[0008] The second object of the present invention is to provide a Cordyceps militaris fermentation product obtained according to the above preparation method.

[0009] The third object of the present invention is to provide the application of the above Cordyceps militaris fermentation product in the preparation of anti-inflammatory drugs.

[0010] The fourth object of the present invention is to provide an anti-inflammatory drug, which is characterized by containing the above Cordyceps militaris fermentation product as an active ingredient

[0011] The Cordyceps militaris is preferably Cordyceps militaris GDMCC 5.269.

[0012] The present invention discovers that the optimized Cordyceps militaris fermentation product has an anti-inflammatory effect and can significantly reduce the generation of the inflammatory factor NO. Therefore, the liquid fermentation of Cordyceps militaris can be applied to the industry of researching and producing soothing cosmetics to enhance the efficacy of the product. Description of the drawings:

[0013] Figure 1 It is the relative content of NO generated by LPS-induced RAW 264.7 by the optimized ethanol extract of the mycelium.

[0014] Figure 2 It is the relative content of NO generated by LPS-induced RAW 264.7 by the optimized aqueous extract of the mycelium.

[0015] Figure 3 It is the relative content of NO generated by LPS-induced RAW 264.7 by the optimized fermentation broth. Detailed implementation manners:

[0016] The following examples are further illustrations of the present invention rather than limitations thereof.

[0017] Example 1:

[0018] 1. Liquid fermentation culture of Cordyceps militaris

[0019] The culture conditions and components of Cordyceps militaris were optimized by single factor and orthogonal methods. The liquid fermentation conditions of Cordyceps militaris were obtained as follows: Fermentation medium: soluble starch concentration 35 g / L, yeast powder concentration 25 g / L, medium pH 6. Cordyceps militaris GDMCC 5.269 was inoculated into the fermentation medium, the liquid loading volume was 100 mL / 250 mL, the shaker speed was 200 r / min, and the culture temperature was 23 °C. The liquid strain was obtained by shake flask culture for 5 d under these conditions. The liquid strain was then inoculated into the fermentation medium at a volume fraction of 14%, with a liquid loading volume of 100 mL / 250 mL, a shaker speed of 200 r / min, and a culture temperature of 23 °C, and cultured for 5 d to obtain the Cordyceps militaris fermentation product.

[0020] 2. Preparation of Cordyceps militaris fermentation broth and extract

[0021] After culture, the fermentation product was centrifuged, and the supernatant was taken and filtered through a 0.22 μm filter membrane to obtain the Cordyceps militaris fermentation broth.

[0022] The precipitate, that is, the mycelium, was dried to a constant weight at 60 °C, pulverized with a pulverizer, and ground with liquid nitrogen added. The process of adding liquid nitrogen and grinding was repeated 3 - 4 times to obtain mycelium powder. Weigh 1 g of mycelium powder, add 20 mL of pure water, extract at room temperature for 30 min, then place it in a 90 °C water bath for heat extraction for 3 h. After centrifugation, the precipitate was extracted again, and the filtrates obtained twice were combined. After concentration to 50% of the original volume at 80 °C, it was filtered through a 0.22 μm filter membrane to obtain the mycelium aqueous extract. Weigh 1 g of mycelium powder, add 8 mL of 70% ethanol, extract at room temperature for 60 min, and place it in an ultrasonic cleaner for 30 min. The ultrasonic power was 240 W, the frequency was 40 kHz, and it was centrifuged at 10000 rpm for 10 min, and then filtered to obtain the mycelium ethanol extract.

[0023] 3. Determination of NO content

[0024] Adjust the cell density of macrophage RAW264.7 to 6.0×10 5 / mL, 200μL was added to each well of a 96-well plate and cultured at 37°C, 5% CO2 for 24h. The sample was adjusted to a safe and non-cytotoxic concentration range for detection. Samples and culture medium (35g / L soluble starch, 25g / L yeast powder, 1g potassium dihydrogen phosphate, 1g magnesium sulfate, water to 1L, natural pH) were added to each well of the sample group. The negative control group (NC) consisted of cells and culture medium, and the blank control group (BC) consisted of pure cell culture medium only. Each group was repeated three times. The sample group was pretreated with samples for 6h and finally treated with LPS for 24h. The supernatant was collected after treatment for the determination of the relative concentration of inflammatory mediator nitric oxide (NO).

[0025] Determination of NO relative content: According to the instructions of the nitric oxide kit, collect 50 μL of supernatant from each well, add 50 μL of reagent A and reagent B respectively, mix thoroughly, react in the dark for 10 minutes, detect the absorbance at 540 nm, record the results and calculate the relative content.

[0026] NO relative content % = A sample / ANC × 100%

[0027] Wherein, Asample refers to the absorbance value of the sample group, and ANC refers to the absorbance value of the negative control group (NC).

[0028] Experimental results:

[0029] Effects of different volume fractions of fermentation products on the relative content of NO produced by RAW 264.7 induced by LPS:

[0030] Human mouse macrophage RAW264.7 was inoculated on a 96-well cell culture plate and cultured in a CO2 culture chamber at 37°C for 24 hours. 0.1% to 20% of the corresponding Cordyceps militaris fermentation product was added, and the sample was pretreated for 6 hours, and finally treated with LPS for 24 hours. After treatment, the supernatant was collected and the relative concentration of inflammatory mediator nitric oxide (NO) was determined.

[0031] The results show that ( Figure 1 , 2 3), the product obtained in Example 1 has a good effect of inhibiting NO generation - anti-inflammatory effect, and the reduction in the amount of inflammatory mediator NO generated indicates that the inflammatory response is weakened and has a soothing effect. It has a significant effect of inhibiting NO generation at some volume fractions. The optimal volume fraction of mycelium alcohol extract for anti-inflammatory is 0.5% to 2%, the optimal volume fraction of water extract for anti-inflammatory is 1% to 20%, and the optimal volume fraction of fermentation broth for anti-inflammatory is 2% to 20%, which means that the method of using Cordyceps militaris extract and fermentation broth fermented with culture broth containing soluble starch can significantly enhance the anti-inflammatory and soothing effects of the product.

Claims

1. A method for preparing a Cordyceps militaris fermentation product, characterized in that: The Cordyceps militaris is fermented to obtain a Cordyceps militaris fermentation liquid, the Cordyceps militaris fermentation liquid is separated to obtain mycelium and a supernatant, the mycelium is crushed and extracted with water and ethanol respectively to obtain a mycelium water extract and a mycelium alcohol extract, and the Cordyceps militaris fermentation product, the mycelium water extract and the mycelium alcohol extract are the Cordyceps militaris fermentation product.

2. The preparation method according to claim 1, characterized in that: The Cordyceps militaris fermentation liquid is centrifuged to obtain a supernatant and a precipitate; the supernatant is filtered through a filter membrane to obtain a Cordyceps militaris supernatant; the precipitate is dried at 60°C to a constant weight, crushed with a powder grinder, and then added with liquid nitrogen for grinding, and the liquid nitrogen is added and ground for 3 to 4 times to obtain mycelium powder; the mycelium powder is weighed, pure water is added, and the powder is extracted at room temperature and placed in a water bath for hot extraction for 1 h, and the mycelium water extract is obtained by concentrating and filtering with a filter membrane; the mycelium powder is weighed, 70% ethanol is added, the powder is extracted at room temperature, ultrasonic extraction is performed, and the mycelium alcohol extract is obtained by filtering.

3. The preparation method according to claim 1, characterized in that: The Cordyceps militaris fermentation liquid is centrifuged to obtain a supernatant and a precipitate; the supernatant is filtered through a 0.22 μm filter membrane to obtain the Cordyceps militaris fermentation liquid; the precipitate is dried at 60°C to a constant weight, crushed with a powder grinder, and then ground with liquid nitrogen, and the grinding is repeated 3 to 4 times to obtain mycelium powder; the mycelium powder is weighed, pure water is added, and the powder is extracted at room temperature for 30 minutes, and then placed in a 90°C water bath for hot extraction for 3 hours, and the precipitate is extracted once after centrifugation, and the filtrates obtained twice are combined, concentrated to 50% of the original volume at 80°C, and filtered through a 0.22 μm filter membrane to obtain a mycelium water extract; the mycelium powder is weighed, 70% ethanol is added, and the powder is extracted at room temperature for 60 minutes, and then placed in an ultrasonic cleaner for 30 minutes, with an ultrasonic power of 240W, a frequency of 40kHz, and centrifuged at 10000rpm for 10 minutes, and filtered to obtain a mycelium alcohol extract.

4. The preparation method according to claim 1, characterized in that: The Cordyceps militaris is Cordyceps militaris militaris)GDMCC 5.

269.

5. A Cordyceps militaris fermentation product obtained according to the preparation method of claim 1, 2, 3, 4 or 5.

6. Use of the Cordyceps militaris fermentation product according to claim 5 in the preparation of anti-inflammatory drugs.

7. An anti-inflammatory drug, characterized in that: Contains the Cordyceps militaris fermentation product according to claim 5 as an active ingredient.

Images