CN113462725B - Cordyceps militaris secondary metabolite of sweet wormwood herb extract as well as preparation method and application thereof - Google Patents
Cordyceps militaris secondary metabolite of sweet wormwood herb extract as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a cordyceps militaris secondary metabolite of an artemisia apiacea extract, and a preparation method and application thereof, and belongs to the technical field of biochemistry. The preparation method comprises the following steps: s1: preparing a sweet wormwood extract solution: taking fresh sweet wormwood, drying, grinding into powder, mixing with water uniformly, homogenizing under high pressure, performing ultrasonic extraction, and performing tangential flow filtration to obtain sweet wormwood extract solution; s2: preparation of sweet wormwood culture medium: adding sucrose, peptone and potassium dihydrogen phosphate into herba Artemisiae Annuae extract solution, mixing, sterilizing, and cooling to room temperature to obtain herba Artemisiae Annuae culture medium; s3: inoculating Cordyceps militaris strain into the herba Artemisiae Annuae culture medium for culturing, and collecting mycelium; s4: washing the mycelium, mixing the mycelium with water, breaking wall, homogenizing under high pressure, centrifuging, collecting supernatant, and lyophilizing to obtain Cordyceps militaris secondary metabolite of herba Artemisiae Annuae extract. The cordyceps militaris secondary metabolite of the sweet wormwood extract is mainly used for preparing cosmetics, and has the effects of removing acnes, repairing and relieving skin.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a cordyceps militaris secondary metabolite of an artemisia apiacea extract, and a preparation method and application thereof.
Background
Herba Artemisiae Annuae (Artemisia carvifolia) is annual herb of Artemisia of Compositae, and has wide distribution, strong adaptability, and effects of clearing heat, cooling blood, relieving steaming, relieving summer heat, dispelling pathogenic wind, and relieving itching, and is a traditional Chinese herbal medicine. The sweet wormwood extract also has a plurality of medicinal values such as anti-inflammatory, antioxidant, antibacterial, immunoregulatory and the like through researches.
Acne is also called comedo, whelk, and the production of facial acne is closely related to factors such as bacterial infection and inflammatory response. It has been reported that the proliferation of propionibacterium acnes causes destruction of the skin's micro-ecological environment and the sebum is decomposed into free acids in a large amount, which causes the skin to develop inflammatory reactions such as redness, swelling, itching, pain, etc., and that the activated inflammatory mediators such as NO, inflammatory factors such as TNF- α and IL-6, etc. induce and enhance local inflammatory reactions during the inflammatory reactions.
Researches show that the sweet wormwood herb extract can be used for treating acne, has no irritation to skin, is economical and practical, but has still to be further improved in treatment effect on skin acne.
Disclosure of Invention
The invention provides a cordyceps militaris secondary metabolite of an artemisia apiacea extract, a preparation method and application thereof, and aims to perform metabolic conversion on the artemisia apiacea extract by using cordyceps militaris, wherein the obtained secondary metabolite can be used as an active ingredient medicament for research and development of skin care products and medicaments.
Cordyceps militaris (Cordyceps militaris) is also called Cordyceps militaris, belongs to Ascomycota, hymenoorder and Clavipitaceae, and is a fungus used as both medicine and food. Cordyceps militaris is rich in Cordyceps polysaccharide, active protein, polypeptide and other pharmacologically active substances, and has obvious effects on respiratory tract diseases, cardiovascular and cerebrovascular diseases, liver and kidney diseases, and has antibacterial, antiviral, antitumor and antiaging effects.
The invention provides a preparation method of a cordyceps militaris secondary metabolite of an sweet wormwood herb extract, which comprises the following steps:
s1: preparing a sweet wormwood extract solution:
taking fresh sweet wormwood, drying, grinding into powder, fully mixing with water, homogenizing under high pressure, performing ultrasonic extraction, and performing tangential flow filtration to obtain sweet wormwood extract solution;
s2: preparation of sweet wormwood culture medium:
adding sucrose, peptone and potassium dihydrogen phosphate into herba Artemisiae Annuae extract solution, mixing, sterilizing, and cooling to room temperature to obtain herba Artemisiae Annuae culture medium;
s3: inoculating Cordyceps militaris strain into the herba Artemisiae Annuae culture medium for culturing, and collecting mycelium;
s4: washing the mycelium, mixing the mycelium with water, breaking wall, homogenizing under high pressure, centrifuging, collecting supernatant, and lyophilizing to obtain Cordyceps militaris secondary metabolite of herba Artemisiae Annuae extract.
Further, in the step S1, the weight ratio of fresh sweet wormwood to water is 1:1.
Further, in the step S1, the condition of ultrasonic extraction is that ultrasonic extraction is carried out for 20-40 minutes at 30-50 ℃.
Preferably, in the step S1, the condition of ultrasonic extraction is ultrasonic extraction at 40 ℃ for 30 minutes.
Further, in the step S2, the ratio of the sweet wormwood extract solution, sucrose, peptone, and potassium dihydrogen phosphate is 1kg:40g:6g:6g.
Further, in the step S1, the sterilization condition is that the sterilization is carried out for 20-30 minutes at the temperature of 110-130 ℃.
Preferably, in the step S1, the sterilization condition is that the sterilization is performed at 121 ℃ for 20-30 minutes.
Further, in the step S2, the inoculation amount is 1:10 according to the mass ratio.
Preferably, in the step S2, the inoculation amount is 1:10 according to the mass ratio.
Further, in the step S2, the conditions for the cultivation are 19℃and 130rpm for 30 days.
Further, in the step S3, the weight ratio of the mycelium to the water is 1:4-6;
in the step S3, the rotational speed of the centrifugation is 12000rpm;
in the step S3, the number of times of washing is 3.
The invention also provides a cordyceps militaris secondary metabolite of the sweet wormwood herb extract prepared by any preparation method. The secondary metabolite of the cordyceps militaris of the sweet wormwood extract mainly utilizes the metabolic function of the cordyceps militaris, and the sweet wormwood extract with similar or synergistic effect is subjected to metabolic conversion to obtain different bioactive substances.
The invention also provides application of the cordyceps militaris secondary metabolite of the sweet wormwood herb extract in cosmetics.
Further, the cosmetic may exist in the form of an aqueous paste, gel, liquid. Preferably, the cosmetic product is in the form of at least one of an aqueous lotion, a water-in-oil or oil-in-water emulsion, an oil or oil alcohol lotion, a vesicular dispersion of an anionic or nonionic amphiphilic lipid, an aqueous gel, a water alcohol gel, an alcohol gel or an oil alcohol gel, a solid stick or an aerosol. The main active ingredient in the cosmetic comprises Cordyceps militaris secondary metabolite of herba Artemisiae Annuae extract.
Further, the cosmetic has at least one of acne removing, repairing and soothing effects. According to cosmetic classification rules and classification catalogues, acne removal mainly refers to the reduction or alleviation of acne (including blackheads or whiteheads); can be used for restoring skin after acne (e.g. regulating hormone-affected, antibacterial and antiinflammatory products, and is not a cosmetic). Repairing mainly means helping to maintain the normal state of the application part (products for curing scar, scald, burn, breakage and other damage parts, not belonging to cosmetics). Soothing mainly means improving the state of skin irritation and the like.
The secondary metabolite of the cordyceps militaris of the sweet wormwood extract is the only active ingredient with the acne removing, repairing and soothing effects. Or the secondary metabolite of Cordyceps militaris of the herba Artemisiae Annuae extract and other substances can be used for preparing skin care preparation with acne removing, repairing and relieving effects. By inhibiting bacteria and inflammation, the acne-removing and repairing cream can effectively improve acne, maintain normal skin state, improve skin irritation and the like, thereby having the effects of removing acnes, repairing and relieving.
The invention has the following advantages:
the cordyceps militaris secondary metabolite of the sweet wormwood extract has the effects of removing acnes, repairing and relieving skin, and the effects of removing acnes, repairing and relieving are obviously superior to those of cordyceps militaris mycelium extract and sweet wormwood extract, and can be used for preparing skin care cosmetics.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 is the effect of Cordyceps militaris secondary metabolite of Artemisia annua extract on inflammatory mediator NO. (n=3, ×p < 0.01)
FIG. 2 is the effect of Cordyceps militaris secondary metabolite of Artemisia annua extract on inflammatory factors TNF- α and IL-1β. (n=3, ×p < 0.01)
FIG. 3 is the effect of Cordyceps militaris secondary metabolite of Artemisia annua extract on mouse auricle swelling. (n=3, ×p < 0.01)
Fig. 4 is MMV and TEWL index improved fractional volunteer distribution.
FIG. 5 is the effect of Cordyceps militaris secondary metabolite of Artemisia annua extract on skin lipid secretion.
FIG. 6 shows the anti-acne effect of the secondary metabolite of Cordyceps militaris of the Artemisia annua extract.
Detailed Description
The invention will be further explained below in connection with specific embodiments, but is not limited to the invention.
In the examples described below, all reagents were obtained from commercial products unless otherwise specified.
Example 1Preparation method of cordyceps militaris secondary metabolite of sweet wormwood extract
Weighing 1kg of fresh sweet wormwood, drying, grinding into powder, fully and uniformly mixing sweet wormwood and water according to the weight ratio of the fresh sweet wormwood to the water of 1:1, homogenizing under high pressure, performing ultrasonic extraction, and performing tangential flow filtration to obtain sweet wormwood extract solution; adding 40g of sucrose, 6g of peptone and 6g of potassium dihydrogen phosphate into 1kg of sweet wormwood extract solution, and fully and uniformly mixing; sterilizing at 121deg.C for 20-30 min to obtain herba Artemisiae Annuae culture medium; after the medium was cooled to room temperature, cordyceps militaris strains were inoculated into 1kg of the sweet wormwood medium in an inoculum size of 1:10 in an ultra clean bench, and after 30 days of culture in a shaker at 19℃and 130rpm, mycelia were collected.
Washing the collected mycelium with purified water for 3 times, adding into a wall breaking machine, fully and uniformly mixing the mycelium and water according to the weight ratio of the mycelium to water of 1:5, breaking the wall, homogenizing under high pressure, and centrifuging at 12000rpm; taking the supernatant after centrifugation, and freeze-drying to obtain 11.9g of white-like powder, which is the cordyceps militaris secondary metabolite of the sweet wormwood extract.
Example 2Inhibition test of Cordyceps militaris secondary metabolite of herba Artemisiae Annuae extract on Propionibacterium acnes
The detection method comprises the following steps: propionibacterium acnes ATCC11827 is selected as a test strain, and is cultured to prepare 10 6 Measuring 100uL of CFU/mL bacterial suspension, respectively adding into 1mL of 0.5% cordyceps militaris secondary metabolite (ACE) solution of sweet wormwood extract and equal amount of cordyceps militaris mycelium extract solution (CE) and sweet wormwood extract (AE) solution with the same concentration, uniformly mixing, adding 9mL of 0.03mol/L PBS after 20min, uniformly mixing, taking 2-3 dilutions, respectively sucking 0.5mL of the dilutions after proper dilution, placing the two plates, pouring 15mL of culture medium cooled to 40-45 ℃ into the plates, rotating the plates to make the plates fully uniform, overturning the plates after agar solidification, and culturing at 35+/-2 ℃ for 24h to count viable bacterial colonies. Meanwhile, a blank control is set, the test is repeated 3 times, the bacteriostasis rate is calculated according to the following formula, and the result is shown in Table 1.
TABLE 1
It can be seen that acne (acne) is related to bacteria, and experimental data shows that the antibacterial effect of Cordyceps militaris secondary metabolite of herba Artemisiae Annuae extract with the same concentration is superior to that of Cordyceps militaris mycelium extract and herba Artemisiae Annuae extract. Namely, the secondary metabolite of the cordyceps militaris of the sweet wormwood extract can effectively improve the acne problem through inhibiting bacteria.
Example 3Detection of influence of cordyceps militaris secondary metabolite of sweet wormwood extract on inflammatory mediators NO, inflammatory factors TNF-alpha and IL-6
The detection method comprises the following steps: the mouse macrophage RAW264.7 is inoculated in a 12-hole plate, cultured for 24 hours in a 5% CO2 incubator at 37 ℃, then a proper amount of 5% ACE, CE and AE are added, and a blank control is not treated; after 2h, an inflammatory response was induced by the addition of 40mg/L Lipopolysaccharide (LPS). After 4h of LPS action, cell supernatants were collected, NO content was determined according to Griess kit instructions, and TNF- α and IL-1β content was determined according to ELISA kit instructions. The results are shown in FIGS. 1 and 2.
Referring to fig. 1 and 2, wherein the LPS: lipopolysaccharide; ACE: a cordyceps militaris secondary metabolite of the sweet wormwood extract; CE: cordyceps militaris mycelium extract; AE: herba Artemisiae Annuae extract. The obtained medicines can inhibit the rise of the contents of inflammatory mediators NO, inflammatory factors TNF-alpha and IL-1 beta caused by LPS induction, the difference from a blank control group is obvious (P is less than 0.01), and the effect of the cordyceps militaris secondary metabolite of the artemisia apiacea extract is obviously superior to that of the cordyceps militaris mycelium extract and the artemisia apiacea extract (P is less than 0.01). Acne (pox) is related to inflammatory response, i.e. the secondary metabolite of cordyceps militaris of the sweet wormwood extract effectively improves the acne problem through inhibiting inflammation.
Example 4Detection of influence of cordyceps militaris secondary metabolite of sweet wormwood extract on mouse auricle swelling
The detection method comprises the following steps: the 1% sodium carboxymethyl cellulose is dispensed to form gel, the blank control group is prepared by uniformly coating 0.2g of corresponding medicine on the inner side and the outer side of the right auricle of each mouse respectively, and the blank control group is prepared by uniformly coating 0.2g of sodium carboxymethyl cellulose on the inner side and the outer side of the right auricle of each mouse, and the blank control group is continuously administered for 7d and is administered for 1 time per day. After 0.5h of last administration on day 7, 50ul of xylene was uniformly applied to the inner and outer sides of the right auricle of each mouse, and the left ear was used as a control. After 20min of inflammation, the human body is killed by removing the neck, cutting the two ears along the auricle line, respectively beating the left and right ear pieces at the same position of the two ears by using a puncher with the diameter of 6mm, weighing, recording data, and carrying out statistical analysis, wherein the mass difference value of the left and right ear pieces is the inflammatory swelling degree. The results are shown in FIG. 3.
Referring to fig. 3, each group of drugs can inhibit the swelling degree of mice auricle caused by dimethylbenzene, the difference between the drugs and a blank group is obvious (P is less than 0.01), and the effect of the cordyceps militaris secondary metabolite of the artemisia apiacea extract is obviously better than that of the cordyceps militaris mycelium extract and the artemisia apiacea extract (P is less than 0.01). It can be seen that the mouse auricle test can further demonstrate the anti-inflammatory effect of the Cordyceps militaris secondary metabolite of the sweet wormwood extract.
Example 5Detection of skin improvement condition by cordyceps militaris secondary metabolite of sweet wormwood extract
The detection method comprises the following steps: 30 healthy, mild acne volunteers, 15 men and women, were recruited for the age of 18-40 years. The product is used by cleaning face before use, and after skin toner, the product is used twice daily in the morning and evening for 7 days continuously for 1 week. Two basic skin condition tests were performed before and after 1 week of continuous use of the product, including skin moisture content (MMV), trans-epidermal moisture loss (TEWL) and VISIA-CR photographs taken with a dermlab instrument, and the face was cleaned and dried before each test, waiting for 30min. The room where the instrument is located is not ventilated, no sunlight is directly irradiated, the room temperature is kept at 24-25 ℃, and the humidity is kept at 40%. And finally, carrying out statistical analysis on the acquired data.
Scoring was done by MMV and TEWL index scores after each volunteer used the product. ( 3, the method comprises the following steps: MMV increases or TEWL decreases > 20%;2, the method comprises the following steps: MMV increase or TEWL decrease by 10% -20%;1, the method comprises the following steps: MMV increases or TEWL decreases by < 10%;0 point: MMV is not increased or TEWL is not decreased )
Wherein the MMV and TEWL index improved fractional volunteer distribution is seen in fig. 4. The amount of porphyrin in the photographs taken by VISIA-CR was used to characterize the secretion of oil, see FIG. 5. The facial acne-removing effect of 2 volunteers 5 days before and after using the product is shown in fig. 6.
The improvement rate of the MMV is 73.33% when the number of people with improvement is 22; the number of the TEWL is 23, the improvement rate reaches 76.67%, which indicates that after the product is used, the moisture in the skin of most subjects is improved, the loss of the moisture through the epidermis is reduced, the skin barrier is repaired to a certain extent, and the skin irritation is improved; the facial porphyrin is obviously reduced, which indicates that propionibacterium acnes are inhibited and the secretion of grease is reduced after the product is used; quick and obvious removal of the acnes on the face. In general, the product has a certain acne removing, repairing and soothing effects.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. The application of the cordyceps militaris secondary metabolite of the sweet wormwood extract in cosmetics is characterized in that the cosmetics have at least one of acne removing, repairing and relieving effects;
the preparation method of the cordyceps militaris secondary metabolite of the sweet wormwood herb extract comprises the following steps:
s1: taking fresh sweet wormwood, drying, grinding into powder, fully mixing with water, homogenizing under high pressure, performing ultrasonic extraction, and performing tangential flow filtration to obtain sweet wormwood extract solution; wherein the condition of ultrasonic extraction is that ultrasonic extraction is carried out for 20-40 minutes at 30-50 ℃;
s2: adding sucrose, peptone and potassium dihydrogen phosphate into herba Artemisiae Annuae extract solution, mixing, sterilizing, and cooling to room temperature to obtain herba Artemisiae Annuae culture medium;
s3: inoculating Cordyceps militaris strain into the herba Artemisiae Annuae culture medium for culturing, and collecting mycelium; culturing at 11deg.C and 130rpm for 30 days;
s4: washing the mycelium, mixing the mycelium with water, breaking wall, homogenizing under high pressure, centrifuging, collecting supernatant, and lyophilizing to obtain Cordyceps militaris secondary metabolite of herba Artemisiae Annuae extract.
2. The use according to claim 1, wherein,
in the step S1, the weight ratio of the fresh sweet wormwood to the water is 1:1;
in the step S1, the ultrasonic extraction condition is that the ultrasonic extraction is carried out for 30 minutes at 40 ℃.
3. The use according to claim 1, wherein,
in the step S2, the ratio of the sweet wormwood extract solution to the sucrose to the peptone to the monopotassium phosphate is 1kg:40g:6g:6g.
4. The use according to claim 1, wherein,
in the step S2, the sterilization condition is that the sterilization is carried out for 20-30 minutes at the temperature of 110-130 ℃.
5. The use according to claim 1, wherein,
in the step S3, the inoculation amount is 1:10 according to the mass ratio.
6. The use according to claim 1, wherein,
in the step S4, the weight ratio of the mycelium to the water is 1:4-6;
in the step S4, the rotational speed of the centrifugation is 12000rpm;
in the step S4, the number of times of washing is 3.
7. The use according to claim 1, wherein,
the cosmetic product is in the form of at least one of an aqueous lotion, a water-in-oil or oil-in-water emulsion, an oil or oil-in-alcohol lotion, a vesicular dispersion of an anionic or nonionic amphiphilic lipid, an aqueous gel, a water-in-alcohol gel, an alcohol gel or an oil-in-alcohol gel, a solid stick or an aerosol.
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| CN106962926A (en) * | 2017-04-28 | 2017-07-21 | 邓和满 | To discard the method that Chinese caterpillar fungus culture medium prepares ferment as raw material |
| CN109722449A (en) * | 2017-10-30 | 2019-05-07 | 湖南新汇制药股份有限公司 | A kind of sweet wormwood bioconversion mycelium and its extract and purposes |
| CN110638688A (en) * | 2019-08-23 | 2020-01-03 | 上海澄穆生物科技有限公司 | Preparation method and application of artemisinin-loaded lentinus edodes polysaccharide compound |
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