Preparation method of saussurea involucrata callus cell extract for efficiently inhibiting propionibacterium acnes

Abstract

The invention relates to a preparation method of a saussurea involucrata callus cell extract for efficiently inhibiting propionibacterium acnes. Through an inhibition zone model, the Chinese herbal medicine saussurea involucrata plant extract obtained through screening has a relatively good inhibition effect on propionibacterium acnes. The saussurea involucrata callus cells are obtained through callus cell induction, the saussurea involucrata callus cell extract obtained under the culture condition of 20g / L betaine induction has a more obvious inhibition effect on propionibacterium acnes, and the inhibition effect reaches 88.5% or above; an anti-aging experiment result based on fibroblasts proves that the betaine-induced callus extract has relatively high oxidation resistance under the condition that the concentration of the betaine-induced callus extract is 0.20 mg / mL, so that the betaine-induced callus extract has a relatively good anti-aging effect.

Description

Technical Field

[0001] The present invention belongs to the field of plant tissue culture and daily chemical industry, and specifically relates to a method for preparing a snow lotus callus extract capable of effectively inhibiting Propionibacterium acnes. Background Art

[0002] Acne is a chronic inflammatory skin disease of the hair follicle sebaceous gland unit, which mainly occurs in adolescents. According to data, acne accounts for about 15% of patients visiting dermatology departments. Epidemiological studies have shown that about 80% to 90% of adolescents suffer from acne to varying degrees. It has a great impact on adolescents' psychology and social life.

[0003] The distribution and number of Propionibacterium acnes in different parts of the body of people of different ages are statistically different. It is most abundant in the head and face, which are rich in sebaceous glands, and least abundant in the middle of the trunk, lower limbs and upper arms. Some scholars have compared the detection rate of Propionibacterium acnes. At the age of 11-15, the ratio of the detection rate of acne patients to the normal population is as high as 15000:1, indicating that there is no difference in the detection rate of Propionibacterium acnes between acne patients and the normal population after puberty.

[0004] The structure, distribution and physiological characteristics of Propionibacterium acnes determine its important role in the pathogenesis of acne, especially the inflammatory damage and severity of acne. At present, there are many acne treatments for Propionibacterium acnes, including the use of tetracyclines, clindamycin, erythromycin and benzoyl peroxide or 5-aminolevulinic acid photodynamic therapy. However, long-term use of antibacterial drugs or hormone drugs to treat acne is prone to adverse consequences such as bacterial resistance and drug dependence. The presence of drug-resistant Propionibacterium acnes has been increasing year by year, among which erythromycin resistance is the most common. It was found that the rate of drug-resistant bacteria carried by patients treated with antibiotics was significantly higher than that of patients who did not receive treatment. The rate of drug-resistant bacteria carried by patients with ineffective treatment or relapse was higher than that of patients with effective treatment, indicating that oral or topical antibiotics can induce drug resistance in Propionibacterium acnes, which is related to treatment failure.

[0005] Acne is a chronic inflammatory skin disease of the pilosebaceous gland unit, which mainly occurs in adolescents and has a great impact on their psychological and social development. Propionibacterium acnes is closely related to the occurrence of acne inflammation. It is very necessary to develop a plant extract that can replace antibiotics to treat acne inflammation.

[0006] The present invention aims to break through the new technology of snow lotus callus cell expansion and suspension culture mass preparation, establish induced culture to promote the inhibitory effect of callus cell extract on Propionibacterium acnes, and expand the application of snow lotus callus cell extract in acne-removing cosmetics. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing a snow lotus callus cell extract that is highly effective in inhibiting Propionibacterium acnes, to establish an effective extraction method, and to study its inhibitory and / or antioxidant effects, and to expand its application in the field of cosmetics.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a method for preparing a snow lotus callus extract capable of efficiently inhibiting Propionibacterium acnes. The method comprises the following steps:

[0010] Step S1: preparing a culture medium for snow lotus callus cells (g / L): 3.2 g of B5 medium, 30 g of sucrose, 0.0002 g of 6-benzylaminopurine (6-BA), 0.004 g of naphthaleneacetic acid (NAA), 2 g of polyvinylpyrrolidone, and 8-10 g of agar powder;

[0011] Step S2: In a sterile clean bench, use tweezers to select loose callus particles, divide them into small pieces, and transfer them to tissue culture bottles for subculture. Subculture is performed every 10 days.

[0012] Preferably, in the subculture in step S2, browned cells are removed during subculture, and calli with good color and condition are selected for subculture.

[0013] Further preferably, in the subculture in step S2, light yellow callus cells are selected and transferred to fresh callus tissue culture medium for amplification and reproduction.

[0014] Preferably, in the subculture in step S2, if the surface of the callus contains a lot of non-embryonic white flocs during the subculture, the white flocs can be scraped off with a scalpel before cutting and culturing.

[0015] Step S3: Add the inducer to the culture medium of the tissue culture bottle according to the required addition ratio, reduce the temperature to about 50 degrees Celsius after sterilization, dispense the culture medium into the tissue culture bottle, and after solidification, transfer the grown callus cell particles into the culture bottle and place it in the incubator for culture.

[0016] Preferably, the inducer in step S3 is betaine, with a mass concentration of 0.5-5%.

[0017] Further preferably, the inducer in step S3 is betaine with a mass concentration of 1-3%.

[0018] More preferably, the inducer in step S3 is betaine with a mass concentration of 2%.

[0019] In another aspect, the present invention further provides a use of the above-mentioned snow lotus callus material in the preparation of cosmetics that inhibit Propionibacterium acnes and / or resist oxidation.

[0020] In another aspect, the present invention also provides a cosmetic for inhibiting Propionibacterium acnes, comprising the above-mentioned snow lotus callus extract.

[0021] In yet another aspect, the present invention also provides an antioxidant cosmetic comprising the above-mentioned snow lotus callus extract.

[0022] The present invention discloses the following technical effects:

[0023] The callus cell extract obtained by using the snow lotus callus obtained by the present invention and inducing culture with betaine has a more obvious inhibitory effect on Propionibacterium acnes, with the inhibitory effect reaching more than 88.5%; and at a concentration of 0.20 mg / mL of betaine-induced callus cell extract, it also has strong antioxidant properties.

[0024] The snow lotus callus cell extract of the present invention can be further developed and used in anti-acne cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 Selection and amplification of callus cells of Saussurea involucrata;

[0027] Figure 2 Normal snow lotus callus and its growth status under the condition of 20g / L betaine inducer;

[0028] Figure 3 The results of the inhibition of Propionibacterium acnes by different snow lotus extracts (control: no extract added, experimental groups: Tianshan snow lotus extract, normal culture callus extract, and betaine culture callus extract were added respectively);

[0029] Figure 4 Antioxidant activity test of callus cell extracts induced by betaine (NT—negative control group, i.e., normal cells; M—model group, i.e., photoaged cells; PC—positive control group (Vc)). DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0035] 1.1 Materials

[0036] 1.1.1 Experimental strains

[0037] Propionibacterium Acnes belongs to bacteria, which is anaerobic or facultative anaerobic. This strain was purchased from China Microbiological Culture Collection Center.

[0038] 1.1.2 Culture medium and culture conditions

[0039] Liquid culture medium and culture conditions of Propionibacterium acnes

[0040] Culture medium (g / L): 3 g of tryptone, 0.5 g of sodium thioglycolate, 0.5 g of L-cystine, 0.001 g of resazurin, 15 g of agar (not added to liquid culture medium), sterilized at 121°C for 15 min. After sterilization, take out the culture bottles and test tubes and tighten the lids in time to reduce air from entering the culture medium.

[0041] Culture conditions: 37°C, anaerobic, shaking on a shaker for 24-36 hours.

[0042] 1.1.3 Experimental equipment and devices

[0043] Experimental equipment: medicine spoon, centrifuge tube (50mL, 10mL), conical flask (250mL), 200 μL pipette tip, 1000 μL pipette tip, EP tube, glycerol, EP tube rack, tweezers, iron box, gloves, sealing film, rope, culture dish, coating stick, scalpel, long-handled tweezers.

[0044] Experimental equipment: incubator, microplate reader, microscope, clean bench, centrifuge, autoclave, etc.

[0045] 1.1.4 Determination method

[0046] Determination of OD value of Propionibacterium acnes concentration: Take the culture medium, dilute it 2 times with deionized water, shake it well, take out 150 μl with a pipette and add it to a 96-well ELISA plate, measure the absorbance at a wavelength of 600 nm, and record the value.

[0047] Determination of the growth of snow lotus callus cells: At the end of the culture of snow lotus callus cells in the culture dish, the cell particles were taken out and weighed on an electronic balance, and then the fresh cells were placed in a 105°C oven to dry. After 24 hours, the dry weight of the callus cells was weighed after the weight was constant.

[0048] Example 1: Snow lotus callus cell culture

[0049] Step 1: Culture medium for snow lotus callus cells (g / L): B5 medium 3.2, sucrose 30, 6-benzylaminopurine (6-BA) 0.0002, naphthaleneacetic acid (NAA) 0.004, polyvinylpyrrolidone 2, agar powder 8-10, pH 5.8-6.0

[0050] Step 2: Subculture of callus tissue. Use tweezers to select loose callus particles in a sterile clean bench, divide them into small pieces, and transfer them to tissue culture bottles for subculture. Remove brown cells during subculture, and select callus with good color and condition for subculture. If the surface of callus tissue contains a lot of non-embryonic white flocs, you can use a scalpel to scrape off the white flocs first and then cut them into pieces for culture. Subculture once every 10 days.

[0051] Step 3: Induced culture with inducers. Make the inducer betaine into a 2% concentrated solution and add it to the culture medium of the tissue culture bottle according to the required addition ratio. After sterilization, reduce the temperature to about 50 degrees Celsius, divide the culture medium into tissue culture bottles, wait for solidification, and transfer the grown callus cell particles to the culture bottle. Put it in the incubator for culture.

[0052] Example 2: Preparation of Saussurea involucrata plant extract

[0053] 500 grams of Snow Lotus tianshanense was purchased from a Chinese herbal medicine store, and 50 grams of the powder was taken after crushing. About 150 mL of a mixed solution of water and ethanol (1:1 v / v) was added to the grinder. After fully crushing for 20 minutes and soaking for 12 hours, the crushed slurry was filtered with filter paper to obtain the extract solution of the Snow Lotus, which was then freeze-dried to obtain the dry powder of the extract for later use.

[0054] Example 3: Obtaining Snow Lotus Callus Cells

[0055] Using tissue culture rapid propagation technology, callus cells are induced on wild snow lotus mother plants on callus induction medium. After 16 days, light yellow callus cells will differentiate and can be independently expanded and grown. The process is shown in Figure 1 .

[0056] Example 4: Plate culture of Propionibacterium acnes

[0057] Prepare solid culture medium according to the culture medium composition of Propionibacterium acnes. After dissolving in water, adjust the pH to 6.5. Add 20g / L agar. Mix well and adjust the volume to 500mL, then put it into 500mL conical flasks, 250mL per bottle. Put it into the autoclave for sterilization. After taking out the sterilized culture medium, wait until the temperature drops to about 50℃, pour the culture medium into a disposable plate in the prepared sterile clean bench, and keep it for later use after cooling.

[0058] Open the purchased Propionibacterium acnes ampoule tube, pour the bacterial powder into a 3mL sterile water test tube, shake and mix to dissolve evenly. Then use a sterile inoculation needle to dip the bacterial solution, streak on the plate culture medium, and then put it in a 35℃ anaerobic culture box, add 2 anaerobic bags, seal it, and remove the oxygen inside. Growth observation shows that after 12 hours of culture, bacterial moss begins to appear on the plate culture medium. After 36 hours of culture, the bacterial moss grows well on the plate culture medium.

[0059] Inoculate Propionibacterium acnes onto the slope of the test tube by streaking. After culturing, place it in a refrigerator at 4°C for later use.

[0060] Example 5: Liquid culture of Propionibacterium acnes

[0061] Prepare the culture medium without adding agar, adjust the pH and dispense into 50mL sterile centrifuge tubes, use an inoculation needle to pick the bacterial moss on the slope of the Propionibacterium acnes test tube and insert it into the liquid centrifuge tube, then cover the lid and place it in a 35℃ anaerobic culture box for about 38-40 hours. Take a sample and dilute it to determine the OD value.

[0062] Effect Experiment

[0063] 1. Screening of the inhibitory effect of snow lotus extract on Propionibacterium acnes

[0064] Weigh 50 mg of the dry extract and dissolve it in 5 mL of aqueous solution to prepare a 1% sample solution. Take 20 uL and add it to the filter paper of the Propionibacterium acnes culture plate. At the same time, the commonly used antibacterial drug erythromycin solution (dissolve erythromycin powder in water to prepare a 500 mg / L solution) and sterile water were selected as controls and added to the filter paper of the Propionibacterium acnes culture plate. Culture at 37°C for 36 hours, observe the size of the inhibition zone, perform 3 parallel experiments for each group of experiments, and calculate the average value of the measurement data of the inhibition zone as the antibacterial effect statistics of the extract. The results are shown in Table 1.

[0065] The experimental results show that the control group of this experiment used water dots to connect to the filter paper. As a comparison group, there was no antibacterial circle around the filter paper, while the antibacterial circle of the western medicine erythromycin solution reached 23.7mm, which significantly inhibited the growth of Propionibacterium acnes and had a good bactericidal effect. Among all the extracts selected in this experiment, the snow lotus extract had the largest antibacterial circle against Propionibacterium acnes, reaching 21.9mm, and had the strongest inhibitory effect on Propionibacterium acnes.

[0066] Data show that the antibacterial effect is generally calculated by the area of ​​the inhibition zone. The data show that the area of ​​the inhibition zone of Tianshan Saussurea extract against Propionibacterium acnes is 85% of that of the Western medicine erythromycin experimental group. It can be seen that the antibacterial effect of the Saussurea extract selected in this experiment is consistent with that of the Western medicine erythromycin.

[0067] Table 1 Inhibitory effects of different plant extracts on Propionibacterium acnes

[0068]

[0069] 2. Effects of different betaine concentrations on the growth of snow lotus callus cells

[0070] By consulting literature related to callus cell culture, we learned that in addition to the hormones added to the culture medium, betaine also has a good regulatory effect on the growth state and metabolite expression of callus cells. Therefore, this experiment investigated the effect of different betaine concentrations added to the culture medium on the growth of snow lotus callus cells.

[0071] Table 2 Effects of different betaine concentrations on the growth status of snow lotus callus cells

[0072]

[0073] The experimental results showed that as the concentration of betaine increased, the color of the cells darkened. When betaine was greater than 10g / L, the growth of callus cells was significantly inhibited. When betaine was 20g / L, the color of callus cells turned red and gradually darkened, and the growth rate of callus cells also slowed down significantly (see Figure 2 ).

[0074] 3. Investigation of the inhibitory effect of snow lotus callus extract on Propionibacterium acnes

[0075] In this experiment, the turbidity method was used to investigate the dynamic inhibition process of snow lotus cell extract on the growth of Propionibacterium acnes after betaine induction. The normal callus cells and the freeze-dried powder of callus cell extract obtained by betaine induction culture were dissolved in deionized water, and then 5 mL was added to the shake flask liquid culture medium inoculated with Propionibacterium acnes. The culture was carried out at 37°C and 100 rpm for 40 hours. The absorbance value of the solution was measured at regular intervals to investigate the inhibitory effect of the extract on Propionibacterium acnes. The results are shown in Figure 3 .

[0076] The experimental results showed that compared with the control, all the snow lotus cell extracts showed an inhibitory effect on Propionibacterium acnes from the beginning of the culture. The results after the growth reached equilibrium after 30 hours also showed that the extracts had a strong inhibitory effect on Propionibacterium acnes. The inhibitory effect of the snow lotus normal callus extract was equivalent to that of the snow lotus tissue extract. The callus extract obtained by culturing on the betaine-induced medium had the best inhibitory effect on Propionibacterium acnes. Compared with the control, the inhibition rate reached more than 88.5%, and its inhibition efficiency was also significantly higher than the inhibition rate of the normal callus extract (69%). The inhibitory efficacy of the callus extract induced by methyl jasmonate was slightly lower than that of the callus extract induced by betaine.

[0077] 4. Antioxidant test of extracts from snow lotus callus induced by betaine

[0078] (1) Cell culture method

[0079] Human fibroblasts (purchased from Zhejiang Sci-Tech University) were cultured using a serum replacement medium containing 2.5% serum replacement, 150 U / mL penicillin, and 120 U / mL streptomycin. The cells were routinely cultured in a 37°C, 5% CO2 incubator, with the medium replaced every 2-3 days, and passaged after 4 cycles of medium replacement.

[0080] (2) Cell senescence model

[0081] D-gal culture medium was diluted with complete culture medium to prepare 0, 4, 10, 20, and 40 mg / mL. Human epidermal fibroblasts in good growth condition were taken and inoculated into 96-well plates at 2×103 / well, with 4 replicates in each group, and cultured in a cell culture incubator at 37°C and 5% CO2. When the cell confluence reaches about 50% to 60%, the old liquid is discarded and washed once with PBS; 200 μL of normal complete culture medium was added to the control group, and 200 μL of complete culture medium with the above drug concentration was added to the experimental group respectively, and the cells were placed in a cell culture incubator and continued to be cultured for 24 hours before CCK-8 detection. The anti-aging effect was tested using a human epidermal cell culture model, and the ultraviolet light aging-induced model (UVA) was used to investigate the test effects of different extract addition concentrations by measuring the content of matrix metalloproteinase-1 (MMP-1). The results are shown in Tables 3 and Figure 4 .

[0082] From the data in Table 3, we can see that the relative content of MMP-1 in the model group (M) was significantly increased compared with the negative control group (NT) (P<0.05); the relative content of MMP-1 in the positive control group was significantly decreased compared with the model group (P<0.05), indicating that the model group was successfully established.

[0083] Table 3 Results of betaine-induced extract anti-aging model

[0084] Betaine-induced extracts and conditions Positive rate (%) Negative control group NT-UVA 71.54±8.62* Model group M+UVA 100# Sample group + UVA + 0.05mg / mL 96.64±8.82* Sample group + UVA + 0.1 mg / mL 78.13±5.43* Sample group + UVA + 0.2mg / mL 37.24±6.83* Positive control group PC+UVA+ascorbic acid 75.81±6.84*

[0085] # indicates that the difference was statistically significant compared with the blank control group (NT) (p < 0.05);

[0086] *Indicates that the difference is statistically significant compared with the model control group (M) (p<0.05)

[0087] At the concentrations of 0.10 mg / mL and 0.20 mg / mL in the callus cell extract group induced by methyl jasmonate, the relative content of MMP-1 in the sample group was significantly reduced by 22.87% and 34.50% compared with the model group. This indicates that the callus cell extract induced by methyl jasmonate can significantly reduce the relative content of MMP-1 in the UVA-induced model, indicating that the extract has a certain anti-aging effect.

[0088] At a concentration of 0.20 mg / mL of betaine-induced callus extract, the relative content of MMP-1 in the sample group was significantly reduced by 62.74% compared with the model group, only 37.24%. This indicates that the sample can significantly reduce the relative content of MMP-1 in the UVA model, indicating that betaine-induced callus extract has a better anti-aging effect.

[0089] The present invention provides a method for preparing a snow lotus callus extract that can effectively inhibit Propionibacterium acnes. The present invention obtains a good inhibitory effect of a Chinese herbal medicine snow lotus plant extract on Propionibacterium acnes through an inhibition zone model. 2) Snow lotus callus cells are obtained by callus induction, and the callus extract obtained by betaine induction culture has a more obvious inhibitory effect on Propionibacterium acnes, with an inhibitory effect of more than 88.5%; 3) The results of anti-aging experiments based on fibroblasts confirm that: at a concentration of 0.20 mg / mL of betaine-induced callus extract, it also has strong antioxidant properties, indicating that betaine-induced callus extract has good anti-aging effects. Snow lotus callus extract can be further developed for use in anti-acne cosmetics.

[0090] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a snow lotus callus extract that effectively inhibits Propionibacterium acnes, characterized in that: The present invention provides a method for preparing a snow lotus callus extract capable of efficiently inhibiting Propionibacterium acnes. The method comprises the following steps: Step S1: preparing the culture medium for the tissue culture flask of snow lotus callus cells. Step S2: In a sterile clean bench, use tweezers to select loose callus particles, divide them into small pieces, and transfer them to tissue culture bottles for subculture. Subculture is performed every 10 days. Step S3: Add the inducer to the culture medium of the tissue culture bottle according to the required addition ratio, reduce the temperature to about 50 degrees Celsius after sterilization, dispense the culture medium into the tissue culture bottle, and after solidification, transfer the grown callus cell particles into the culture bottle and place it in the incubator for culture.

2. The method according to claim 1, characterized in that The composition of the culture medium used is: (g / L): B5 culture medium 3.2, sucrose 30, 6-benzylaminopurine (6-BA) 0.0002, naphthaleneacetic acid (NAA) 0.004, polyvinylpyrrolidone 2, agar powder 8-10.

3. The method according to claim 1, characterized in that In the subculture described in step S2, browned cells are removed during subculture, and calli with good color and condition are selected for subculture.

4. The method according to claim 3, characterized in that During subculture, light yellow callus cells are selected and transferred to fresh callus tissue culture medium for amplification and reproduction.

5. The method according to claim 1, characterized in that In the subculture described in step S2, if the surface of the callus tissue contains a lot of non-embryonic white floccules during the subculture, the white floccules can be scraped off with a scalpel and then cut into pieces for culture.

6. The method according to claim 1, characterized in that The inducing agent in step S3 is betaine, and the mass concentration is 0.5-5%.

7. The method according to claim 6, characterized in that The inducer in step S3 is betaine with a mass concentration of 1-3%.

8. The method according to claim 7, characterized in that The inducer in step S3 is betaine, with a mass concentration of 2%.

9. Use of the snow lotus callus extract according to any one of claims 1 to 8 in inhibiting Propionibacterium acnes and / or in antioxidant cosmetics.

10. A cosmetic for inhibiting Propionibacterium acnes, characterized in that: The invention comprises the snow lotus callus extract according to any one of claims 1 to 8.

11. An antioxidant cosmetic, characterized in that: The invention comprises the snow lotus callus extract according to any one of claims 1 to 8.

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