Preparation method and application of plum blossom callus extract

Abstract

The invention relates to the technical field of plant extraction, in particular to a preparation method and application of a plum blossom callus extract, and the key point of the technical scheme is that the preparation method comprises the following steps: S1, culture: cutting a plum blossom plant to obtain plum blossom tissue blocks; performing induction dark culture and proliferation dark culture on the plum blossom tissue blocks to obtain plum blossom calluses; s2, freeze-drying: pre-freezing the plum blossom callus, performing vacuum freeze-drying for 48-72 hours to obtain freeze-dried plum blossom callus, and storing the freeze-dried plum blossom callus at low temperature in a dark place; s3, extraction: adding liquid nitrogen into a grinding tool for pre-cooling, then adding the freeze-dried plum blossom callus, and grinding the freeze-dried plum blossom callus into powder to obtain plum blossom callus powder; mixing 50% ethanol with the plum blossom callus powder, carrying out ultrasonic extraction, and filtering to obtain a filtrate; performing rotary evaporation on the filtrate to obtain a target product. The method is not limited by the growth cycle and the growth season of plum blossom plants, and has the advantages of being high in efficacy and wide in application range.

Description

Technical Field

[0001] The invention relates to the technical field of plant extraction, and more specifically to a method for preparing a plum blossom callus extract. Background Art

[0002] Plum is a woody plant belonging to the genus Prunus and the family Rosaceae. It blooms in winter and spring and bears fruit from May to June. In my country, plum has been cultivated for more than 3,000 years, and there are many varieties of plums, both for ornamental and fruit trees. Plums have high ornamental and medicinal value, and also contain a variety of active substances, such as total flavonoids and total polyphenols. In addition, old plums with a long history have higher content and more types of active substances, which can be used in the field of cosmetics.

[0003] However, in the prior art, plum blossom extracts have not been widely used in the preparation of cosmetics, and the reasons for this are as follows: the extraction of plum plant extracts is limited by the production cycle and growing season of plum blossoms, and the extraction is relatively time-consuming; in addition, the extraction of plum plant extracts requires a large number of plum plants to extract its effective ingredients, which causes damage to a large number of plum plants and is not conducive to the sustainable development of plum plants. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for preparing a plum blossom callus extract, which has the advantages of not being restricted by the growth cycle and having a wide range of applications.

[0005] The above technical purpose of the present invention is achieved through the following technical scheme: a method for preparing a plum callus extract, comprising the following steps: S1. Cultivation: cutting ancient plum blossom plants to obtain plum blossom tissue blocks; Carrying out induction dark culture and proliferation dark culture on the plum blossom tissue block to obtain plum blossom callus; S2, freeze-drying: pre-freeze the plum blossom callus, and then vacuum freeze-dry for 48-72 hours to obtain freeze-dried plum blossom callus, and store it at low temperature and away from light; S3, extraction: adding liquid nitrogen to a grinding tool for precooling, then adding the freeze-dried plum blossom callus and grinding into powder to obtain plum blossom callus powder; Mixing 50% ethanol with the plum blossom callus powder, performing ultrasonic extraction, and filtering to obtain a filtrate; The filtrate is subjected to rotary evaporation to obtain the target product; The culture medium for the induction dark culture and the proliferation dark culture is 2-4 g / L MS culture medium, 25-35 g / L sucrose, 6-8 g / L agar, 0.5 mg / L 6-BA, 2 mg / L TDZ and 1 mg / L NAA, and the pH is 5-6.5.

[0006] In one embodiment, the plum blossom plant is an ancient plum blossom plant.

[0007] In one embodiment, the step S1 further includes sterilizing the ancient plum plant, and the specific steps are: The ancient plum blossom plant was cleaned, then immersed in 70% ethanol for 30 seconds, and then washed with sterile water; Soak the washed ancient plum plants in 10% sodium hypochlorite solution for 2 minutes, wash with sterile water, and absorb the moisture.

[0008] In one embodiment, the induction dark culture time is 20-30 days, and the proliferation dark culture time is 20-25 days.

[0009] In one embodiment, the vacuum freeze drying conditions are: cold trap temperature -80°C, freezing chamber temperature -45 to -35°C, vacuum degree 0-10 Pa; In S2, the storage temperature of the freeze-dried plum callus is -80 to -20°C.

[0010] In one embodiment, the solid-liquid ratio of the plum blossom callus powder to the 50% ethanol is 1:10; The conditions of the ultrasonic extraction are: ultrasonic power 2000w, ultrasonic temperature 25-45°C, ultrasonic time 30min, and ultrasonic extraction 3 times.

[0011] In one embodiment, in S3, the rotary evaporation is divided into three times; The first rotary evaporation conditions are 35-42°C, 80-120 rpm, and 35-40 min. The second rotary evaporation conditions are 48°C, 80-120 rpm, and 30 min. The third rotary evaporation conditions are 48°C, a rotation speed of 80-120 rpm, and rotary evaporation for 30-45 minutes.

[0012] A plum blossom callus extract is prepared by the preparation method of the plum blossom callus extract.

[0013] In one embodiment, the active ingredients include chlorogenic acid, cryptochlorogenic acid, quercetin, rutin, hyperoside and isoquercetin.

[0014] The plum callus extract is used in the preparation of skin care products with anti-aging, anti-inflammatory, soothing, antioxidant and skin lightening effects.

[0015] The above-mentioned method for preparing the plum callus extract has the following beneficial effects: Firstly, the present invention uses plum blossom plants as raw materials, uses a specific culture medium to induce proliferation and culture, obtains plum blossom callus, and then freeze-dries and extracts, thereby avoiding damage to the plum plants and obtaining plum blossom callus extracts with richer active substances. The extracts are not limited by the growth cycle and growing season of the plum blossom plants, and are helpful to expand the application scope of the plum blossom extracts. Secondly, the present invention can comprehensively obtain and enrich the effective ingredients in plum blossoms. Compared with plum blossom plant extracts, the plum blossom callus extract prepared by the present invention has stronger efficacy, and has anti-aging, anti-inflammatory, soothing, antioxidant and skin-lightening effects, and has broad cosmetic application prospects and potential economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a chromatogram of active substances of the plum callus extract in Example 1; Figure 2 is the active substance chromatogram of the product in Comparative Example 1; Figure 3 is the active substance chromatogram of the product in Comparative Example 2; Figure 4 is the active substance chromatogram of the product in Comparative Example 3; Figure 5 is a statistical graph of gene expression of collagen-related genes regulated by the product in Example 1; Figure 6 is a statistical graph of gene expression levels of matrix metalloproteinase-related genes regulated by the product in Example 1; Figure 7 is a statistical graph of the gene expression of barrier protein-related genes regulated by the product in Example 1; Figure 8 is a statistical graph of gene expression levels of cellular antioxidant enzyme-related genes regulated by the product in Example 1; Fig. 9 This is a statistical graph of the gene expression of cytokine genes related to cell inflammation regulated by the product in Example 1; Fig.10 is a graph showing the results of measuring cell survival rates of the products in Example 1 and Comparative Example 1 after UVB irradiation; Fig.11 It is a graph showing the free radical scavenging test results of the products in Example 1 and Comparative Example 1; Fig.12 This is a test result diagram of the product in Example 1 regulating the content of cellular melanin; Fig.13 It is a test result diagram of the product in Example 1, carnosine and red phaff in inhibiting CML. DETAILED DESCRIPTION

[0017] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0018] A method for preparing a plum blossom callus extract comprises the following steps: S1. Cultivation: cutting plum blossom plants to obtain plum blossom tissue blocks; performing induction dark culture and proliferation dark culture on the plum blossom tissue blocks to obtain plum blossom callus; In the culture step, the plum blossom plant is cut into tissue blocks of about 10 mm using a scalpel, and is inoculated into an induction medium for induction dark culture, and then inoculated into a proliferation medium for proliferation dark culture, so as to effectively obtain plum blossom callus.

[0019] In actual preparation, the culture temperature of induction dark culture and proliferation dark culture is 20-25°C, and the specific compositions of induction medium and proliferation medium are: 2-4g / L MS medium, 25-35g / L sucrose, 6-8g / L agar, 0.5mg / L 6-BA (6-benzylaminopurine), 2mg / L TDZ (thidiazuron) and 1mg / L NAA (naphthaleneacetic acid), and the pH of the two culture media is 5-6.5.

[0020] S2, freeze-drying: pre-freeze the plum blossom callus, and then vacuum freeze-dry for 48-72 hours to obtain freeze-dried plum blossom callus, and store it at low temperature and away from light; In actual preparation, a freeze dryer is used to freeze-dry the plum blossom callus. Pre-freezing it in advance helps to cool down the freezing chamber of the freeze dryer, reduce the cooling time, and ensure the freshness of the callus.

[0021] S3, extraction: adding liquid nitrogen to a grinding tool for precooling, then adding freeze-dried plum blossom callus and grinding into powder to obtain plum blossom callus powder; In actual preparation, the grinding tools are a mortar and a grinding pestle. Liquid nitrogen is added to the mortar for precooling. The amount of liquid nitrogen added is two-thirds of the mortar, and the grinding pestle is used for stirring. After the liquid nitrogen evaporates, the operation is repeated to precool the mortar and the grinding pestle. Mix 50% ethanol with plum blossom callus powder, perform ultrasonic extraction, and filter to obtain a filtrate; perform rotary evaporation on the filtrate to remove the ethanol solvent in the solution to obtain a plum blossom callus extract, which needs to be stored in the dark at -20 to 4°C.

[0022] The invention uses plum blossom plants as raw materials, uses a specific culture medium to induce proliferation culture, obtains plum blossom callus, and then freeze-dries and extracts to obtain a plum blossom callus extract with richer active substances. The extract is not limited by the growth cycle and growing season of the plum blossom plant, and is helpful to expand the application scope of the plum blossom extract.

[0023] Specifically, the plum blossom plant is an ancient plum blossom plant. An ancient plum blossom plant refers to a plum blossom tree that is over 100 years old. Compared with conventional plum blossom plants, ancient plum blossom plants have more types and higher active ingredients, making the efficacy of the obtained plum blossom callus stronger. The present invention extracts plum blossom callus extracts from ancient plum blossom plants, while protecting the ancient plum blossom plants as much as possible, so that the product has stronger efficacy and sustainable development characteristics.

[0024] Furthermore, S1 also includes sterilizing the ancient plum blossom plants, and the specific steps are as follows: The ancient plum blossom plants were cleaned, immersed in 70% ethanol for 30 seconds, and then washed with sterile water; Soak the washed ancient plum plants in 10% sodium hypochlorite solution (0.1% Tween 20 can be added) for 2 minutes, wash with sterile water, and absorb the moisture.

[0025] Specifically, the induction dark culture time is 20-30 days, and the proliferation dark culture time is 20-25 days.

[0026] Specifically, the conditions for vacuum freeze drying are: cold trap temperature -80°C, freezing chamber temperature -45 to -35°C, vacuum degree 0-10pa; In S2, the storage temperature of freeze-dried plum callus is -80 to -20°C.

[0027] Specifically, the solid-liquid ratio of plum blossom callus powder and 50% ethanol is 1:10; The conditions of ultrasonic extraction were as follows: ultrasonic power 2000w, ultrasonic temperature 25-45°C, ultrasonic time 30min, ultrasonic extraction 3 times, and the 3 extracts were combined for filtration.

[0028] Specifically, in S3, the rotary evaporation is divided into three times; The first rotary evaporation conditions are 35-42°C, 80-120 rpm, and 35-40 min to prevent the liquid from overflowing the rotary evaporation bottle during the rotary evaporation process; The second rotary evaporation conditions are 48°C, 80-120 rpm, and 30 min. The third rotary evaporation conditions are 48°C, a rotation speed of 80-120 rpm, and rotary evaporation for 30-45 minutes.

[0029] In actual preparation, after rotary evaporation, the alcohol content of the solution can be determined using an alcohol meter reference table to ensure that the residual ethanol content is within 0-3%.

[0030] A plum blossom callus extract is prepared by the preparation method of the plum blossom callus extract.

[0031] Specifically, it contains the following active ingredients: chlorogenic acid, cryptochlorogenic acid, quercetin, rutin, hyperoside and isoquercetin.

[0032] The above-mentioned plum callus extract is used in the preparation of skin care products with anti-aging, anti-inflammatory, soothing, anti-oxidation and skin lightening effects. Compared with plum plant extracts, plum callus extracts have stronger efficacy, broad cosmetic application prospects and potential economic benefits. Example 1

[0033] A plum blossom callus extract is prepared by the following steps: S1. Cultivation: Cut the sterilized ancient plum plants to obtain tissue blocks of about 10 mm, and then inoculate them into induction medium (2g / L MS medium, 30g / L sucrose, 7g / L agar, 0.5mg / L 6-BA, 0.5mg / L NAA, 2mg / L TDZ). The pH of the induction medium is 5.8, and the dark reaction is carried out at 25°C for 20-30 days. The induced callus tissue was then inoculated into a proliferation medium (2 g / L MS medium, 30 g / L sucrose, 7 g / L agar, 0.5 mg / L 6-BA, 0.5 mg / L NAA, 2 mg / L TDZ), the pH of the proliferation medium was 5.8, and a dark reaction was performed at 25°C for 20-25 days to obtain plum blossom callus tissue; S2. Freeze-drying: The callus of plum blossom was pre-frozen in a constant temperature refrigerator at -30℃ for 24h, and then placed in a vacuum freeze dryer for vacuum freeze drying, wherein the cold trap temperature was -80℃, the freezing chamber temperature was -45~-35℃, and the vacuum degree was 0-10pa; During this process, the temperature and vacuum degree of the freezing chamber gradually changed with the freezing time, showing the following trends: Program 1 (1h, -45℃, vacuum degree 0), Program 2 (0.5h, -45℃, vacuum degree 0), Program 3 (0.5h, -45℃, vacuum degree 0-10Pa), Program 4 (0.5h, -30℃, vacuum degree 0-10Pa), Program 5 (1h, -10℃, vacuum degree 0-10Pa), Program 6 (5h, 0℃, vacuum degree 0-10Pa), Program 7 (4h, 5℃, vacuum degree 0-10Pa), program 8 (3h, 10℃, vacuum degree 0-10Pa), program 9 (5h, 15℃, vacuum degree 0-10Pa), program 10 (5h, 20℃, vacuum degree 0-10Pa), program 11 (5h, 25℃, vacuum degree 0-10Pa), program 12 (5h, 30℃, vacuum degree 0-10Pa), program 13 (5h, 30℃, vacuum degree 0-10Pa), program 14 (7.5h, 35℃, vacuum degree 0-10Pa); After freeze drying, freeze-dried plum callus tissue was obtained and temporarily stored in a refrigerator at -80 to -20°C away from light.

[0034] S3, Extraction: Pre-cool the mortar and pestle, add the freeze-dried plum callus and grind into powder; Weigh 1 kg of powdered plum callus, add 10 kg of 50% ethanol at a (m / m) solid-liquid ratio of 1:10, and ultrasonicate the mixture for 30 min at an ultrasonic power of 2000 W and an action temperature of 25-45 ° C. Repeat the extraction 3 times, and filter the obtained extracts with a Buchner funnel, and then filter them with a 0.22 μm filter membrane; The filtrate is subjected to three-step rotary evaporation using a rotary evaporator, wherein the first step is carried out under the conditions of 35-42° C., 80-120 rpm, and 35-40 min; the second step is carried out under the conditions of 48° C., 80-120 rpm, and 30 min; and the third step is carried out under the conditions of 48° C., 80-120 rpm, and 30-45 min to remove the solvent from the filtrate, thereby obtaining a plum blossom callus extract, which is stored in a refrigerator at -20 to 4° C. away from light.

[0035] Comparative Example 1 Different from Example 1, a plum blossom plant extract is obtained by directly freeze-drying and extracting the ancient plum blossom plant, and the specific preparation steps are as follows: S1. Freeze-drying: The ancient plum blossom plants were pre-frozen in a -30°C constant temperature refrigerator for 24 hours, and then placed in a vacuum freeze dryer for vacuum freeze drying, wherein the cold trap temperature was -80°C, the freezing chamber temperature was -45 to -35°C, and the vacuum degree was 0-10pa; During this process, the temperature and vacuum degree of the freezing chamber gradually changed with the freezing time, showing the following trends: Program 1 (1h, -45℃, vacuum degree 0), Program 2 (0.5h, -45℃, vacuum degree 0), Program 3 (0.5h, -45℃, vacuum degree 0-10Pa), Program 4 (0.5h, -30℃, vacuum degree 0-10Pa), Program 5 (1h, -10℃, vacuum degree 0-10Pa), Program 6 (5h, 0℃, vacuum degree 0-10Pa), Program 7 (4h, 5℃, vacuum degree 0-10Pa), program 8 (3h, 10℃, vacuum degree 0-10Pa), program 9 (5h, 15℃, vacuum degree 0-10Pa), program 10 (5h, 20℃, vacuum degree 0-10Pa), program 11 (5h, 25℃, vacuum degree 0-10Pa), program 12 (5h, 30℃, vacuum degree 0-10Pa), program 13 (5h, 30℃, vacuum degree 0-10Pa), program 14 (7.5h, 35℃, vacuum degree 0-10Pa); After freeze-drying, freeze-dried ancient plums were obtained and temporarily stored in a refrigerator at -80 to -20°C away from light.

[0036] S2. Extraction: Pre-cool the mortar and pestle, add freeze-dried gumei and grind into powder; Weigh 1 kg of freeze-dried ancient plum powder, add 10 kg of 50% ethanol at a (m / m) material-liquid ratio of 1:10, and ultrasonicate the mixture for 30 min at an ultrasonic power of 2000 W and an action temperature of 25-45 ° C. Repeat the extraction 3 times, and filter the obtained extracts with a Buchner funnel, and then filter them with a 0.22 μm filter membrane; The filtrate was subjected to three-step rotary evaporation using a rotary evaporator, wherein the first step was carried out under the conditions of 35-42°C, 80-120 rpm, and 35-40 min; the second step was carried out under the conditions of 48°C, 80-120 rpm, and 30 min; the third step was carried out under the conditions of 48°C, 80-120 rpm, and 30-45 min to remove the solvent from the filtrate, thereby obtaining a plum blossom plant extract, which was then stored in a refrigerator at -20 to 4°C away from light.

[0037] Comparative Example 2 Different from Example 1, this comparative example uses ethyl acetate instead of 50% ethanol as an extraction reagent to obtain a plum callus extract.

[0038] Comparative Example 3 This comparative example is a commercially available plum blossom plant extract purchased from outside.

[0039] Experimental analysis and testing were carried out on the products of Example 1 and Comparative Examples 1-3.

[0040] Test 1: Active ingredient test The obtained products of Example 1 and Comparative Examples 1-3 were used as detection raw materials, and qualitative analysis was performed under light-proof conditions.

[0041] Dissolve 1 mL of the test raw material in methanol solution, use an ultrasonic instrument to shake for 20 minutes to promote dissolution, shake and centrifuge, repeat this operation 3 times, and pass the supernatant obtained 3 times through an organic membrane; Binary gradient elution was used, with acetonitrile as mobile phase A and double distilled water (0.2% phosphoric acid) as mobile phase B, column temperature at 25°C, injection volume of 20 μL, flow rate of 1 mL / min, and detection wavelength of 360 nm; elution program: 0-4 min, 80%-76% mobile phase B; 4-20 min, 76%-69% mobile phase B; 20-25 min, 69%-60% mobile phase B; repeated 3 times to obtain the chromatograms of the active substances in each test raw material.

[0042] Test analysis: Figure 1 As shown, the product obtained in Example 1 showed 6 characteristic peaks. The chromatographic retention time and peak area of ​​the chromatogram of the plum callus extract were compared with the active substance standard peaks, and the 6 characteristic peaks were found to be chlorogenic acid, cryptochlorogenic acid, quercetin, rutin, hyperoside, and isoquercetin.

[0043] like Figure 2 As shown, the product obtained in Comparative Example 1 showed two characteristic peaks. The chromatographic retention time and peak area of ​​the chromatogram of the plum blossom plant extract were compared using the active substance standard peak, and it was found that the two characteristic peaks were quercetin and rutin.

[0044] like Figure 3 As shown, the product obtained in Comparative Example 2 showed 4 characteristic peaks. The chromatographic retention time and peak area were compared with the peaks of the active substance standard product, and the characteristic peaks were found to be: quercetin, rutin, hyperoside, and isoquercetin.

[0045] like Figure 4 As described above, the commercially available plum plant extract in Comparative Example 3 showed two characteristic peaks. The chromatographic retention time and peak area were compared using the active substance standard peak, and it was found that one of the characteristic peaks was chlorogenic acid, with a retention time of 23.763.

[0046] The chromatographic results showed that the types of active substances in the plum callus extract of Example 1 were far more than those in Comparative Examples 1-3.

[0047] Test 2: Anti-aging efficacy test (1) diluting the product obtained in Example 1 with DMEM culture medium to obtain a DMEM culture medium containing 0.5% plum callus extract; (2) Take HSF cells (fibroblasts) with good growth status and culture them at 5×10 per well. 3 The cells were inoculated at a density of 100 in a 6-well culture plate and cultured overnight at 37°C in a 5% CO2 environment. (3) The culture medium was discarded and the cells were washed twice with PBS (phosphate buffer solution). Except for the blank control group, the model group and the sample group were given UVB 50mJ / cm 2 The irradiation dose was used for modeling; (4) After modeling, the cells were treated accordingly according to the control group and sample group (sample group: 2 mL of DMEM culture medium containing 0.5% plum callus extract; control group: 2 mL of DMEM culture medium), with 3 replicates for each treatment group, and incubated for 24 h after addition; (5) Remove the cell culture medium, collect the cells, add lysis buffer, extract total cell RNA, detect RNA concentration and purity, and use RT-qPCR technology to measure the gene expression levels of barrier-related genes COL1A1 (type I collagen) and ELN (elastin); (6) GraphPad Prism 8 software was used for data analysis, and one-way analysis of variance was used to analyze the differences between the control group and the model group. P <0.05 (#) was considered to be significantly different. P <0.01 (##) was considered to be extremely significantly different. P <0.001 (###) was considered to be a highly significant difference; the difference between the sample group and the model group was analyzed using one-way analysis of variance. P <0.05 (*) was considered to be significantly different. P <0.01 (**) was considered to be extremely significantly different. P <0.001 (***) was considered a highly significant difference.

[0048] Test analysis: Figure 5 As shown in the figure, compared with the blank control group, the relative expression of COL1A1 and ELN genes in the model group after UVB stimulation decreased, indicating that the modeling of this experiment was successful and the model group was effective. Compared with the model group, adding 0.5% plum callus extract after UVB treatment can promote the expression of collagen and elastin related genes. 0.5% plum callus extract can upregulate the expression of elastin gene ELN gene by 96.82% ( P <0.001); can upregulate the expression of collagen gene COL1A1 by 32.30% ( P<0.01). In vitro cell experiments show that plum callus extract can promote the expression of collagen genes in fibroblasts, increase the collagen content and elastin content inside cells, reduce the loss of type I and type III collagen caused by aging, reduce skin elasticity, and delay the aging process of skin.

[0049] Test 3: Anti-wrinkle efficacy test (1) diluting the product obtained in Example 1 with DMEM culture medium to obtain a DMEM culture medium containing 0.5% plum callus extract; (2) Take HaCaT cells (human immortalized keratinocytes) in good growth condition and plate 5×10 cells per well. 3 The cells were inoculated at a density of 100 in a 6-well culture plate and cultured overnight at 37°C in a 5% CO2 environment. (3) After the culture medium was discarded and the cells were washed twice with PBS, the model group and the sample group were given UVB 50 mJ / cm 2 The irradiation dose was used for modeling; (4) After modeling, the cells were treated accordingly according to the control group and sample group (sample group: 2 mL of DMEM culture medium containing 0.5% plum callus extract; control group: 2 mL of DMEM culture medium), with 3 replicates for each treatment, and incubated for 24 h after addition; (5) The cell culture medium was removed, the cells were collected, lysis buffer was added, total cell RNA was extracted, RNA concentration and purity were detected, and RT-qPCR technology was used to determine the gene expression levels of collagen degradation-related genes MMP1 (matrix metalloproteinase 1) and MMP3 (matrix metalloproteinase 3); (6) GraphPad Prism 8 software was used for data analysis, and one-way analysis of variance was used to analyze the differences between the control group and the model group. P <0.05 (#) was considered to be significantly different. P <0.01 (##) was considered to be extremely significantly different. P <0.001 (###) was considered to be a highly significant difference; the difference between the sample group and the model group was analyzed using one-way analysis of variance. P <0.05 (*) was considered to be significantly different. P <0.01 (**) was considered to be extremely significantly different. P <0.001 (***) was considered a highly significant difference.

[0050] Test analysis: Figure 6As shown in the figure, compared with the blank control group, the relative expression of MMP1 and MMP3 genes in the model group cells after UVB stimulation increased significantly, indicating that the modeling of this experiment was successful and the model group was effective. Compared with the model group, 0.5% plum callus extract can significantly inhibit the expression of MMP1 and MMP3 genes, and the inhibition rate of MMP1 gene expression is 17.08% ( P <0.05); the inhibition rate of MMP3 gene expression was 9.71% ( P <0.05). In vitro cell experiments show that the plum callus extract of the present invention can inhibit the expression of cell matrix metalloproteinases MMP1 and MMP3 genes.

[0051] Test 4: Soothing effect test The test analysis method was the same as test 3. The barrier-related genes KRT1 (keratin 1), KRT16 (keratin 16), and TJP1 (tight junction protein) were measured and data analyzed in the model group, sample group, and blank control group.

[0052] Test analysis: Figure 7 As shown in the figure, compared with the blank control group, the relative expression of KRT1, KRT16, and TJP1 genes in the model group cells after UVB stimulation decreased significantly, indicating that the modeling of this experiment was successful and the model group was effective. Compared with the model group, 0.5% plum callus extract can significantly upregulate the expression of KRT1, KRT16, and TJP1 genes, and the expression of KRT1 gene increased by 405.76% ( P <0.001); KRT16 gene expression was upregulated by 159.12% ( P <0.01); TJP1 gene expression was upregulated by 291.68% ( P <0.001). KRT1, KRT16 and TJP1 are closely related to skin barrier function. Plum callus extract can promote the expression of skin keratin, promote the expression of skin tight junction structural protein, and enhance skin defense.

[0053] Test 5: Antioxidant efficacy test (1) diluting the product obtained in Example 1 with DMEM culture medium to obtain a DMEM culture medium containing 0.5% plum callus extract; (2) Take HaCaT cells (human immortalized keratinocytes) in good growth condition and plate 5×10 cells per well. 3 The cells were inoculated at a density of 100 in a 6-well culture plate and cultured overnight at 37°C in a 5% CO2 environment. (3) Remove the waste culture medium and perform corresponding treatments according to the control group and sample group (sample group: 2 mL of DMEM culture medium containing 0.5% plum callus extract; control group: 2 mL of DMEM culture medium), with 3 replicates for each treatment, and incubate for 24 h after addition; (4) Remove the cell culture medium, collect the cells, add lysis buffer, extract the total cell RNA, and detect the RNA concentration and purity; The qualified RNA was processed by SQK-PCB109 kit for gene library establishment, and then the MinION nanopore sequencer was used to measure the gene expression. The Nanopore software was used to perform quality analysis on the obtained whole gene sequence file, and the DESeq2 software was selected to compare the expression of the reference genome of the cell gene samples and calculate the difference in gene expression. Finally, DESeq2 was used to perform gene expression difference analysis to obtain the expression of genes related to the cellular antioxidant enzyme system, including SOD3 (superoxide dismutase 3), NQO1 (NAD(P)H quinone dehydrogenase 1), NFE2L1 (nuclear factor erythroid 2-related factor 1), GPX3 (glutathione peroxidase), and HMOX1 (heme oxygenase 1).

[0054] Test analysis: Figure 8 As shown, compared with the blank control group, 1% plum callus extract treatment increased SOD3 gene expression by 151.37%, NQO1 gene expression by 31.73%, NFE2L1 gene expression by 25.60%, GPX3 gene expression by 21.21%, and HMOX1 gene expression by 43.79%. Plum callus extract can increase the antioxidant enzyme activity of HaCaT cells, remove excess free radicals in skin cells, and alleviate oxidative damage to cells. It can be used as an antioxidant raw material in skin care products.

[0055] Test 6: Anti-inflammatory efficacy test The test analysis method was the same as test 3. The inflammation-related cytokine genes IL-1β (interleukin 1β), IL-6 (interleukin 6), TNF-α (tumor necrosis factor), and PGE2 (prostaglandin E2) were measured and data analyzed in the model group, sample group, and blank control group.

[0056] Test analysis: Fig. 9As shown in the figure, compared with the blank control group, after UVB irradiation, the gene expression of inflammatory cytokines IL-1β, IL-6, TNF-α, and PGE2 in the cells increased, indicating that the model group of this experiment was effective. Compared with the model group, the gene expression of IL-1β, IL-6, TNF-α, and PGE2 was inhibited by 0.5% of the plum callus extract in the cells, and the inhibition rate of IL-1β gene expression was 80.08% ( P <0.001); the inhibition rate of IL-6 gene expression was 80.78% ( P <0.001); the inhibition rate of TNF-α gene expression was 79.66% ( P <0.001); the inhibition rate of PGE2 gene expression was 61.74% ( P <0.05). The plum callus extract of the present invention can inhibit the secretion of inflammation-related cytokines caused by UVB and slow down the occurrence of immune inflammation.

[0057] Test 7: Cell viability test (1) The products obtained in Example 1 and Comparative Example 1 were diluted with DMEM culture medium to obtain the following test samples: DMEM culture medium containing 0.1% plum callus extract, DMEM culture medium containing 0.5% plum callus extract, DMEM culture medium containing 0.1% plum plant extract, and DMEM culture medium containing 0.5% plum plant extract; (2) Preparation of HaCaT cells: Take cells in logarithmic growth phase and add (8-10)×10 3 The cells were inoculated at a density of 1000 and 1000 μg / mL in a 6-well plate and incubated in a 37°C, 5% CO2 incubator for 24 h. (3) CCK-8 method can be used to determine the number of viable cells in cells: discard the culture medium, set up a sample group (6 replicates) and a blank control group for comparative experiments (using 90 mJ / cm 2 UVB irradiation, the sample groups were added with the test samples of step (1), and the blank control group was added with 100 μL DMEM basal culture medium; 100 μL was added to each sample group and incubated for 24 h; 10 μL CCK-8 reagent was added and incubated for 1 h, and the OD value at 450 nm was measured); (4) The data were statistically analyzed using GraphPad Prism 8 software. One-way ANOVA was used to determine the significance of the differences between the groups. “*” indicates a significant difference. P <0.05, “**” indicates extremely significant difference, P <0.01, "***" indicates a very significant difference, P <0.001.

[0058] Test analysis: Fig.10 As shown in the figure, compared with the blank control group, after 24 hours of co-culture of 0.1% and 0.5% plum callus extracts with HaCaT cells, the cell survival rate was above 80%, and there was a very significant difference compared with the model group, indicating that within this concentration threshold, the plum callus extract alleviated the cell damage and death after UVB irradiation. At the same concentration, the plum callus extract showed a higher cell viability effect than the plum plant extract.

[0059] Test 8: DPPH clearance test (1) The products obtained in Example 1 and Comparative Example 1 were diluted with DMEM culture medium to obtain the following test samples: DMEM culture medium containing 0.1% plum blossom callus extract, DMEM culture medium containing 1% plum blossom callus extract, DMEM culture medium containing 5% plum blossom callus extract, DMEM culture medium containing 0.1% plum blossom plant extract, DMEM culture medium containing 1% plum blossom plant extract, and DMEM culture medium containing 0.1% plum blossom plant extract; (2) Determination of free radical scavenging rate by DPPH method: The DPPH scavenging rate index is used to evaluate the antioxidant capacity of the test sample. The greater the scavenging rate, the stronger the antioxidant capacity; Weigh a certain amount of DPPH and prepare a 0.04 mg / mL DPPH solution with anhydrous ethanol. Take 2 mL of the solutions of different test samples respectively, add 2 mL of DPPH solution to each group of samples, mix well, leave at room temperature for 30 minutes, and centrifuge at 5000 r / min for 10 minutes; take the supernatant and measure the absorbance at 517 nm. Use 1% concentration of VC solution as a positive control.

[0060] The scavenging rate of the sample on DPPH free radicals was calculated using the following formula: Inhibition rate = (1-(A 样品 -A 空白 ) / A 控制 )X100%. The data were tested using T-test for paired differences between samples at the same concentration, where P <0.05 (*) was considered to be significantly different. P <0.01 (**) was considered to be extremely significantly different. P <0.005 (***) was considered a highly significant difference.

[0061] Test analysis: Fig.11 As shown in Figure 2, the DPPH scavenging rate of 0.1% plum plant extract was 1.73%; the DPPH scavenging rate of the same proportion of plum callus extract was 24.60% ( P<0.001); the DPPH scavenging rate of 1% plum blossom plant extract was 16.97%, and the DPPH scavenging rate of the same proportion of plum blossom callus extract was 48.46% ( P <0.001); the DPPH scavenging rate of 5% plum blossom plant extract was 54.23%, and the DPPH scavenging rate of the same proportion of plum blossom callus extract was 85.04% ( P <0.001). Compared with the blank control group (treated with 1% VC), the DPPH scavenging rate of the 5% plum callus extract was close to that of the 1% VC. Under the same concentration conditions, the plum callus extract of the present invention has a stronger DPPH scavenging effect than the plum plant extract.

[0062] Test 9: Skin brightening effect test (1) diluting the product obtained in Example 1 with DMEM culture medium to obtain a DMEM culture medium with a concentration of 0.5% of plum blossom callus extract; (2) Take B16-F10 cells and add 1×10 cells per well. 6 The cells were inoculated at a density of 100 μg / mL in a 6-well culture plate. After overnight culture, the culture medium was discarded and the sample obtained in (1) was added and cultured for another 24 h. (3) After the culture is completed, the cells are digested with trypsin and collected. 1 mL of 1 mol / L NaOH solution containing 10% DMSO is added. The mixture is incubated in an 80°C water bath for 2 h and vortexed to obtain a melanin solution. (4) Take 200 μL of melanin solution and add it to a 96-well plate. Set up three replicates for each sample and measure A405nm. (5) GraphPad Prism 8 software was used for data analysis, and the differences between the sample group and the control group were analyzed using one-way analysis of variance. P <0.05 (*) was considered to be significantly different. P <0.01 (**) was considered to be extremely significantly different. P <0.001 (***) was considered a highly significant difference.

[0063] Test analysis: Fig.12 As shown in the figure, compared with the blank control group, the relative content of melanin decreased by 43.88% after the cells were treated with 0.5% plum callus extract ( P <0.01). The plum callus extract of the present invention can reduce the relative content of melanin.

[0064] Test 10: Efficacy test on reducing AGEs (advanced glycation end products) The ability of a product to resist the effects of glycation can be evaluated by testing CML (carboxymethyl lysine, a glycation product), as follows: (1) Preparation of test samples: The product obtained in Example 1, carnosine and red phaff were diluted with DMEM culture medium to obtain the following samples: 0.5% DMEM culture medium containing plum callus extract, 5% red phaff and 50 mM carnosine; (2) Take HSF cells at 1×10 per well. 6 The cells were inoculated at a density of 1000 in a 6-well culture plate. After overnight culture, the culture medium was discarded, and the test samples were added and cultured for another 19 h. (3) After the incubation is completed, replace the test sample in the culture plate and continue incubation for 5 hours; (4) Collect cells by trypsin digestion, centrifuge at 2000g for 5 min at room temperature, add 400 μL PBS for resuspending and count; (5) Place the resuspended cell solution in liquid nitrogen for 3 minutes, and then thaw it in a 37°C constant temperature water bath for 5 minutes; (6) Repeat step (5) three times to obtain a cell lysate containing CML, centrifuge at 10,000 rpm for 5 min, and take the supernatant to detect the CML content; (7) Detecting the CML content using a CML ELISA detection kit, and dividing the content detected by the kit by the number of cells in the corresponding sample to obtain the CML content per million cells; (8) GraphPad Prism 8 software was used for data analysis, and one-way analysis of variance was used to analyze the differences between the control group and the model group. P <0.05 (#) was considered to be significantly different. P <0.01 (##) was considered to be extremely significantly different. P <0.001 (###) was considered to be a highly significant difference; the difference between the sample group and the model group was analyzed using one-way analysis of variance. P <0.05 (*) was considered to be significantly different. P <0.01 (**) was considered to be extremely significantly different. P <0.001 (***) was considered a highly significant difference.

[0065] Test results: Fig.13 As shown in the figure, compared with the blank control group, the CML content in the model group stimulated by MGO increased significantly, indicating that the model group was effective. Compared with the model group, the use of 0.5% plum callus extract could significantly inhibit 45.79% CML ( P <0.001), 50mM carnosine can significantly inhibit 49.83% CML ( P <0.001), using 5% red phaff (astaxanthin) can significantly inhibit 40% CML ( P <0.001).

[0066] Example 2, Comparative Examples 4-9 Example 2 and Comparative Examples 4-9 all provide a kind of plum blossom callus, and the difference lies in: the components and contents of the proliferation medium are different. The components of the proliferation medium in each example are specifically shown in the following table: Components Example 2 Comparative Example 4 Comparative Example 5 Comparative Example 6 Comparative Example 7 Comparative Example 8 Comparative Example 9 MS medium (g / L) 2 2 4 4 4 4 4 Sucrose (g / L) 30 30 30 30 30 30 30 Agar (g / L) 7 7 7 7 7 7 7 BA (mg / L) 0.5 1 / 1 / / / TDZ (mg / L) 2 2 / / 2 / / NAA (mg / L) 0.5 1 / / / 1 / 2,4-D (mg / L) / / / / / / 1 pH 5.8 5.8 5.8 5.8 5.8 5.8 5.8 Table 1 The specific preparation steps of Example 2 and Comparative Examples 4-9 are as follows: The ancient plum blossom plants were cleaned, soaked in 70% ethanol for 30 seconds, washed 5 times with sterile water, soaked in 10% sodium hypochlorite solution (with 0.1% Tween 20) for 2 minutes, washed 5 times with sterile water, and then the surface moisture of the plants was dried with sterile filter paper; Cut the sterilized ancient plum plant, cut out a tissue block of about 10 mm, and then inoculate it into an induction medium (4g / LMS medium, 30g / L sucrose, 7 / L agar, 0.5mg / L 6-BA, 0.5mg / L NAA, 2mg / L TDZ). The pH of the induction medium is 5.8, and the dark reaction is carried out at 25°C for 20-30 days. Then 5 g of the induced callus was inoculated into a proliferation medium and subjected to a dark reaction at 25° C. for 20-25 days to obtain plum blossom callus.

[0067] The contents of total flavonoids and total polyphenols in the callus of plum blossom cultured for 25 days were determined.

[0068] The test method for total flavonoids is as follows: take 5 mg of the sample to be tested and place it in a 25 mL volumetric flask (0.5 mL of 50% ethanol as a blank control), add 1 mL of 5% NaNO2 solution and 1 mL of 10% Al (NO3) 3 solution in sequence, shake well and let stand for 6 minutes; then add 10 mL of 4% NaOH solution and shake to mix; finally add 50% ethanol to make up to 25 mL, shake well and let stand for 20 minutes, and measure the absorbance at the maximum absorption wavelength of 510 nm.

[0069] The total flavonoid content in the filtrate was calculated by regression equation, and the calculation formula was: Total flavonoid content (μg / mL) = calculated concentration (μg / mL) * filtrate dilution multiple The test method for total flavonoids is as follows: take 5 mg of the sample to be tested and place it in a 25 mL volumetric flask, add 6 mL of distilled water, then add 1 mL of Folin phenol reagent and shake to mix, add 3 mL of 7.5% Na2CO3 within 8 minutes, react in the dark for 2 hours, and measure the absorbance value at a wavelength of 765 nm.

[0070] The total polyphenol content in the filtrate was calculated by regression equation, and the calculation formula is as follows: Total polyphenol content (μg / mL) = calculated concentration of standard curve (μg / mL) * filtrate dilution multiple The test results are shown in the following table: Serial number Total flavonoids (mg / g) Total polyphenols (mg / g) Example 2 183.57±1.64 71.73±0.02 Comparative Example 4 108.70±2.31 24.77±0.03 Comparative Example 5 41.98±0.69 10.42±0.06 Comparative Example 6 76.34±7.70 15.81±0.16 Comparative Example 7 61.98±2.26 15.14±0.16 Comparative Example 8 53.13±4.07 19.03±0.20 Comparative Example 9 27.18±2.52 2.73±0.02 Table 2 As shown in Table 2, the total flavonoids and total polyphenols content of the plum blossom callus in Example 2 is the highest, which is 4.4 times the total flavonoids content and 6.9 times the total polyphenols content of Comparative Example 5 (without adding phytonutrients). Compared with Example 2, although Comparative Example 4 has a similar proliferation medium formula, the content of total flavonoids and total polyphenols is weaker than that of Example 2 due to the different content of phytonutrients.

[0071] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a plum blossom callus extract, characterized in that: The following steps are involved: S1. Cultivation: cutting plum blossom plants to obtain plum blossom tissue blocks; Carrying out induction dark culture and proliferation dark culture on the plum blossom tissue block to obtain plum blossom callus; S2, freeze-drying: pre-freeze the plum blossom callus, and then vacuum freeze-dry for 48-72 hours to obtain freeze-dried plum blossom callus, and store it at low temperature and away from light; S3, extraction: adding liquid nitrogen to a grinding tool for precooling, then adding the freeze-dried plum blossom callus and grinding into powder to obtain plum blossom callus powder; Mixing 50% ethanol with the plum blossom callus powder, performing ultrasonic extraction, and filtering to obtain a filtrate; The filtrate is subjected to rotary evaporation to obtain the target product; The culture medium for the induction dark culture and the proliferation dark culture is 2-4 g / L MS culture medium, 25-35 g / L sucrose, 6-8 g / L agar, 0.5 mg / L 6-BA, 2 mg / L TDZ and 1 mg / L NAA, and the pH is 5-6.

5.

2. The method for preparing a plum callus extract according to claim 1, characterized in that: The plum blossom plant is an ancient plum blossom plant.

3. The method for preparing a plum blossom callus extract according to claim 2, characterized in that: In S1, the ancient plum blossom plant is sterilized, and the specific steps are as follows: The ancient plum blossom plant was cleaned, then immersed in 70% ethanol for 30 seconds, and then washed with sterile water; Soak the washed ancient plum plants in 10% sodium hypochlorite solution for 2 minutes, wash with sterile water, and absorb the moisture.

4. The method for preparing a plum callus extract according to claim 1, characterized in that: The induction dark culture time is 20-30 days, and the proliferation dark culture time is 20-25 days.

5. The method for preparing a plum callus extract according to claim 1, characterized in that: The vacuum freeze drying conditions are: cold trap temperature -80°C, freezing chamber temperature -45 to -35°C, vacuum degree 0-10pa; In S2, the storage temperature of the freeze-dried plum callus is -80 to -20°C.

6. The method for preparing a plum callus extract according to claim 1, characterized in that: The solid-liquid ratio of the plum blossom callus powder to the 50% ethanol is 1:10; The conditions of the ultrasonic extraction are as follows: ultrasonic power 2000w, ultrasonic temperature 25-45°C, ultrasonic time 30min, and ultrasonic extraction 3 times.

7. The method for preparing a plum callus extract according to claim 1, characterized in that: In S3, the rotary evaporation is divided into three times; The first rotary evaporation conditions are 35-42°C, 80-120 rpm, and 35-40 min. The second rotary evaporation conditions are 48°C, 80-120 rpm, and 30 min. The third rotary evaporation conditions are 48°C, a rotation speed of 80-120 rpm, and rotary evaporation for 30-45 minutes.

8. A plum callus extract, characterized in that: The extract is prepared according to the method for preparing a plum callus extract according to any one of claims 1 to 7.

9. The plum blossom callus extract according to claim 8, characterized in that: Contains the following active ingredients: chlorogenic acid, cryptochlorogenic acid, quercetin, rutin, hyperoside and isoquercetin.

10. Use of the plum callus extract according to claim 8 in the preparation of skin care products with anti-aging, anti-inflammatory, soothing, anti-oxidation and skin lightening effects.

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