Preparation method, product and application of synotis erythropappa extract

Abstract

The present invention relates to a preparation method, product and application of an extract of Synotis erythropappa. The preparation method includes mixing a Synotis erythropappa raw material with a composite enzyme for enzymatic hydrolysis, and subjecting the enzymatic hydrolysis reaction solution to high-voltage pulsed electric field extraction to obtain the Synotis erythropappa extract; the composite enzyme includes a combination of cellulase, hemicellulase, pectinase and tannase. The present invention uses an enzymatic hydrolysis method combined with high-voltage pulsed electric field for extraction, and further uses a specific enzyme combination, and it is found that the combination of these four enzymes can interact with each other, and has a synergistic promoting effect in improving the enzymatic hydrolysis extraction effect and thus enhancing the antioxidant effect of the extract. The prepared extract is very suitable for skin care products, is mild and non-irritating, and has good stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extracts, and relates to a preparation method, product and application of Synotis erythropappa extract. Background Art

[0002] Synotis erythropappa is a plant of the genus Synotis in the family Asteraceae, a herbaceous plant. The stem is erect, the leaves are mostly ovate and oblong-lanceolate, and the fruits are cylindrical. It is mainly produced in southeastern Tibet, western Hubei, Sichuan, Yunnan and other places in China. It is a perennial herb with a rhizome. The rhizome is woody, erect or ascending obliquely, with villous fibrous roots. The stem is solitary or several, erect or rarely prostrate, up to 100 cm tall, usually with inflorescence branches in the upper part, without leaves in the lower part during the flowering period, covered with yellow-brown pubescence, arachnoid pubescence or nearly glabrous. The leaves have long petioles, ovate, ovate-lanceolate or oblong-lanceolate, with the apex gradually pointed or caudate-acuminate, the base cordate, subtruncate, rounded or cuneate, the margin with regular dense to coarse unequal shallow to deep serrations or teeth, papery or thinly papery, pubescent to glabrous on the upper surface, especially pubescent to nearly glabrous along the veins on the lower surface, pinnately veined, with 3-5 pairs of lateral veins, arcuately ascending obliquely, the veins obvious on the lower surface; the petiole is 2-6 cm long, pubescent or nearly glabrous; the leaves on the upper part and branches are smaller, narrowly lanceolate, with short petioles.

[0003] Synotis erythropappa has high medicinal value, dispelling wind and removing dampness, clearing heat and detoxifying, and relieving itching. It can be used for acute red and swollen eyes, sores, dermatitis, and traumatic injuries. In addition, studies have shown that the extract of Synotis can inhibit the activity of tyrosinase and thus block the production of melanin, thereby achieving the effect of whitening. Moreover, Synotis erythropappa is rich in flavonoid active substances, which can be added to products as raw materials for antioxidant cosmetics to effectively improve the related efficacy of the products. Therefore, it is very necessary to provide a new, safe, environmentally friendly and efficient preparation method for Synotis erythropappa extract. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a preparation method, product and application of Synotis erythropappa extract.

[0005] To achieve the purpose of this invention, the following technical solutions are adopted:

[0006] In the first aspect, the present invention provides a preparation method of Synotis erythropappa extract, and the preparation method includes the following steps:

[0007] Mix the Synotis erythropappa raw material with a composite enzyme for enzymatic hydrolysis, perform high-voltage pulsed electric field extraction on the enzymatic hydrolysis reaction solution, and after centrifugation and filtration, collect the filtrate to obtain the Synotis erythropappa extract;

[0008] The composite enzyme includes a combination of cellulase, hemicellulase, pectinase and tannase.

[0009] The present invention creatively discovers that a composite enzyme composed of four enzymes, namely cellulase, hemicellulase, pectinase and tannase, can interact with each other and have a positive mutual influence, improving the extraction efficiency of Synotis erythropappa, thereby increasing the active ingredients in the extract product.

[0010] Moreover, in the present invention, enzymatic hydrolysis and high-voltage pulsed electric field technology are used for the synergistic extraction of Synotis erythropappa raw materials, which can efficiently extract the effective components in Synotis erythropappa, prepare a high-activity Synotis erythropappa extract product, and can further be applied to cosmetics to improve the antioxidant efficacy of the product.

[0011] Preferably, the Synotis erythropappa raw material is the crushed Synotis erythropappa obtained by crushing and sieving Synotis erythropappa.

[0012] Preferably, the mesh number of the sieve used in the sieving process is not less than 20 mesh, such as 20 mesh, 25 mesh, 30 mesh, 35 mesh, 40 mesh, 45 mesh, 50 mesh, etc. Other specific point values within this numerical range can be selected and will not be elaborated here one by one.

[0013] Preferably, the mass ratio of the cellulase, hemicellulase, pectinase and tannase is (3 - 6):(1 - 5):(1 - 4):(2 - 7).

[0014] Among them, the specific values in the above "3 - 6" can be, for example, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.

[0015] The specific values in the above "1 - 5" can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.

[0016] The specific values in the above "1 - 4" can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, etc.

[0017] The specific values in the above "2 - 7" can be, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, etc. Other specific point values within this numerical range can be selected and will not be elaborated here one by one.

[0018] In the present invention, when the dosage ratio of each enzyme in the adopted composite enzyme combination is within the above specific range, the enzymatic hydrolysis extraction effect on Synotis erythropappa is better.

[0019] Preferably, before enzymatic hydrolysis, the Synotis erythropappa material is mixed with water, and the solid-liquid ratio is 1:(10 - 50) g / mL. For example, it can be 1:10 g / mL, 1:12 g / mL, 1:15 g / mL, 1:18 g / mL, 1:20 g / mL, 1:22 g / mL, 1:25 g / mL, 1:28 g / mL, 1:30 g / mL, 1:32 g / mL, 1:35 g / mL, 1:38 g / mL, 1:40 g / mL, 1:42 g / mL, 1:45 g / mL, 1:50 g / mL, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0020] In the present invention, when the material-liquid ratio of the Synotis erythropappa material and water is within the above specific range, a better enzymatic hydrolysis effect is achieved.

[0021] Preferably, the addition amount of the composite enzyme is 0.1‰ - 0.5‰ of the mass of the Synotis erythropappa material. For example, it can be 0.1‰, 0.15‰, 0.2‰, 0.25‰, 0.3‰, 0.35‰, 0.4‰, 0.45‰, 0.5‰, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0022] In the present invention, when the addition amount of the composite enzyme is within the above specific range, a better extraction effect on the Synotis erythropappa material is achieved.

[0023] Preferably, the pH of the enzymatic hydrolysis is 4 - 6. For example, it can be 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0024] Preferably, water bath heating is used during the enzymatic hydrolysis, and the temperature is 35 - 55°C. For example, it can be 35°C, 38°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0025] Preferably, the time of the enzymatic hydrolysis is 1 - 3 h. For example, it can be 1 h, 1.2 h, 1.5 h, 1.8 h, 2 h, 2.3 h, 2.5 h, 2.8 h, 3 h, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0026] Preferably, the field strength of the high-voltage pulsed electric field is 30 - 50 kv / m. For example, it can be 30 kv / m, 32 kv / m, 35 kv / m, 38 kv / m, 40 kv / m, 42 kv / m, 45 kv / m, 48 kv / m, 50 kv / m, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0027] Preferably, the number of electric shocks of the high-voltage pulsed electric field is 60 - 140 times. For example, it can be 60 times, 70 times, 80 times, 90 times, 100 times, 110 times, 120 times, 130 times, 140 times, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0028] In the present invention, the high-voltage pulsed electric field extraction carried out with the field strength and the number of electric shocks within the above specific parameter ranges can not only gently inactivate the residual enzymes during the enzymatic hydrolysis process, but also further extract the active ingredients in Synotis erythropappa, with a better extraction effect.

[0029] Preferably, stirring treatment is carried out after the high-voltage pulsed electric field extraction.

[0030] Preferably, the rotation speed of the stirring is 50 - 80 rpm. For example, it can be 50 rpm, 60 rpm, 70 rpm, 80 rpm, etc.; the time is 20 - 40 min. For example, it can be 20 min, 22 min, 25 min, 28 min, 30 min, 32 min, 35 min, 38 min, 40 min, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0031] Preferably, the rotation speed of the centrifugation is 8000 - 12000 rpm. For example, it can be 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm, 10000 rpm, 10500 rpm, 11000 rpm, 11500 rpm, 12000 rpm, etc.; the time is 3 - 8 min. For example, it can be 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 min, etc.

[0032] In the second aspect, the present invention provides an extract of Synotis erythropappa, and the extract of Synotis erythropappa is prepared by the preparation method described in the first aspect.

[0033] In the third aspect, the present invention provides an application of the extract of Synotis erythropappa described in the second aspect in cosmetics.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention has developed a completely new method for preparing extracts of **Synotis erythropappa**, creatively using an enzymatic hydrolysis method combined with high-voltage pulsed electric field technology to extract **Synotis erythropappa** raw materials, and further using a composite enzyme combination containing four enzymes, namely cellulase, hemicellulase, pectinase, and tannase, for enzymatic hydrolysis. The combination of the four enzymes can interact with each other and have a positive mutual influence, improving the extraction efficiency of **Synotis erythropappa** and thus increasing the active ingredients in the extract product. The extraction method of the present invention can efficiently extract the active ingredients in **Synotis erythropappa**, prepare a highly active **Synotis erythropappa** extract product, and can further be applied to cosmetics to improve the antioxidant efficacy of the product. **Specific Embodiments**

[0036] To further elaborate on the technical means and effects adopted by the present invention, the following preferred embodiments of the present invention are used to further illustrate the technical solutions of the present invention, but the present invention is not limited to the scope of the embodiments.

[0037] The cellulase involved in the following embodiments is a product with the model S10041 purchased from Shanghai Yuanye Bio-Technology Co., Ltd.;

[0038] The hemicellulase is a product with the model S10045 purchased from Shanghai Yuanye Bio-Technology Co., Ltd.;

[0039] The pectinase is a product with the model S10007 purchased from Shanghai Yuanye Bio-Technology Co., Ltd.;

[0040] The tannase is a product with the model S10207 purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0041] **Example 1**

[0042] This example provides an extract of **Synotis erythropappa**, and its preparation method is as follows:

[0043] Wash, dry, and pulverize the stems and leaves of **Synotis erythropappa**, and pass through a 40-mesh sieve. Mix the pulverized **Synotis erythropappa** with pure water, with a solid-liquid ratio of 1:30 g / mL. Mix and enzymatically hydrolyze with a composite enzyme (composed of 4 parts of cellulase, 4 parts of hemicellulase, 4 parts of pectinase, and 4 parts by weight) at a temperature of 40 °C and a pH of 5.5 for 2 h, with water bath heating. The addition amount of the composite enzyme is 0.3‰ of the mass of the pulverized **Synotis erythropappa** flower. Perform high-voltage pulsed electric field extraction on the enzymatically hydrolyzed reaction solution after enzymatic hydrolysis. The extraction conditions are 40 kv / m, the number of electric shocks is 100 times, stir at 80 rpm for 30 min after the reaction, centrifuge at 10,000 rpm for 5 min, and filter the filtrate through a 0.22-μm filter paper, and collect the filtrate and freeze-dry to obtain the **Synotis erythropappa** extract.

[0044] **Example 2**

[0045] This embodiment provides a synotis erythropappa extract, and its preparation method is as follows:

[0046] Wash, dry, and pulverize the stems and leaves of synotis erythropappa, and then pass through a 20-mesh sieve. Take the pulverized synotis erythropappa and mix it evenly with pure water. The solid-liquid ratio is 1:20 g / mL. Mix and enzymatically hydrolyze it with a complex enzyme (composed of 5 parts of cellulase, 3 parts of hemicellulase, 1 part of pectinase, and 7 parts of tannase by weight) at a temperature of 35 °C and a pH of 5 for 2.5 h, and heat it in a water bath. The addition amount of the complex enzyme is 0.4‰ of the mass of the pulverized synotis erythropappa. Perform high-voltage pulsed electric field extraction on the enzymolysis reaction solution after enzymolysis is completed. The extraction conditions are 30 kv / m, the number of electric shocks is 120 times, stir at 50 rpm for 40 min after the reaction, centrifuge at 8000 rpm for 8 min, and then filter through a 0.22-μm filter paper. After collecting the filtrate, freeze-dry it to obtain the synotis erythropappa extract.

[0047] Example 3

[0048] This embodiment provides a synotis erythropappa extract, and its preparation method is as follows:

[0049] Wash, dry, and pulverize the stems and leaves of synotis erythropappa, and then pass through a 30-mesh sieve. Take the pulverized synotis erythropappa and mix it evenly with pure water. The solid-liquid ratio is 1:40 g / mL. Mix and enzymatically hydrolyze it with a complex enzyme (composed of 6 parts of cellulase, 5 parts of hemicellulase, 3 parts of pectinase, and 2 parts of tannase by weight) at a temperature of 45 °C and a pH of 4.5 for 4.5 h, and heat it in a water bath. The addition amount of the complex enzyme is 0.2‰ of the mass of the pulverized synotis erythropappa. Perform high-voltage pulsed electric field extraction on the enzymolysis reaction solution after enzymolysis is completed. The extraction conditions are 50 kv / m, the number of electric shocks is 80 times, stir at 60 rpm for 36 min after the reaction, centrifuge at 12000 rpm for 3 min, and then filter through a 0.22-μm filter paper. After collecting the filtrate, freeze-dry it to obtain the synotis erythropappa extract.

[0050] Example 4

[0051] This embodiment provides a synotis erythropappa extract, using the dried and pulverized synotis erythropappa raw material of Example 1. The difference in its preparation method from that of Example 1 is only that the addition amount of the complex enzyme is 1‰ of the pulverized synotis erythropappa.

[0052] Example 5

[0053] This embodiment provides a synotis erythropappa extract, using the dried and pulverized synotis erythropappa raw material of Example 1. The difference in its preparation method from that of Example 1 is only that the addition amount of the complex enzyme is 0.05‰ of the pulverized synotis erythropappa.

[0054] Example 6

[0055] This example provides a synotis erythropappa extract, using the synotis erythropappa raw material dried and pulverized in Example 1. The difference in its preparation method from Example 1 is only that the solid-liquid ratio of the synotis erythropappa pulverized product to pure water is 1:5 g / mL.

[0056] Example 7

[0057] This example provides a synotis erythropappa extract, using the synotis erythropappa raw material dried and pulverized in Example 1. The difference in its preparation method from Example 1 is only that the solid-liquid ratio of the synotis erythropappa pulverized product to pure water is 1:60 g / mL.

[0058] Example 8

[0059] This example provides a synotis erythropappa extract, using the enzymatic hydrolysis reaction solution after the enzymatic hydrolysis in Example 1. The difference in its preparation method from Example 1 is only that the electric field strength of the high-voltage pulsed electric field is 20 kv / m.

[0060] Example 9

[0061] This example provides a synotis erythropappa extract, using the enzymatic hydrolysis reaction solution after the enzymatic hydrolysis in Example 1. The difference in its preparation method from Example 1 is only that the electric field strength of the high-voltage pulsed electric field is 70 kv / m.

[0062] Example 10

[0063] This example provides a synotis erythropappa extract, using the enzymatic hydrolysis reaction solution after the enzymatic hydrolysis in Example 1. The difference in its preparation method from Example 1 is only that the number of electric shocks of the high-voltage pulsed electric field is 40 times.

[0064] Example 11

[0065] This example provides a synotis erythropappa extract, using the enzymatic hydrolysis reaction solution after the enzymatic hydrolysis in Example 1. The difference in its preparation method from Example 1 is only that the number of electric shocks of the high-voltage pulsed electric field is 180 times.

[0066] Comparative Example 1

[0067] This comparative example provides a synotis erythropappa extract, using the synotis erythropappa raw material dried and pulverized in Example 1. The difference in its preparation method from Example 1 is only that the composite enzyme does not contain cellulase, and the reduced amount is proportionally distributed to hemicellulase, pectinase, and tannase.

[0068] Comparative Example 2

[0069] This comparative example provides a synotis erythropappa extract, using the synotis erythropappa raw material after drying and pulverizing in Example 1. The difference in its preparation method from that of Example 1 is only that the complex enzyme does not contain hemicellulase, and the reduced amount is proportionally distributed to cellulase, pectinase, and tannase.

[0070] Comparative Example 3

[0071] This comparative example provides a synotis erythropappa extract, using the synotis erythropappa raw material after drying and pulverizing in Example 1. The difference in its preparation method from that of Example 1 is only that the complex enzyme does not contain pectinase, and the reduced amount is proportionally distributed to cellulase, hemicellulase, and tannase.

[0072] Comparative Example 4

[0073] This comparative example provides a synotis erythropappa extract, using the synotis erythropappa raw material after drying and pulverizing in Example 1. The difference in its preparation method from that of Example 1 is only that the complex enzyme does not contain tannase, and the reduced amount is proportionally distributed to cellulase, hemicellulase, and pectinase.

[0074] Comparative Example 5

[0075] This comparative example provides a synotis erythropappa extract, using the synotis erythropappa raw material after drying and pulverizing in Example 1. The difference in its preparation method from that of Example 1 is only that no enzymolysis treatment is carried out. The synotis erythropappa pulverized product is mixed with pure water, heated in a water bath at 40 °C for 2 h, then directly subjected to high-voltage pulsed electric field extraction, and then the filtrate is collected by stirring, centrifugal filtration and freeze-dried to obtain the synotis erythropappa extract.

[0076] Comparative Example 6

[0077] This comparative example provides a synotis erythropappa extract, using the enzymolysis reaction solution after enzymolysis in Example 1. The difference in its preparation method from that of Example 1 is only that no high-voltage pulsed electric field extraction is carried out. The enzymolysis reaction solution is subjected to hot water extraction, the extraction temperature is 75 °C, the extraction time is 60 min, and then the filtrate is collected by stirring, centrifugal filtration and freeze-dried to obtain the synotis erythropappa extract.

[0078] Test Example 1

[0079] Antioxidant effect test

[0080] (1) DPPH-radical scavenging

[0081] Using a 96-well plate, three replicates were set for each group, and the system was 200 μL. Sample group: Take 100 μg of the extract samples prepared in Examples 1-11 and Comparative Examples 1-6 respectively, mix them with 100 μL of pure water, and add 100 μL of 0.1 mM DPPH solution to the reaction system; Control group: Take 100 μL of pure water and then add 100 μL of 0.1 mM DPPH solution. After the reaction system was constructed, it was shaken in the dark for 10 min, and the absorbance at 517 nm was measured using a microplate reader.

[0082] Calculation method of scavenging rate: Scavenging rate (%) = [(A0 - Ax) / A0] × 100%, where A0 is the absorbance of the control group and Ax is the absorbance of the sample group.

[0083] (2) ABTS-radical scavenging

[0084] Preparation of ABTS working solution: Mix ABTS and potassium persulfate solution to prepare a 7 mM ABTS stock solution, store it in the dark for 12 h, shake well and mix it with 10 mM PBS (pH = 7.4) so that the absorbance value of the solution at 734 nm is 0.8 to obtain the ABTS working solution.

[0085] Using a 96-well plate, three replicates were set for each group, and the system was 200 μL. Sample group: Take 100 μg of the extract samples prepared in Examples 1-11 and Comparative Examples 1-6 respectively, mix them with 10 μL of pure water, and add 190 μL of the ABTS working solution to the reaction system; Control group: Take 10 μL of pure water and then add 190 μL of the ABTS working solution. After the reaction system was constructed, it was shaken well and incubated for 6 min, and the absorbance at 734 nm was measured using a microplate reader.

[0086] Calculation method of scavenging rate: Scavenging rate (%) = [(A0 - Ax) / A0] × 100%, where A0 is the absorbance of the control group and Ax is the absorbance of the sample group.

[0087] The test results are shown in Table 1.

[0088] Table 1

[0089]

[0090]

[0091] It can be seen from the data in the table that

[0092] (1) It can be seen from Examples 1 to 3 that the synotis erythropappa extract prepared by the present invention has excellent DPPH-radical and ABTS-radical scavenging effects, showing significant antioxidant effects. Among them, the effect of Example 1 is the best.

[0093] (2) By comparing Example 1 with Examples 4 - 5, it can be seen that when the addition amount of the complex enzyme is within a specific range, the antioxidant effect of the synotis erythropappa extract prepared therefrom is better.

[0094] (3) By comparing Example 1 with Examples 6 - 7, it can be seen that when the ratio of synotis erythropappa to pure water in the mixed feed liquid is not within a specific range, the scavenging effects of the synotis erythropappa extract prepared therefrom on DPPH free radicals and ABTS free radicals are worse than those in Example 1. It can be seen that when within the specific ratio range of the feed liquid used in the enzymatic hydrolysis of the present invention, the enzymatic hydrolysis extraction effect on synotis erythropappa is better.

[0095] (4) By comparing Example 1 with Examples 8 - 11, it can be seen that when the parameters of the electric field strength and the number of electric shocks of the high - voltage pulsed electric field used in the extraction are selected within a specific range, the extraction effect on synotis erythropappa is better, and a synotis erythropappa extract with a higher antioxidant effect is prepared.

[0096] (5) By comparing Example 1 with Comparative Examples 1 - 4, it can be seen that the present invention uses a compound of four enzymes, namely cellulase, hemicellulase, pectinase and tannase, and has an excellent enzymatic hydrolysis extraction effect on synotis erythropappa. When any one of the four enzymes, cellulase, hemicellulase, pectinase and tannase, is absent in Comparative Examples 1 - 4 respectively, the antioxidant effect of the synotis erythropappa extract prepared therefrom decreases compared with that in Example 1, indicating that the four enzymes, cellulase, hemicellulase, pectinase and tannase, have a synergistic promoting effect when applied to the extraction of active ingredients in synotis erythropappa.

[0097] (6) By comparing Example 1 with Comparative Examples 5 - 6, it can be seen that the present invention uses an enzymatic hydrolysis method combined with a high - voltage pulsed electric field technology to extract synotis erythropappa raw materials, and the two synergistically enhance the extraction effect on synotis erythropappa and increase the content of active ingredients in the finally prepared synotis erythropappa extract.

[0098] Test Example 2

[0099] Determination of the total flavonoid content in synotis erythropappa extract by ultraviolet spectrophotometry

[0100] (1) Take 2.4 mL of rutin solutions with different concentration gradients (0 - 0.2 mg / mL), add 0.4 mL of 5% NaNO2 solution respectively, mix well by shaking, and let stand for 6 min; then add 0.4 mL of 10% Al(NO3)3 solution, mix well by shaking, and let stand for 6 min; then add 4 mL of 4% NaOH solution, make up the volume to 10 mL with pure water, mix well by shaking, and let stand for 15 min. Zero with the corresponding solvent blank, measure the absorbance value at 510 nm. Take the rutin concentration as the abscissa and the absorbance at 510 nm as the ordinate to draw a standard curve, and the standard curve is y = 0.0078x - 0.0026.

[0101] (2) Take 2.4 mg of the extract samples prepared in Examples 1 - 3 respectively, mix them with 2.4 mL of pure water, then add 0.4 mL of 5% NaNO2 solution respectively, mix well by shaking, and let stand for 6 min; then add 0.4 mL of 10% Al(NO3)3 solution, mix well by shaking, and let stand for 6 min; then add 4 mL of 4% NaOH solution, make up the volume to 10 mL with pure water, mix well by shaking, and let stand for 15 min. Zero with the corresponding solvent blank, measure the absorbance value at 510 nm. Take three parallel samples for each sample, calculate the total flavonoid content in the extract sample, and the formula is as follows: Total flavonoid content (mg / mL) = (Cx × 10) / V × K × 0.001, where the value 10 is the test volume in mL, 0.001 is for unit conversion. Cx is the concentration calculated by substituting the measured absorbance value into the standard curve, in μg / mL. V is the volume of the sample taken for testing, in mL. K is the sample dilution factor. The results are shown in Table 2.

[0102] Table 2

[0103] Total flavonoid content (mg / mL) Example 1 15.36 Example 2 15.21 Example 3 14.91

[0104] From the data in the table, it can be seen that the Synotis erythropappa extract prepared by the present invention has a high content of flavonoid active substances, has high antioxidant activity, and can be used as a cosmetic raw material to greatly improve its antioxidant effect.

[0105] Test Example 3

[0106] Product safety test

[0107] Fifteen females and fifteen males were selected according to the subject inclusion criteria to participate in the experiment. They were randomly divided into 3 groups (5 males and 5 females in each group), and were respectively given the extract products of Examples 1-3 (after the extract was mixed with pure water to adjust the extract concentration to 10%, it was tested) for trial use. 0.025 mL of the sample was taken with a syringe and placed in the medicine chamber of the patch tape. Immediately, the patch tape with the sample was applied to the normal skin of the left forearm of the subject. At the same time, the tape was gently pressed with the palm to make it evenly adhere to the skin. The test site was marked for observation. The patch was applied once every 24 hours. Half an hour after removing the patch, the inspection was carried out according to the terms of the CTFA guidelines. A total of 6 patch tests were carried out. On the sixth day after the skin application was completed, an additional inspection was carried out to observe the delayed reaction. The data were compared using the results of the last inspection, and the results are shown in Table 3.

[0108] Table 3

[0109]

[0110] From the results in the table, it can be seen that the synotis erythropappa extract product prepared by the present invention has no irritation to the skin and no positive reaction, which proves that the product has a high safety level, low irritation and is relatively mild. To sum up, the present invention has developed a brand-new preparation method for synotis erythropappa extract, and creatively uses the enzymatic hydrolysis method combined with the high-voltage pulsed electric field technology to extract the synotis erythropappa raw material, and further uses a composite enzyme combination containing four enzymes, namely cellulase, hemicellulase, pectinase and tannase, for enzymatic hydrolysis. The combination of the four enzymes can interact with each other and have a positive mutual influence, improving the extraction efficiency of synotis erythropappa, and thus increasing the active ingredients in the extract product. The extraction method of the present invention can efficiently extract the effective components in synotis erythropappa, prepare a high-activity synotis erythropappa extract product, and can be further applied to cosmetics to improve the antioxidant efficacy of the product.

[0111] The applicant declares that the present invention uses the above-mentioned examples to illustrate the technical solutions of the present invention, but the present invention is not limited to the above-mentioned examples, that is, it does not mean that the present invention must rely on the above-mentioned examples to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0112] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0113] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any appropriate manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A preparation method of Synotis erythropappa extract, characterized in that The preparation method comprises the following steps: Mix the Synotis erythropappa raw material with a composite enzyme for enzymatic hydrolysis, subject the enzymatic hydrolysis reaction solution to high-voltage pulsed electric field extraction, and after centrifugation and filtration, collect the filtrate to obtain the Synotis erythropappa extract; The composite enzyme comprises a combination of cellulase, hemicellulase, pectinase and tannase with a mass ratio of (3-6):(1-5):(1-4):(2-7); Before enzymatic hydrolysis, mix the Synotis erythropappa raw material with water, and the solid-liquid ratio is 1:(10-50) g / mL; The field strength of the high-voltage pulsed electric field is 30-50 kv / m; the number of electric shock times is 60-140 times.

2. The preparation method of the synotis erythropappa extract according to claim 1, wherein, The Synotis erythropappa raw material is the Synotis erythropappa pulverized product after pulverizing and sieving the Synotis erythropappa.

3. The preparation method of the synotis erythropappa extract according to claim 2, wherein The mesh number of the sieve used during the sieving process is not less than 20 meshes.

4. The preparation method of the synotis erythropappa extract according to claim 1, characterized in that, The addition amount of the composite enzyme is 0.1‰-0.5‰ of the mass of the Synotis erythropappa raw material; 5. The preparation method of the synotis erythropappa extract according to claim 1, characterized in that, The pH of the enzymatic hydrolysis is 4-6; 6. The preparation method of the synotis erythropappa extract according to claim 1, characterized in that, During enzymatic hydrolysis, water bath heating is adopted, and the temperature is 35-55°C; 7. The preparation method of the synotis erythropappa extract according to claim 1, characterized in that, The time of enzymatic hydrolysis is 1-3 h; 8. The preparation method of the synotis erythropappa extract according to claim 1, characterized in that, Stirring treatment is also carried out after the high-voltage pulsed electric field extraction; 9. The preparation method of the synotis erythropappa extract according to claim 8, characterized in that, The rotation speed of the stirring is 50-80 rpm, and the time is 20-40 min; 10. The preparation method of the synotis erythropappa extract according to claim 1, characterized in that, The rotation speed of the centrifugation is 8000-12000 rpm, and the time is 3-8 min; 11. A synotis erythropappa extract, characterized in that, The Synotis erythropappa extract is prepared by the preparation method according to any one of claims 1-10; 12. Use of the Synotis erythropappa extract according to claim 11 in cosmetics.

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