Preparation method of rhodomyrtus tomentosa fruit extract and product and application of rhodomyrtus tomentosa fruit extract

Abstract

The invention relates to a preparation method of a rhodomyrtus tomentosa fruit extract as well as a product and application of the rhodomyrtus tomentosa fruit extract. The preparation method comprises the following steps: mixing a rhodomyrtus tomentosa fruit raw material with a compound enzyme for enzymolysis, adding an ethanol aqueous solution into an enzymolysis reaction solution after enzymolysis is completed, performing negative pressure cavitation extraction, filtering and collecting filtrate to obtain the rhodomyrtus tomentosa fruit extract, the compound enzyme comprises a combination of cellulase, bromelain, alpha-amylase and tannase. According to the method, the effective components in the longan pulp are extracted by adopting the specific compound enzyme, the related components in the rhodomyrtus tomentosa fruits can be efficiently extracted, a potential synergistic relationship exists among bromelain, alpha-amylase and tannase, the extraction effect can be remarkably improved after the bromelain, the alpha-amylase and the tannase are combined, and the method is suitable for industrial production. The prepared extract product has the effects of resisting inflammation, relieving, resisting oxidation and the like.

Description

Technical Field

[0001] This invention belongs to the field of plant extract technology, and relates to a method for preparing myrtle fruit extract, its products, and applications. Background Technology

[0002] Myrtle (Rhodomyrtus tomentosa (Ait.) Hassk.), a member of the Myrtaceae family and the Rhodomyrtus genus, is an evergreen shrub that can reach a height of 2 meters. Young branches are densely covered with soft hairs. Leaves are opposite, elliptical or obovate. Flowers are long-stalked, usually solitary, purplish-red, with round anthers. The fruit is a berry, ovoid-urn-shaped, turning purplish-black when ripe. Flowering occurs from April to May. According to the *Illustrated Compendium of Chinese Herbal Medicine*, myrtle has the effects of nourishing blood and stopping bleeding, astringing the intestines and consolidating menstruation. It can be used to treat symptoms such as blood deficiency and weakness, hematemesis, epistaxis, hemoptysis due to overexertion, hematochezia, seminal emission, burns, and external bleeding. The pigments contained in myrtle have excellent light and heat stability, making them promising natural pigments.

[0003] Myrtle is not only a traditional Chinese medicine with good pharmacological activity and health benefits, but it also contains a variety of active ingredients such as isovaleraldehyde, eucalyptol, limonene, ketones, myrtle aldehyde, and myrtle acetate. These ingredients can alter skin permeability, allowing the active ingredients to penetrate deeper into the skin and better exert their anti-inflammatory, soothing, antioxidant, and moisturizing effects. Therefore, the various plant-based active components abundant in myrtle can effectively help improve skin quality, nourish and protect the skin, and prevent and alleviate some symptoms related to skin problems. Therefore, it is essential to develop a new, safe, environmentally friendly, and highly effective method for preparing myrtle extract. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing myrtle fruit extract, its products and applications, and in particular, a method for preparing myrtle fruit extract, its products and applications in cosmetics.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing a myrtle fruit extract, the method comprising the following steps:

[0007] The raw material of myrtle fruit was mixed with a compound enzyme for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, an aqueous ethanol solution was added to the enzymatic hydrolysis reaction solution for negative pressure cavitation extraction. The filtrate was collected by filtration to obtain the myrtle fruit extract.

[0008] The complex enzyme comprises a combination of cellulase, bromelain, α-amylase, and tanninase.

[0009] This invention creatively discovers a complex enzyme composed of four enzymes: cellulase, bromelain, α-amylase, and tanninase. Furthermore, it discovers a potential synergistic relationship among bromelain, α-amylase, and tanninase, which can interact and positively influence each other to improve the extraction efficiency of myrtle fruit raw materials, thereby increasing the active ingredients in the extracted product.

[0010] Furthermore, this invention is the first to employ negative pressure cavitation extraction technology to achieve efficient and rapid extraction of active ingredients from myrtle fruit. Compared with extracts obtained by conventional water extraction, the extract obtained by negative pressure cavitation has an increased total sugar content and possesses advantages such as antibacterial, antioxidant, and long shelf life. This is of great significance for the production of myrtle fruit extracts with excellent biological activity.

[0011] Preferably, the mass ratio of cellulase, bromelain, α-amylase and tanninase is (1-5):(0.5-2):(1-3):(0.5-2);

[0012] Among them, the above "1-5" can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.;

[0013] The "0.5-2" mentioned above can be, for example, 0.5, 0.6, 0.8, 1, 1.2, 1.5, 1.6, 1.8, 2, etc.;

[0014] The "1-3" mentioned above can be, for example, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, etc. Other specific point values ​​within this range can be selected, which will not be elaborated here.

[0015] In this invention, when the proportion of each enzyme in the composite enzyme combination is within the above-mentioned specific range, its enzymatic extraction effect on the active ingredients in myrtle fruit is better.

[0016] Preferably, before enzymatic hydrolysis, the myrtle fruit raw material is mixed with water, and the solid-liquid ratio is 1:(15-50) g / mL. For example, it can be 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 values ​​within this range can be selected, and will not be elaborated here.

[0017] In this invention, when the ratio of myrtle fruit raw material to water is within the above-mentioned specific range, the enzymatic hydrolysis effect is better.

[0018] Preferably, the amount of the compound enzyme added is 0.2‰-0.8‰ of the weight of the myrtle fruit raw material, for example, it can be 0.2‰, 0.25‰, 0.3‰, 0.35‰, 0.4‰, 0.45‰, 0.5‰, 0.6‰, 0.8‰, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0019] In this invention, when the amount of compound enzyme added is within the above-mentioned specific range, the extraction effect on myrtle fruit raw material is better.

[0020] Preferably, the enzymatic hydrolysis temperature is 40-60℃, for example, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 52℃, 55℃, 58℃, 59℃, 60℃, etc.; the time is 1-3 h, for example, 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 values ​​within this range can be selected, and will not be elaborated here.

[0021] Preferably, the solid-liquid ratio of the myrtle fruit raw material to the ethanol aqueous solution is 1:(80-200) g / mL, for example, it can be 1:80 g / mL, 1:100 g / mL, 1:120 g / mL, 1:150 g / mL, 1:180 g / mL, 1:200 g / mL, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0022] Preferably, the ethanol aqueous solution contains 80%-98% by mass, for example, 80%, 82%, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0023] Preferably, the negative pressure cavitation refers to introducing a vacuum system into the reflux extraction reaction device.

[0024] Preferably, the vacuum degree of the negative pressure cavitation is 65-100 mbar, such as 65 mbar, 70 mbar, 75 mbar, 80 mbar, 85 mbar, 90 mbar, 95 mbar, 100 mbar, etc.; the temperature is 50-70℃, such as 50℃, 52℃, 55℃, 58℃, 60℃, 62℃, 65℃, 68℃, 70℃, etc.; the time is 1-3 h, such as 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 values ​​within this range can be selected, and will not be elaborated here.

[0025] Preferably, the filtrate is further concentrated and purified.

[0026] Preferably, the purification step includes dissolving the concentrated crude extract of myrtle fruit in an alcohol solution, filtering, and collecting the filtrate.

[0027] Preferably, the ratio of the crude myrtle extract to the alcohol solution is 1:(8-15) g / mL, for example, it can be 1:8 g / mL, 1:10 g / mL, 1:12 g / mL, 1:13 g / mL, 1:14 g / mL, 1:15 g / mL, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0028] Preferably, the alcohol solution comprises an aqueous solution of ethanol and / or an aqueous solution of butanediol.

[0029] Preferably, the mass percentage of ethanol in the ethanol-water solution is 50%-95%, for example, it can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0030] Preferably, the mass percentage of butanediol in the butanediol aqueous solution is 50%-95%, for example, it can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0031] In a second aspect, the present invention provides a myrtle fruit extract, which is prepared by the preparation method described in the first aspect.

[0032] Thirdly, the present invention provides the application of the myrtle fruit extract according to the second aspect in cosmetics.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention develops a novel method for preparing myrtle fruit extract. It creatively employs an enzymatic hydrolysis combined with negative pressure cavitation extraction to extract myrtle fruit raw materials. Furthermore, it utilizes a complex enzyme combination containing cellulase, bromelain, α-amylase, and tanninase for enzymatic hydrolysis. The study discovered a potential synergistic effect among bromelain, α-amylase, and tanninase, which interact and positively influence each other, improving the extraction efficiency of myrtle fruit and thus increasing the active ingredients in the extract. This extraction method can efficiently extract the effective components from myrtle fruit, preparing a highly active myrtle fruit extract product, which can be further applied to cosmetics to enhance their antioxidant and anti-inflammatory effects. Detailed Implementation

[0035] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0036] The cellulase, bromelain, α-amylase and tanninase involved in the following examples were purchased from Shanghai Yuanye Biotechnology Co., Ltd. and were model numbers S10041, S10009, S10003 and S10207, respectively.

[0037] Example 1

[0038] (1) The washed myrtle fruit was dried, ground and crushed. The crushed myrtle fruit was mixed with pure water at a solid-liquid ratio of 1:30 g / mL. The mixture was then hydrolyzed with a compound enzyme (composed of cellulase, bromelain, α-amylase and tanninase in a mass ratio of 3:1:1.5:1) at 50℃ for 2 h. The amount of compound enzyme added was 0.3‰ of the crushed myrtle fruit. After the reaction was completed, the hydrolysate was obtained.

[0039] (2) A vacuum system was introduced into the atmospheric pressure reflux extraction device with a vacuum degree of 80 mbar. The enzymatic hydrolysate was mixed with 90% ethanol aqueous solution and then refluxed for extraction. The solid-liquid ratio of the myrtle fruit powder to the ethanol aqueous solution was 1:120 g / mL. The extraction conditions were: extraction at 60℃ for 2 h. The filtrate was collected through a 200-mesh sieve and concentrated under reduced pressure. The concentrated crude myrtle fruit extract was dissolved in an alcohol solution (70% ethanol aqueous solution and 70% butanediol aqueous solution, volume ratio 1:1) with a material-liquid ratio of 1:10 g / mL. After thorough mixing, the solution was filtered through a 0.22 μm microporous membrane, the filtrate was collected, concentrated and freeze-dried to obtain the myrtle fruit extract.

[0040] Example 2

[0041] (1) The washed myrtle fruit was dried, ground and crushed. The crushed myrtle fruit was mixed with pure water at a solid-liquid ratio of 1:18 g / mL. The mixture was then hydrolyzed with a compound enzyme (composed of cellulase, bromelain, α-amylase and tanninase in a mass ratio of 1:2:1:2) at 42℃ for 2.8 h. The amount of compound enzyme added was 0.1‰ of the crushed myrtle fruit. After the reaction was completed, the hydrolysate was obtained.

[0042] (2) A vacuum system was introduced into the atmospheric pressure reflux extraction device with a vacuum degree of 70 mbar. The enzymatic hydrolysate was mixed with 85% ethanol aqueous solution and then refluxed for extraction. The solid-liquid ratio of the myrtle fruit powder to the ethanol aqueous solution was 1:100 g / mL. The extraction conditions were: 50℃ for 3 h, the filtrate was collected through a 200-mesh sieve, concentrated under reduced pressure, and the concentrated crude myrtle fruit extract was dissolved in an alcohol solution (50% ethanol aqueous solution and 95% butanediol aqueous solution, volume ratio 1:1) with a material-liquid ratio of 1:8 g / mL. After thorough mixing, the solution was filtered through a 0.22 μm microporous membrane, the filtrate was collected, concentrated and freeze-dried to obtain the myrtle fruit extract.

[0043] Example 3

[0044] (1) The washed myrtle fruit was dried, ground and crushed. The crushed myrtle fruit was mixed with pure water at a solid-liquid ratio of 1:45 g / mL. The mixture was then hydrolyzed with a compound enzyme (composed of cellulase, bromelain, α-amylase and tanninase in a mass ratio of 5:0.5:3:0.5) at 58℃ for 1.2 h. The amount of compound enzyme added was 0.5‰ of the crushed myrtle fruit. After the reaction was completed, the hydrolysate was obtained.

[0045] (2) A vacuum system was introduced into the atmospheric pressure reflux extraction device with a vacuum degree of 90 mbar. The enzymatic hydrolysate was mixed with 95% ethanol aqueous solution and then refluxed for extraction. The solid-liquid ratio of the myrtle fruit powder to the ethanol aqueous solution was 1:180 g / mL. The extraction conditions were: extraction at 70℃ for 1 h. The filtrate was collected through a 200-mesh sieve and concentrated under reduced pressure. The concentrated crude myrtle fruit extract was dissolved in an alcohol solution (95% ethanol aqueous solution and 50% butanediol aqueous solution, volume ratio 1:1) with a material-liquid ratio of 1:15 g / mL. After thorough mixing, the solution was filtered through a 0.22 μm microporous membrane, the filtrate was collected, concentrated and freeze-dried to obtain the myrtle fruit extract.

[0046] Example 4

[0047] This embodiment provides a myrtle fruit extract, which uses the myrtle fruit extract of Example 1. The only difference between this and Example 1 is that the amount of compound enzyme added is 0.02‰ of the myrtle fruit powder. All other steps and process parameters are the same as in Example 1.

[0048] Example 5

[0049] This embodiment provides a myrtle fruit extract, which uses the myrtle fruit extract of Example 1. The only difference between this and Example 1 is that the amount of compound enzyme added is 2‰ of the myrtle fruit powder. All other steps and process parameters are the same as in Example 1.

[0050] Example 6

[0051] This embodiment provides a myrtle fruit extract, which is prepared by using the crude myrtle fruit extract obtained from step (2) of Example 1 through negative pressure cavitation extraction, filtration, and concentration. The only difference between this preparation method and Example 1 is that the crude myrtle fruit extract is dissolved in a 70% ethanol aqueous solution at a material-to-liquid ratio of 1:10 g / mL. After thorough mixing, the solution is filtered through a 0.22 μm microporous membrane, the filtrate is collected, concentrated, and freeze-dried to obtain the myrtle fruit extract. All other steps and process parameters are the same as in Example 1.

[0052] Example 7

[0053] This embodiment provides a myrtle fruit extract, prepared by negative pressure cavitation extraction, filtration and concentration of crude myrtle fruit extract obtained in step (2) of Example 1. The only difference between this preparation method and Example 1 is that the crude myrtle fruit extract is dissolved in a 70% butanediol aqueous solution at a material-to-liquid ratio of 1:10 g / mL. After thorough mixing, the solution is filtered through a 0.22 μm microporous membrane, the filtrate is collected, concentrated and freeze-dried to obtain the myrtle fruit extract. All other steps and process parameters are the same as in Example 1.

[0054] Example 8

[0055] This embodiment provides a myrtle fruit extract, which is obtained by using the crude myrtle fruit extract extracted by negative pressure cavitation extraction, filtration and concentration in step (2) of embodiment 1. The only difference between this embodiment and embodiment 1 is that no subsequent purification process is performed, and the crude myrtle fruit extract is directly freeze-dried to obtain the myrtle fruit extract.

[0056] Comparative Example 1

[0057] This comparative example provides a myrtle fruit extract, using the myrtle fruit pulverized material from Example 1. The only difference between this preparation method and Example 1 is that the compound enzyme does not contain bromelain, and its reduced proportion is allocated to α-amylase and tanninase. All other steps and process parameters are the same as in Example 1.

[0058] Comparative Example 2

[0059] This comparative example provides a myrtle fruit extract, which uses the myrtle fruit pulverized material from Example 1. The only difference between this preparation method and Example 1 is that the compound enzyme does not contain α-amylase, and its reduced amount is proportionally allocated to bromelain and tanninase. All other steps and process parameters are the same as in Example 1.

[0060] Comparative Example 3

[0061] This comparative example provides a myrtle fruit extract, which uses the myrtle fruit pulverized material from Example 1. The only difference between this preparation method and Example 1 is that the compound enzyme does not contain tanninase, and its reduced proportion is allocated to bromelain and α-amylase. All other steps and process parameters are the same as in Example 1.

[0062] Comparative Example 4

[0063] This embodiment provides a myrtle fruit extract, using the enzymatic hydrolysate prepared in step (1) of Example 1. The only difference between this embodiment and Example 1 is that a vacuum system is not introduced in the atmospheric pressure reflux extraction device in step (2). The solid-liquid ratio of the myrtle fruit powder to the ethanol aqueous solution is 1:120 g / mL. The enzymatic hydrolysate is mixed with 90% ethanol aqueous solution and then refluxed for extraction. The extraction conditions are: 60℃ for 2 h. The subsequent concentration, purification and other process flow and parameters are all the same as in Example 1.

[0064] Comparative Example 5

[0065] This embodiment provides a myrtle fruit extract, which uses the enzymatic hydrolysate prepared in step (1) of Example 1. The only difference between this embodiment and Example 1 is that the negative pressure cavitation extraction in step (2) is replaced with conventional hydrothermal extraction. The extraction conditions are as follows: the enzymatic hydrolysate is mixed with deionized water and then refluxed for extraction. The solid-liquid ratio of myrtle fruit powder to deionized water is 1:10 g / mL. The extraction conditions are as follows: extraction at 80℃ for 3 h. The subsequent concentration, purification and other process flow and parameters are all the same as in Example 1.

[0066] Test Example 1

[0067] Anti-inflammatory experiment

[0068] Test method: RAW264.7 mouse macrophages in logarithmic growth phase with good morphology were seeded in DMEM culture medium containing 10% FBS fetal bovine serum and incubated for 24 h, at a concentration of 2×10⁻⁶ cells / mL. 5 Cells were seeded at a density of cells / well in 24-well plates and incubated at 37°C with 5% CO2 for 2 h. Cells were then stimulated with 1 μg / mL lipopolysaccharide for 24 h to establish a cell inflammation model.

[0069] 1% of the myrtle fruit extract prepared in Examples 1-8 and Comparative Examples 1-5 was added to each group of cells. After incubation at 37°C for 24 h, the cell supernatant was collected and the ELISA kit instructions were followed to detect the contents of TNF-α and IL-6 in the collected cell supernatant. The experimental results were analyzed using data processing software, and the results are shown in Table 1.

[0070] Table 1

[0071]

[0072] TNF-α and IL-6 are important pro-inflammatory mediators that mediate various inflammatory responses. Reducing their levels can inhibit inflammatory responses, thereby achieving an anti-inflammatory effect. The data in the table show that:

[0073] (1) As can be seen from Examples 1 to 3, the preparation method of myrtle fruit extract provided by the present invention has a significant inhibitory effect on two pro-inflammatory mediators. Among them, Example 1 is the best.

[0074] (2) By comparing Example 1 with Examples 4-5, it can be seen that when the amount of compound enzyme added is within a specific range, the myrtle fruit extract prepared by it has a better effect in anti-inflammatory and soothing.

[0075] (3) By comparing Example 1 with Examples 6-8, it can be seen that when the crude extract of myrtle fruit is further purified by alcohol precipitation, the resulting myrtle fruit extract has a better effect in related anti-inflammatory and soothing aspects. Furthermore, it was found that when a composite alcohol solution composed of ethanol aqueous solution and butanediol aqueous solution is used as the alcohol precipitation solvent, the effect of the prepared myrtle fruit extract is further improved.

[0076] (4) A comparison between Example 1 and Comparative Examples 1-3 shows that the present invention uses a complex enzyme with a specific composition to treat myrtle fruit. The combined use of bromelain, α-amylase, and tanninase has an excellent enzymatic extraction effect on myrtle fruit. When Comparative Examples 1-3 do not contain any of these three enzymes, the myrtle fruit extracts prepared from them have a lower effect on inhibiting the secretion of the two pro-inflammatory mediators than in Example 1, indicating that bromelain, α-amylase, and tanninase have a synergistic promoting effect when applied to the extraction of active ingredients from myrtle fruit.

[0077] (5) By comparing Example 1 and Comparative Example 5, it can be seen that the negative pressure cavitation extraction technology used in this invention to further extract the enzymatically hydrolyzed myrtle fruit system has a better effect than conventional hydrothermal extraction, and a myrtle fruit extract with better anti-inflammatory effect was prepared.

[0078] Test Example 2

[0079] Antioxidant effect test

[0080] (1) DPPH-free radical scavenging

[0081] A 96-well plate was used, with three replicates per group, and the total volume was 200 μL. For the sample group: 100 μg of the extracts prepared in Examples 1-8 and Comparative Examples 1-5 were mixed with 100 μL of pure water, and 100 μL of 0.1 mM DPPH solution was added to the reaction system. For the control group: 100 μL of pure water was mixed with 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] The clearance rate is calculated as follows: clearance 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-free radical scavenging

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

[0085] A 96-well plate was used, with three replicates per group, and the total volume was 200 μL. For the sample group, 100 μg of the extracts prepared in Examples 1-8 and Comparative Examples 1-5 were mixed with 10 μL of purified water, and 190 μL of ABTS working solution was added to the reaction system. For the control group, 10 μL of purified water was mixed with 190 μL of ABTS working solution. After the reaction system was constructed, it was thoroughly shaken and incubated for 6 min. The absorbance at 734 nm was measured using a microplate reader.

[0086] The clearance rate is calculated as follows: clearance 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 2.

[0088] Table 2

[0089]

[0090] As can be seen from the data in the table, the myrtle fruit extract prepared by the method provided by the present invention has excellent DPPH- and ABTS- free radical scavenging effects, and exhibits significant antioxidant effects. Among them, the antioxidant effect of Example 1 is better than that of Examples 4-8 and Comparative Examples 1-5, indicating that the specific preparation conditions of the myrtle fruit extract used in the present invention have different degrees of influence on the antioxidant efficacy of the final extract.

[0091] Test Example 3

[0092] Product safety testing

[0093] Eighty participants were selected according to the inclusion criteria, with a male-to-female ratio of 1:1, and randomly divided into 8 groups (5 males and 5 females in each group) to receive the trial. An appropriate amount of the extract sample prepared in Examples 1-8 (the extract was mixed with pure water to adjust the extract concentration to 5% before testing) was placed in the drug compartment of the test strip. The test strip containing the sample was then applied to the flexor surface of the subject's forearm with medical tape for 24 hours. Thirty minutes after removing the test strip, and after the indentation disappeared, the skin reaction was observed. For those with negative results, the skin reaction was observed again at 24 hours and 48 hours after the trial, and the results were recorded. The grading criteria for the degree of reaction are shown in the table below.

[0094]

[0095] The results showed that the products prepared in Examples 1-8 all exhibited negative reactions at 24 h and 48 h after the test, with no irritation or erythema. Therefore, the myrtle fruit extract product prepared by this invention is non-irritating to the skin and showed no positive reaction, proving that the product is highly safe, has low irritation, and is relatively mild.

[0096] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0097] 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 solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0098] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing a myrtle fruit extract, characterized in that, The preparation method includes the following steps: The raw material of myrtle fruit was mixed with a compound enzyme for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, an aqueous ethanol solution was added to the enzymatic hydrolysis reaction solution for negative pressure cavitation extraction. The filtrate was collected by filtration to obtain the myrtle fruit extract. The complex enzyme comprises a combination of cellulase, bromelain, α-amylase, and tanninase.

2. The preparation method according to claim 1, characterized in that, The mass ratio of cellulase, bromelain, α-amylase and tanninase is (1-5):(0.5-2):(1-3):(0.5-2).

3. The preparation method according to claim 1, characterized in that, Before enzymatic hydrolysis, the myrtle fruit raw material is mixed with water at a solid-liquid ratio of 1:(15-50) g / mL.

4. The preparation method according to claim 1, characterized in that, The amount of the compound enzyme added is 0.1‰-0.5‰ of the weight of the myrtle fruit raw material; Preferably, the enzymatic hydrolysis is performed at a temperature of 40-60°C for 1-3 hours.

5. The preparation method according to claim 1, characterized in that, The solid-liquid ratio of the myrtle fruit raw material to the ethanol aqueous solution is 1:(80-200) g / mL; Preferably, the ethanol aqueous solution contains 80%-98% ethanol by mass.

6. The preparation method according to claim 1, characterized in that, The vacuum degree of the negative pressure cavitation is 65-100 mbar, the temperature is 50-70℃, and the time is 1-3 h.

7. The preparation method according to claim 1, characterized in that, The filtrate is further concentrated and purified.

8. The preparation method according to claim 7, characterized in that, The purification step includes dissolving the concentrated crude extract of myrtle fruit in an alcohol solution, filtering, and collecting the filtrate. Preferably, the ratio of the crude myrtle extract to the alcohol solution is 1:(8-15) g / mL; Preferably, the alcohol solution comprises an aqueous solution of ethanol and / or an aqueous solution of butanediol.

9. A myrtle fruit extract, characterized in that, The myrtle fruit extract is prepared by the preparation method according to any one of claims 1-8.

10. The application of the myrtle fruit extract according to claim 9 in cosmetics.

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