Preparation method of aquilaria sinensis extract as well as product and application of aquilaria sinensis extract

Abstract

The invention belongs to the technical field of substance extraction, and relates to a preparation method of an aquilaria sinensis extract as well as a product and application of the aquilaria sinensis extract, the preparation method comprises the following steps: mixing aquilaria sinensis powder with water to obtain an aquilaria sinensis mixed solution; mixing the aquilaria sinensis mixed solution with a compound enzyme, performing enzymolysis, performing enzyme deactivation, and performing negative-pressure cavitation extraction to obtain an extracting solution; mixing the extracting solution with aquilaria sinensis powder, and performing solid-liquid separation after treatment to obtain a treatment solution; and mixing the treating fluid with a flocculating agent, carrying out solid-liquid separation, taking liquid, and drying to obtain the flocculant. The aquilaria sinensis extract provided by the invention can inhibit melanogenesis and tyrosinase activity in B16F10 cells, also can inhibit melanogenesis of zebra fish, inhibits ERK1, MITF and TYR gene expression, and has a remarkable whitening effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of substance extraction, and relates to a preparation method of white camphor extract, a product thereof and application. BACKGROUND

[0002] With the prevalence of whitening, people pay more and more attention to the whiteness of the skin. The key to whitening lies in the inhibition of melanin. The common melanin inhibiting ingredients at present are mostly chemical ingredients, such as arbutin, nicotinamide, kojic acid, etc. People have great concerns about the safety of them. Arbutin has poor stability, and will be converted into hydroquinone under the action of human skin microorganisms or glucosidase, while hydroquinone has the risk of causing exogenous ochronosis and vitiligo, and even the risk of sensitization and carcinogenesis, and is a banned ingredient in China. Moreover, it is mentioned in "Study on the cytotoxicity of four whitening agents on skin melanocytes" (Journal of Ningxia Medical University, 2006, 28(2): 114-116+119) that arbutin has cytotoxicity at 70 μg / mL or above. Nicotinamide has high overall safety, but some people may still experience transient skin irritation after using it, such as redness, tingling, dryness or desquamation, etc. However, it is reported in "Toxicity study and safety evaluation of kojic acid on animals" (Food Science, 2007, 28(9): 536-540) that long-term high-dose (500 mg / kg bw) use of kojic acid may affect the growth and development of experimental animals and change the structure and function of organs.

[0003] Therefore, it is an urgent problem to be solved to seek a natural and non-irritating whitening ingredient. Plant extracts, as natural source of functional ingredients, are deeply loved by the public due to their safety and green environmental protection. It has been found that plant extracts have whitening effects, such as Morus alba water extract can resist the damage of oxygen free radicals and ultraviolet rays to the skin and improve skin pigmentation ("Study on the whitening effect of Morus alba water extract on guinea pig skin pigmentation model", Pharmaceutical and Clinical Research, 2024, 32(4): 313-315), 400 μg / mL of total phenethyl alcohol glycosides of Cistanche can effectively inhibit the tyrosinase activity in the melanin deposition model of human skin melanocytes caused by UVB radiation ("Study on the effect of total phenethyl alcohol glycosides of Cistanche on melanin synthesis in human skin melanocytes and its mechanism", Xinjiang Medical University, 2016), and 0.5 mg / mL of Bletilla striata flower extract can inhibit the tyrosinase activity in B16 cells ("Study on the whitening, antioxidant and anti-aging effects of Bletilla striata flower extract", Yunnan Chemical Industry, 2024, 51(10): 100-104).

[0004] Aquilaria sinensis (Lour.) is also called Aquilaria sinensis (Lour.) and daughter incense, which is a tree of Thymelaeaceae. The incense part of Aquilaria sinensis (Lour.) is agarwood, and the white wood part without incense is Aquilaria sinensis (Lour.). Flavonoids, lignans, phenylpropanoids, steroids, triterpenes and other components are the main components of Aquilaria sinensis (Lour.), which have anti-inflammatory, antibacterial, antioxidant and other effects. At present, the research on Aquilaria sinensis (Lour.) mainly focuses on the components of volatile oil, the ability to form incense, the antibacterial ability and the chemical components, and the related efficacy research is only related to antioxidant and anti-inflammatory. In patent CN103405371A, the extract is obtained by extracting the pericarp of Aquilaria sinensis (Lour.) with ethyl acetate, and it is found that the extract can inhibit 9.4%, 23.1%, 45.3%, 61.8% and 80.1% of tyrosinase activity at 20, 50, 100, 200 and 500 μg / mL, respectively, indicating that the pericarp of Aquilaria sinensis (Lour.) may have a certain whitening ability. In patent CN104523520A, ethanol water is used as the extraction solvent, and the agarwood leaf extract is obtained by macroporous resin separation, and it is found that the inhibition rate of B16F10 cell melanin is 12.3% and the inhibition rate of intracellular tyrosinase is 33.9% at 140 μg / mL. And it is found that the market price of agarwood is expensive, and the price of ordinary agarwood is 800-1500 yuan / g, while the price of Aquilaria sinensis (Lour.) is only 20-100 yuan / kg, so Aquilaria sinensis (Lour.) has more economic value than agarwood.

[0005] Although agarwood and Aquilaria sinensis (Lour.) belong to the wood part of Aquilaria sinensis (Lour.), agarwood is usually a high-fat incense part, and the Pharmacopoeia 2025 edition stipulates that the agarwood tetrol content in agarwood medicinal materials shall not be less than 0.1%. The Aquilaria sinensis (Lour.) medicinal material involved in the present application is the white wood part other than the incense part, and the Aquilaria sinensis (Lour.) extract involved in the present application does not contain agarwood tetrol, and the two are not the same in terms of part, component and efficacy. The above research on Aquilaria sinensis (Lour.) in whitening uses organic reagents such as ethanol and ethyl acetate as extraction solvents, which can stimulate or have other adverse reactions on the skin, and the effective amount is high, which cannot achieve the green, safe and efficient demand. SUMMARY

[0006] In view of the deficiencies in the prior art, the purpose of the present application is to provide a preparation method of Aquilaria sinensis (Lour.) extract and its product and application.

[0007] In order to achieve the purpose of the present application, the following technical solutions are adopted:

[0008] In the first aspect, the present application provides a preparation method of Aquilaria sinensis (Lour.) extract, which comprises:

[0009] (1) mixing Aquilaria sinensis (Lour.) powder with water to obtain an Aquilaria sinensis (Lour.) mixture;

[0010] (2) mixing the liquid of Aquilaria sinensis with complex enzymes for enzymolysis, deactivating the enzymes, and extracting by negative pressure cavitation to obtain an extract;

[0011] (3) mixing the extract with the powder of Aquilaria sinensis in step (1) for treatment, and separating the solid and the liquid after the treatment to obtain a treatment liquid;

[0012] (4) mixing the treatment liquid with a flocculating agent, separating the solid and the liquid to obtain a liquid, and drying the liquid to obtain the product.

[0013] The active ingredients in the medicinal material of Aquilaria sinensis are enriched to the greatest extent by enzymolysis and negative pressure cavitation extraction, and the extraction solvent is pure water, which maximizes the safety of the extract. The efficacy of the extract of Aquilaria sinensis is detected by cell tests and zebra fish tests, and the extract is shown to have excellent whitening efficacy.

[0014] Preferably, the ratio of the powder of Aquilaria sinensis to water in step (1) is 1: (5-20) mL, and the specific point values in (5-20) can be selected as 5, 8, 10, 12, 15, 18, 20, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0015] Preferably, the temperature of the mixing in step (1) is 10-30℃, and the time is 1-3 h.

[0016] The temperature can be selected as 10℃, 12℃, 15℃, 18℃, 20℃, 22℃, 25℃, 28℃, 30℃, and the time can be selected as 1 h, 1.2 h, 1.5 h, 1.8 h, 2 h, 2.2 h, 2.5 h, 2.8 h, 3 h, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0017] Preferably, the pH of the liquid of Aquilaria sinensis is adjusted to 4-6, for example, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0018] Preferably, the adjusting agent used for the pH adjustment includes 0.01-0.2 M acetic acid buffer, 0.01-0.2 M citric acid buffer, and 0.01-0.2 M phosphoric acid buffer.

[0019] The specific point values in 0.01-0.2 M can be selected as 0.01 M, 0.02 M, 0.05 M, 0.08 M, 0.1 M, 0.12 M, 0.15 M, 0.18 M, 0.2 M, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0020] Preferably, the mass of the complex enzyme is 1-4% of the mass of the liquid mixture of Aquilaria sinensis, such as 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, and the like, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0021] Preferably, the complex enzyme is a combination of cellulase and pectinase.

[0022] Preferably, the mass ratio of the cellulase and pectinase is (10-30):(1-5), and the specific point values in (10-30) can be selected as 10, 12, 15, 18, 20, 22, 25, 28, 30, and the like, and the specific point values in (1-5) can be selected as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, and the like, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0023] Preferably, the temperature of the enzymatic hydrolysis is 40-55°C, and the time is 1-3 h.

[0024] The temperature can be selected as 40°C, 42°C, 45°C, 48°C, 50°C, 52°C, 55°C, and the like, and the time can be selected as 1 h, 1.2 h, 1.5 h, 1.8 h, 2 h, 2.2 h, 2.5 h, 2.8 h, 3 h, and the like, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0025] Preferably, the temperature of the enzyme inactivation is 80-90°C, and the time is 5-15 min.

[0026] The temperature can be selected as 80°C, 82°C, 84°C, 86°C, 88°C, 90°C, and the like, and the time can be selected as 5 min, 8 min, 10 min, 12 min, 15 min, and the like, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0027] Preferably, the temperature of the negative pressure cavitation extraction is 30-70°C, the pressure is 90-150 mbar, the time is 1-3 h, and the number of times is 1-3 times.

[0028] The temperature can be selected from 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, etc., the pressure can be selected from 90 mbar, 100 mbar, 110 mbar, 120 mbar, 130 mbar, 140 mbar, 150 mbar, etc., the time can be selected from 1 h, 1.2 h, 1.5 h, 1.8 h, 2 h, 2.2 h, 2.5 h, 2.8 h, 3 h, etc., and the number of times is 1, 2, 3, etc. Other specific point values within the above numerical range can be selected, and here they will not be repeated one by one.

[0029] Preferably, the solid-liquid ratio of the white mulberry powder to the extraction liquid in step (3) is (0.001-0.01) g:1 mL. The specific point values in (0.001-0.01) can be selected from 0.001, 0.002, 0.004, 0.006, 0.008, 0.01, etc. Other specific point values within the above numerical range can be selected, and here they will not be repeated one by one.

[0030] Preferably, the temperature of the mixing treatment in step (3) is 40-70℃, and the time is 5-30 min.

[0031] The temperature can be selected from 40℃, 42℃, 45℃, 48℃, 50℃, 52℃, 55℃, 58℃, 60℃, 62℃, 65℃, 68℃, 70℃, etc., and the time can be selected from 5 min, 8 min, 10 min, 12 min, 15 min, 18 min, 20 min, 22 min, 25 min, 28 min, 30 min, etc. Other specific point values within the above numerical range can be selected, and here they will not be repeated one by one.

[0032] Preferably, the flocculating agent is a combination of gellan gum and sordarium gum.

[0033] Preferably, the mass ratio of the gellan gum to the sordarium gum is (1-10):(1-10).

[0034] The specific point values in (1-10) can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. Other specific point values within the above numerical range can be selected, and here they will not be repeated one by one.

[0035] Preferably, the mass of the flocculating agent is 0.5-4% of the mass of the treatment liquid, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, etc. Other specific point values within the above numerical range can be selected, and here they will not be repeated one by one.

[0036] Preferably, the temperature for mixing with the flocculating agent is 25-60℃, and the time is 0.5-3 h.

[0037] The temperature can be selected from 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, etc., and the time can be selected from 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0038] Preferably, the solid-liquid separation method includes centrifugation, static sedimentation or plate and frame filtration.

[0039] Preferably, the drying method includes vacuum drying, freeze-drying or spray drying.

[0040] In a second aspect, the present application provides a white mulberry extract prepared by the preparation method of the white mulberry extract according to the first aspect.

[0041] In a third aspect, the present application provides a use of the white mulberry extract according to the second aspect in the preparation of a product with whitening effect.

[0042] Preferably, the product includes a cosmetic or skin care product.

[0043] Preferably, the dosage form of the skin care product includes a skin care cream or serum.

[0044] Preferably, the mass percentage of the white mulberry extract in the product is 0.001-0.008%, for example, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0045] In a fourth aspect, the present application provides a use of the white mulberry extract according to the second aspect in the preparation of a cell melanin inhibitor, an ERK1 gene expression inhibitor, an MITF gene expression inhibitor, a tyrosinase inhibitor or a zebrafish melanin inhibitor.

[0046] Melanin is produced by melanocytes, in which melanin granules exist. When the skin is exposed to external stimuli such as ultraviolet light, the relevant signaling pathways are activated, and through the catalysis of tyrosinase and other enzymes, melanin is generated. The MAPK signaling pathway is a common whitening pathway, mainly composed of Ras / MEK / ERK / MITF. Generally, inhibiting any one target in the pathway can inhibit the final expression of the MAPK pathway. ERK includes ERK1 and ERK2, so inhibiting any one of the two can inhibit the expression of the ERK gene, and then inhibit the expression of the MAPK signaling pathway. Rasayana down-regulates FOXM1 through the JNK / P38 MAPK signaling pathway to inhibit the proliferation and migration of melanoma cells ("Research on the molecular mechanism of Rasayana inhibiting FOXM1 to inhibit melanoma through the PI3K / AKT, JNK / P38 MAPK pathway", North Sichuan Medical College, 2024). A coumarin glycoside derivative extracted from Angelica root can inhibit melanin production in B16F10 cells by inhibiting ERK / MSK1 ("Nodakenin Inhibits Melanogenesis Via the ERK / MSK1 Signaling Pathway", Pharmazie, 2023, 78(1): 6-12).

[0047] Zebrafish genes have high homology with human genes and can exhibit similar physiological characteristics to humans. In experiments, zebrafish models are often used to study diseases such as inflammation, cancer, diabetes, and fatty liver. At the same time, due to their fast melanin production and ease of observation, they are often used for qualitative evaluation of melanin inhibition effects. The patent "Application of recombinant short peptide in anti-wrinkle, whitening, skin lightening, and antioxidant aspects" (CN118415908B) verifies that the recombinant short peptide has whitening effect through zebrafish whitening model; The phenolic ketone components in the non-medical part of Salvia miltiorrhiza, Salvia miltiorrhiza flower, were detected for their potential whitening activity through zebrafish model ("Exploration of the whitening and antioxidant effects of phenolic ketone components in Salvia miltiorrhiza flower based on zebrafish model", Times Journal of Traditional Chinese Medicine and Pharmacy, 2024, 3(8): 1868-1871); Psoralen was verified by zebrafish model to have whitening effect ("Evaluation of the efficacy of Psoralen in the field of cosmetics based on zebrafish model", Guangdong Chemical Industry, 2023, 50(7): 70-72); The green synthesis of nanometer platinum by Nymphaea tetragona extract was detected by cell and biochemical experiments to have whitening effect, and its whitening effect was further verified by zebrafish model ("Green synthesis of nanometer platinum by Nymphaea tetragona extract and its whitening and anti-aging effects", Anhui Medical University, 2023).

[0048] According to the research results of the present application, the prepared Aquilaria sinensis extract has the effects of significantly inhibiting the expression of ERK1 and MITF genes, can inhibit the expression of cell melanin, and inhibit the activity of tyrosinase, so that the prepared Aquilaria sinensis extract can be used as an in vitro preparation, such as for improving inflammation or UV caused skin pigmentation, or for treating pigment related diseases; can also be used for scientific basic research, such as inhibiting gene expression, or for inhibiting melanin production; and can also be used for biological field research, such as tyrosinase inhibitors.

[0049] Compared with the prior art, the present application has the following beneficial effects:

[0050] (1) The present application first extracts Aquilaria sinensis extract by mixed enzyme enzymolysis and negative pressure cavitation method, and the extraction process does not involve organic reagents, which is safer and more reliable;

[0051] (2) The preparation method of the Aquilaria sinensis extract of the present application can effectively inhibit the melanin production of B16F10 cells, specifically by inhibiting the melanin content of B16F10 cells and the activity of tyrosinase in cells;

[0052] (3) The Aquilaria sinensis extract of the present application can significantly inhibit the expression of ERK1 gene, thereby inhibiting the expression of MITF and the production of melanin, and has excellent whitening effect;

[0053] (4) The Aquilaria sinensis extract of the present application can significantly inhibit the melanin production of zebrafish, and has whitening effect;

[0054] (5) The Aquilaria sinensis extract of the present application can significantly inhibit the expression of TNF-α gene, and has anti-inflammatory effect;

[0055] (6) The safety of the Aquilaria sinensis extract of the present application is verified, and it can be safely applied to related products. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 is a flow chart of the preparation method of Aquilaria sinensis extract.

[0057] Figure 2 is the liquid chromatogram of Aquilaria sinensis extract, Aquilaria sinensis extract and linalyl acetate.

[0058] Figure 3 is a difference chart in the extraction process according to Example 1.

[0059] Figure 4 is a chart showing the effect of Aquilaria sinensis extract on the expression of ERK1, TYR and MITF genes.

[0060] Figure 5 is a chart showing the results of zebrafish melanin inhibition test of Aquilaria sinensis extract.

[0061] Figure 6 Figure is a graph showing the effect of bai-muxiang extract on inflammatory factors. DETAILED DESCRIPTION

[0062] The technical solutions of the present application are further illustrated below by means of specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations on the present application.

[0063] The cellulase is a product with the trade name of cellulase purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0064] The pectinase is a product with the trade name of pectinase purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0065] The gellan gum is a product with the trade name of Gelzan™ CM gellan gum purchased from Merck Chemical Co., Ltd.;

[0066] The siraclot is a product with the trade name of siraclot purchased from Guangdong Mingtong Biotechnology Co., Ltd.

[0067] In the table and figure: * / # represents p<0.05, ** / ## represents p<0.01, *** / ### represents p<0.001, **** / #### represents p<0.0001.

[0068] Experiment 1

[0069] Bai-muxiang and agarwood were prepared according to the provisions of the Pharmacopoeia 2025 Edition, and bai-muxiang extract and agarwood extract were obtained. According to the provisions of the Pharmacopoeia 2025 Edition for agarwood medicinal materials, agarwood is the wood containing resin of Aquilaria sinensis (Lour.) of the family Thymelaeaceae. It can be harvested throughout the year, and the wood containing resin is cut and taken, and the part not containing resin is removed and dried. Agarwood medicinal materials contain agarwood tetrol not less than 0.1% according to dry product. Through ultra-high performance liquid chromatography, Agilent-Zorbax SB-AQ-C18 is used for UPLC detection. Mobile phase conditions: A is 0.1% acetic acid aqueous solution, B is acetonitrile; gradient elution conditions: 0~2 min, 2% B; 2~8 min, 2~20% B; 8~18 min, 20~35% B; 18~23 min, 35~80% B; 23~27 min, 80~80% B; 27~27.1 min, 80~2% B; 27.1~30 min, 2% B; injection volume 5 μL, flow rate 0.4 mL / min, column temperature 35℃, detection wavelength 252 nm.

[0070] From Figure 2It can be seen that there is no Linalyl Acetate in the extract of Aquilaria sinensis, and the chromatogram is quite different from that of the extract of Aquilaria sinensis.

[0071] Example 1

[0072] The present example provides a preparation method of an extract of Aquilaria sinensis, which comprises:

[0073] (1) crushing the medicinal material of Aquilaria sinensis to 60 mesh, to obtain the medicinal material powder A of Aquilaria sinensis; mixing the medicinal material powder A of Aquilaria sinensis with water, the solid-liquid ratio being 1:10 g / mL, and standing at room temperature for 1 h to obtain a mixed solution of the medicinal material powder A of Aquilaria sinensis;

[0074] (2) adjusting the pH of the mixed solution of the medicinal material powder A of Aquilaria sinensis to 5, and mixing with a composite enzyme, the amount of the composite enzyme being 2.3% of the mass of the mixed solution, the composite enzyme being a fibrozyme and a pectinase with a mass ratio of 20:3, and enzymolysis being carried out at 50°C for 2 h to obtain an enzymolysis solution;

[0075] (3) inactivating the enzymolysis solution at 90°C for 10 min, and carrying out negative pressure cavitation extraction, the extraction pressure being 125 mbar, the temperature being 50°C, and the extraction time being 2 h, and the extraction being carried out twice, and the filtrates being combined to obtain an extract of Aquilaria sinensis;

[0076] (4) mixing the medicinal material powder A of Aquilaria sinensis with the extract of Aquilaria sinensis at a solid-liquid ratio of 0.01:1 g / mL, and reacting at 50°C for 10 min, and then carrying out plate and frame filtration to obtain a treatment solution;

[0077] (5) mixing the treatment solution with a flocculating agent at 30°C for 2 h, the flocculating agent being a curdlan and a sclerotium gum with a mass ratio of 1:1, and the mass of the flocculating agent being 2% of the mass of the treatment solution, and then carrying out plate and frame filtration, and vacuum suction filtration of the filtrate, and freeze-drying after concentration to obtain the extract of Aquilaria sinensis.

[0078] The state of the sample during the extraction process is shown in Figure 3 The figure A is the extract of Aquilaria sinensis prepared after step (3) of negative pressure cavitation extraction, B is the treatment solution obtained in step (4), and C is the filtrate obtained after flocculation and plate and frame filtration in step (5).

[0079] Example 2

[0080] The present example provides a preparation method of an extract of Aquilaria sinensis, which comprises:

[0081] (1) crushing the medicinal material of Aquilaria sinensis to 60 mesh, to obtain the medicinal material powder A of Aquilaria sinensis; mixing the medicinal material powder A of Aquilaria sinensis with water, the solid-liquid ratio being 1:5 g / mL, and standing at room temperature for 2 h to obtain a mixed solution of the medicinal material powder A of Aquilaria sinensis;

[0082] (2) the pH of the powder A mixture solution is adjusted to 4, then mixed with a compound enzyme, the compound enzyme is added in an amount of 1% of the mass of the mixture solution, the compound enzyme is a mixture of a fibrolytic enzyme and a pectic enzyme in a mass ratio of 1:1, and enzymolysis is carried out at 40℃ for 3 hours to obtain an enzymolysis solution;

[0083] (3) the enzymolysis solution is inactivated at 85℃ for 5 minutes, then subjected to negative pressure cavitation extraction, the extraction pressure is 90 mbar, the temperature is 30℃, the extraction time is 3 hours, and the extraction is carried out twice, and the filtrates are combined to obtain an extract of A. balsamifera;

[0084] (4) the powder A and the extract of A. balsamifera are mixed in a solid-liquid ratio of 0.005:1 g / mL, and reacted at 40℃ for 30 minutes, then subjected to plate and frame filtration to obtain a treatment solution;

[0085] (5) the treatment solution is mixed with a flocculating agent at 60℃ for 0.5 hours, the flocculating agent is a mixture of a gellan gum and a sclerotium gum in a mass ratio of 10:1, and the mass of the flocculating agent is 4% of the mass of the treatment solution, then subjected to plate and frame filtration, the filtrate is subjected to vacuum filtration, concentrated and freeze-dried to obtain the product.

[0086] Example 3

[0087] The embodiment provides a preparation method of an extract of A. balsamifera, and the preparation method comprises the following steps:

[0088] (1) A. balsamifera is crushed to 60 mesh to obtain a powder A; the powder A is mixed with water in a solid-liquid ratio of 1:20 g / mL, and then placed at room temperature for 3 hours to obtain a powder A mixture solution;

[0089] (2) the pH of the powder A mixture solution is adjusted to 6, then mixed with a compound enzyme, the compound enzyme is added in an amount of 4% of the mass of the mixture solution, the compound enzyme is a mixture of a fibrolytic enzyme and a pectic enzyme in a mass ratio of 9:1, and enzymolysis is carried out at 55℃ for 1 hour to obtain an enzymolysis solution;

[0090] (3) the enzymolysis solution is inactivated at 80℃ for 15 minutes, then subjected to negative pressure cavitation extraction, the extraction pressure is 150 mbar, the temperature is 70℃, the extraction time is 1 hour, and the extraction is carried out twice, and the filtrates are combined to obtain an extract of A. balsamifera;

[0091] (4) the powder A and the extract of A. balsamifera are mixed in a solid-liquid ratio of 0.01:1 g / mL, and reacted at 70℃ for 5 minutes, then subjected to plate and frame filtration to obtain a treatment solution;

[0092] (5) the treatment solution is mixed with a flocculating agent at 25℃ for 3 hours, the flocculating agent is a mixture of a gellan gum and a sclerotium gum in a mass ratio of 1:10, and the mass of the flocculating agent is 1% of the mass of the treatment solution, then subjected to plate and frame filtration, the filtrate is subjected to vacuum filtration, concentrated and freeze-dried to obtain the product.

[0093] Example 4

[0094] The present example provides a preparation method of extract of A. woodii, which is different from example 1 only in that step (2) is "adjusting the pH of the A. woodii medicinal material powder A mixture solution to 7, then mixing with a composite enzyme, the addition amount of the composite enzyme is 2.3% of the mass of the mixture solution, the composite enzyme is a fibrozyme and a pectinase with a mass ratio of 20:3, and the enzymolysis is carried out at 50℃ for 2 h to obtain an enzymolysis solution", and other operations remain unchanged.

[0095] Example 5

[0096] The present example provides a preparation method of extract of A. woodii, which is different from example 1 only in that step (2) is "adjusting the pH of the A. woodii medicinal material powder A mixture solution to 3, then mixing with a composite enzyme, the addition amount of the composite enzyme is 2.3% of the mass of the mixture solution, the composite enzyme is a fibrozyme and a pectinase with a mass ratio of 20:3, and the enzymolysis is carried out at 50℃ for 2 h to obtain an enzymolysis solution", and other operations remain unchanged.

[0097] Example 6

[0098] The present example provides a preparation method of extract of A. woodii, which is different from example 1 only in that step (5) is "mixing the treatment solution with a flocculating agent at 30℃ for 2 h, the flocculating agent is Sclerotium gum, and the mass of the flocculating agent is 2% of the mass of the treatment solution, then performing plate and frame filtration, reducing pressure suction filtration of the filtrate, and freeze-drying after concentration", and other operations remain unchanged.

[0099] Example 7

[0100] The present example provides a preparation method of extract of A. woodii, which is different from example 1 only in that step (5) is "mixing the treatment solution with a flocculating agent at 30℃ for 2 h, the flocculating agent is gellan gum, and the mass of the flocculating agent is 2% of the mass of the treatment solution, then performing plate and frame filtration, reducing pressure suction filtration of the filtrate, and freeze-drying after concentration", and other operations remain unchanged.

[0101] Comparative Example 1

[0102] The present comparative example provides a preparation method of extract of A. woodii, which is different from example 1 only in that step (3) is "inactivating the enzymolysis solution at 90℃ for 10 min, then performing reflux extraction, and the extraction time is 2 h, and the extraction is performed twice, and the filtrate is combined to obtain an A. woodii extract", and other operations remain unchanged.

[0103] Comparative Example 2

[0104] The present comparative example provides a preparation method of extract of A. woodii, which is different from example 1 only in that it does not contain step (2), and other operations remain unchanged.

[0105] Comparative Example 3

[0106] The present comparative example provides a preparation method of a A. bidwilli extract, which is different from Example 1 only in that step (4) is "mixing the activated carbon and the A. bidwilli extraction liquid at a solid-liquid ratio of 0.01:1 g / mL, reacting at 50°C for 10 min, and obtaining a treated liquid by plate and frame filtration after the reaction", and other operations remain unchanged.

[0107] Comparative Example 4

[0108] The present comparative example provides a preparation method of a A. bidwilli extract, which is different from Example 1 only in that step (4) is "mixing the activated carbon and the A. bidwilli extraction liquid at a solid-liquid ratio of 0.01:1 g / mL, reacting at 50°C for 10 min, and obtaining a treated liquid by plate and frame filtration after the reaction", and other operations remain unchanged.

[0109] Comparative Example 5

[0110] The present comparative example provides a preparation method of a A. bidwilli extract, which is different from Example 1 only in that step (5) is "concentrating and freeze-drying the treated liquid after vacuum filtration, to obtain the A. bidwilli extract", and other operations remain unchanged.

[0111] Comparative Example 6

[0112] The present comparative example provides a preparation method of a A. bidwilli extract, which comprises:

[0113] (1) crushing A. bidwilli medicinal materials to 60 mesh to obtain A. bidwilli medicinal material powder A; mixing the A. bidwilli medicinal material powder A with water at a solid-liquid ratio of 1:10 g / mL, and standing at room temperature for 1 h to obtain an A. bidwilli medicinal material powder A mixture;

[0114] (2) adjusting the pH of the A. bidwilli medicinal material powder A mixture to 5, and mixing with a composite enzyme, the composite enzyme being added at an amount of 2.3% of the mass of the mixture, the composite enzyme being a fibrozyme and a pectinase at a mass ratio of 20:3, and enzymolysis at 50°C for 2 h to obtain an enzymolysis liquid;

[0115] (3) inactivating the enzymolysis liquid at 90°C for 10 min, and performing plate and frame filtration to obtain A. bidwilli medicinal material powder B;

[0116] (4) mixing the A. bidwilli medicinal material powder B with water at a solid-liquid ratio of 1:10 g / mL, and performing negative pressure cavitation extraction, the extraction pressure being 125 mbar, the temperature being 50°C, and the extraction time being 2 h, and the extraction being performed twice, and the filtrates being combined to obtain an A. bidwilli extraction liquid;

[0117] (5) mixing the A. bidwilli medicinal material powder A with the A. bidwilli extraction liquid at a solid-liquid ratio of 0.01:1 g / mL, and reacting at 50°C for 10 min, and obtaining a treated liquid by plate and frame filtration after the reaction;

[0118] (6) mixing the treatment liquid with flocculants at 30℃ for 2 h, the flocculants being gellan gum and sordarium gum at a mass ratio of 1:1, the mass of the flocculants being 2% of the mass of the treatment liquid, performing plate and frame filtration, performing vacuum suction filtration on the filtrate, and freeze-drying after concentration, to obtain the product.

[0119] Test Example 1

[0120] B16F10 mouse melanoma cell melanin content test

[0121] Test method: blank group, model group (α-melanocyte stimulating hormone), sample group (α-melanocyte stimulating hormone + white camphor extract) were set. Except the blank group, 0.1% α-melanocyte stimulating hormone was added to the rest of the groups. The B16F10 cells in the sample group were treated with white camphor extract (120 μg / mL) for 48 h, and then the cells were digested and transferred to centrifuge tubes, centrifuged at 13000 r for 5 min, the supernatant was discarded, NaOH (containing 10% DMSO) was added, vortexed thoroughly, and then placed in a metal bath for 2 h. The absorbance was measured at 405 nm. The color group only added NaOH (containing 10% DMSO). The melanin content was calculated. In the table, "*" represents comparison with the model group, and "#" represents comparison with Example 1.

[0122] Melanin content (%) = (A sample - A color deduction) / (A model - A color deduction) x 100%

[0123] In the formula: A sample is the absorbance of the sample solution; A color deduction is the absorbance of the color deduction group; A model is the absorbance of the model group.

[0124] Table 1

[0125]

[0126] From the results in Table 1, compared with the α-melanocyte stimulating hormone group (model group), the white camphor extract prepared in Examples 1-3 can greatly inhibit the melanin content of B16F10 cells, and the effect is better than that of arbutin, indicating that the white camphor extract prepared in the application has a significant whitening effect on B16F10 cells.

[0127] Test Example 2

[0128] B16F10 mouse melanoma cell tyrosinase activity test

[0129] Test method: Set blank group, model group (alpha-melanocyte stimulating hormone), sample group (alpha-melanocyte stimulating hormone + white camphor extract). Except the blank group, the rest of the groups were added with 0.1% alpha-melanocyte stimulating hormone, the sample group B16F10 cells were treated with white camphor extract (120 μg / mL), and were cultured for 48 h. After the cells in the 6-hole plate were washed with PBS, cell lysis solution was added to each hole, and the supernatant was obtained by centrifugation, mixed with 0.1% L-DOPA, and incubated at 37°C for 1 h. The absorbance value was measured at 475 nm, and the model group was normalized, and the tyrosinase activity was calculated by combining the absorbance of the sample group. In the table, "*" represents comparison with the model group, and "#" represents comparison with Example 1.

[0130] Tyrosinase activity (%) = Ssample / Smodel x 100%

[0131] In the formula: Ssample is the absorbance of the sample solution; Smodel is the absorbance of the model group.

[0132] Table 2

[0133]

[0134] From the results in Table 2, compared with the alpha-melanocyte stimulating hormone group (model group), the white camphor extract prepared in Examples 1-3 has a significantly stronger inhibitory effect on the tyrosinase activity in cells than the white camphor extract prepared in other examples and comparative examples, and the inhibitory effect is better than that of arbutin, indicating that the white camphor extract prepared in the present application can significantly inhibit the tyrosinase activity in cells, and thus achieve the whitening effect of inhibiting melanin.

[0135] Test Example 3

[0136] Whitening-related gene expression test

[0137] Test method: Set blank group, model group (alpha-melanocyte stimulating hormone), sample group (alpha-melanocyte stimulating hormone + white camphor extract). Except the blank group, the rest of the groups were added with 0.1% alpha-melanocyte stimulating hormone, the sample group B16F10 cells were treated with white camphor extract (120 μg / mL), and were cultured for 48 h. After the cells in the 6-hole plate were washed with PBS, cell lysis solution was added to each hole, and the supernatant was obtained by centrifugation, mixed with 0.1% L-DOPA, and incubated at 37°C for 1 h. The absorbance value was measured at 475 nm, and the model group was normalized, and the tyrosinase activity was calculated by combining the absorbance of the sample group. In the table, "*" represents comparison with the model group, and "#" represents comparison with Example 1.

[0138] Gene expression was detected by real-time fluorescent quantitative PCR. The PCR amplification was performed by using Roche LightCycler 480 instrument and sequence detection software for fluorescent quantitative PCR analysis. ERK1, MITF and TYR were detected respectively, and the primers were synthesized by Nanjing Nasen Biotechnology Co., Ltd. 2 −∆∆Ct The method quantifies and expresses the level of the gene. Figure 4 In the table, "*" represents comparison with the model group, and "#" represents comparison with Example 1.

[0139] Table 3

[0140]

[0141] As can be seen from the results in Table 3, compared with the alpha-melanocyte stimulating hormone group (model group), the white camphor extracts prepared in Examples 1 and 2 can significantly inhibit the expression of ERK1, MITF and TYR genes, and the inhibition effect is more excellent compared with the white camphor extracts prepared in Example 4 and Comparative Example 3. The inhibition capacity of the white camphor extract prepared in Example 1 on ERK1 and TYR is about 2 times that of Comparative Example 3, which shows that the white camphor extract prepared in the present application can significantly inhibit the expression of melanin synthesis related genes (ERK1, MITF, TYR), and then play its whitening effect.

[0142] Test Example 4

[0143] Zebrafish whitening model test

[0144] Test method: A sufficient number of 6 hfp zebrafish embryos with consistent development were pre-screened, and the zebrafish embryos were randomly divided into 6-well plates. The standard dilution water in the well plate was removed, 3 mL of solution containing white camphor extract (30 μg / mL) was quickly added to each well. After mixing thoroughly, the culture plate was covered and wrapped with aluminum foil, and incubated in a 28℃ biochemical incubator in the dark to 72 hpf. After incubation, zebrafish with normal phenotype and behavior were selected, fixed with 3% methyl cellulose, observed under a microscope and photographed. Propylthiouracil (PTU) was used as a positive control. Figure 5 In the table, "*" represents comparison with the blank group, and "#" represents comparison with Example 1.

[0145] Zebrafish melanin content (%) = (Fblank-Fsample) / Fblankx100%

[0146] In the formula, Fblank is the melanin signal intensity of the zebrafish embryo blank group, and Fsample is the melanin signal intensity of the zebrafish embryo after sample treatment.

[0147] Table 4

[0148]

[0149] As can be seen from the results in Table 4, compared with the blank group, the extract of Aquilaria sinensis prepared in Example 1 can significantly inhibit melanin production in zebrafish, with an inhibition rate of 52.31%, and the inhibition effect is better than that of the extract of Aquilaria sinensis prepared in Example 4 and Comparative Example 3, indicating that the extract of Aquilaria sinensis prepared in the application has excellent whitening effect.

[0150] Test Example 5

[0151] Inflammation factor inhibition test

[0152] Logarithmic growth period HaCaT cells with good morphology were inoculated in a 12-well plate and incubated in a 37°C incubator for 24 h. The blank group, model group (UVB), positive control group (5 μM dexamethasone, Dex) and sample group (Aquilaria sinensis extract 120 μg / mL) were incubated in a 37°C incubator for 24 h, and then UVB (67.81 mJ / cm 2 ) irradiation was performed on the blank group. Figure 6 “*” represents comparison with the model group, and “#” represents comparison with Example 1.

[0153] Table 5

[0154]

[0155] As can be seen from the results in Table 5, compared with the model group (UVB), the extracts of Aquilaria sinensis prepared in Examples 1 and 2 can significantly inhibit the expression of inflammation factor TNF-α gene, and have better anti-inflammatory effect than the extract of Aquilaria sinensis prepared in Example 4, while the anti-inflammatory effect of Comparative Example 3 is not significant, which indicates that the extract of Aquilaria sinensis prepared in the application can significantly inhibit the expression of inflammation factor TNF-α and has certain anti-inflammatory effect.

[0156] Test Example 6

[0157] According to the Cosmetic Safety Technical Specifications (2015), the extracts of Aquilaria sinensis prepared in Examples 1-3 were subjected to acute eye irritation / corrosion test, skin irritation / corrosion test, skin allergy test, skin phototoxicity test, hormone, pesticide residue, microorganism, heavy metal and H-Clat sensitization test, and the safety of the extract of Aquilaria sinensis was comprehensively evaluated.

[0158] Table 6

[0159]

[0160] The test results are shown in Table 6, and the extracts of Aquilaria sinensis obtained in Examples 1-3 have no irritation, no skin allergy, no sensitization and no phototoxicity, and no hormones, pesticide residues, etc. are detected.

[0161] The above merely describes the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and it should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily conceived by those skilled in the art, and all such changes and replacements fall within the protection scope and disclosure scope of the present application.

Claims

1. A method for preparing an extract of Aquilaria sinensis, characterized in that, The preparation method comprises: (1) mixing the powder of Aquilaria sinensis with water to obtain an Aquilaria sinensis mixture; (2) mixing the Aquilaria sinensis mixture with a composite enzyme for enzymolysis, inactivating the enzyme, and performing negative pressure cavitation extraction to obtain an extract; (3) mixing the extract with the powder of Aquilaria sinensis in step (1) for treatment, and then performing solid-liquid separation to obtain a treatment liquid; (4) mixing the treatment liquid with a flocculating agent, performing solid-liquid separation to obtain a liquid, and then drying the liquid to obtain the product.

2. The method of claim 1, wherein the white mulberry extract is prepared by the steps of: The ratio of the powder of Aquilaria sinensis to water in step (1) is 1:(5-20) mL; ​ Preferably, the temperature for mixing in step (1) is 10-30℃, and the time is 1-3 h; Preferably, before the enzymolysis with the composite enzyme, the pH of the Aquilaria sinensis mixture is adjusted to 4-6; Preferably, the pH adjusting agent used for the pH adjustment comprises 0.01-0.2 M acetic acid buffer, 0.01-0.2 M citric acid buffer, or 0.01-0.2 M phosphoric acid buffer.

3. The method for preparing the Aucklandia lappa extract according to claim 1 or 2, characterized in that, The mass of the composite enzyme is 1-4% of the mass of the Aquilaria sinensis mixture; Preferably, the composite enzyme is a combination of cellulase and pectinase; Preferably, the mass ratio of the cellulase to the pectinase is (10-30):(1-5).

4. The method of preparing extract of A. calonea according to any one of claims 1-3, characterized in that, The temperature for the enzymolysis is 40-55℃, and the time is 1-3 h; Preferably, the temperature for inactivating the enzyme is 80-90℃, and the time is 5-15 min.

5. The method of preparing extract of A. calonea according to any one of claims 1-4, characterized in that, The temperature for the negative pressure cavitation extraction is 30-70℃, the pressure is 90-150 mbar, the time is 1-3 h, and the number of times is 1-3; Preferably, the ratio of the powder of Aquilaria sinensis to the extract in step (3) is (0.001-0.01) g:1 mL; Preferably, the temperature for the mixing treatment in step (3) is 40-70℃, and the time is 5-30 min.

6. The method of preparing extract of A. calonea according to any one of claims 1-5, characterized in that, The flocculating agent is a combination of gellan gum and sordariin gum; Preferably, the mass ratio of the gellan gum to the sordariin gum is (1-10):(1-10); Preferably, the mass of the flocculating agent is 0.5-4% of the mass of the treatment liquid; Preferably, the temperature for mixing with the flocculating agent is 25-60℃, and the time is 0.5-3 h; Preferably, the solid-liquid separation is performed by centrifugation, static sedimentation, or plate-and-frame filtration; Preferably, the drying is performed by vacuum drying, freeze drying, or spray drying.

7. An Aquilaria sinensis extract prepared by the preparation method of the Aquilaria sinensis extract according to any one of claims 1-6.

8. Use of the Aquilaria sinensis extract according to claim 7 in the preparation of a product having a whitening effect.

9. Use according to claim 8, characterized in that, The product comprises a cosmetic or a skin care product; Preferably, the dosage form of the skin care product comprises a skin care cream or an essence; Preferably, the mass percentage of the Aquilaria sinensis extract in the product is 0.001-0.008%.

10. Use of the Aquilaria sinensis extract according to claim 8 in the preparation of a cell melanin inhibitor, an ERK1 gene expression inhibitor, an MITF gene expression inhibitor, a tyrosinase inhibitor, or a zebrafish melanin inhibitor.

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