Phyllanthus emblica active ingredient, and separation method and application thereof

Abstract

The application provides a phyllium active ingredient and a separation method and application thereof. The method first adopts subcritical water to pretreat phyllium, then uses a macroporous resin chromatographic column to separate and purify a crude extract of the phyllium, and creatively divides the macroporous resin used in the macroporous resin chromatographic column into two layers, and the types of the two layers of macroporous resin are respectively specifically selected, so that three important active ingredients (phyllium polysaccharide, phyllium polyphenol and phyllium flavone) in the phyllium can be separated out in a high efficiency and high purity in a one-time column chromatography process, and the raw material utilization rate of the phyllium is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics. More particularly, it relates to a phyllanthus emblica active ingredient, a separation method and application thereof. BACKGROUND

[0002] Phyllanthus emblica is the dried fruit of Phyllanthus emblica L. of Euphorbiaceae. It is sour and sweet, and tastes sweet after a long time. It is also known as oil citron, oil citron, amolai, and Dian olive. Phyllanthus emblica has important value in health care and medical treatment, and is widely used in traditional Chinese medicine, Tibetan medicine, Mongolian medicine, white medicine and Zhuang medicine.

[0003] Phyllanthus emblica is rich in active ingredients such as polysaccharides, polyphenols (including chebulagic acid, chebulinic acid, corilagin, mucic acid, oil citron acid, ellagic acid, corilagin, gallic acid and pyrogallol acid) and flavonoids (including quercetin, rutin, kaempferol, eriodictyol, wild forsythia and Han forsythia). These active ingredients have excellent whitening and antioxidant effects. In order to improve the utilization rate of phyllanthus emblica, the efficient extraction method of its active ingredients has gradually become one of the research hotspots in the field of cosmetics. For example, the prior art separates phyllanthus emblica polysaccharides by hot water soaking, beating, heating refluxing and concentrating, but it cannot separate phyllanthus emblica polyphenols and phyllanthus emblica flavonoids.

[0004] Therefore, in order to more efficiently and fully utilize phyllanthus emblica, it is urgent to find a method that can separate its main active ingredients (i.e. polysaccharides, polyphenols and flavonoids) at one time. SUMMARY

[0005] The present application aims to provide a separation method of phyllanthus emblica active ingredients, which can efficiently and highly purify polysaccharides, polyphenols and flavonoids in phyllanthus emblica by one column chromatography, and significantly improve the utilization rate of phyllanthus emblica raw materials.

[0006] The first object of the present application is to provide a separation method of phyllanthus emblica active ingredients.

[0007] The second object of the present application is to provide a phyllanthus emblica active ingredient separated by the above method.

[0008] The third object of the present application is to provide an application of the above phyllanthus emblica active ingredient in the preparation of cosmetics.

[0009] The fourth object of the present application is to provide a cosmetic containing the above phyllanthus emblica active ingredient.

[0010] The above objects of the present application are achieved by the following technical solutions:

[0011] The present application provides a separation method of phyllanthus emblica active ingredients, comprising the following steps:

[0012] S1. crushing the phyllanthus emblica and sequentially performing subcritical water extraction, separation and concentration to obtain a crude extract of phyllanthus emblica;

[0013] S2. adding the crude extract of phyllanthus emblica into a macroporous resin chromatographic column and collecting the effluent;

[0014] S3. continuing column chromatography with water as the elution solvent, collecting water eluate of 0BV→(0.8-1.6)BV, mixing the water eluate with the effluent, and sequentially performing concentration, alcohol precipitation, separation, drying the precipitate obtained by separation to obtain phyllanthus emblica polysaccharide;

[0015] S4. continuing column chromatography with water as the elution solvent, collecting water eluate of 0BV→(0.8-1.6)BV, mixing the water eluate with the effluent, and sequentially performing concentration, alcohol precipitation, separation, drying the precipitate obtained by separation to obtain phyllanthus emblica polysaccharide;

[0016] S5. replacing the elution solvent with ethanol solution, collecting ethanol eluate of 0BV→(2.5-4.5)BV, and drying to obtain phyllanthus emblica flavone;

[0017] In the macroporous resin chromatographic column of S2, the macroporous resin is 2 layers, the upper layer macroporous resin is D101, AB-8 and / or HPD100 macroporous resin, and the lower layer macroporous resin is ADS-17, BS45 and / or BS75 macroporous resin.

[0018] The term "upper layer macroporous resin" refers to the macroporous resin that first contacts the elution solvent in the separation and purification process; the term "lower layer macroporous resin" refers to the macroporous resin that later contacts the elution solvent in the separation and purification process; and the term "BV" refers to the volume multiple of the macroporous resin filled in the macroporous resin chromatographic column.

[0019] The present application specifically studies the properties of phyllanthus emblica, first pretreats phyllanthus emblica with subcritical water, then separates and purifies the crude extract of phyllanthus emblica by using a macroporous resin chromatographic column, and creatively divides the macroporous resin used in the macroporous resin chromatographic column into two layers, and specifically selects the types of the two layers of macroporous resin, so that the three important active ingredients in phyllanthus emblica (phyllanthus emblica polysaccharide, phyllanthus emblica polyphenol and phyllanthus emblica flavone) can be efficiently and highly purified in one column chromatography process.

[0020] Preferably, the crushing in S1 is crushing to 40-80 mesh.

[0021] Preferably, the mass ratio of phyllanthus emblica to subcritical water in S1 is 1:(8-15).

[0022] Preferably, the extraction temperature in S1 is 105-115°C.

[0023] Preferably, the extraction time in S1 is 1-4h.

[0024] Preferably, the end point of the concentration in S1 is that the mass ratio of the crude extract of Phyllanthus emblica to Phyllanthus emblica is (3-5.5):1.

[0025] Preferably, the ratio of diameter to height of the macroporous resin chromatographic column in S2 is 1:(4-8).

[0026] Preferably, the volume ratio of the upper layer macroporous resin to the lower layer macroporous resin is (2-4):1.

[0027] Preferably, the ratio of the amount of the crude extract of Phyllanthus emblica to the amount of macroporous resin in the macroporous resin chromatographic column in S2 is (1-2)g:1mL.

[0028] Preferably, the flow rate of the elution solvent in S3 is 1-1.5BV / h.

[0029] Preferably, the end point of the concentration in S3 is that the ratio of the amount of the concentrated product to the amount of Phyllanthus emblica is 1mL:(1-2.2)g.

[0030] Preferably, the reagent used in the alcohol precipitation in S3 is ethanol, such as anhydrous ethanol.

[0031] Further preferably, the volume ratio of the ethanol to the concentrated product is (3-5):1.

[0032] Preferably, the temperature of the alcohol precipitation in S3 is 3-5℃.

[0033] Preferably, the time of the alcohol precipitation in S3 is 10-14h.

[0034] Preferably, the flow rate of the elution solvent in S4 is 1-1.5BV / h.

[0035] Preferably, the concentration of the ethanol solution in S5 is 60%(v / v)-95%(v / v).

[0036] Preferably, the flow rate of the ethanol solution in S5 is 1-1.5BV / h.

[0037] The Phyllanthus emblica polysaccharide, the Phyllanthus emblica polyphenol and the Phyllanthus emblica flavone obtained by the method of the present application have high purity and excellent antioxidant effect, and therefore, the active ingredients of Phyllanthus emblica obtained by the above method, the application of the active ingredients of Phyllanthus emblica in the preparation of cosmetics, and the cosmetics containing the active ingredients of Phyllanthus emblica should be within the protection scope of the present application.

[0038] Preferably, the active ingredients of Phyllanthus emblica include one or more of Phyllanthus emblica polysaccharide, Phyllanthus emblica polyphenol and Phyllanthus emblica flavone.

[0039] The present application has the following beneficial effects:

[0040] 1.The present application first pretreats Phyllanthus emblica by subcritical water, then separates and purifies the crude extract of Phyllanthus emblica by using a macroporous resin chromatographic column, and creatively divides the macroporous resin used in the macroporous resin chromatographic column into two layers, and specifically selects the types of the two layers of macroporous resin, so that three important active ingredients in Phyllanthus emblica (Phyllanthus emblica polysaccharide, Phyllanthus emblica polyphenol, and Phyllanthus emblica flavone) can be separated in one column chromatography process with high efficiency and high purity.

[0041] 2.The method has the advantages of low cost, easy operation, stable process, etc., is suitable for batch production, and is beneficial to greatly improving the raw material utilization rate of Phyllanthus emblica. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 The test results of the antioxidant properties of Phyllanthus emblica polysaccharide.

[0043] Figure 2 The test results of the antioxidant properties of Phyllanthus emblica polyphenol, Phyllanthus emblica flavone, and VC. DETAILED DESCRIPTION

[0044] The present application will be further described below in combination with the drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present application are conventional reagents, methods, and equipment in the technical field.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] Example 1: Separation of active ingredients of Phyllanthus emblica

[0047] S1. Wash the dried Phyllanthus emblica fruit with pure water, crush it with a traditional Chinese medicine powdering machine, and pass it through a 70-mesh sieve to obtain Phyllanthus emblica powder. Then, extract 20 g of the Phyllanthus emblica powder in 200 g of subcritical water at 110℃ for 1 h, perform solid-liquid separation, and concentrate the obtained liquid to 80 g to obtain a crude extract of Phyllanthus emblica;

[0048] S2. Add 80 g of the crude extract of Phyllanthus emblica to a macroporous resin chromatographic column (column diameter to height ratio is 1:6, and the volume of the filled macroporous resin is 40 mL, with D101 macroporous resin in the upper layer and ADS-17 macroporous resin in the lower layer, and the volume ratio of the upper layer to the lower layer is 3:1) at a speed of 1.2 BV / h to perform adsorption, and collect the effluent;

[0049] S3. Continue column chromatography with water as the elution solvent (the flow rate remains unchanged), collect the water eluent from 0 BV to 1.2 BV, mix the eluent with the effluent, concentrate to 20 mL, add 80 mL of anhydrous ethanol, mix well, place in a 4℃ refrigerator for 12 h, centrifuge to collect the precipitate, and dry the obtained light gray powder to obtain Phyllanthus emblica polysaccharide;

[0050] S4. Continue column chromatography with water as elution solvent (flow rate remains unchanged), collect the water eluate of 2BV-5BV, dry the light beige powder obtained to obtain the phyllium polyphenol;

[0051] S5. Change the elution solvent to 60% (v / v) ethanol solution (flow rate remains unchanged), collect the ethanol eluate of 0BV-3.5BV, concentrate to 1 / 5 of the original volume and vacuum freeze-dry to obtain the yellow powder, which is the phyllium flavone.

[0052] Example 2 Separation of active ingredients of phyllium

[0053] S1. Wash the dried phyllium fruit with pure water, then crush it with a traditional Chinese medicine powdering machine, pass it through a 40-mesh sieve to obtain phyllium powder, then extract 20g of the phyllium powder in 300g subcritical water at 105°C for 4h, perform solid-liquid separation, and concentrate the separated liquid to 110g to obtain the phyllium crude extract;

[0054] S2. Add the 110g phyllium crude extract to a macroporous resin chromatography column (column diameter to height ratio is 1:8, the volume of the filled macroporous resin is 110mL, the upper layer is AB-8 macroporous resin, and the lower layer is BS45 macroporous resin, the volume ratio of the upper layer to the lower layer is 4:1) to perform adsorption at a speed of 1BV / h, and collect the effluent;

[0055] S3. Continue column chromatography with water as elution solvent (flow rate remains unchanged), collect the water eluate of 0BV-1.6BV, mix it with the effluent, concentrate to 9.1mL, then add it to 45.5mL of anhydrous ethanol, mix well, place it in a 3°C refrigerator for 10h, then centrifuge to collect the precipitate, and dry the light gray powder obtained to obtain the phyllium polysaccharide;

[0056] S4. Continue column chromatography with water as elution solvent (flow rate remains unchanged), collect the water eluate of 2BV-5BV, dry the light beige powder obtained to obtain the phyllium polyphenol;

[0057] S5. Change the elution solvent to 95% (v / v) ethanol solution (flow rate remains unchanged), collect the ethanol eluate of 0BV-2.5BV, concentrate to 1 / 5 of the original volume and vacuum freeze-dry to obtain the yellow powder, which is the phyllium flavone.

[0058] Example 3 Separation of active ingredients of phyllium

[0059] S1. Wash the dried phyllium fruit with pure water, then crush it with a traditional Chinese medicine powdering machine, pass it through an 80-mesh sieve to obtain phyllium powder, then extract 20g of the phyllium powder in 160g subcritical water at 115°C for 1h, perform solid-liquid separation, and concentrate the separated liquid to 60g to obtain the phyllium crude extract;

[0060] S2. The 60 g crude extract of Phyllanthus emblica was added to a macroporous resin chromatographic column (column diameter to height ratio of 1:4, macroporous resin volume filled of 30 mL, upper layer of HPD100 macroporous resin, lower layer of BS75 macroporous resin, volume ratio of upper layer to lower layer of 2:1) at a speed of 1.5 BV / h for adsorption, and the effluent was collected;

[0061] S3. Column chromatography was continued with water as the elution solvent (flow rate remained unchanged), and the water eluate of 0 BV→0.8 BV was collected. After mixing with the effluent, it was concentrated to 20 mL, then added to 60 mL of anhydrous ethanol, mixed, and placed in a 5°C refrigerator for 14 h. After centrifugal collection of the precipitate, the light gray powder obtained was dried to obtain the Phyllanthus emblica polysaccharide;

[0062] S4. Column chromatography was continued with water as the elution solvent (flow rate remained unchanged), and the water eluate of 0 BV→3 BV was collected. The light beige powder obtained after drying was the Phyllanthus emblica polyphenol;

[0063] S5. The elution solvent was replaced with 60% (v / v) ethanol solution (flow rate remained unchanged), and the ethanol eluate of 0 BV→4.5 BV was collected. After concentration to 1 / 5 of the original volume and vacuum freeze-drying, the yellow powder obtained was the Phyllanthus emblica flavone.

[0064] Comparative Example 1

[0065] The same as in Example 1, except that the elution solvent of S5 was replaced with water, and the water eluate of 1 BV→4.5 BV was collected. That is, S5 was as follows:

[0066] S5. Column chromatography was continued with water as the elution solvent (flow rate remained unchanged), and the water eluate of 1 BV→4.5 BV was collected. After concentration to 1 / 5 of the original volume and vacuum freeze-drying, the Phyllanthus emblica flavone was obtained.

[0067] Comparative Example 2

[0068] The same as in Example 1, except that the elution solvents of S3 and S4 were replaced with 60% (v / v) ethanol solution, and the ethanol eluate of S3 was 0.3 BV→1.5 BV, and the ethanol eluate of S4 was 2 BV→5 BV. That is, S3-S5 were as follows:

[0069] S3. Column chromatography was continued with 60% (v / v) ethanol solution as the elution solvent (flow rate remained unchanged), and the ethanol eluate of 0.3 BV→1.5 BV was collected. After mixing with the effluent, it was concentrated to 20 mL, then added to 80 mL of anhydrous ethanol, mixed, and placed in a 4°C refrigerator for 12 h. After centrifugal collection of the precipitate, it was dried to obtain the Phyllanthus emblica polysaccharide;

[0070] S4. Continue column chromatography with 60% (v / v) ethanol solution as elution solvent (flow rate remains unchanged), collect ethanol eluate of 2 BV→5 BV, dry to obtain phyllium polyphenol;

[0071] S5. Continue column chromatography with 60% (v / v) ethanol solution as elution solvent (flow rate remains unchanged), collect ethanol eluate of 0 BV→3.5 BV, concentrate to 1 / 5 of the original volume and vacuum freeze-dry to obtain phyllium flavone.

[0072] Comparative Example 3

[0073] The same as Example 1, except that the elution solvent of S4 is replaced with 60% (v / v) ethanol solution, and the ethanol eluate of 2 BV→5 BV is collected. That is, S4 and S5 are as follows:

[0074] S4. Continue column chromatography with 60% (v / v) ethanol solution as elution solvent (flow rate remains unchanged), collect ethanol eluate of 2 BV→5 BV, dry to obtain phyllium polyphenol;

[0075] S5. Continue column chromatography with 60% (v / v) ethanol solution as elution solvent (flow rate remains unchanged), collect ethanol eluate of 0 BV→3.5 BV, concentrate to 1 / 5 of the original volume and vacuum freeze-dry to obtain phyllium flavone.

[0076] Comparative Example 4

[0077] The same as Example 1, except that the macroporous resin chromatography column has only one layer of macroporous resin, which is D101 macroporous resin; and S3 collects water eluate of 0.3 BV→1.5 BV, S4 collects water eluate of 3 BV→6 BV, and S5 collects ethanol eluate of 0 BV→3.5 BV.

[0078] Comparative Example 5

[0079] The same as Example 1, except that the macroporous resin chromatography column has only one layer of macroporous resin, which is ADS-17 macroporous resin; and S3 collects water eluate of 0 BV→1.2 BV, S4 collects water eluate of 3 BV→6 BV, and S5 collects ethanol eluate of 0 BV→3.5 BV.

[0080] Comparative Example 6

[0081] The same as Example 1, except that the upper layer of macroporous resin in the macroporous resin chromatography column is ADS-17 macroporous resin, and the lower layer of macroporous resin is D101 macroporous resin; and S3 collects water eluate of 0.3 BV→1.5 BV, S4 collects water eluate of 2 BV→5 BV, and S5 collects ethanol eluate of 0 BV→3.5 BV.

[0082] Comparative Example 7

[0083] The same as Example 1, except that the subcritical water of 110°C in S1 is replaced by water of 80°C.

[0084] Test Example 1 Determination of extraction effect of active ingredients of Phyllanthus emblica

[0085] I. Detection method

[0086] The Phyllanthus emblica polysaccharides, Phyllanthus emblica polyphenols and Phyllanthus emblica flavonoids obtained in Examples 1-3 and Comparative Examples 1-7 are weighed and recorded, and then the ratio of the total mass of the three active ingredients to the mass of the Phyllanthus emblica raw material (i.e. 20 g) is calculated to obtain the total extraction rate. Then the content of each of the three active ingredients is determined according to the following method to characterize the purity (Note: The starting point and the end point of the collection of the eluent in Examples 1-3 and Comparative Examples 1-7 are also determined by the following method, the starting point is when the content of the active ingredient is greater than 0, and the end point is when the content is infinitely close to 0):

[0087] (1) Phyllanthus emblica polysaccharides

[0088] ① Preparation of standard curve

[0089] Glucose is dried at 105°C to constant weight to obtain anhydrous glucose, and then 0.5 g of the anhydrous glucose is dissolved in water and diluted to 500 mL in a volumetric flask to obtain a glucose stock solution with a concentration of 1 mg / mL. 2, 4, 6, 8 and 10 mL of the glucose stock solution are respectively measured and diluted to 100 mL to obtain glucose standard solutions with concentrations of 20-100 μg / mL.

[0090] 1 mL of each concentration gradient of the glucose standard solution is mixed with 1 mL of a phenol reagent with a concentration of 5% (v / v), and then 5 mL of concentrated sulfuric acid with a concentration of 98.3 wt% is added, and the mixture is mixed uniformly. When the temperature cools to 25°C, the absorbance value at 490 nm is measured by an enzyme marker. The absorbance value is taken as the vertical coordinate, and the concentration of the glucose standard solution is taken as the horizontal coordinate to draw a standard curve graph and calculate the standard curve equation of the polysaccharide.

[0091] ② Sample detection

[0092] The Phyllanthus emblica polysaccharides obtained in Examples 1-3 and Comparative Examples 1-7 are dissolved in water to prepare solutions with a concentration of 100 μg / mL, and the absorbance value at 490 nm is measured by an enzyme marker. The purity of the Phyllanthus emblica polysaccharides is determined by referring to the standard curve graph and the standard curve equation.

[0093] (2) Phyllanthus emblica polyphenols

[0094] ① Preparation of standard curve

[0095] Dilute 1.0 mg / mL of gallic acid standard solution to 0.1, 0.2, 0.3, 0.4 and 0.5 mg / mL standard solution dilutions.

[0096] Add 200 μL of each concentration gradient standard solution dilution to a 10 mL centrifuge tube, and sequentially add 2 mL of distilled water and 200 μL of Folin-phenol reagent, mix well, then add 600 μL of 20 wt% sodium carbonate solution, add distilled water to make up the volume to 5 mL, and after 2 h of reaction at 30°C, measure the absorbance value at 760 nm using an enzyme marker. Plot the standard curve graph with the absorbance value as the ordinate and the concentration of the standard solution dilution as the abscissa, and calculate the standard curve equation of the polyphenol.

[0097] ②Sample detection

[0098] Dissolve the phyllium polyphenols obtained in Examples 1-3 and Comparative Examples 1-7 in water to prepare a 0.5 mg / mL solution, measure the absorbance value at 760 nm using an enzyme marker, and determine the purity of the phyllium polyphenol by referring to the standard curve graph and the standard curve equation.

[0099] (3) Phyllium flavone

[0100] ①Standard curve plotting

[0101] Dissolve 50 mg of rutin reference material in a 50 mL volumetric flask using methanol to obtain a 1 mg / mL rutin standard solution. Measure 1, 2, 4, 6 and 8 mL of the rutin standard solution, respectively, and make up to 10 mL to obtain a 0.1, 0.2, 0.4, 0.6 and 0.8 mg / mL rutin standard solution.

[0102] Measure 1 mL of the 0.1, 0.2, 0.4, 0.6 and 0.8 mg / mL rutin standard solution, respectively, into a 10 mL cuvette, add 0.5 mL of 5 wt% sodium nitrite solution, mix well and stand for 6 min, then add 0.5 mL of 10 wt% aluminum nitrate solution, mix well and stand for 6 min, and then add 4 mL of 4 wt% sodium hydroxide solution, stand for 15 min, and then measure the absorbance value at 510 nm using spectrophotometry. Plot the standard curve graph with the absorbance value as the ordinate and the concentration of the rutin standard solution as the abscissa, and calculate the standard curve equation of the flavone.

[0103] ②Sample detection

[0104] Dissolve the phyllium flavones obtained in Examples 1-3 and Comparative Examples 1-7 in water to prepare a 0.8 mg / mL solution, measure the absorbance value at 510 nm, and determine the purity of the phyllium flavone by referring to the standard curve graph and the standard curve equation.

[0105] II. Test Results

[0106] The results of the quality, purity and total extraction rate of the active ingredients of Phyllanthus emblica obtained in Examples 1-3 and Comparative Examples 1-7 are shown in Table 1.

[0107] Table 1 Extraction effect of active ingredients of Phyllanthus emblica

[0108]

[0109]

[0110] It can be seen that the total extraction rate of the active ingredients of Phyllanthus emblica obtained in Examples 1-3 is significantly higher than that of Comparative Examples 1-7, and although the quality of Phyllanthus emblica polyphenols in Comparative Examples 2-3 is slightly improved compared with Examples 1-3, the purity is significantly reduced, and overall the extraction effect of Phyllanthus emblica polyphenols is still significantly poorer than that of Examples 1-3. It shows that it is the method of the present application that pretreats Phyllanthus emblica with subcritical water and separates and purifies the crude extract of Phyllanthus emblica by using a macroporous resin chromatographic column, and the specific selection of the type and layering design of the macroporous resin used in the macroporous resin chromatographic column, that can efficiently and highly purify the three important active ingredients (Phyllanthus emblica polysaccharide, Phyllanthus emblica polyphenol, Phyllanthus emblica flavone) in Phyllanthus emblica in one column chromatography process.

[0111] Test Example 2 Whitening performance test of Phyllanthus emblica polyphenol

[0112] I. Test Method

[0113] Phyllanthus emblica polyphenols obtained in Example 1 and Comparative Examples 4-6 were dissolved in disodium hydrogen phosphate-citric acid buffer (pH = 6.8) to prepare test solutions with a concentration of 2 mg / mL; α-arbutin was dissolved in disodium hydrogen phosphate-citric acid buffer (pH = 6.8) to prepare a test solution with a concentration of 2 mg / mL. Sample tubes, sample blank tubes, enzyme reaction tubes and solvent blank tubes were set up.

[0114] Sample tube: 1 mL of test solution and 0.5 mL of tyrosinase solution (enzyme activity of tyrosinase is 100 U / mL) were added to a 10 mL test tube;

[0115] Sample blank tube: 1 mL of test solution and 0.5 mL of disodium hydrogen phosphate-citric acid buffer (pH = 6.8) were added to a 10 mL test tube;

[0116] Enzyme reaction tube: 1 mL of disodium hydrogen phosphate-citric acid buffer (pH = 6.8) and 0.5 mL of tyrosinase solution (enzyme activity of tyrosinase is 100 U / mL) were added to a 10 mL test tube;

[0117] Solvent blank tube: 1.5 mL of disodium hydrogen phosphate-citric acid buffer (pH = 6.8) was added into a 10 mL test tube.

[0118] Each tube was incubated in a 37℃ water bath for 10 min, then 2 mL of levodopa solution (1 mg / mL) was added, and after 5 min of reaction, the solution in the test tube was transferred into a cuvette, and the absorbance value was measured at 475 nm (3 parallel samples were set for each tube, and the average value was taken).

[0119] II. Test results

[0120] The inhibition rate of the phyllium polysaccharide and α-arbutin on tyrosinase was calculated according to the formula "tyrosinase inhibition rate (%) = [1-(T-T0) / (C-C0)]x100%" (wherein T, T0, C, C0 are the absorbance values of the sample tube, the sample blank tube, the enzyme reaction tube, and the solvent blank tube, respectively), and the results are shown in Table 2.

[0121] Table 2 Test results of tyrosinase inhibition rate

[0122] Sample Inhibition rate / % Example 1 89.4 Comparative Example 4 53.6 Comparative Example 5 48.9 Comparative Example 6 61.2 Alpha-matrimonyvine glucoside 79.3

[0123] It can be seen that the inhibition rate of the phyllium polysaccharide obtained in Example 1 on tyrosinase is significantly better than that of α-arbutin and Comparative Examples 4-6, indicating that the phyllium polysaccharide separated by the method of the present application has excellent whitening performance.

[0124] Test Example 3 Antioxidant performance test of active ingredients of phyllium

[0125] I. Test method

[0126] The phyllium polysaccharide obtained in Example 1 was dissolved in water to prepare test solutions with concentrations of 1.0, 0.5, 0.25, 0.125, and 0.0625 mg / mL; the phyllium polysaccharide obtained in Example 1 was dissolved in water to prepare test solutions with concentrations of 50, 25, 12.5, 6.25, and 3.125 μg / mL; the phyllium flavone obtained in Example 1 was dissolved in water to prepare test solutions with concentrations of 50, 25, 12.5, 6.25, and 3.125 μg / mL; and VC was dissolved in water to prepare test solutions with concentrations of 50, 25, 12.5, 6.25, and 3.125 μg / mL. Sample groups, control groups, and blank groups were set.

[0127] Sample group: 2 mL of the aforementioned test solution was mixed with 2 mL of DPPH solution (0.05 mmol / L), shaken well, and left to stand for 30 min, and the absorbance value at 517 nm was measured with 60% (v / v) ethanol as the zero point, and recorded as A sample.

[0128] Control group: 2 mL of the aforementioned sample solution was mixed with 2 mL of 60% (v / v) ethanol, and then the absorbance value at 517 nm was measured, and recorded as Acontrol.

[0129] Blank group: 2 mL of the DPPH solution (0.05 mmol / L) was mixed with 2 mL of 60% (v / v) ethanol, and then the absorbance value at 517 nm was measured, and recorded as Ablank.

[0130] II. Test results

[0131] The DPPH scavenging rates of the three active ingredients and VC were calculated according to the formula "DPPH scavenging rate (%) = [1-(A sample-A control) / A blank]x100%", and the results are shown in Figures 1-2 Table 3 and Table 4. Among them, Figure 1 Table 3 is the test results of the antioxidant properties of the Phyllanthus emblica polysaccharides, Figure 2 Table 4 is the test results of the antioxidant properties of the Phyllanthus emblica polyphenols, Phyllanthus emblica flavones and VC.

[0132] Table 3 Test results of DPPH scavenging rate-Phyllanthus emblica polysaccharides

[0133] Concentration (mg / mL) DPPH scavenging rate / % 0.0625 56.5 0.125 85.1 0.25 95.8 0.5 96.9 1 97.1

[0134] Table 4 Test results of DPPH scavenging rate-Phyllanthus emblica polyphenols, Phyllanthus emblica flavones and VC

[0135]

[0136] It can be seen that the three active ingredients of Phyllanthus emblica all have excellent antioxidant properties, and the antioxidant properties of the low-concentration Phyllanthus emblica polyphenols and Phyllanthus emblica flavones are significantly better than those of the same concentration VC. Even the antioxidant properties of the high-concentration (50 μg / mL) Phyllanthus emblica polyphenols and Phyllanthus emblica flavones can keep the same level as the same concentration VC, indicating that the active ingredients of Phyllanthus emblica separated by the method of the present application all have excellent antioxidant properties.

[0137] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement modes, and all shall be included in the protection scope of the present application.

Claims

1. A method for isolating active ingredients of Phyllanthus emblica, characterized by, Comprising the following steps: S1. The crushed Phyllanthus emblica is subjected to subcritical water extraction, separation and concentration in sequence to obtain crude Phyllanthus emblica extract; S2. The crude Phyllanthus emblica extract is added to a macroporous resin chromatographic column, and the effluent is collected; S3. Column chromatography is continued with water as the elution solvent, and the 0 BV→(0.8-1.6) BV water eluate is collected, mixed with the effluent, and then subjected to concentration, alcohol precipitation, separation, drying of the separated precipitate, to obtain Phyllanthus emblica polysaccharide; S4. Column chromatography is continued with water as the elution solvent, and the 0-2 BV→(3-5) BV water eluate is collected and dried to obtain Phyllanthus emblica polyphenol; S5. The elution solvent is replaced with an ethanol solution, and the 0 BV→(2.5-4.5) BV ethanol eluate is collected and dried to obtain Phyllanthus emblica flavone. In S1, the extraction temperature is 105-115 ℃; in S2, the macroporous resin chromatographic column uses 2 layers of macroporous resin, the upper layer of macroporous resin is D101, AB-8 and / or HPD100 macroporous resin, and the lower layer of macroporous resin is ADS-17, BS45 and / or BS75 macroporous resin; the volume ratio of the upper layer of macroporous resin to the lower layer of macroporous resin is (2-4):1; in S5, the concentration of the ethanol solution is 60%(v / v)-95%(v / v).

2. The separation method according to claim 1, characterized in that, In S1, the mass ratio of Phyllanthus emblica to subcritical water is 1:(8-15); and the extraction time in S1 is 1-4 h.

3. The separation method according to claim 1, characterized in that, In S3, the flow rate of the elution solvent is 1-1.5 BV / h.

4. The separation method according to claim 1, characterized in that, In S4, the flow rate of the elution solvent is 1-1.5 BV / h.

5. The separation method according to claim 1, characterized in that, In S5, the flow rate of the ethanol solution is 1-1.5 BV / h.

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