Purslane extract, preparation method and application thereof

Abstract

The invention relates to a purslane extract and a preparation method and application thereof. The preparation method comprises the following steps: (1) weighing dried and crushed purslane powder, heating and refluxing under ultrasonic conditions for alcohol extraction to obtain a crude purslane extract; (2) firstly adsorbing the crude purslane extract and a macroporous resin, filtering, and retaining the macroporous resin; then sequentially adding ethanol solutions of different concentrations, respectively performing a first elution, a second elution, and a third elution, collecting the filtrates of the second elution and the third elution, and rotary evaporating to obtain the purslane extract; the method can increase the content of purslanamide A and E in the extract, significantly improve the soothing, anti-inflammatory, skin barrier repairing and other effects of the purslane extract, and has stable performance. The obtained extract can be applied to cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of cosmetics, and in particular relates to a purslane extract and a preparation method and application thereof. Background Art

[0002] Portulaca oleracea L. is an annual fleshy herb of the Portulacaceae family. It is rich in resources and widely distributed in temperate and tropical regions around the world. In addition to secondary metabolites such as organic acids, flavonoids, coumarins and terpenes, alkaloids are an important class of chemical components in Portulaca oleracea, and there are many structural types, including oleracein A, B, C, D, E, F, G, etc. Among them, oleracein E, also known as tropaeoline, is a tetrahydroisoquinoline alkaloid isolated from Portulaca oleracea. It has many biological activities, especially in antibacterial, antiviral, antioxidant, etc. It shows potential application value. Oleraciamide A shows potential application value in antioxidation, anti-inflammatory and whitening.

[0003] There are many methods for preparing purslane extract, including boiling, alcohol extraction, enzymatic hydrolysis and ultrasonic extraction, but different extraction methods will extract different effective ingredients, which will affect the specific efficacy of the extract. Summary of the invention

[0004] The present invention conducts targeted research on the properties of purslane, improves the preparation method of purslane extract, increases the content of active ingredients, especially purslanamide A and purslanamide E, and significantly improves the soothing, anti-inflammatory, skin barrier repairing and other effects of purslane extract, so that it can be better applied in the field of cosmetics.

[0005] The first aspect of the present invention provides a method for preparing a purslane extract, comprising the following steps:

[0006] (1) Weighing dried and crushed purslane powder, heating and refluxing under ultrasonic conditions for alcohol extraction, filtering the filtrate, concentrating it by rotary evaporation, and then fixing the volume with deionized water to obtain a crude purslane extract; wherein the reagent used in the alcohol extraction is anhydrous ethanol-methanol, and the volume ratio thereof is 2-5:1;

[0007] (2) Adsorption: Mix the crude extract of Purslane with a macroporous resin, adsorb at a rotation speed of 30-50 rpm, filter, and retain the macroporous resin; the macroporous resin model is one or more of AB-8, NKA-9, and HPD400;

[0008] First elution: add 30% ethanol to the adsorbed macroporous resin, perform the first elution at a speed of 200-400 rpm, filter, and retain the macroporous resin;

[0009] Second elution: add 95% ethanol to the macroporous resin after the first elution, perform the second elution at a rotation speed of 200-500 rpm, filter, collect the filtrate I, and retain the macroporous resin;

[0010] The third elution: add 95% ethanol to the macroporous resin after the second elution, perform the third elution at a rotation speed of 200-500 rpm, and collect the filtrate II;

[0011] The filtrate I and the filtrate II were combined and rotary evaporated to obtain the purslane extract.

[0012] The active ingredients of purslane were extracted by using a combination of anhydrous ethanol and methanol, and two specific simple processes, ultrasound and reflux, were combined to greatly improve the dissolution of the active ingredients, especially the dissolution of purslanamide A and purslanamide E. The adsorption and elution extraction were further combined with macroporous resins. Weakly polar macroporous resins such as HPD400, AB-8, and NKA-9 have excellent adsorption capacity for alkaloids. On the one hand, they can purify and enrich alkaloids and increase their content; on the other hand, they can remove other impurities, such as polysaccharides, peptides and other active ingredients, which can improve the stability of the extract and reduce the accumulation of harmful substances. Finally, purslanamide A and purslanamide E and a purslane extract with good stability were obtained.

[0013] Preferably, the purslane powder has a particle size of 10 to 60 meshes. In some preferred embodiments, the purslane powder has a particle size of 40 meshes.

[0014] Preferably, the ratio of the purslane powder to anhydrous ethanol-methanol is 1 g: 5-20 mL. In some preferred embodiments, the ratio of the purslane powder to anhydrous ethanol-methanol is 1 g: 10 mL.

[0015] Preferably, the ultrasonic power is 100-500W, the heating reflux temperature is 40-60°C, and the ultrasonic heating reflux time is 30-90min. In some preferred embodiments, the ultrasonic power is 500W, the heating reflux temperature is 60°C, and the ultrasonic heating reflux time is 30min.

[0016] Preferably, the rotary evaporation concentration temperature in step (1) is 50° C., which is beneficial to protecting the effective active ingredients and fully volatilizing the organic solvent.

[0017] Preferably, the crude extract of Purslane: macroporous resin = 50~100mL: 500g.

[0018] Preferably, the adsorption time is 10 min to 30 min. In some preferred embodiments, the adsorption process of the macroporous resin has a rotation speed of 50 rpm and an adsorption time of 30 min.

[0019] Preferably, the first elution time is 20 to 40 minutes at a rotation speed of 200 to 400 rpm. In some preferred embodiments, the first elution process of the macroporous resin has a rotation speed of 300 rpm and an adsorption time of 20 minutes.

[0020] Preferably, the second elution time is 20 to 40 minutes at a rotation speed of 200 to 500 rpm. In some preferred embodiments, the second elution process of the macroporous resin has a rotation speed of 400 rpm and an adsorption time of 40 minutes.

[0021] Preferably, the third elution time is 20-30 min at a rotation speed of 200-500 rpm. In some preferred embodiments, the third elution process of the macroporous resin has a rotation speed of 300 rpm and an adsorption time of 20 min.

[0022] Preferably, the rotary evaporation temperature in step (2) is 50° C., which can effectively retain the active ingredients and remove organic alcohols.

[0023] The second aspect of the present invention also provides a purslane extract prepared by the preparation method.

[0024] The third aspect of the present invention also provides a cosmetic comprising the above-mentioned purslane extract.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The invention optimizes the extraction process, utilizes a combination of anhydrous ethanol and methanol to extract the active ingredients of purslane, and combines ultrasound and reflux to enhance the dissolution of the active ingredients; further combined with the selection of macroporous resin, suitable adsorption and elution processes are adopted to purify and enrich the alkaloids in the extract, remove other impurities, enhance the stability of the extract, and reduce the accumulation of harmful substances. The results show that the method can increase the contents of purslanamide A and purslanamide E in the extract, significantly enhance the soothing, anti-inflammatory, skin barrier repairing and other effects of the purslane extract, and has stable performance. The obtained extract can more effectively improve the problems of burning, redness, itching and the like on the skin, thereby restoring the skin to a stable and healthy state, and can be applied to cosmetics. DETAILED DESCRIPTION

[0027] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0028] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0029] Example 1

[0030] A method for preparing a purslane extract comprises the following steps:

[0031] (1) 10 g of purslane powder that had been dried and crushed to 40 mesh was added to 100 mL of anhydrous ethanol-methanol solution (anhydrous ethanol:methanol = 2:1 V / V), and heated under reflux at an ultrasonic power of 500 W and a temperature of 60°C for 30 min. The filtrate was filtered and concentrated by rotary evaporation at 50°C and then fixed to 100 mL with deionized water to obtain a crude purslane extract;

[0032] (2) 2-1 Adsorption: 100 mL of the crude extract of Portulaca oleracea obtained in step (1) and 500 g of HPD400 macroporous resin were adsorbed at a rotation speed of 50 rpm for 30 min, filtered, and the HPD400 macroporous resin was retained;

[0033] 2-2 First elution: Add 100 mL of 30% ethanol to the HPD400 macroporous resin obtained in step 2-1, perform the first elution at a speed of 300 rpm for 20 min, filter, and retain the HPD400 macroporous resin;

[0034] 2-3 Second elution: Add 100 mL of 95% ethanol to the HPD400 macroporous resin obtained in step 2-2, perform a second elution at a rotation speed of 400 rpm for 40 min, filter, collect the filtrate I, and retain the HPD400 macroporous resin;

[0035] 2-4 Third elution: Add 100 mL of 95% ethanol to the HPD400 macroporous resin obtained in step 2-3, perform the third elution at a rotation speed of 300 rpm for 20 min, and collect the filtrate II;

[0036] 2-5 The above filtrate I and filtrate II were combined, and organic alcohol was removed by rotary evaporation at 50°C to obtain a purslane extract.

[0037] Example 2

[0038] A method for preparing a purslane extract comprises the following steps:

[0039] (1) 10 g of purslane powder, dried and crushed to 20 mesh, was added to 50 mL of anhydrous ethanol-methanol solution (anhydrous ethanol: methanol = 4:1 V / V), heated and refluxed at an ultrasonic power of 200 W and a temperature of 45°C for 90 min, the filtrate was filtered, concentrated by rotary evaporation at 50°C, and then fixed to 50 mL with deionized water to obtain a crude purslane extract;

[0040] (2) 2-1 Adsorption: 50 mL of the crude extract of Portulaca oleracea obtained in step (1) and 500 g of AB-8 macroporous resin were adsorbed at a rotation speed of 30 rpm for 20 min, filtered, and the AB-8 macroporous resin was retained;

[0041] 2-2 First elution: Add 50 mL of 30% ethanol to the AB-8 macroporous resin obtained in step 2-1, perform the first elution at a rotation speed of 400 rpm for 30 min, filter, and retain the AB-8 macroporous resin;

[0042] 2-3 Second elution: Add 50 mL of 95% ethanol to the AB-8 macroporous resin obtained in step 2-2, perform a second elution at a speed of 200 rpm for 30 min, filter, collect the filtrate I, and retain the AB-8 macroporous resin;

[0043] 2-4 Third elution: Add 50 mL of 95% ethanol to the AB-8 macroporous resin obtained in step 2-3, perform the third elution at a rotation speed of 500 rpm for 25 min, and collect the filtrate II;

[0044] 2-5 The above filtrate I and filtrate II were combined, and organic alcohol was removed by rotary evaporation at 50°C to obtain a purslane extract.

[0045] Example 3

[0046] A method for preparing a purslane extract comprises the following steps:

[0047] (1) 5 g of purslane powder that had been dried and crushed to 60 mesh was added to 100 mL of anhydrous ethanol-methanol solution (anhydrous ethanol:methanol = 5:1 V / V), and heated under reflux at an ultrasonic power of 400 W and a temperature of 40°C for 60 min. The filtrate was filtered and concentrated by rotary evaporation at 50°C, and then the volume was fixed to 100 mL with deionized water to obtain a crude purslane extract;

[0048] (2) 2-1 Adsorption: 100 mL of the crude extract of Portulaca oleracea obtained in step (1) and 500 g of NKA-9 macroporous resin were adsorbed at a rotation speed of 40 rpm for 100 min, filtered, and the NKA-9 macroporous resin was retained;

[0049] 2-2 First elution: Add 100 mL of 30% ethanol to the NKA-9 macroporous resin obtained in step 2-1, perform the first elution at a speed of 200 rpm for 40 min, filter, and retain the NKA-9 macroporous resin;

[0050] 2-3 Second elution: Add 100 mL of 95% ethanol to the NKA-9 macroporous resin obtained in step 2-2, perform a second elution at a speed of 500 rpm for 20 min, filter, collect the filtrate I, and retain the AB-8 macroporous resin;

[0051] 2-4 Third elution: Add 50 mL of 95% ethanol to the NKA-9 macroporous resin obtained in step 2-3, perform the third elution at a rotation speed of 200 rpm for 30 min, and collect the filtrate II;

[0052] 2-5 The above filtrate I and filtrate II were combined, and organic alcohol was removed by rotary evaporation at 50°C to obtain a purslane extract.

[0053] Comparative Example 1

[0054] Same as Example 1, except that the HPD400 macroporous resin is replaced by LSA-20 macroporous resin.

[0055] Comparative Example 2

[0056] Same as Example 1, except that the HPD400 macroporous resin is replaced by HPD100 macroporous resin.

[0057] Comparative Example 3

[0058] Same as Example 1, except that anhydrous ethanol-methanol is replaced with anhydrous ethanol-water of equal volume and proportion.

[0059] Comparative Example 4

[0060] Same as Example 1, except that anhydrous ethanol-methanol is replaced with an equal volume of pure water.

[0061] Comparative Example 5

[0062] Same as Example 1, except that anhydrous ethanol-methanol = 2:1 V / V is replaced by anhydrous ethanol-methanol = 1:1 V / V.

[0063] Comparative Example 6

[0064] Same as Example 1, except that no ultrasonic process is performed.

[0065] Comparative Example 7

[0066] Same as Example 1, except that the usage ratio of purslane powder to anhydrous ethanol-methanol is changed from 1 g:10 mL to 1 g:30 mL.

[0067] Comparative Example 8

[0068] The same as Example 1, except that the crude purslane extract: macroporous resin = 100 mL: 500 g is replaced by the crude purslane extract: macroporous resin = 200 mL: 500 g.

[0069] Comparative Example 9

[0070] Same as Example 1, except that three elutions (first elution with 30% ethanol, second elution with 95% ethanol, third elution with 95% ethanol) are replaced by two elutions (first elution with 30% ethanol, second elution with 95% ethanol, and the second eluate is collected).

[0071] Comparative Example 10

[0072] The same as Example 1, except that the rotation speed of the purslane extract and the macroporous resin at 50 rpm is replaced by 10 rpm.

[0073] Comparative Example 11

[0074] The same as Example 1, except that the rotation speeds of the three elutions of the crude purslane extract and the macroporous resin are all replaced with 100 rpm.

[0075] Comparative Example 12

[0076] The same as Example 1, except that the 95% ethanol used in the second and third elutions of the crude extract of Purslane with the macroporous resin is replaced by 60% ethanol.

[0077] The purslane extracts obtained in the embodiments and comparative examples were tested as follows:

[0078] Test Example 1: Purslaneamide A content

[0079] HPLC test conditions: chromatographic column: Eclipse XDB-C18 (4.6×250mm, 5μm), mobile phase: acetonitrile (a)-water (b)-formic acid (20:80:0.1%) for gradient elution: (a) 11%-15% (0-12min), (a) 15%-19% (12-30min), (a) 19% (30-36min), (a) 19%-20% (36-40min), (a) 20% (40-55min), (a) 20%-100% (55-60min), flow rate: 0.8mL / min, detection wavelength: 336nm, column temperature: 30℃, injection: 10μL; all samples were filtered through 0.45μm microporous membrane before injection.

[0080] The test results are shown in Table 1.

[0081] Test Example 2: Content of Purslaneamide E

[0082] HPLC test conditions: chromatographic column: ODS-80Ts C18 chromatographic column (4.6mm×250mm, 5μm), column temperature: 25℃, mobile phase: methanol-water-phosphoric acid (45:55:0.1%), flow rate: 0.8mL / min, detection wavelength: 283nm, injection volume: 10μL; all samples were filtered through a 0.45μm microporous filter membrane before injection.

[0083] The test results are shown in Table 1.

[0084] Table 1 Test results of purslanamide A and E content in the extract

[0085]

[0086] It can be seen that the contents of purslane amide A and E extracted from the purslane extracts of Examples 1 to 3 are significantly higher than those of Comparative Examples 1 to 12, indicating that it is the present invention that extracts purslane according to specific extraction process conditions and parameters (anhydrous ethanol-methanol, ultrasound, heating), and combines the HPD400 macroporous resin process for elution, purification and enrichment, which significantly improves the purslane amide A and E in purslane, thereby significantly improving the raw material utilization rate of purslane.

[0087] Test Example 3: Stability test of purslane extract

[0088] The purslane extracts obtained in Examples 1 to 3 and Comparative Examples 1 to 12 were placed in an environment of -18°C, 4°C, room temperature (25°C), 45°C, and light for a period of 3 months to investigate the appearance stability. The degree of change in the appearance of the samples before and after the stability test was observed, and whether precipitation was generated and the color change of the solution was recorded. The experimental results are shown in Table 2.

[0089] Table 2 Stability test results

[0090]

[0091] It can be seen that the purslane extracts obtained in Examples 1 to 3 can still remain clear and transparent after 3 months under -18°C, 4°C, room temperature, 45°C and light conditions, and have good stability; while the purslane extracts obtained in Comparative Examples 4 to 8 and the purslane extracts obtained in Comparative Example 12 are unstable after being placed under the same conditions for 3 months. This shows that the purslane extract obtained by the present invention is extracted according to specific extraction process conditions and parameters (anhydrous ethanol-methanol, ultrasound, heating), and is eluted, purified and enriched in combination with the HPD400 macroporous resin process, so that the purslane extract obtained has excellent stability.

[0092] Test Example 4: Detection of inflammatory factors IL-6, TNF-α, and NO content

[0093] RAW264.7 cells in the logarithmic growth phase were seeded in 96-well plates at a cell concentration of 1×10 4 Each well was incubated overnight in an incubator (37°C, 5% CO2). The working solution of the test substance was prepared according to Table 3. When the cell plating rate in the 96-well plate reached 80%, the samples were added in groups, 100 μL per well, incubated for 1 hour, LPS was added to each well, and incubated for another 4 hours. Three replicates were set for each group. The cell supernatant was collected and the contents of IL-6, TNF-α, and NO were detected according to the instructions of the kit.

[0094] Table 3 Test conditions for inflammatory factor content

[0095]

[0096] The experimental results are shown in Table 4.

[0097] Table 4 Test results of inflammatory factor content

[0098]

[0099] It can be seen that compared with the negative control group, the Portulaca oleracea extract obtained in Example 1 has a significant anti-inflammatory effect (P < 0.001, statistically significant), and its anti-inflammatory efficacy in reducing the content of inflammatory factors IL-6, TNF-α and NO is better than that of Comparative Examples 1 to 3 and Comparative Example 9, and is better than commercially available cosmetic products.

[0100] Test Example 5: Detection of the expression of TRPV-1 protein content

[0101] The cell concentration was 1.8 × 10 5 Human immortalized epidermal cells (HaCaT) were inoculated into 6-well plates at 100 μl / well and incubated overnight in an incubator (37°C, 5% CO2). The test working solutions were prepared according to Table 5. When the cell plating rate in the 6-well plate reached 80%, the samples were added in groups, 2 mL per well, and 3 replicates were set up in each group. After the addition of samples, the 6-well plate was placed in an incubator (37°C, 5% CO2) and incubated for 24 hours. The cell supernatant was collected and the TRPV-1 content was detected according to the instructions of the kit.

[0102] Table 5 TRPV-1 protein content expression test conditions

[0103]

[0104] The experimental results are shown in Table 6.

[0105] Table 6 TRPV-1 protein content expression test results

[0106]

[0107] It can be seen that compared with the negative control group, the purslane extract obtained in Example 1 has a significant effect on reducing the expression of TRPV-1 protein content (P < 0.001, statistically significant), and is better than comparative examples 1 to 3 and comparative example 9, and is better than commercially available cosmetic products, indicating that the purslane extract obtained in Example 1 has good soothing effect.

[0108] Test Example 6: TEWL value test

[0109] Twenty-six healthy male and female subjects aged 18 to 60 were recruited. The test sites were free of hair, dermatitis and other influencing factors. The facial skin of the subjects was cleaned. After cleaning, the subjects sat quietly for 30 minutes in a test environment with constant temperature and humidity (humidity 50% ± 10%, 21℃ ± 1℃). The test area was selected on the inner side of the forearm in advance, and the area of ​​each area was 3cm × 3cm. The selected area was pasted with low-allergenic tape, pressed for 2 seconds and then slowly peeled off, and continuously torn off until visible erythema appeared. The CK skin tester Tewameter probe was used to measure the TEWL value before using the product (0h). The test sample was evenly applied once on the designated test area that had been torn off to erythema using a disposable latex finger cot, and the TEWL value after use (20min) was measured. Each area was measured 3 times, and the average value was taken to calculate the TEWL change rate. The calculation formula is: TEWL value change rate (%) = (after use-before use) / before use.

[0110] The experimental results are shown in Table 7.

[0111] Table 7 TEWL value test results

[0112]

[0113] It can be seen that compared with 0 h before use, the TEWL value change rate of the test area 20 minutes after applying the Portulaca oleracea extract obtained in Example 1 was 24.06% (P<0.001, statistically significant), which was better than Comparative Examples 1 to 3, Comparative Example 9 and commercially available cosmetic products, indicating that the Portulaca oleracea extract obtained in Example 1 has excellent skin barrier repairing effect.

[0114] In summary, it can be seen that the extraction method of the embodiment of the present invention can increase the content of purslanamide A and E in the extract, significantly improve the soothing, anti-inflammatory, skin barrier repairing and other effects of the purslane extract, and has stable performance. The obtained extract can be used in cosmetics.

[0115] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0116] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a purslane extract, characterized in that: The following steps are involved: (1) Weighing dried and crushed purslane powder, heating and refluxing under ultrasonic conditions for alcohol extraction, filtering the filtrate, concentrating it by rotary evaporation, and then fixing the volume with deionized water to obtain a crude purslane extract; wherein the reagent used for the alcohol extraction is anhydrous ethanol-methanol, and the volume ratio thereof is 2-5:1; and the amount ratio of the purslane powder to the anhydrous ethanol-methanol is 1 g: 5-20 mL; (2) Adsorption: The crude extract of purslane and the macroporous resin are mixed according to the ratio of crude extract of purslane to macroporous resin = 50-100 mL: 500 g, and adsorption and filtration are performed at a rotation speed of 30-50 rpm to retain the macroporous resin; the model of the macroporous resin is one or more of AB-8, NKA-9, and HPD400; First elution: add 30% ethanol to the adsorbed macroporous resin, perform the first elution at a speed of 200-400 rpm, filter, and retain the macroporous resin; Second elution: add 95% ethanol to the macroporous resin after the first elution, perform the second elution at a rotation speed of 200-500 rpm, filter, collect the filtrate I, and retain the macroporous resin; The third elution: add 95% ethanol to the macroporous resin after the second elution, perform the third elution at a rotation speed of 200-500 rpm, and collect the filtrate II; The filtrate I and the filtrate II were combined and rotary evaporated to obtain the purslane extract.

2. The preparation method according to claim 1, characterized in that: The purslane powder has a particle size of 10 to 60 meshes.

3. The preparation method according to claim 1, characterized in that: The ultrasonic power is 200-500W; the heating reflux temperature is 40-60°C; and the ultrasonic heating reflux time is 30-90min.

4. The preparation method according to claim 1, characterized in that: The rotary evaporation concentration temperature in step (1) is 50°C.

5. The preparation method according to claim 1, characterized in that: The adsorption time is 10min~30min; The first elution time is 20 to 40 minutes; The second elution time is 20 to 40 minutes; The third elution time is 20 to 30 minutes.

6. The preparation method according to claim 1, characterized in that: The rotary evaporation temperature in step (2) is 50°C.

7. The purslane extract prepared by the preparation method according to any one of claims 1 to 6.

8. A cosmetic, characterized in that: The invention comprises the purslane extract as claimed in claim 7.

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