Pine pollen flavonoids and extraction method and application thereof

Abstract

The invention discloses pine pollen flavonoids and an extraction method and application thereof. The invention obtains high-quality pine pollen flavonoids based on extraction with a low eutectic solvent and purification by adsorption with cross-linked polyvinylpyrrolidone, and studies the firming and anti-wrinkle effects of the pine pollen flavonoids. The method is simple and easy to implement, and has a high extraction rate. Cross-linked polyvinylpyrrolidone is a pharmaceutical excipient and can also be used as a beer clarifier. It has good safety when used for separation and purification of ingredients in beauty and skin care products, and the separation process is more economical and easy to scale up and continuously operate. The obtained pine pollen flavonoids have application prospects in skin care products.

Description

Technical Field

[0001] The invention relates to pine pollen flavonoids and an extraction method and application thereof. Background Art

[0002] Pine pollen (English: POLLEN PINI), also known as pine flower and pine yellow, refers to the dry pollen produced by the stamens of pine plants such as Masson pine, Chinese pine, Korean pine, Armand pine and Scotch pine. It was first recorded in the Eastern Han Dynasty's "Shennong's Herbal Classic". It is a bright yellow or light yellow fine powder with a sweet and non-toxic taste. It is a traditional Chinese food. Some traditional Chinese foods such as pine flower cake, pine flower dumplings, and pine flower wine still add pine pollen. It has great medicinal value and is effective for skin-related diseases. It has been included in 37 pharmacopoeias since ancient times, including the "Pharmacopoeia of the People's Republic of China".

[0003] Pine pollen, as the genetic material of pine plants, contains not only general nutrients such as protein, fat, and carbohydrates, but also more than 200 nutrients and biologically active substances such as flavonoids, steroids, enzymes, and fatty acids.

[0004] The water-soluble flavonoids in pine pollen have good biological activity, but are currently mainly extracted using organic solvents such as ethanol and water. The disadvantages of these methods are that they are time-consuming, have low extraction rates, and the quality of the extracted flavonoids is not high. At the same time, there is a lack of research on the activity of the obtained pine pollen flavonoids in skin care products that can be used to firm and anti-wrinkle skin care products. Summary of the invention

[0005] The present invention aims to provide a pine pollen flavonoids and an extraction method and application thereof. The present invention obtains high-quality pine pollen flavonoids based on extraction with a low eutectic solvent and purification by adsorption with cross-linked polyvinylpyrrolidone.

[0006] The process of the present invention has a large processing capacity, low energy consumption, low production cost, high purification yield, good product purity, low equipment investment, cross-linked polyvinylpyrrolidone as an adsorption separation material does not cause flavonoid inactivation or denaturation, no organic solvent residue exists, the separation process is more economical, and it is easy to scale up the project and operate continuously. The obtained pine pollen flavonoids have application prospects in skin care products.

[0007] The technical solution of the present invention is as follows:

[0008] A method for extracting pine pollen flavonoids comprises the following steps:

[0009] (1) mixing choline chloride, citric acid and water, and stirring at 70-90° C. (preferably 80° C.) until transparent and uniform, to obtain a low eutectic solvent (stored at room temperature for future use);

[0010] The mass ratio of choline chloride to citric acid is 1:1.5-2.5, preferably 1:2;

[0011] Based on the total mass of the deep eutectic solvent, water accounts for 30-50%, preferably 40%;

[0012] (2) Put the broken pine pollen in an extraction tank, add the low eutectic solvent obtained in step (1), heat to 60-80° C. (preferably 70° C.), stir and extract for 2-3 h (preferably 3 h), then let stand to room temperature, centrifuge, and collect the supernatant for later use;

[0013] The solid-liquid ratio of the broken pine pollen to the low eutectic solvent is 1:20-30, g / mL; preferably 1:25, g / mL;

[0014] The centrifugal conditions are: 4000-8000 rpm, 15-30 min;

[0015] (3) taking the supernatant obtained in step (2), adding food-grade cross-linked polyvinylpyrrolidone at room temperature, stirring for 0.5-3 hours, filtering, washing with pure water, and obtaining cross-linked polyvinylpyrrolidone loaded with pine pollen flavonoids; then eluting with 85% ethanol aqueous solution by volume, and evaporating the eluate under reduced pressure to dryness to obtain pine pollen flavonoids;

[0016] The mass of the supernatant is counted as M, and the amount of cross-linked polyvinylpyrrolidone is 0.001-0.01M, preferably 0.005M;

[0017] Specifically, the mixture is washed with pure water for 3 times, each time with a dosage of 0.01-0.05 M, preferably 0.03 M; the purpose of the pure water washing is to remove the low eutectic solvent and other water-soluble impurities;

[0018] Specifically, elution is performed twice with 85% ethanol aqueous solution, each time with an amount of 0.01-0.5M, preferably 0.03M.

[0019] The invention relates to pine pollen flavonoids obtained by the extraction method, or cross-linked polyvinylpyrrolidone loaded with pine pollen flavonoids.

[0020] The pine pollen flavonoids or cross-linked polyvinylpyrrolidone loaded with pine pollen flavonoids can be used in skin care products; a specific application method is, for example, adding pine pollen flavonoids or cross-linked polyvinylpyrrolidone loaded with pine pollen flavonoids to ordinary moisturizing liquid and mixing well, which will have a good firming and anti-wrinkle skin care effect.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0022] (1) The extractant used in the method for preparing pine pollen flavonoids of the present invention has good safety performance. Cross-linked polyvinylpyrrolidone is a pharmaceutical excipient and can also be used as a beer clarifier. It is safe for separation and purification of ingredients in beauty and skin care products.

[0023] (2) The pine pollen obtained by the method of the present invention has a flavonoid content of 81-89%, and has a simple process, convenient operation, high flavonoid content, and low equipment investment.

[0024] (3) Cross-linked polyvinylpyrrolidone loaded with pine pollen flavonoids has a sustained release effect on flavonoids and has good biocompatibility, and can be directly used in cosmetics, skin care products, and health care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Results of type I collagen content detection (Note: compared with the model, * indicates P<0.05, significant difference; ** indicates P<0.01).

[0026] Figure 2 :Fluorescence analysis of pine pollen flavonoids' inhibition on elastase.

[0027] Figure 3 : Release effect of pine pollen flavonoids.

[0028] Figure 4 : Standard curve of flavonoid concentration and absorbance.

[0029] Figure 5 : Liquid phase analysis results of pine pollen flavonoids extract. DETAILED DESCRIPTION

[0030] The present invention is further described below by means of specific embodiments, but the protection scope of the present invention is not limited thereto.

[0031] In the following embodiments,

[0032] Broken wall pine pollen: Zhejiang Yalin Biotechnology Co., Ltd., batch number 20241010;

[0033] Cross-linked polyvinylpyrrolidone: UPS grade, Shanghai Yiji Industrial Co., Ltd.

[0034] Example 1

[0035] Preparation of low eutectic solvent: 10 kg of choline chloride and 15 kg of citric acid were placed in a sealed container containing 12 kg of water and stirred continuously at 70° C. until a transparent and uniform liquid was produced. The prepared low eutectic solvent was stored at room temperature before use.

[0036] 1.3 kg of broken-wall pine pollen powder was put into an extraction tank, 37 kg of the low eutectic solvent prepared above was added, the mixture was heated to 70° C., and the mixture was stirred and extracted for 2.5 h to obtain 38.3 kg of pine pollen low eutectic solvent extract.

[0037] Take about 12L of the supernatant of the above extract, add 120g of cross-linked polyvinylpyrrolidone, stir for 2h, filter and elute three times with 0.75L of pure water to obtain 173g of loaded cross-linked polyvinylpyrrolidone. Then elute twice with 0.8L of 85% ethanol, combine the 85% ethanol eluate, remove the solvent by reduced pressure distillation to obtain about 36.5g of pine pollen flavonoid extract, in which the flavonoid content is 89%.

[0038] Example 2

[0039] Preparation of low eutectic solvent: 1 kg of choline chloride and 2 kg of citric acid were placed in a sealed container containing 3 kg of water and stirred continuously at 80° C. until a transparent and uniform liquid was produced. The prepared low eutectic solvent was stored at room temperature before use.

[0040] 330 g of broken-wall pine pollen powder was placed in an extraction tank, and the low eutectic solvent prepared above was added, heated to 80° C., and stirred for extraction for 3 h to obtain 6.3 kg of pine pollen low eutectic solvent extract;

[0041] Take about 3L of the supernatant of the above extract, add 20g of cross-linked polyvinylpyrrolidone, stir for 3h, filter and elute three times with 0.3L of pure water to obtain 23g of loaded cross-linked polyvinylpyrrolidone. Then elute twice with 0.3L of 85% ethanol, combine the 85% ethanol eluate, remove the solvent by reduced pressure distillation to obtain about 8.6g of pine pollen flavonoid extract, in which the flavonoid content is 87%.

[0042] Example 3

[0043] Effect of pine pollen flavonoids extract on the secretion of type I collagen by fibroblasts:

[0044] After incubation overnight, 3T3 cells were inoculated and washed with PBS, and then divided into five groups according to the experiment: blank group, model control group, positive control group (TGF-β1 (100 ng / mL)), and sample group (two groups of pine pollen flavonoids prepared in Example 1 at 0.1 μg / mL and 0.3 μg / mL). All groups except the blank group were subjected to 30 J / cm 2 According to the cell viability test results obtained by MTT assay, 100 μL of sample was added to each well, and 3 replicate wells were set up for each group.

[0045] like Figure 1 As shown, compared with the model control group, the upregulation rate of type I collagen content in the positive control group was 30.2%, and the upregulation rate of type I collagen content in the 0.1 μg / mL pine pollen flavonoids was 26.5%, which was significantly different from the model control group (P<0.05), proving that 0.1 μg / mL pine pollen flavonoids has the ability to promote type I collagen synthesis in the UVA-damaged mouse 3T3 cell model.

[0046] Elastase inhibition test:

[0047] Elastase was used as the enzyme target and MeOSuc-Ala-Ala-Pro-Val-AMC was used as the enzyme reaction substrate. According to the enzyme reaction principle, an in vitro target enzyme drug screening model was established by measuring the relative fluorescence intensity, and drug screening and evaluation were performed. 50 μL of 0.5 μM elastase solution was mixed with 50 μL of 20 μM fluorescent substrate, and Tris-HCl buffer solution was added to make the total volume 200 μL, with 200 μL buffer as blank. 298K was cultured for 15 min, oscillated for 300 s, and the excitation wavelength was set to 380 nm, the emission wavelength was set to 420-540 nm, and the step length was 5 nm. The relative fluorescence value of pure enzyme activity was recorded. 20 μL of pine pollen 0.1 μg / mL, 0.3 μg / mL flavonoids (Example 1), and 0.1 μg / mL tea polyphenols were added respectively, and their relative fluorescence values ​​were measured under the same conditions.

[0048] Table 1: Inhibition rate of elastase by pine pollen flavonoids at different concentrations

[0049]

[0050] The results are as follows Figure 2 As shown in Table 1. Compared with the model control group, the positive control 0.1μg / mL tea polyphenol elastase inhibition rate was 50.8%, and the sample group 0.1μg / mL and 0.3μg / mL pine pollen flavonoids elastase inhibition rates were 45.8% and 72.4%, respectively. Among them, the elastase inhibition rates of 0.1μg / mL pine pollen flavonoids and 0.1μg / mL tea polyphenols were close, and the effects were equivalent. This shows that pine pollen flavonoids have a certain inhibitory effect on elastase.

[0051] Example 4

[0052] Study on the sustained release effect of cross-linked polyvinylpyrrolidone loaded with pine pollen flavonoids in vitro

[0053] According to the paddle method in Appendix ⅩC of the Chinese Pharmacopoeia 2020

[0054] Release of flavonoids in deionized water: 100 mg of cross-linked polyvinylpyrrolidone loaded with pine pollen flavonoids (containing 20.5 mg of pine pollen flavonoids) was weighed, and 900 mL of degassed deionized water was added. The mixture was incubated at 37.0 °C and 75 r / min. -1 Under the conditions, the test was carried out according to the release rate investigation method, and samples were collected and analyzed at different time points of 10min, 20min, 30min, 40min, 50min, 60min, 90min, 120min, 180min, and 240min to investigate the sustained release effect in deionized water solution. The results showed that the cross-linked polyvinylpyrrolidone loaded with pine pollen flavonoids was released slowly in deionized water.

[0055] Appendix 1: Flavonoids content determination method

[0056] Weigh 20.0 mg of naringenin reference substance dried to constant weight at 105°C accurately, dissolve it in 70% ethanol and dilute it in a 100 mL volumetric flask to prepare a rutin standard solution with a concentration of 0.2 mg / mL. Accurately pipette 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL of naringenin standard solution respectively, place it in a 10 mL volumetric flask, add 2 mL of purified water, add 500 μL of 10% sodium nitrite solution and shake it well, let it stand, and react for 6 minutes; then add 500 μL of 10% aluminum nitrate solution and shake it well, let it stand and react for 6 minutes; finally, add 2.5 mL of 5% sodium hydroxide solution, mix and react for 15 minutes, dilute it to 10 mL with purified water, measure its absorbance at a wavelength of 510 nm with purified water as the blank control solution, draw a standard curve, and obtain the regression equation of y=11.914x-0.003(R 2 =0.9992), such as Figure 4 As shown in the figure, within a certain range, the concentration of naringenin and its absorbance value showed a good linear relationship.

[0057] Appendix 2: HPLC analysis method of pine pollen flavonoids extract

[0058] The above-mentioned pine pollen flavonoid extract sample and each standard substance were prepared to a concentration of about 1 μg / mL, Agilent Eclipse XDB-C18 was used as a chromatographic column, acetonitrile-0.1% formic acid was used as a mobile phase at a flow rate of 1 ml / min, the column temperature was 35° C., the injection volume was 10 μL, detection was performed at 280 nm, and the gradient elution conditions were as follows: 0-4 min (acetonitrile: 5%-25%), 4-6 min (acetonitrile: 25%-40%), 6-8 min (acetonitrile: 40%-60%), 8-12 min (acetonitrile: 60%-80%), 12-16 min (acetonitrile: 80%), 16-18 min (acetonitrile: 80%-100%), 18-20 min (acetonitrile: 100%), 20-22 min (acetonitrile: 100%-5%), 22-24 min (acetonitrile: 5%). (Standard samples were purchased from Sichuan Weiqi Biological Co., Ltd.), the specific results are as follows Figure 5 shown.

Claims

1. A method for extracting pine pollen flavonoids, characterized in that: The steps include: (1) mixing choline chloride, citric acid and water, and stirring at 70-90° C. until the mixture becomes transparent and uniform, to obtain a deep eutectic solvent; (2) Put the broken pine pollen in an extraction tank, add the low eutectic solvent obtained in step (1), heat to 60-80° C., stir and extract for 2-3 hours, then let stand to room temperature, centrifuge, and collect the supernatant for later use; (3) Take the supernatant obtained in step (2), add food-grade cross-linked polyvinylpyrrolidone at room temperature, stir for 0.5-3h, filter, wash with pure water, and obtain cross-linked polyvinylpyrrolidone loaded with pine pollen flavonoids; then elute with 85% ethanol aqueous solution, evaporate the eluate under reduced pressure to dryness, and obtain pine pollen flavonoids.

2. The extraction method according to claim 1, characterized in that In step (1), the mass ratio of choline chloride to citric acid is 1:1.5-2.

5.

3. The extraction method according to claim 1, characterized in that In step (1), water accounts for 30-50% based on the total mass of the deep eutectic solvent.

4. The extraction method according to claim 1, characterized in that In step (2), the solid-to-liquid ratio of the broken pine pollen to the low eutectic solvent is 1:20-30, g / mL.

5. The extraction method according to claim 1, characterized in that In step (2), the centrifugal conditions are: 4000-8000 rpm, 15-30 min.

6. The extraction method according to claim 1, characterized in that In step (3), the mass of the supernatant is calculated as M, the amount of cross-linked polyvinylpyrrolidone is 0.001-0.01M; the mixture is washed with pure water for 3 times, each time with an amount of 0.01-0.05M; The solution was eluted twice with 85% ethanol aqueous solution, each time using 0.01-0.5 M ethanol.

7. The pine pollen flavonoids obtained by the extraction method according to any one of claims 1 to 6, or the cross-linked polyvinylpyrrolidone loaded with pine pollen flavonoids.

8. Use of the pine pollen flavonoids as claimed in claim 7, or cross-linked polyvinylpyrrolidone loaded with pine pollen flavonoids in skin care products.

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