CN113499358B

CN113499358B - Preparation process and application of sanguinarine extract gel

Info

Publication number: CN113499358B
Application number: CN202110757765.XA
Authority: CN
Inventors: 傅建; 吴静澜; 杨姗; 张永萍; 徐剑
Original assignee: Guizhou University of Traditional Chinese Medicine
Current assignee: Guizhou University of Traditional Chinese Medicine
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2022-08-30
Anticipated expiration: 2041-07-05
Also published as: CN113499358A

Abstract

The invention discloses a preparation process and application of a sanguinea root extract gel. The process comprises the following steps: (1) scattering carbomer 940 in glycerol, adding deionized water for swelling, heating in water bath, stirring for dissolving, adding sodium hyaluronate, allantoin and azone, stirring to obtain colloid, taking off, and cooling to room temperature to obtain phase A; (2) dissolving the blood ginseng extract powder with 10mL of 60% ethanol, adding propylene glycol and methyl paraben, and stirring for dissolving to obtain phase B; (3) slowly adding phase B into phase A, stirring, adding triethanolamine to adjust pH, adding deionized water, and stirring to obtain XUERENSHEN extract gel. The sanguinarine extract gel prepared by the invention has no granular sensation, good transparency, moderate viscosity and easy spreading, and the pH value is 6.30, so that the sanguinarine gel is a sanguine gel. The sanguinea root extract gel provided by the invention has good whitening and freckle removing effects.

Description

Preparation process and application of sanguinarine extract gel

Technical Field

The invention relates to the technical field of blood-applied ginseng, in particular to a preparation process and application of a blood-applied ginseng extract gel.

Background

Xuezhen, name of traditional Chinese medicine. Is dried root of Indigofera stachyoide Lindl (Indigofera stachyoide Lindl) belonging to genus Indigofera of family Leguminosae. Has the effects of clearing away heat, relieving exterior syndrome, eliminating phlegm, promoting diuresis, promoting blood circulation and relieving pain.

The flavone components of Ginseng radix include epicatechin, catechin, protocatechuic aldehyde, protocatechuic acid, and gallic acid. In the research of the extraction process of the total flavone of the panax ginseng, the content of the total flavone in crude drugs is low, the purity of the total flavone is only 4.33 percent, and the effect of active ingredients is not favorably exerted. At present, the development and application of sanguinary ginseng only stay in the aspect of utilizing the traditional effect of sanguinary ginseng as a traditional Chinese medicinal material, and other applications are not developed.

Disclosure of Invention

The invention aims to provide a preparation process and application of a sanguinarine extract gel. The sanguinarine extract gel prepared by the invention has no granular sensation, good transparency, moderate viscosity and easy spreading, and the pH value is 6.30, so that the sanguinarine gel is a sanguine gel. The invention also provides a method for separating and purifying the blood ginseng extract, and the obtained high-purity blood ginseng extract powder is used as a raw material to ensure the effect of the blood ginseng extract gel. The sanguinea root extract gel provided by the invention has good whitening and freckle removing effects.

The technical scheme of the invention is as follows: a preparation process and application of a sanguinea root extract gel comprise the following steps:

(1) scattering carbomer 940 in glycerol, adding deionized water for swelling, heating in water bath, stirring for dissolving, adding sodium hyaluronate, allantoin and azone, stirring to obtain colloid, taking off, and cooling to room temperature to obtain phase A;

(2) dissolving the powder of the blood ginseng extract with ethanol, adding propylene glycol and methyl paraben, and stirring for dissolving to obtain phase B;

(3) slowly adding phase B into phase A, stirring, adding triethanolamine to adjust pH, adding deionized water, and stirring to obtain XUERENSHEN extract gel.

The preparation process of the sanguinea root extract gel comprises the following steps:

(1) according to the weight portion, 0.38-0.42 portion of carbomer 940 is scattered into 18-19 portions of glycerin, 45-55 portions of deionized water are added for swelling for 22-26h, the mixture is heated in a water bath at 80-90 ℃ and stirred for dissolving, 0.8-0.12 portion of sodium hyaluronate, 0.12-0.18 portion of allantoin and 0.8-1.2 portion of azone are added, the mixture is stirred for 0.8-1.2h to be colloidal, and the colloidal mixture is cooled to room temperature after being taken down to obtain phase A;

(2) dissolving 4.8-5.2 parts of blood ginseng extract powder in 8-12 parts of 50% -70% ethanol, adding 4-6 parts of propylene glycol and 0.08-0.12 part of methyl hydroxybenzoate, and stirring for dissolving to obtain phase B;

(3) slowly adding phase B into phase A, stirring, adding 0.27-0.31 part triethanolamine to adjust pH, adding 95-105 parts deionized water, and stirring to obtain XUERENSHEN extract gel.

(1) according to the weight portion, 0.4 portion of carbomer 940 is scattered into 18.35 portions of glycerin, 50 portions of deionized water are added for swelling for 24 hours, the mixture is heated in a water bath at 85 ℃ and stirred for dissolving, 0.1 portion of sodium hyaluronate, 0.15 portion of allantoin and 1 portion of azone are added, the mixture is stirred for 1 hour to be colloidal, and the colloidal mixture is cooled to room temperature after being taken down to obtain phase A;

(2) dissolving 5 parts of blood ginseng extract powder with 10 parts of 60% ethanol, adding 5 parts of propylene glycol and 0.1 part of methyl hydroxybenzoate, and stirring for dissolving to obtain phase B;

(3) slowly adding phase B into phase A, stirring, adding 0.29 part triethanolamine to adjust pH, adding 100 parts deionized water, and stirring to obtain XUERENSHEN extract gel.

In the preparation process of the sanguinary extract gel, the preparation method of the sanguinary extract powder in the step (2) comprises the following steps:

(1) extracting Ginseng radix powder with 50-70% ethanol for 3 times, each for 1.5-2.5 hr, filtering the extractive solution, and recovering ethanol from the filtrate under reduced pressure at 40-50 deg.C in a rotary evaporator to obtain concentrated solution;

(2) diluting the concentrated solution obtained in the step (1) by adding 0.8-1.5 times of water, extracting petroleum ether with the volume of 0.8-1.2 times for 1-2 times, discarding the petroleum ether layer, continuously adding ethyl acetate with the volume of 0.8-1.2 times into the water layer, extracting for 5-7 times, discarding the water layer, concentrating the ethyl acetate extract under reduced pressure to recover ethyl acetate, and drying in a vacuum drying oven to obtain an ethyl acetate part extract;

(3) pretreatment of macroporous adsorption resin: taking AB-8 type macroporous adsorption resin, adding 2BV 95% ethanol, soaking for 24h to fully swell the resin, repeatedly washing the resin with distilled water after filtering until no white turbidity and no ethanol smell exist, then respectively soaking the resin with 2-5% hydrochloric acid solution and 2-5% NaOH solution for 2.5-3.5h, repeatedly washing the resin with distilled water until the solution is neutral, finally preserving the resin with distilled water, and storing the resin in a sealed manner to obtain pretreated AB-8 type macroporous adsorption resin;

(4) preparation of a sample solution: adding distilled water into the ethyl acetate part extract obtained in the step (2) for ultrasonic dissolution to prepare a blood ginseng extract solution with the concentration of 5-5.5 mg/mL;

(5) and (3) purification: and (3) taking 8-12g of the pretreated AB-8 type macroporous adsorption resin obtained in the step (3), filling the column by a wet method, slowly filling the column into a chromatographic column, taking 65-75mL4.7BV of the blood ginseng extract solution obtained in the step (4) with the pH value adjusted to 3.5-4.5, sampling at the volume flow of 0.8-1.2mL/min, removing impurities by pure water after adsorbing for 2.5-3.5h, eluting with 110-130mL of 65-75% ethanol of 8BV at the volume flow of 2.5-3.5mL/min, collecting the concentration of eluent and drying to obtain the blood ginseng extract powder.

(1) weighing radix Ginseng Indici powder, extracting with 60% ethanol for 3 times, each for 2 hr, filtering the extractive solution, and recovering ethanol from the filtrate under reduced pressure at 45 deg.C in a rotary evaporator until no alcohol smell exists to obtain concentrated solution;

(2) diluting the concentrated solution obtained in the step (1) by adding 1 time of water, extracting petroleum ether with the same volume for 1 time, removing a petroleum ether layer, continuously adding ethyl acetate with the same volume into a water layer for extracting for 6 times, removing a water layer, concentrating an ethyl acetate extract under reduced pressure to recover ethyl acetate, and drying in a vacuum drying oven to obtain an ethyl acetate part extract;

(3) pretreatment of macroporous adsorption resin: taking AB-8 type macroporous adsorption resin, adding 2BV 95% ethanol, soaking for 24h to fully swell the resin, repeatedly washing with distilled water after filtering until no white turbidity and no ethanol smell exist, then respectively soaking with 5% hydrochloric acid solution and 5% NaOH solution for 3h, repeatedly washing with distilled water until the solution is neutral, finally preserving with distilled water, sealing and storing to obtain pretreated AB-8 type macroporous adsorption resin;

(4) preparation of sample solution: taking the ethyl acetate part extract obtained in the step (2), adding a proper amount of distilled water, and performing ultrasonic dissolution to prepare a blood ginseng extract solution with the concentration of 5.38 mg/mL;

(5) and (3) purification: precisely weighing 10.0g of the pretreated AB-8 type macroporous adsorption resin obtained in the step (3), filling the mixture into a column by a wet method, slowly filling the column with the size of 15mm multiplied by 300mm into a chromatographic column, taking 70mL4.7BV of the blood ginseng extract solution obtained in the step (4) with the pH value adjusted to be 4, sampling at the volume flow of 1mL/min, removing impurities by pure water after adsorbing for 3h, eluting at the volume flow of 3mL/min by using 120mL of 8BV 70% ethanol, collecting the concentration of eluent and drying to obtain the blood ginseng extract powder.

The application of the preparation process of the sanguinea root extract gel is to prepare the sanguinea root extract gel for whitening and removing freckles.

Compared with the prior art, the invention has the following beneficial effects:

1. the preparation process of the optimal sanguinea ginseng extract gel is determined through a large number of experiments, according to an optimal prescription, 0.4 part of carbomer 940 is weighed and is scattered into 18.35 parts of glycerin, 50 parts of deionized water is added for swelling for 24 hours, water bath heating at 85 ℃ is carried out, stirring and dissolving are carried out, 0.1 part of sodium hyaluronate, 0.15 part of allantoin and 1 part of azone are added, stirring is carried out for 1 hour, the mixture is made into a gel shape, and the gel is taken down to be cooled and is used as phase A for standby; weighing 5 parts of sanguinea ginseng extract powder, dissolving with a proper amount of solvent, adding 5 parts of propylene glycol and 0.1 part of methylparaben, and stirring for dissolving to obtain a B phase for later use. Slowly adding the phase B into the phase A, uniformly stirring, adding 0.29 part of triethanolamine to adjust the pH value, adding 100 parts of deionized water, and uniformly stirring to obtain the sanguinary extract gel, wherein the prepared gel is fine, smooth, granular-free, good in transparency, moderate in viscosity, easy to spread and capable of having the pH value of 6.30, and is a sanguine gel.

2. The preparation process of the blood ginseng extract gel provided by the invention is simple, the prescription is stable and feasible, and the method is reliable.

3. The preparation of the sanguinary ginseng extract gel needs sanguinary ginseng extract powder, and on the basis that the purity of the sanguinary ginseng extract obtained by the prior art is only 4.33 percent, the AB-8 type macroporous adsorption resin is provided by the invention as the optimal type of macroporous adsorption resin for purifying the sanguinary ginseng extract. The optimal purification conditions are as follows: the purity of the total flavone in the panax notoginseng extract can be increased from 29.20% to 46.01% by the sample loading volume of 70mL (4.7BV), the sample loading concentration of 5.38mg/mL, the sample loading flow volume of 1mL/min, the eluent volume flow of 3mL/min, the eluent of 70% ethanol and the eluent volume of 120mL (8 BV). The quality of the blood ginseng extract gel is guaranteed by the high-purity ginseng total flavone powder.

4. The preparation process of the blood ginseng extract gel has good whitening and freckle removing effects. The research of the efficacy is derived from the research (1) that the panax sanguinea extractive has the inhibition effect on the activities of tyrosine monophenolase and diphenolase, can effectively inhibit the activities of tyrosine monophenolase and diphenolase, and the inhibition rate is increased along with the increase of the mass concentration of total flavonoids, and the IC is ₅₀ 88.78mg/L and 34.46mg/L respectively. The result of the analysis of the inhibition and braking mechanical parameters of the tyrosinase shows that V can be known by the Mie's equation _max Decreases with increasing concentration of Panax schinseng extract, K _m The value is kept unchanged along with the increase of the mass concentration of the panax sanguinea extract, so that the inhibition type of the panax sanguinea extract on tyrosinase can be judged to be reversible non-competitive inhibition, and the inhibition constant K of the panax sanguinea extract is _i It was 15.25 mg/L. Therefore, the panax sanguinea extractive has a certain inhibition effect on tyrosinase and is dose-dependent, and the inhibition type is reversible non-competitive inhibition. (2) The Panax schinseng extract has certain inhibition effect on mouse B16 melanoma cell proliferation, intracellular tyrosinase activity and melanin synthesis. The cytomorphological results show that when the concentration of the blood ginseng extract is less than 5mg/L, the change of the cell morphology and the number of the cells is not obvious, and when the cells reach 50mg/L, the cells almost all die. The panax sanguinea extract has concentration and time dependence on the proliferation of mouse B16 melanoma cells, the activity of tyrosinase in cells and the inhibition effect of melanin synthesis. The ginseng extract can inhibit the proliferation of melanoma cells, the tyrosinase activity and the melanin synthesis.

In summary, the following steps: the sanguinarine extract gel prepared by the invention has no granular sensation, good transparency, moderate viscosity and easy spreading, and the pH value is 6.30, so that the sanguinarine gel is a sanguine gel. The invention also provides a method for separating and purifying the sanguinarine extract, which can obtain the high-purity sanguinarine extract powder with the purity of the sanguinarine total flavonoids being more than 46 percent as a raw material to ensure the effect of the sanguinarine extract gel. The sanguinea root extract gel provided by the invention has good whitening and freckle removing effects.

Drawings

FIG. 1 is a UV full wavelength scan;

FIG. 2 is a graph of a standard epicatechin control;

FIG. 3 is a graph of static adsorption;

FIG. 4 is a static resolution graph;

FIG. 5 is a leak graph;

FIG. 6 is a graph showing the effect of different concentrations on the adsorption rate of a blood ginseng extract;

FIG. 7 is a graph of the effect of different sample loading volume flow rates on the adsorption rate of a blood ginseng extract;

FIG. 8 is a graph of the effect of different elution volume flow rates on total flavone resolution;

FIG. 9 is a graph of the effect of different volume fractions of eluent on total flavone resolution;

FIG. 10 is a view for examining the volume of an elution solvent;

FIG. 11 is a graph showing the progression of the action of sanguineous ginseng extract on monophenolase;

FIG. 12 is a graph showing the inhibitory effect of Panax schinseng extract on tyrosine monophenolase;

FIG. 13 is a graph showing the progression of the action of a sanguinarine extract on tyrosine diphenolase;

FIG. 14 is a graph showing the inhibitory effect of a Panax schinseng extract on tyrosinase;

FIG. 15 is a graph of tyrosinase concentration versus enzyme activity for various mass concentrations of Panax schinseng C.A.Meyer extract;

FIG. 16 is a Lineweaver-Burk plot of inhibition of tyrosinase-catalyzed oxidation of L-DOPA by Panax schinseng extract;

FIG. 17 is a Dixon plot of concentration of Panax schinseng extract versus tyrosine diphenolase;

FIG. 18 is a first image of the morphology of mouse B16 melanoma cells observed with blood Panax ginseng extract;

FIG. 19 is a second image of the morphology of mouse B16 melanoma cells observed with the extract of Panax schinseng C.A.Meyer;

FIG. 20 is a graph showing the effect of a Panax schinseng extract on the rate of inhibition of B16 melanoma cell proliferation;

FIG. 21 is a graph showing the effect of Panax schinseng extract on the inhibition of tyrosinase activity in B16 melanoma cells;

FIG. 22 is a graph showing the effect of Panax schinseng extract on the rate of inhibition of melanin formation in B16 melanoma cells;

FIG. 23 is a graph of the effect of carbomer 940 dosage on viscosity of a sanguinarine extract gel;

FIG. 24 is a graph of the effect of sodium hyaluronate dosage on viscosity of a blood ginseng extract gel;

FIG. 25 is a graph of the effect of glycerol dosage on viscosity of a blood ginseng extract gel;

FIG. 26 is a graph of the effect of triethanolamine dosage on viscosity of a blood ginseng extract gel;

FIG. 27 is a graph of the effect of propylene glycol dosage on viscosity of a blood ginseng extract gel;

FIG. 28 is a graph showing the results of analysis of the surface of response of carbomer interaction with glycerol;

FIG. 29 is a graph showing the results of analysis of the response surface of carbomer interacting with triethanolamine;

FIG. 30 is a graph showing the results of analysis of the interaction response surface of glycerol with triethanolamine;

FIG. 31 is a graph showing the verification test of the sanguinarine extract gel of the present invention.

Description of the drawings: in fig. 18 to 19, a: a control group; b: 2.5 μ g/mL total flavonoids; c: 5 μ g/mL total flavonoids; d: 10 μ g/mL total flavonoids; e: 15 μ g/mL total flavonoids; f: 20 μ g/mL total flavonoids; g: 30 μ g/mL total flavonoids; h: 40 μ g/mL total flavonoids; i: 50 μ g/mL total flavonoids; in FIG. 1, A is a control and B is a sample.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

Example 1. A preparation process of a sanguinarine extract gel comprises the following steps:

the preparation method of the blood ginseng extract powder comprises the following steps:

(1) extracting 4kg of blood Ginseng radix powder with 50% ethanol for 3 times, each time for 1.5 hr, filtering the extractive solution, and recovering ethanol from the filtrate at 40 deg.C under reduced pressure in a rotary evaporator until no alcohol smell exists to obtain 4L concentrated solution;

(2) diluting the concentrated solution obtained in the step (1) by adding 0.8 time of water, extracting 0.8 time of petroleum ether for 1 time, removing a petroleum ether layer, continuously adding 0.8 time of ethyl acetate into a water layer, extracting for 5 times, removing a water layer, concentrating an ethyl acetate extract under reduced pressure, recovering ethyl acetate, and drying in a vacuum drying oven to obtain an ethyl acetate part extract;

(3) pretreatment of macroporous adsorption resin: taking AB-8 type macroporous adsorption resin, adding 2BV 95% ethanol, soaking for 24h to fully swell the resin, repeatedly washing the resin with distilled water after filtering until no white turbidity and no ethanol smell exist, then respectively soaking the resin with 2% hydrochloric acid solution and 2% NaOH solution for 2.55h, repeatedly washing the resin with distilled water until the resin is neutral, finally preserving the resin with distilled water, and storing the resin in a sealed manner to obtain pretreated AB-8 type macroporous adsorption resin;

(4) preparation of sample solution: adding distilled water into the ethyl acetate part extract obtained in the step (2) for ultrasonic dissolution, and preparing a sanguinarine extract solution with the concentration of 5 mg/mL;

(5) and (3) purification: and (3) taking 8g of the pretreated AB-8 type macroporous adsorption resin obtained in the step (3), carrying out wet column packing, slowly loading into a chromatographic column, taking 65mL4.7BV of the blood ginseng extract solution obtained in the step (4) with the pH value adjusted to 3.5, loading the blood ginseng extract solution into a sample at the volume flow of 0.8mL/min, removing impurities by pure water after adsorbing for 2.5h, eluting with 1108BV of 65% ethanol at the volume flow of 2.5mL/min, collecting the concentration of eluent and drying to obtain blood ginseng extract powder.

The preparation method of the sanguinarine extract gel comprises the following steps: (1) spreading 0.38g of carbomer 940 in 18g of glycerol, adding 45mL of deionized water to swell for 22h, heating in a water bath at 80 ℃, stirring and dissolving, adding 0.8g of sodium hyaluronate, 0.12g of allantoin and 0.8g of azone, stirring for 0.8h to form a colloid, taking down, and cooling to room temperature to obtain a phase A;

(2) dissolving 4.8g of blood ginseng extract powder in 10mL of 60% ethanol, adding 4.8g of propylene glycol and 0.1g of methylparaben, and stirring for dissolving to obtain phase B;

(3) slowly adding phase B into phase A, stirring, adding 0.27mL triethanolamine to adjust pH, adding 95g deionized water, and stirring to obtain XUERENSHEN extract gel.

The using method comprises the following steps: the appropriate amount of the blood ginseng extract gel is taken and smeared once every night before bedtime.

Example 2. A preparation process of a sanguinarine extract gel comprises the following steps:

the preparation method of the Xuezhen extract powder comprises the following steps:

(1) extracting 6kg of blood Ginseng radix powder with 70% ethanol for 3 times, each time for 2.5 hr, filtering the extractive solution, and recovering ethanol from the filtrate at 50 deg.C under reduced pressure in a rotary evaporator until no ethanol smell exists to obtain 6L concentrated solution;

(2) diluting the concentrated solution obtained in the step (1) by adding 1.5 times of water, extracting petroleum ether with the volume of 1.2 times for 2 times, removing a petroleum ether layer, continuously adding ethyl acetate with the volume of 1.2 times into a water layer, extracting for 7 times, removing the water layer, concentrating an ethyl acetate extract under reduced pressure, recovering ethyl acetate, and drying in a vacuum drying oven to obtain an ethyl acetate part extract;

(3) pretreatment of macroporous adsorption resin: taking AB-8 type macroporous adsorption resin, adding 2BV 95% ethanol, soaking for 24h to fully swell the resin, repeatedly washing the resin with distilled water after filtering until no white turbidity and no ethanol smell exist, then respectively soaking the resin with 5% hydrochloric acid solution and 5% NaOH solution for 3.5h, repeatedly washing the resin with distilled water until the resin is neutral, finally preserving the resin with distilled water, and sealing and storing the resin to obtain pretreated AB-8 type macroporous adsorption resin;

(4) preparation of a sample solution: adding distilled water into the ethyl acetate part extract obtained in the step (2) for ultrasonic dissolution, and preparing a blood ginseng extract solution with the concentration of 5.5 mg/mL;

(5) and (3) purification: and (3) taking 12g of the pretreated AB-8 type macroporous adsorption resin obtained in the step (3), carrying out wet column packing, slowly loading into a chromatographic column, taking 75mL4.7BV of the blood ginseng extract solution obtained in the step (4) with the pH value adjusted to 4.5, loading the sample at the volume flow of 1.2mL/min, removing impurities by pure water after adsorption for 3.5h, eluting by using 130mL of 8BV 75% ethanol at the volume flow of 3.5mL/min, collecting the concentration of eluent and drying to obtain blood ginseng extract powder.

Example 3. A preparation process of a sanguinarine extract gel comprises the following steps:

(1) weighing 5kg of radix Ginseng Indici powder, extracting with 60% ethanol for 3 times, each for 2 hr, filtering the extractive solution, and recovering ethanol from the filtrate under reduced pressure at 45 deg.C in a rotary evaporator until no ethanol smell exists to obtain 5L of concentrated solution;

(4) preparation of a sample solution: taking the ethyl acetate part extract obtained in the step (2), adding a proper amount of distilled water, and ultrasonically dissolving to prepare a blood ginseng extract solution with the concentration of 5.38 mg/mL;

Example 4. A preparation process of a sanguinarine extract gel comprises the following steps:

(2) diluting the concentrated solution obtained in the step (1) by adding 0.8 time of water, extracting 0.8 time of petroleum ether for 1 time, removing a petroleum ether layer, continuously adding 0.8 time of ethyl acetate into a water layer for extraction for 5 times, removing a water layer, concentrating an ethyl acetate extract under reduced pressure, recovering ethyl acetate, and drying in a vacuum drying oven to obtain an ethyl acetate part extract;

(3) pretreatment of macroporous adsorption resin: taking AB-8 type macroporous adsorption resin, adding 2BV 95% ethanol, soaking for 24h to fully swell the resin, repeatedly washing with distilled water after filtering until no white turbidity and no ethanol smell exist, then respectively soaking with 2% hydrochloric acid solution and 2% NaOH solution for 2.55h, repeatedly washing with distilled water until the solution is neutral, finally preserving with distilled water, sealing and storing to obtain pretreated AB-8 type macroporous adsorption resin;

(4) preparation of a sample solution: adding distilled water into the ethyl acetate part extract obtained in the step (2) for ultrasonic dissolution, and preparing a blood ginseng extract solution with the concentration of 5 mg/mL;

The preparation method of the sanguinarine extract gel comprises the following steps: (1) spreading 0.42g of carbomer 940 in 19g of glycerol, adding 55mL of deionized water to swell for 26h, heating in a water bath at 90 ℃, stirring for dissolving, adding 0.12g of sodium hyaluronate, 0.18g of allantoin and 1.2g of azone, stirring for 1.2h to obtain a colloid, taking down, and cooling to room temperature to obtain a phase A;

(2) dissolving 5.2g of blood ginseng extract powder in 10mL of 60% ethanol, adding 5.2g of propylene glycol and 0.1g of methylparaben, and stirring for dissolving to obtain phase B;

(3) slowly adding phase B into phase A, stirring, adding 0.31mL triethanolamine to adjust pH, adding 105g deionized water, and stirring to obtain XUERENSHEN extract gel.

Example 5. A preparation process of a sanguinarine extract gel comprises the following steps:

(3) pretreatment of macroporous adsorption resin: taking AB-8 type macroporous adsorption resin, adding 2BV 95% ethanol, soaking for 24h to fully swell the resin, repeatedly washing the resin with distilled water after filtering until no white turbidity and no ethanol smell exist, then respectively soaking the resin with 5% hydrochloric acid solution and 5% NaOH solution for 3.5h, repeatedly washing the resin with distilled water until the solution is neutral, finally preserving the resin with distilled water, and storing the resin in a sealed manner to obtain pretreated AB-8 type macroporous adsorption resin;

(5) and (3) purification: and (2) taking 12g of the pretreated AB-8 type macroporous adsorption resin obtained in the step (3), filling the mixture into a wet method column, slowly filling the column into a chromatographic column, taking 75mL4.7BV of the blood ginseng extract solution obtained in the step (4) with the pH value adjusted to 4.5, sampling at the volume flow of 1.2mL/min, removing impurities by using pure water after adsorbing for 3.5 hours, eluting with 130mL8BV of 75% ethanol at the volume flow of 3.5mL/min, collecting the concentration of eluent and drying to obtain blood ginseng extract powder.

Example 6. A preparation process of a sanguinarine extract gel comprises the following steps:

The preparation method of the sanguinarine extract gel comprises the following steps: (1) spreading 0.42g of carbomer 940 in 19g of glycerol, adding 55mL of deionized water to swell for 26h, heating in a water bath at 90 ℃, stirring and dissolving, adding 0.12g of sodium hyaluronate, 0.18g of allantoin and 1.2g of azone, stirring for 1.2h to obtain a colloid, taking down, and cooling to room temperature to obtain a phase A;

Example 7. A preparation process of a sanguinarine extract gel comprises the following steps:

The preparation method of the sanguinarine extract gel comprises the following steps: (1) spreading 0.4g of carbomer 940 in 18.35g of glycerol, adding 50mL of deionized water to swell for 24h, heating in a water bath at 85 ℃, stirring and dissolving, adding 0.1g of sodium hyaluronate, 0.15g of allantoin and 1g of azone, stirring for 1h to obtain a colloid, taking down, and cooling to room temperature to obtain a phase A;

(2) dissolving 5g of blood ginseng extract powder in 10mL of 60% ethanol, adding 5g of propylene glycol and 0.1g of methylparaben, and stirring for dissolving to obtain a phase B;

(3) slowly adding phase B into phase A, stirring, adding 0.29mL triethanolamine to adjust pH, adding 100g deionized water, and stirring to obtain XUERENSHEN extract gel.

Example 8. A preparation process of a sanguinarine extract gel comprises the following steps:

Example 9. A preparation process of a sanguinarine extract gel comprises the following steps:

In the process of researching the scheme of the invention, a large number of tests are carried out, and part of the tests are recorded as follows:

experimental example 1 research on process for separating and purifying blood ginseng extract by macroporous adsorption resin

1 background

The flavone components of Ginseng radix include epicatechin, catechin, protocatechuic aldehyde, protocatechuic acid, and gallic acid. In the research of the extraction process of the total flavone of the panax ginseng, the content of the total flavone in crude drugs is low, the purity of the total flavone is only 4.33 percent, and the effect of active ingredients is not favorably exerted. In earlier researches, the flavone components of the blood ginseng are mainly concentrated at the ethyl acetate part, and the purity of the total flavone of the blood ginseng can only reach 29.20 percent after the blood ginseng extract is primarily purified by ethyl acetate. In order to improve the purity of the blood ginseng extract, the experiment takes the extract of the ethyl acetate part of the blood ginseng as a research object, and uses macroporous adsorption resin to purify the extract, so that the purification process of the blood ginseng extract is optimized, and an experimental basis is provided for further development and application of the blood ginseng.

2 method

2.1 preparation of the solution

2.1.1 preparation of Standard solution

Accurately weighing appropriate amount of epicatechin reference substance, adding appropriate amount of 60% ethanol solution, ultrasonic dissolving, cooling, and fixing volume to scale mark to obtain epicatechin reference substance solution with mass concentration of 0.20 mg/mL.

2.1.2 preparation of sample solution before applying blood Ginseng to column

Weighing 5.0kg of crude powder of the blood ginseng medicinal material, carrying out reflux extraction for three times with 30 times of 60% ethanol for 2 hours each time, combining the extracting solutions and carrying out suction filtration. Recovering ethanol from the filtrate at 45 deg.C under reduced pressure until the medicinal liquid has no alcohol smell, and the final volume is 5L. Diluting the concentrated solution with double distilled water, extracting once with petroleum ether with the same volume, extracting for 5-7 times with ethyl acetate solution with the same volume, recovering ethyl acetate to obtain a thick extract, and drying in a vacuum drying oven at 45 ℃ to obtain extract powder with the total mass of 228.05 g. Weighing appropriate amount of ethyl acetate part extract powder of Panax ginseng C.A.Meyer, adding water, ultrasonic dissolving, and preparing into required solubility according to experiment.

2.2 selection of measurement wavelength

Accurately sucking appropriate amount of epicatechin reference solution and test solution, performing color development treatment by using a classical flavone color development method (NaNO2-Al (NO3)3-NaOH), scanning within 400-700 nm, and measuring the maximum absorption wavelength.

2.3 methodological inspection

2.3.1 drawing of Standard Curve

Accurately weighing epicatechin reference substance solutions 0.2mL, 0.4mL, 0.6 mL, 0.8mL, 1.0mL and 1.2mL respectively, placing in 10mL volumetric flasks, adding 60% ethanol respectively to make up to 5 mL; precisely adding 0.3mL of 5% sodium nitrite solution, shaking up, and standing for 6 min; adding 0.3mL of 10% aluminum nitrate solution, shaking up, and standing for 6 min; adding 4mL of 4% sodium hydroxide solution, adding 60% ethanol to a constant volume to a scale, shaking up, standing for 15min to obtain reference substance solutions with a series of mass concentrations, measuring the absorbance of epicatechin at a wavelength of 503nm by using an ultraviolet spectrophotometry, and drawing a standard curve by taking the mass concentration (X) of the reference substance as an abscissa and the absorbance (Y) as an ordinate.

2.3.2 precision review

0.4mL of epicatechin control solution was precisely measured in a 10mL volumetric flask, color-developed according to the method under item 2.3.1, continuously measured 6 times at a wavelength of 503nm, and the absorbance value was recorded to calculate the RSD value.

2.3.3 stability Studies

Precisely measuring appropriate amount of sample solution, performing color development treatment according to the method under item 2.3.1, measuring at 503nm wavelength, measuring once per minute, continuously measuring for 15min, recording absorbance value, and calculating RSD value.

2.3.4 repeatability test

Accurately weighing 6 parts of 10mg of blood ginseng ethyl acetate part extract powder in a 10mL volumetric flask, adding distilled water for ultrasonic dissolution, respectively taking 0.2mL in the 10mL volumetric flask, performing color development treatment according to the method under item 2.3.1, measuring at 503nm wavelength, recording absorbance value A, and calculating RSD value.

2.3.5 sample recovery Studies

Precisely weighing 9 parts of 10mg blood ginseng ethyl acetate part extract powder 9 parts, respectively putting the extract powder into a 25mL volumetric flask, respectively adding 1.5mg epicatechin reference substances into No. 1-3, adding 3mg epicatechin reference substances into No. 4-6, adding 4.5mg epicatechin reference substances into No. 7-9, respectively adding distilled water to dissolve, then taking 0.2mL to perform color development treatment according to the method under item 2.3.1, measuring at the wavelength of 503nm, and calculating the recovery rate.

2.4 static adsorption-desorption test

2.4.1 pretreatment of macroporous adsorbent resins

Soaking macroporous adsorbent resin with 95% ethanol for 24 hr for swelling, filtering, washing with pure water repeatedly until there is no white turbidity and no ethanol smell, soaking with 5% hydrochloric acid solution and 5% NaOH solution for 3 hr, washing with pure water repeatedly until the solution is neutral, keeping fresh with pure water, sealing, and storing.

2.4.2 screening of macroporous adsorbent resin types

2.4.2.1 investigation of adsorption Properties of Panax schinseng extract by different types of macroporous adsorbent resins

Respectively weighing 2.0g of pretreated AB-8, D-101, HPD-100, HP-20, D-301 and H-103 macroporous adsorption resin into a 100mL conical flask with a plug, respectively adding 50mL of sample solution (1.78mg/mL), shaking in a constant-temperature oscillation water tank at room temperature for 24H (25 ℃, 100rpm/min), filtering to obtain an adsorption solution, sampling, performing color development treatment according to the method under item 2.3.1, determining at the wavelength of 503nm, and calculating the adsorption amount and the adsorption rate.

Static adsorption capacity (mg/g) ═ C ₀ -C ₁ )×V ₁ /W

Adsorption rate (%) - (C) ₀ -C ₁ )/C ₀ ×100％

Note: c ₀ Initial solubility (mg/mL); c ₁ As equilibrium concentration (mg/mL); v ₁ Sample fluid volume (mL); w is the resin weight (g).

2.4.2.2 investigation of analytic Properties of blood Ginseng extract by different types of macroporous adsorbent resins

Washing the saturated macroporous adsorbent resin in each conical flask with pure water to remove the solution and impurities on the resin surface, adding 50mL 50% ethanol for desorption, shaking in a constant-temperature oscillation water tank at room temperature for 24h (25 ℃, 100rpm/min) to fully desorb, sampling, performing color development treatment according to the method under item 2.3.1, measuring at 503nm wavelength, and calculating the content and the resolution of total flavonoids after desorption.

Desorption amount (mg/g) ═ C ₂ ×V ₂ /W

Desorption rate (%) ═ C ₂ ×V ₂ /(C ₀ -C ₁ )×V ₁ ×100％

Note: c ₂ The mass concentration of the desorption solution (mg/mL); v ₂ Is the volume of the desorption solution; w is the resin weight (g); v ₁ Is the volume of solution (mL).

2.4.3 static adsorption kinetics curves

Precisely weighing 2.0g of the pretreated ideal resin into a 100mL conical flask with a plug, adding 50mL of sample solution, shaking in a constant-temperature shaking water tank at room temperature for 12h (25 ℃, 100rpm/min), sampling at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12h respectively, performing color development treatment according to the method under item 2.3.1, determining at 503nm wavelength, and drawing a static adsorption kinetic curve of concentration and time.

2.4.4 static analytical kinetics curves

Washing the macroporous adsorption resin with saturated adsorption with pure water to remove the solution and impurities on the surface of the resin, precisely adding 50mL of 50% ethanol for desorption, placing the resin in a shaking table at room temperature for shaking for 12h (25 ℃, 100rpm/min), sampling at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12h respectively, performing color development treatment according to the method under item 2.3.1, performing measurement at the wavelength of 503nm, and establishing a static desorption kinetic curve of concentration and time.

2.4.5 influence of pH value of sample liquid on adsorption and resolution

Weighing 2.0g of pretreated ideal macroporous adsorption resin into 100mL conical bottles with stoppers, adding 1mol/L HCl solution and 1mol/L NaOH solution to adjust the pH values to 50mL of sample solutions of 2, 3, 4, 5, 6, 7 and 8, shaking in a constant-temperature oscillation water tank at room temperature for 24h (25 ℃, 100rpm/min), filtering to obtain adsorption solution, sampling, performing color development treatment according to the method under item 2.3.1, determining at 503nm wavelength, and calculating the adsorption amount and adsorption rate. Washing the saturated resin adsorbed in each bottle with pure water to remove the solution and impurities on the surface, adding 50mL of 50% ethanol precisely for desorption, placing in a constant-temperature oscillation water tank at room temperature, shaking for 24h (25 ℃, 100rpm/min) to fully desorb, sampling, performing color development treatment according to the method under item 2.3.1, measuring at 503nm wavelength, calculating the content and the resolution of the total flavone after desorption, and calculating the recovery rate of the total flavone according to the adsorption rate and the resolution.

2.5 dynamic adsorption-desorption test

2.5.1 plotting of leakage curves

Taking 3 parts of pretreated AB-8 type macroporous adsorption resin 10.0g, precisely weighing, slowly loading into a chromatographic column (15mm multiplied by 300mm) by a wet method, respectively taking 250mL sample solutions with total flavone concentrations of 1.73mg/mL, 3.50mg/mL and 5.38mg/mL, adjusting the pH value to 4 at room temperature, adding into the resin column at a volume flow of 1.0mL/min, sectionally collecting the effluent liquid, collecting 1 part of the effluent liquid per 10mL, taking each effluent liquid to perform color development treatment according to a method under item 2.3.1, measuring at a wavelength of 503nm, calculating the total flavone content, respectively drawing a leakage curve, and determining the optimal sample loading volume.

2.5.2 investigation of the concentration of the sample liquid

Taking 10.0g of pretreated AB-8 type macroporous adsorption resin, packing the pretreated AB-8 type macroporous adsorption resin into a column (15mm multiplied by 300mm) by a wet method, taking 70mL of sample solution with the concentrations of 1.96, 5.38, 9.45, 12.83 and 14.31mg/mL respectively, adjusting the pH value to be 4, sampling at the flow rate of 1.0mL/min, respectively collecting filtrate, performing color development treatment according to the method under item 2.3.1, measuring at the wavelength of 503nm, calculating the adsorption rate under each mass concentration, and determining the mass concentration of the sample solution.

2.5.3 Effect of sample Loading volume flow on dynamic adsorption Performance

Loading pretreated AB-8 type macroporous adsorbent resin 10.0g into a column (15mm × 300mm) by wet method, taking 70mL of sample loading solution, adjusting pH to 4, loading at volume flow rates of 1.0, 2.0, 3.0 and 4.0mL/min, collecting filtrates, performing color development treatment according to the method under item 2.3.1, measuring at 503nm wavelength, calculating adsorption rate at each volume flow rate, and determining sample loading volume flow rate.

2.5.4 Effect of elution volume flow on elution Effect

Taking 10.0g of pretreated AB-8 type macroporous adsorption resin, filling the pretreated AB-8 type macroporous adsorption resin into a column (15mm multiplied by 300mm) by a wet method, taking 70mL of a sample solution, adjusting the pH value to 4, loading the sample at the flow rate of 1.0mL/min, after the sample is subjected to special adsorption saturation, eluting the sample solution by 50mL of 70% ethanol at the volume flow rates of 1.0, 2.0, 3.0 and 4.0mL/min respectively, collecting eluent, carrying out color development treatment according to the method under item 2.3.1, measuring at the wavelength of 503nm, calculating the elution rate, and determining the optimal elution volume flow rate.

2.5.5 influence of elution solvent on AB-8 macroporous resin elution effect of Panax ginseng C.A.Meyer extract

Accurately weighing 10 parts of pretreated AB-8 type macroporous adsorption resin 10.0g, loading the resin into a wet column (15mm multiplied by 300mm), taking 70mL of a sample solution, adjusting the pH value to 4, loading the resin at a flow rate of 1.0mL/min, after the resin is subjected to special adsorption saturation, sequentially eluting the resin by using ultrapure water and 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 95% ethanol solutions of 120mL respectively, respectively collecting eluates, performing color development treatment according to a method under '2.3.1', measuring at a wavelength of 503nm, and determining an optimal elution solvent.

2.5.6 influence of elution solvent dosage on elution effect

Taking 10.0g of the pretreated AB-8 type macroporous adsorption resin, sampling and removing impurities according to the method, performing gradient elution by using 70% ethanol solution (200mL) at the volume flow of 3mL/min, respectively collecting eluent, performing color development treatment by using a method under item 2.3.1 for every 10mL, determining at the wavelength of 503nm, calculating the content of total flavonoids in each eluent, drawing an elution curve, and determining the optimal eluent volume.

3 results of

3.1 selection of measurement wavelength

As shown in FIG. 1, both the control and the sample had an absorption maximum at 503nm, and thus 503nm was determined as the maximum absorption wavelength.

3.2 methodological observations

3.2.1 drawing of Standard Curve

And (3) taking the mass concentration (X) of the control line as an abscissa and the absorbance (Y) as an ordinate to obtain a linear regression equation: y is 39.754X +0.0361, and the correlation coefficient r is 1, which shows that the epicatechin control has good linear relationship in the range of 4.00-24.00 mg/L, as shown in FIG. 2.

3.2.2 precision test

The results of the precision tests are shown in Table 1, where RSD was calculated to be 0.31%, indicating that the precision of the instrument is good.

TABLE 1 precision test results

3.2.3 stability test

The stability results are shown in table 2, with calculated RSD of 1.39%, indicating that the sample solutions are stable well within 15 min.

Table 2 stability results

Time (min)	Absorbance of the solution	Time (min)	Absorbance of the solution
				0	0.7847	8	0.7685
1	0.7840	9	0.7669
				2	0.7845	10	0.7658
3	0.7826	11	0.7618
				4	0.7807	12	0.7604
5	0.7735	13	0.7563
				6	0.7719	14	0.7570
7	0.7698	15	0.7540

3.2.4 repeatability test

The results of the 6 sample repeatability tests are shown in table 3, and the calculated RSD was 0.91%, indicating that the method was highly reproducible.

TABLE 3 results of the repeatability tests

3.2.5 sample recovery test

The sample recovery test results are shown in table 4, the average recovery rate is 98.68%, and is between 95% and 105%, and the RSD is 2.04%, which indicates that the recovery rate of the method is good.

TABLE 4 sample application recovery test results

3.3 results of static adsorption-desorption test

3.3.1 screening results of macroporous adsorbent resin type

The physicochemical property of the macroporous adsorption resin can obviously influence the adsorption, separation and purification effects of target components, and is mainly related to factors such as the pore diameter, the specific surface area and the like of the macroporous adsorption resin, wherein the pore diameter influences the diffusion of adsorbed substances, and the specific surface area influences the quantity of the adsorbed substances. In the experiment, 6 different types of macroporous adsorption resins are selected to be compared through static adsorption and static analysis tests, as can be seen from Table 5, the adsorption rates are respectively H-103 > AB-8 > D-101 > HPD-100 > HP-20 > D-301 from large to small, the adsorption rates are respectively HPD-100 > AB-8 > D-101 > HP-20 > H-103 > D-301 from large to small, although the adsorption rate of H-103 is larger than AB-8, the analysis rate is smaller, so that the recovery rate and the cost of total flavonoids are integrated, and AB-8 is selected as the optimal resin type of the purified panax notoginseng extract.

TABLE 5 adsorption rate, resolution rate and total recovery rate of blood Ginseng extract by different types of macroporous adsorbent resin

3.3.2 drawing of static adsorption Curve

As can be seen from the graph 3, within 0-6 h, the adsorption rate of the blood ginseng extract is obviously increased, and with the increase of the adsorption time, the adsorption rate of the blood ginseng extract slowly increases after 6h and tends to be balanced. Therefore, the adsorption of the AB-8 macroporous adsorption resin on the panax japonicus extract is basically saturated within 6 hours.

3.3.3 drawing of static analytical Curve

As can be seen from figure 4, the AB-8 macroporous adsorption resin has the resolution rate of 83.20% in 1h for static resolution of the sanguinary extract, the elution amount is large in 3h, the elution amount is slowly increased after 3h and is quickly balanced, so that the sanguinary extract is quickly balanced in static resolution.

3.3.4 determination of pH value of sample liquid

By comparing the influence of different pH values of the sample solution on the adsorption rate and the resolution ratio of the total flavonoids, the results are shown in Table 6, the adsorption rate is smaller and smaller as the pH value is increased, and the adsorption rate is the largest when the pH value is 2, but the resolution ratio is the lowest when the pH value is 2 and the resolution ratio is the highest when the pH value is 4, so that the pH value of the optimal sample solution is selected as the pH value of the total flavonoids.

TABLE 6 adsorption rate, resolution ratio and total flavone recovery rate of Panax schinseng extract at different pH values

Numbering	Adsorption Rate (%)	Resolution (%)	Total flavone recovery (%)
				pH2	92.53	74.71	69.13
pH3	88.50	88.44	78.27
				pH4	88.51	90.25	79.88
pH5	87.93	89.89	79.04
				pH6	86.78	87.37	75.82
pH7	85.06	88.30	75.11
				pH8	79.31	76.14	60.39

3.4 dynamic adsorption-desorption test results

3.4.1 plotting of leakage curves

When the mass concentration of the total flavonoids in the effluent liquid reaches one tenth of the mass concentration of the total flavonoids in the sample loading liquid, a leakage point is reached, and the optimal sample loading volume is considered. The experiment examined the leakage curves of 3 sample solutions with different concentrations, as shown in FIG. 5, when the concentration is 1.73mg/mL, the leakage starts in the 16 th effluent, and the sample loading volume is 160mL (10.7 BV); at a concentration of 3.50mg/mL, the leak started in the 11 th effluent and the loading volume was 110mL (7.3 BV); at a concentration of 5.38mg/mL, a leak started in the 7 th effluent. To take the amount and time of loading into consideration, the concentration of the loading solution was selected to be 5.38mg/mL and the loading volume was selected to be 70mL (4.6 BV).

3.4.2 determination of the concentration of the sample liquid

As shown in fig. 6, when the concentrations of the sample solutions are 1.96, 5.38, 9.45, 12.83 and 14.31mg/mL, the adsorption rates are 99.97%, 97.86%, 94.04%, 80.93% and 78.61%, respectively, and the adsorption rate decreases with the increase of the concentration of the sample solution, and when the concentration is 1.96mg/mL, the adsorption rate approaches 100%, but too low concentration increases the sample amount, which prolongs the production cycle, and too high concentration decreases the adsorption rate, which results in waste of the sample and the solvent. The concentration of 5.38mg/mL is preferably selected so that the adsorption rate is 97.86%, and the concentration of the sample loading solution is 5.38 mg/mL.

3.4.3 sample Loading liquid volume flow determination

As shown in FIG. 7, the adsorption rate decreases with the increase of the sample volume flow, the decrease is more obvious when the volume flow is 1-3 mL/min, and the decrease tends to be gentle when the volume flow is 3-4 mL/min. When the volume flow is 1mL/min, the adsorption rate is the highest and is 97.86%, so that the optimal sample loading volume flow is determined to be 1 mL/min.

3.4.4 determination of volume flow of elution solvent

As shown in FIG. 8, the elution solvent volume flow rate was selected to be 3mL/min, since the resolution rate increased with an increase in the volume flow rate, the resolution rate decreased with a volume flow rate of 4mL/min, and the elution rate at 3mL/min was 88.60%.

3.4.5 determination of elution solvent

By examining water and ethanol eluents of different concentrations, as shown in fig. 9, the results indicated that the elution rates of water, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 95% ethanol on the sanguinea ginseng extract were 5.85%, 21.87%, 40.05%, 40.25%, 56.08%, 90.03%, 93.65%, 94.22%, 92.23% and 90.74%, respectively. Therefore, 70% of ethanol has the largest elution rate on the total flavonoids, so 70% of ethanol is selected as the best elution solvent

3.4.6 plotting elution volume curves of eluents

As shown in FIG. 10, the elution profile of the blood ginseng extract was relatively good, a small amount of flavone eluted in the effluent of fraction 1, the flavone content of the effluent of fraction 2 was the highest, and the elution of the blood ginseng extract was almost complete when fraction 12 was reached, so that it was determined that the amount of elution solvent of the blood ginseng extract was 120mL (8 BV).

3.5 proof test

Respectively weighing 3 parts of 10.0g of pretreated AB-8 type macroporous resin, loading the pretreated AB-8 type macroporous resin on a column by a wet method, taking 3 parts of 70mL of sample solution, adjusting the pH to 4, loading the sample at a volume flow of 1mL/min, collecting effluent, adsorbing for 3 hours, removing impurities by pure water, eluting by using 120mL (8BV) of 70% ethanol at a volume flow of 3mL/min, collecting eluent, taking the eluent to carry out color development treatment according to a method under item 2.3.1, measuring absorbance at a wavelength of 503nm, calculating the content of total flavonoids in the eluent, and drying the rest of the eluent into powder in a vacuum drying box. The results showed that the average adsorption rate was 97.48% (RSD% was 0.32%), the average resolution was 91.97% (RSD% was 2.77%), and the purity was 46.01% (RSD% was 2.23).

TABLE 7 verifiability test results

Number of	1	2	3	Mean value of	RSD(％)
						Purity of Total Flavonoids (%)	44.83	46.52	46.68	46.01	2.23

4 conclusion and discussion

Xuezhen is a commonly used herb in minority nationality in Guizhou province and is widely distributed in Guiyang, Anshun, southeast Qian, southwest Qian and the like. At present, the research on the panax ginseng is mainly the research on pharmacological action and chemical components, and the purification and separation of the panax ginseng extract are not reported. In the research report of the extraction process of the blood ginseng extract, the content of the total flavone is found to be lower, which is not beneficial to the exertion of the drug effect of the active ingredients. In earlier researches, flavone components of the panax japonicus are mainly concentrated on ethyl acetate parts, so that the ethyl acetate is adopted for primarily purifying the extracting solution. For further research, on the basis of the previous test, the separation and purification technology of the panax sanguinea extractive is adopted to separate and purify the panax sanguinea extractive.

The macroporous adsorption resin is a solid high molecular material which has no dissociable group, has a porous structure and is insoluble in water, and is a common medium for separating and purifying flavonoid compounds. The macroporous adsorption resins of different types have different adsorption capacities and desorption capacities for the same substance due to different polarities, specific surface areas, pore diameters and the like, so that different adsorption effects are generated. The experiment integrates the adsorption capacity and the desorption capacity, 6 kinds of macroporous adsorption resins with different models are investigated, and the AB-8 type macroporous adsorption resin is determined to be the best type of macroporous adsorption resin for purifying the panax sanguinea extract. The optimal purification conditions are as follows: the loading volume was 70mL (4.7BV), the loading concentration was 5.38mg/mL, the loading flow volume was 1mL/min, the eluent volume was 3mL/min, the eluent was 70% ethanol, and the eluent volume was 120mL (8 BV). Through a verification test, the result shows that the purity of the total flavone in the panax ginseng extract is increased from 29.20% to 46.01%. Although the purity of the total flavone after purification is improved, the purity is not up to 50 percent, different impurity removal modes are tried, and the purity of the total flavone after polyamide purification is not improved.

In conclusion, the purification process of the sanguinaria ginseng extract optimized by the static and dynamic adsorption-desorption tests of the macroporous adsorption resin is stable and reliable, can effectively improve the purity of the total flavone, and provides a certain reference for the development and utilization of the sanguinaria ginseng.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Test example 2 study on inhibition of tyrosinase by Panax schinseng extract

1 background of the invention

Tyrosinase, also known as polyphenol oxidase, is a copper-containing metalloenzyme with a complex structure, and widely exists in human bodies, animals, plants and microorganisms. Tyrosinase plays a key role in the process of melanin formation, can catalyze the hydroxylation of L-tyrosine into L-DOPA, oxidizes the L-DOPA into dopaquinone, and forms melanin through a series of complex reactions. The abnormal over-expression of tyrosinase can cause skin pigmentation diseases such as freckle, chloasma and senile plaque, so the synthesis of melanin can be reduced by inhibiting the activity of tyrosinase, thereby achieving the effects of whitening and removing freckles. The flavonoid compound has multiple physiological and pharmacological activities such as oxidation resistance, anti-inflammatory activity, antiviral activity, anticancer activity and the like, and also has safe and efficient tyrosinase activity inhibition capability. In recent years, tyrosinase inhibitors are widely applied to the cosmetic market as whitening and freckle-removing agents, but some traditional tyrosinase inhibitors have the problems of anaphylaxis, adverse reaction and the like, so that the development of more natural, safe and efficient tyrosinase inhibitors in whitening and freckle-removing cosmetics has a good research prospect. The research uses the blood ginseng extract as a raw material, researches the inhibition effect of the blood ginseng extract on the activity of tyrosine monophenolase and diphenolase, the inhibition kinetics and the inhibition mechanism of the blood ginseng extract on the tyrosinase, and provides reference for the development and the application of the blood ginseng extract in cosmetics.

2 method

2.1 preparation of reagents

Preparation of 2.1.150mmol/L PBS buffer (pH 6.8)

Accurately weighing NaH ₂ PO ₄ 7.80g, dissolving with deionized water, and fixing the volume to 1000mL to be used as mother liquor 1 for standby; precisely weighing Na ₂ HPO ₄ 17.90g, dissolved by deionized water and then fixed to 1000mL as mother liquor 2 for standby. 51mL of mother liquor 1 and 49mL of mother liquor 2 are respectively measured and mixed when needed, and PBS buffer solution with the concentration of 50mmol/LpH 6.8.8 is obtained.

Preparation of 2.1.2L-tyrosine solution

Accurately weighing 45.30mg of L-tyrosine, adding PBS buffer solution (50mmol/L) with pH of 6.8 to dissolve, diluting to 50mL to obtain L-tyrosine solution with concentration of 5mmol/L, and storing at 4 ℃ in dark place for later use.

Preparation of 2.1.3L-dopa solution

Accurately weighing 49.33mg of L-DOPA (L-DOPA), placing in a 50mL volumetric flask, adding PBS buffer solution (50mmol/L) with pH of 6.8 to dissolve, fixing the volume to scale to obtain L-DOPA solution with concentration of 5mmol/L, and storing at 4 ℃ in dark place for later use.

2.1.4 preparation of Tyrosinase (TYR) solution

Precisely weighing 1.0mg of tyrosinase in a 10mL volumetric flask, dissolving the tyrosinase in a PBS buffer solution (50mmol/L) with the pH value of 6.8, and fixing the volume to a scale to obtain a tyrosinase solution with the concentration of 0.1mg/mL, and storing the tyrosinase solution at the temperature of 4 ℃ in a dark place.

2.1.5 preparation of blood Ginseng extract solution

Precisely weighing a proper amount of the blood ginseng extract, dissolving the blood ginseng extract with deionized water by ultrasonic waves to prepare sample solutions with the concentration of 5.0mg/mL, and adding deionized water into sample solutions with different volumes in volumetric flasks to respectively dilute the sample solutions with the different volumes to obtain sample solutions with the concentrations of 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 and 5.0mg/mL for later use.

2.2 inhibition of tyrosine monophenolase Activity by Panax schinseng extracts of different concentrations

Referring to the methods of Liu-Lin, etc., and slightly adjusting, using L-tyrosine (5mmol/L) as a substrate, respectively measuring the influence of blood ginseng extracts with different mass concentrations (final concentrations are 0, 16.67, 33.33, 50.00, 66.67, 100.00, 133.33 and 166.67mg/L) on the activity of tyrosine monophenol enzyme, respectively sucking 50mmol/L of PBS solution with pH 6.8, 10. mu.L of sample solutions with different concentrations or deionized water and 40. mu.L of tyrosinase solution into a 96-well microplate, placing the microplate in a microplate reader for incubation and shaking at 30 ℃ for 10min, then adding 100. mu. L L-tyrosine solution (5mmol/L), and measuring the absorbance value at 475nm (reading and recording data once per minute, continuously measuring for 10 min). Calculating the inhibition rate of the Panax schinseng extract on tyrosinase according to the formula (1), plotting the relative inhibition rate of the enzyme on the concentration of the sample and calculating IC ₅₀ 。

Inhibition rate (%) ([ 1- (A) ] ₃ -A ₄ )/(A ₁ -A ₂ )]×100％

TABLE 8 composition and amount of reaction solution for determination of tyrosine monophenolase Activity

2.3 inhibition of tyrosinase Activity by Panax schinseng extracts of different concentrations

Referring to the method of Liu-Lin, etc., and slightly adjusting, using L-DOPA (5mmol/L) as a substrate, respectively measuring the effect of the blood ginseng extracts with different concentrations (final concentrations are 0, 16.67, 33.33, 50.00, 66.67, 100.00, 133.33 and 166.67mg/L) on the tyrosinase activity, respectively sucking 50mmol/L of PBS solution with pH 6.8, 10 μ L of sample solution with different concentrations or deionized water, 20 μ L of tyrosinase solution into a 96-well microplate, placing the microplate in a microplate reader for incubation and shaking at 30 ℃ for 10min, then adding 100 μ L-DOPA solution (5mmol/L), and measuring the absorbance value at 475nm (reading the recorded data once per minute and continuously measuring for 10 min). Calculating the inhibition rate of the Panax schinseng extract on tyrosinase according to the formula (1), plotting the relative inhibition rate of the enzyme on the concentration of the sample and calculating IC ₅₀ 。

TABLE 9 composition and amount of reaction solution for determination of tyrosinase Activity

2.4 mechanism of action of Ginsengseng extract on tyrosinase Activity

In the experiment, the tyrosinase concentration (final concentration is 0, 0.8, 1.6, 2.4 and 3.2mg/L respectively), the concentration of a fixed substrate L-DOPA is 5mmol/L, the influence of the blood ginseng extract (0, 16.67, 33.33, 50.00 and 66.67mg/L) with different concentrations on the tyrosinase activity is measured, the enzyme mass concentration and the relative enzyme activity are plotted, whether the inhibition effect of the blood ginseng extract on the tyrosinase is reversible inhibition or irreversible inhibition is judged, and when a group of straight lines passing through the origin is obtained, the inhibition belongs to reversible inhibition; if a set of parallel straight lines is obtained, irreversible inhibition is attributed.

2.5 kinetics of inhibition of tyrosinase Activity by Henry Ginseng extract

In the experiment, the substrate L-DOPA concentration was varied (0.25, 0.5, 1, 2, 4 mm)ol/L), the concentration of the fixed tyrosinase is 0.1mg/mL, blood ginseng extract solutions with different mass concentrations (the final concentrations are 0, 16.67, 33.33, 50.00 and 66.67mg/L respectively) are added, and the influence of the blood ginseng extracts with different mass concentrations on the activity of the tyrosinase is measured through the change of the absorbance value of a reaction system along with time. And (3) by using a Lineweaver-Burk double reciprocal mapping method, and by using a reciprocal of the reaction rate to a reciprocal of the concentration of a substrate L-DOPA to map, judging the inhibition type of the blood ginseng extract on tyrosinase. If a group of straight lines intersecting with the Y axis is obtained in a Lineweaver-Burk reciprocal diagram, the inhibition type of the reaction is competitive inhibition, and the kinetic parameter is represented as V _max The value remains unchanged, K _m The value is increased; if a group of straight lines intersecting the X axis is obtained, the inhibition belongs to non-competitive inhibition, and the kinetic parameter is represented as V _max Value decreases, K _m The value remains unchanged; if a group of straight lines intersecting the second quadrant is obtained, mixed suppression is performed, and the kinetic parameter is represented as V _max Value decreases, K _m The value is increased; if a group of parallel straight lines is obtained, the inhibition belongs to the anti-competitive inhibition, and the kinetic parameter is represented as V _max Value decreases, K _m The value decreases. And (3) carrying out secondary mapping by using a Dixon mapping method and taking the concentration of the total flavone as an abscissa and the reciprocal (1/V) of the reaction rate as an ordinate to judge the inhibition constant of the panax sanguinea extract on the tyrosine diphenolase.

3 results of

3.1 inhibition of tyrosine monophenolase Activity by blood Ginseng extracts of different concentrations

The action process curve of the panax sanguinea extracts with different mass concentrations on the tyrosine monophenolase activity is shown in figure 11, and it can be seen from the figure that the absorbance values of all reaction systems are increased along with the increase of the reaction time, but the absorbance values are slowly increased in the initial stage of the reaction, and a certain hysteresis effect exists. The enzyme reaction rate gradually decreases along with the increase of the mass concentration of the blood ginseng extract at the same time. The result shows that the blood ginseng extract can inhibit the enzymatic oxidation reaction of tyrosine monophenol enzyme. The relative inhibition rate of the blood ginseng extracts with different mass concentrations on the tyrosine monophenolase is shown in fig. 12, the inhibition effect of the blood ginseng extracts on the tyrosine monophenolase is positively correlated with the concentration of the blood ginseng extracts, namely, the inhibition rate of the blood ginseng extracts on the tyrosine monophenolase activity is increased along with the increase of the mass concentration of the blood ginseng extracts. The concentration of the Panax schinseng extract is 88.78mg/L when the inhibition rate of the Panax schinseng extract on the tyrosine monophenolase activity reaches 50%.

3.2 inhibition of tyrosinase Activity by Henry Ginseng extracts at different concentrations

The action progress curve of the blood ginseng extract with different mass concentrations on tyrosine diphenolase is shown in fig. 13, and the absorbance value of each reaction system is gradually increased along with the increase of time. At the same time, the absorbance value of the reaction system is reduced, namely the reaction rate of the enzyme is reduced along with the increase of the mass concentration of the panax sanguinea extract. Thus, the ginseng radix extract can inhibit the enzymatic oxidation of L-DOPA. The relative inhibition rate of the panax japonicus extract on the tyrosine diphenolase with different mass concentrations is shown in fig. 14, and the result shows that the panax japonicus extract has a certain inhibition effect on the tyrosine diphenolase, and the inhibition effect is positively correlated with the concentration of the panax japonicus extract, namely the inhibition rate on the tyrosine diphenolase activity is increased along with the increase of the mass concentration of the panax japonicus extract. The concentration of the Panax schinseng extract is 36.46mg/L when the inhibition rate of the Panax schinseng extract on the enzymatic activity of tyrosine dioxygenase reaches 50%, namely IC ₅₀ It was 36.46 mg/L.

3.3 mechanism of inhibition of tyrosinase Activity by Panax ginseng C.A.Meyer extract

The relationship between the mass concentration of tyrosinase and the enzyme activity in the sanguineous ginseng extract solution with different mass concentrations is shown in figure 15, a group of straight lines passing through the origin are obtained after the mass concentration of tyrosinase and the enzyme activity are plotted, and the slope of the straight lines is gradually reduced along with the increase of the mass concentration of the sanguineous ginseng extract, so that the inhibition of the sanguineous ginseng extract on tyrosinase belongs to reversible inhibition, namely the inhibition of the sanguineous ginseng extract on the enzyme activity leads to the reduction of the enzyme catalysis efficiency, but not caused by the reduction of the effective enzyme amount.

3.4 kinetics of inhibition of tyrosinase Activity by Henry Ginseng extract

By changing the concentration of a substrate L-DOPA to study the inhibition types of the panax sanguinea extracts with different mass concentrations on tyrosinase, a Lineweaver-Burk double reciprocal diagram is shown in figure 16, a group of straight lines intersecting on the abscissa axis of a second quadrant are obtained, a Mie equation can be obtained according to the straight lines, and a K equation can be obtained through the Mie equation _m Value sum V _max The values and results are shown in Table 10. K increases with the concentration of the Panax schinseng extract _m Constant value, V _max The value is gradually reduced, namely only the maximum rate of the enzyme catalytic reaction is influenced, the Michaelis constant is not obviously influenced, and the kinetic parameters of non-competitive inhibition are met. The inhibition type of the panax japonicus extract on tyrosinase is reversible non-competitive inhibition, and the inhibitor does not preempt the active center of enzyme with substrate, but inhibits the activity of the enzyme by combining with essential groups except the active center.

TABLE 10 inhibition kinetics parameters results of the extract of Panax schinseng for tyrosinase

Plotting the concentration of Panax Ginseng extract against the reciprocal of the reaction rate (1/V) at different concentrations of L-DOPA as shown in Dixon plot (FIG. 17) yields a set of lines intersecting the X-axis with the absolute value of the abscissa of the intersection being the inhibition constant K _i Fitting equation suppression constant K _i As shown in Table 11, K _i It was 15.25 mg/L.

TABLE 11 inhibition constants for inhibition of tyrosinase activity by sanguinarine extract

4 conclusion and discussion

The color of the skin can be determined by 4 chromophores in the human body, namely carotenoids, haemoglobin, oxyhaemoglobin and melanin, of which melanin is the most predominant component. The excessive accumulation of melanin can cause a plurality of skin diseases, such as freckles, age spots, chloasma and the like, tyrosinase is a main rate-limiting enzyme in the process of forming melanin, and a tyrosinase inhibitor mainly influences the generation of melanin by inhibiting the activity of the tyrosinase, so that better whitening and freckle removing effects are achieved.

Experimental results show that the Panax schinseng extract has inhibition effect on the activities of tyrosine monophenolase and diphenolase, can effectively inhibit the activities of tyrosine monophenolase and diphenolase, and the inhibition rate is increased along with the increase of the mass concentration of total flavonoids, and the IC is ₅₀ 88.78mg/L and 34.46mg/L respectively. The result of the analysis of the inhibition and braking mechanical parameters of the tyrosinase shows that V can be known by the Mie's equation _max Decreases with increasing concentration of Panax schinseng extract, K _m The value is kept constant along with the increase of the mass concentration of the panax sanguinea extractive, so that the inhibition type of the panax sanguinea extractive on the tyrosinase can be judged to be reversible non-competitive inhibition, and the inhibition constant K is _i It was 15.25 mg/L. In conclusion, the sanguinarine extract has a certain inhibition effect on tyrosinase, is dose-dependent, and has reversible non-competitive inhibition type, so that the research provides a certain theoretical basis for the development and application of the sanguinarine as a tyrosinase inhibitor.

Test example 3 Effect of Panax ginseng C.A.Meyer extract on the synthesis of melanin in mouse B16 melanoma cells and the study of the mechanism thereof

1 background of the invention

The color of skin mainly depends on melanin produced by human body, and when the melanin is over-expressed and can not be metabolized in time, pigmentation symptoms such as color spots, chloasma and the like appear on the skin. Therefore, inhibiting or blocking the production of melanin is important for skin whitening. The mouse B16 melanoma cell is a tumor cell for screening drugs capable of inhibiting melanin synthesis, the melanin synthesis function of the tumor cell is basically consistent with that of a normal human melanocyte, and the cell strain is widely used as a test cell for determining the efficacy of a whitening and freckle-removing agent because the primary skin melanoma cell of a human body is very difficult to culture. The blood ginseng extract is used as an effector, the B16 melanoma cells are used as research objects, the influence of the blood ginseng extract on the proliferation activity, the intracellular tyrosinase activity and the melanin synthesis of the B16 melanoma cells is researched, and a certain theoretical basis is provided for the whitening and freckle removing effects of the blood ginseng extract.

2 method

2.1 preparation of solvent

2.1.1 preparation of blood Ginseng extract solution

Precisely weighing blood Ginseng radix extract, adding RPMI-1640 culture medium, ultrasonically dissolving, and diluting with culture medium to obtain sample solutions with concentrations of 2.5, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0 and 50.0 mg/L.

2.1.2 preparation of MTT solution

An appropriate amount of MTT was precisely weighed and prepared into a 5mg/mL MTT solution using 1 XPBS solution.

Preparation of 2.1.31% Triton X-100 solution

Accurately weighing an appropriate amount of Triton X-100, adding 1 XPBS solution (pH 7.4) to prepare 10% Triton X-100 solution as mother solution for standby, and diluting the mother solution with 1 XPBS solution to 1% Triton X-100 solution when in need.

2.2 culture of cells

Inoculating mouse B16 melanoma cell into RPMI-1640 culture solution containing 10% fetal calf serum under aseptic condition, standing at 37 deg.C and 5% CO ₂ And (4) carrying out constant-temperature culture in an incubator, and changing the culture solution for 1 to 2 days for 1 time.

2.3 Effect of Panax ginseng C.A.Meyer extract on the morphology of melanoma cells in mice B16

Collecting mouse B16 melanoma cells in logarithmic growth phase, digesting with 0.25% trypsin, and adjusting cell concentration to 5 × 10 ⁴ And (3) inoculating 100 mu L of the extract per mL of the extract into a 96-well plate, culturing in an incubator for 24h, removing the supernatant, adding blood ginseng extract solutions with the concentrations of 2.5, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0 and 50.0mg/L respectively, adding only RPMI-1640 culture solution with the same volume into a blank control group, setting 6 multiple wells in each group, culturing for 72h respectively, placing under an inverted microscope, observing the cell size, morphology, growth density, dendritic morphology, fusion state and the like of the control group and the experimental group, and photographing and recording.

2.4 Effect of Henry Ginseng extract on the inhibition of mouse B16 melanoma cell proliferation

Collecting mouse B16 melanoma cells in logarithmic growth phase, digesting with 0.25% trypsin, and adjusting cell concentration to 5 × 10 ⁴ Inoculating 100 μ L of the extract into 96-well plate per well, culturing in incubator for 24 hr, discarding supernatant, adding 2.5, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0 and 50.0mg/L of herba Polygoni Cymosi extract solution, adding equal volume of RPMI-1640 culture medium into blank control group, culturing for 24 hr, 48 hr and 72 hr, adding 20 μ L of MTT solution into each well, culturing at 37 deg.C and 5% CO for 24 hr ₂ Culturing for 4h at constant temperature in a cell incubator, discarding the supernatant, adding 150 mu L DMSO solution into each hole, shaking for 10min, and measuring the absorbance at 490nm of an enzyme-labeling instrument.

The calculation formula is as follows: inhibition (%) [1- (sample absorbance/blank absorbance) ] × 100%

2.5 Effect of Panax schinseng extract on the inhibition of tyrosinase Activity in mouse B16 melanoma cells

The cells were collected by digesting mouse B16 melanoma cells in logarithmic growth phase with 0.25% trypsin, and the cell concentration was adjusted to 1X 10 ⁵ each/mL, 100 μ L of cell suspension is inoculated into a 96-well plate, the plate is placed in an incubator to be cultured for 24h, then the supernatant is discarded, blood ginseng extract solutions with different concentrations (2.5, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0 and 50.0mg/L) are respectively added, only equal volume of RPMI-1640 culture medium is added into a blank control group, and 6 multiple wells are arranged in each group. Standing at 37 deg.C for 5% CO ₂ Respectively culturing in a constant temperature incubator for 24h, 48h and 72h, discarding the culture solution, washing with PBS solution for 3 times, adding 100 μ L of 1% Triton X-100 solution, freezing at-80 deg.C for 30min, thawing at room temperature, preheating at 37 deg.C, adding 100 μ L of 1mg/mL L-DOPA solution, reacting at 37 deg.C for 2h, and measuring absorbance at 475nm of an enzyme labeling instrument.

2.6 Effect of Panax schinseng extract on the inhibition of melanin synthesis in mouse B16 melanoma cells

0.25% of eggs for selection of melanoma cells of mice in the logarithmic growth phaseDigesting with white enzyme solution to collect cells, adjusting cell concentration to 1 × 10 ⁵ one/mL, inoculated in 6-well cell culture plates, 2mL per well, at 37 ℃ with 5% CO ₂ Continuously culturing for 24h in an incubator, discarding culture solution, respectively adding blood ginseng extract solutions with different concentrations (2.5, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0 and 50.0mg/L), only adding equal volume of RPMI-1640 culture medium to a blank control group, respectively treating for 24, 48 and 72h, washing for 3 times with PBS solution, digesting and collecting cells, centrifuging cell suspension at 1200rpm/min for 10min, then discarding supernatant, adding 1mL of 1mol/L NaOH solution containing 10% DMSO, carrying out water bath at 80 ℃ for 1h to crack cells and dissolve melanin in cells, respectively taking 200 mu L of supernatant, transferring to a 96-well cell culture plate, and measuring absorbance value at 405nm of an enzyme labeling instrument.

The calculation formula is as follows: inhibition (%) [ 1-sample (absorbance in wells/absorbance in blank control) ] × 100%

3 results of

3.1 Effect of Henry Ginseng extract on mouse B16 melanoma cell morphology results

As can be seen in FIGS. 18-19, the control cells grew well and were morphologically normal. When blood ginseng extract solutions with different mass concentrations (2.5, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0 and 50.0mg/L) are added, the cell morphology and the number change to different degrees, and when the concentration of the blood ginseng extract is less than 5mg/L, the cell morphology and the number change are not obvious; along with the increase of the mass concentration of the blood ginseng extract, the cell distribution begins to become sparse, the cell dendrites are reduced or disappear, and the cell dendrites cannot be fused with each other to form a net structure; when the concentration of the blood ginseng extract reaches 20mg/L, the number of cells is obviously reduced, and the change of cell morphology is obvious; when the concentration of the blood ginseng extract reaches 50mg/L, almost all cells die. Therefore, when the mass concentration of the panax sanguinea extract is less than 5mg/L, the cells can grow normally.

3.2 Effect of Panax ginseng C.A.Meyer extract on the inhibition of proliferation of melanoma cells in mouse B16

As shown in table 12 and fig. 20, the blood ginseng extracts have an inhibitory effect on cell proliferation compared to the control group after being applied to mouse B16 melanoma cells with different concentrations of the blood ginseng extracts. The inhibition rates of the blood ginseng extract at different mass concentrations and the same time and the inhibition rates of the blood ginseng extract at different mass concentrations and the same time on cell proliferation are compared and analyzed. The results show that the inhibition rate of the blood ginseng extract on the proliferation of mouse B16 melanoma cells is increased along with the increase of time within different time periods of the same mass concentration; the inhibition rate of the blood ginseng extract on the proliferation of the melanoma cells of the mouse B16 is increased along with the increase of the mass concentration under different mass concentrations in the same time. The experimental result shows that the panax sanguinea extract has stronger inhibition capacity on the proliferation of B16 melanoma cells, and the mass concentration of the panax sanguinea extract is increased along with the increase of time.

TABLE 12 influence of Panax schinseng extract on the proliferation inhibition rate of mouse B16 melanoma cells

Total Flavonoids concentration (mg/L)	24h	48h	72h
				2.5	3.20±1.26 ^aA	12.60±0.10 ^aB	20.94±0.51 ^aC
5	5.59±0.76 ^bA	25.03±0.57 ^bB	43.04±0.37 ^bC
				10	17.62±0.18 ^cA	41.02±1.75 ^cB	58.82±1.12 ^cC
15	30.65±0.46 ^dA	45.26±1.04 ^dB	70.80±0.16 ^dC
				20	40.41±0.53 ^eA	54.53±0.94 ^eB	73.09±1.35 ^eC
30	46.53±0.90 ^fA	62.84±0.20 ^fB	75.29±1.44 ^fC
				40	50.09±0.78 ^gA	69.61±0.69 ^gB	77.08±0.77 ^gC
50	56.63±1.01 ^hA	71.94±1.20 ^hB	78.13±1.11 ^gC

Note: a. b, c, d, e, f, g and h represent the difference of different sample concentrations at the same time, and letters represent that significant difference exists; A. b, C shows the difference of the concentration of the same sample at different times, and the difference of letters shows that there is a significant difference (P < 0.05).

3.3 Effect of Panax ginseng C.A.Meyer extract on the inhibition of tyrosinase Activity in mouse B16 melanoma cells

As shown in table 13 and fig. 21, when the sanguinary ginseng extract with different mass concentrations was applied to mouse B16 melanoma cells, the sanguinary ginseng extract had a significant inhibitory effect on the intracellular tyrosinase activity compared to the blank control group. Respectively comparing and analyzing the inhibition rate of different mass concentrations at the same time and the inhibition rate of the same mass concentration at different times. The results show that the inhibition rate of the blood ginseng extract on the tyrosinase activity in mouse B16 melanoma cells is increased along with the increase of time within different time periods of the same mass concentration; the inhibition rate of the blood ginseng extract on the tyrosinase activity in the mouse B16 melanoma cells at different mass concentrations in the same time is increased along with the increase of the mass concentration. The experimental result shows that the panax sanguinea extract has stronger inhibition capacity on tyrosinase activity in mouse B16 melanoma cells, and the tyrosinase activity is increased along with the increase of mass concentration and the increase of time.

TABLE 13 influence of Panax schinseng extract on the inhibition rate of tyrosinase activity in B16 melanoma cells

Note: a. b, c, d, e, f, g and h represent the difference of different sample concentrations at the same time, and letters represent that significant difference exists; A. b, C shows the difference of the concentration of the same sample at different times, and the difference of letters shows that the difference is significant (P < 0.05)

3.4 Effect of Panax ginseng C.A.Meyer extract on the inhibition of melanin synthesis in melanoma cells of mouse B16

As can be seen from table 14 and fig. 22, when the sanguinea root extract was applied to mouse B16 melanoma cells at different mass concentrations, it had a certain inhibitory effect on the formation of melanin in B16 melanoma cells. Respectively comparing and analyzing the inhibition rate of different mass concentrations at the same time and the inhibition rate of the same mass concentration at different times. The results show that the inhibition rate of the blood ginseng extract on the formation of melanin in mouse B16 melanoma cells is increased along with the increase of time within different time periods of the same mass concentration; the inhibition rate of the blood ginseng extract on the formation of melanin in mouse B16 melanoma cells under different mass concentrations in the same time is increased along with the increase of the mass concentration. The experimental result shows that the panax sanguinea extract has stronger inhibiting capability on the formation of melanin in mouse B16 melanoma cells, and is positively correlated with time and concentration.

TABLE 14 influence of Panax schinseng extract on the rate of inhibition of melanogenesis in B16 melanoma cells

Concentration of Total Flavonoids	24h	48h	72h
				2.5	3.93±1.96 ^aA	8.74±1.37 ^aB	12.20±2.38 ^aB
5	5.90±1.44 ^aA	13.11±0.62 ^bB	18.50±1.14 ^bC
				10	15.73±1.05 ^bA	21.43±1.13 ^cB	27.85±0.92 ^cC
15	21.10±0.47cA	28.65±1.93 ^dB	36.24±1.67 ^dC
				20	28.96±1.97 ^dA	37.33±0.50 ^eB	45.26±0.73 ^eC
30	43.51±0.52 ^eA	49.77±0.88 ^fB	57.49±1.08 ^fC
				40	47.84±0.66 ^fA	57.26±0.94 ^gB	67.72±0.54 ^gC
50	49.54±0.26 ^fA	62.13±0.19 ^hB	72.15±0.58 ^hC

4 conclusion and discussion

The skin whitening and freckle removing are hot points of attention of modern people, the requirements on cosmetics are higher and higher, and the pursuit of more natural, safer and efficient cosmetics becomes the current hot trend. The color of the skin is mainly determined by the content and distribution of skin pigments (melanin, carotene, etc.), and melanin is the most important determinant. Therefore, inhibiting the formation of melanin is one of the main ways of whitening and removing freckles. Because melanocytes cultured in vitro are normally slow to produce and have extremely limited proliferation capacity, and are easily polluted by keratinocytes and fibroblasts, the amount of the obtained melanocytes is small, and the clinical application of the melanocytes is limited. The mouse B16 melanoma cell is derived from the murine skin melanoma with high metastatic capacity, has higher similarity with human epidermal cells in gene composition, is easier to culture than the human epidermal melanoma cells, can be subjected to multiple passages and grow fast, and is a preferred cell model for testing the influence of a test object on the biological function of the melanocytes.

The experimental result shows that the panax sanguinea extract has certain inhibition effect on the proliferation of melanoma cells of mouse B16, the activity of tyrosinase in the cells and the synthesis of melanin. The cytomorphological results show that when the concentration of the blood ginseng extract is less than 5mg/L, the change of the cell morphology and the number of the cells is not obvious, and when the cells reach 50mg/L, the cells almost all die. The panax sanguinea extract has concentration and time dependence on the proliferation of mouse B16 melanoma cells, the activity of tyrosinase in cells and the inhibition effect of melanin synthesis. The ginseng extract can inhibit the proliferation of melanoma cells, the tyrosinase activity and the melanin synthesis.

Test example 4 response surface method for optimizing forming process of sanguinea ginseng extract gel

1 background of the invention

The gel is an external preparation prepared by taking a water-soluble polymer material as a main matrix and adding medicines, and belongs to a transdermal drug delivery system. The gel has the advantages of high drug loading, accurate dosage, good moisture retention, no sensitization and irritation, convenient use, comfort and the like. The blood ginseng extract can be dissolved in water, the molecular weight of the known components is less than 400, the research takes the blood ginseng extract as a raw material, the blood ginseng extract is prepared into blood ginseng extract gel, and the preparation process of the blood ginseng extract gel is optimized by adopting a response surface method to determine the optimal preparation process of the blood ginseng extract.

2 method

2.1 blood Ginseng extract gel matrix prophase screening

When selecting a hydrogel matrix, a matrix suitable for diffusion and extrusion should be selected. The gel matrix has moderate viscosity, good stability, no chemical change with main drug, no irritation to skin, and good drug release performance. The experiment carries out early experimental investigation on hydrogel matrix carbomer 940, carbomer 934, carbomer 941, sodium alginate and polyvinyl alcohol 124 (PVA-124). The polyvinyl alcohol (PVA-124) has high mechanical strength and high consistency, a layer of strong elastic film is easily formed on the surface of the substrate after the polyvinyl alcohol (PVA-124) is placed, and the mud rubbing phenomenon is easily caused after the polyvinyl alcohol (PVA-124) is coated. Sodium alginate has high consistency, is easy to form a film after being smeared, has a mud rubbing phenomenon, has high mechanical strength, forms gel with poor stability, is spongy after being placed for a period of time, is easy to dehydrate and dry, and is not suitable for being prepared into an easy-to-wash gel preparation. Carbomer is hygroscopic white powder, has slight odor and acidity, is easily soluble in water and glycerol, has high content of carboxyl group in molecule, is acidic after being dissolved in water, and can be neutralized with triethanolamine to make into gel preparation in medicine or cosmetic field. Carbomer 934 is stable under high viscosity conditions for thick gels, whereas carbomer 941 has a long rheology, high clarity, and slightly less viscosity when applied to the back of the hand. Carbomer 940 has short rheological property, high viscosity, medium clarity, uniform and fine smeared hand back, moderate viscosity, no mud rubbing phenomenon and no film forming phenomenon after being placed for a period of time, and the result of preliminary screening shows that carbomer 940 is ideal, so carbomer 940 is selected as a matrix.

2.2 selection of preservatives

Because the sanguinarine extract gel is a water-soluble gel, and is easy to mildew after being placed at room temperature for a long time, a proper amount of preservative is required to be added. The methyl paraben serving as a common preservative does not react with main drugs, does not separate out obviously, has no influence on the formation of the preparation and has obvious preservative effect. Therefore, 0.1g of methyl paraben was chosen as preservative in this experiment.

2.3 preparation of gel of blood ginseng extract

Weighing carbomer 940 according to the formula amount, scattering on the surface of a proper amount of glycerol, adding 50mL of deionized water, uniformly stirring, swelling at normal temperature for 24 hours, and then placing in a water bath at 85 ℃ for heating, stirring and dissolving. Weighing appropriate amount of sodium hyaluronate, allantoin and azone in sequence, stirring for dissolving for 1 hr, taking down, and cooling to obtain phase A. Weighing herba Macaranthi Cordatae extract powder, adding appropriate amount of solvent for dissolving, adding appropriate amount of propylene glycol and methyl hydroxybenzoate, and making into phase B.

Slowly adding phase B into phase A, stirring, adding appropriate amount of triethanolamine to adjust pH, adding deionized water to 100g, and stirring to obtain XUERENSHEN extract gel.

2.4 Single factor test investigation

2.4.1 examination of the amount of carbomer 940

Respectively weighing 0.1g, 0.2g, 0.3 g, 0.4g and 0.5g of carbomer 940 in a beaker, fixing other dosage, preparing according to the preparation method under item 2.3, taking the appearance, the spreadability and the viscosity as evaluation indexes, and investigating the influence of the dosage of carbomer 940 on gel forming.

2.4.2 examination of the amount of sodium hyaluronate

Fixing other dosage, respectively adding 0.02, 0.04, 0.06, 0.08 and 0.10g of sodium hyaluronate, preparing according to the preparation method under item 2.3, taking appearance, spreadability and viscosity as evaluation indexes, and investigating the influence of the dosage of the sodium hyaluronate on gel forming.

2.4.3 examination of the amount of Glycerol used

Respectively weighing 5g, 10g, 15g, 20 g and 25g of glycerol in a beaker, fixing other dosages, preparing according to the preparation method under item 2.3, and taking appearance, spreadability and viscosity as evaluation indexes to examine the influence of the glycerol dosage on gel forming.

2.4.4 examination of the amount of triethanolamine used

Fixing other dosage, respectively adding 0, 0.1, 0.2, 0.3 and 0.4mL of triethanolamine, preparing according to the preparation method under item 2.3, and taking appearance, spreadability and viscosity as evaluation indexes to examine the influence of the dosage of triethanolamine on gel forming.

2.4.5 investigation of propylene glycol dosage

The other dosage is fixed, 1, 3, 5, 7 and 9g of propylene glycol are respectively added, the preparation is carried out according to the preparation method under item 2.3, the appearance, the spreading property and the viscosity are used as evaluation indexes, and the influence of the dosage of the propylene glycol on gel forming is examined.

2.5 survey index determination

2.5.1 measurement of appearance Properties

The smoothness, transparency, color, fineness and the presence or absence of bubbles of the blood ginseng extract gel are observed by naked eyes, and the appearance of the blood ginseng extract gel is evaluated.

2.5.2 spreadability assay

0.2mL of the prepared blood ginseng extract gel is applied to a position of 4cm multiplied by 4cm on the back of the hand, and the evaluation is carried out according to the viscosity of the gel, whether the phenomenon of mud rubbing occurs or not and whether the gel is easy to apply or not.

2.5.3 viscosity determination

Keeping the prepared blood ginseng extract gel at the constant temperature of 25 +/-2 ℃, selecting a proper rotor by using an NDJ-5S numerical viscometer, immersing the gel into a beaker with the diameter of not less than 80mm, screwing the rotor into a screw, putting the rotor into a gel, making the scale of the rotor flush with the surface of liquid, starting an instrument for measurement, and recording the measured viscosity value.

2.6 design of response surface test

According to the results of the single-factor test, the dosage of carbomer, the dosage of glycerin and the dosage of triethanolamine have great influence on the formation of the sanguinary extract gel, and a response surface factor level table is designed by using the three factors, as shown in table 15. The evaluation indexes of viscosity, spreadability and appearance are shown in table 16, and the total score is appearance × 30% + spreadability × 30% + viscosity × 40%. Analyzing and designing the test by adopting Design Expert 8.0.6 system analysis software, and preferably selecting the optimal prescription proportion of the gel mask.

Table 15 level table for responding to surface factors

TABLE 16 blood Ginseng extract gel examination index scores

3 results

3.1 Single factor test results

3.1.1 amount of carbomer 940

The gel viscosity increased with increasing amounts of carbomer 940, as shown in fig. 23, but the color clarity and smoothness decreased. When the dosage is 0.1g or 0.2g, the prepared gel has too thin viscosity and does not form gel; when the dosage is 0.3 g and 0.4g, the prepared gel has moderate viscosity, can be uniformly coated on the back of the hand, is uniform and fine, and has no adhesive feeling; when the amount is 0.5g, the gel is too thick to be easily applied. Therefore, the proper amount of carbomer is 0.3-0.4 g.

3.1.2 examination of the amount of sodium hyaluronate

As shown in FIG. 24, the viscosity of the gel was not significantly affected and remained substantially stable as the amount of sodium hyaluronate was increased. And the fineness of the gel is increased along with the increase of the dosage, and when the dosage is 0.08-0.10 g, the fineness is the same, so that the dosage of the sodium hyaluronate is determined to be 0.10 g.

3.1.3 examination of Glycerol consumption

As shown in fig. 25, the viscosity and spreadability of the gel system did not significantly affect with increasing glycerol levels. The color and transparency of the gel prepared by using different amounts of glycerin have obvious influence, the color and the luster of the gel are yellow and have no transparent feeling when the amounts of the glycerin are 5.0g and 10.0g, and the color and the luster of the gel are red and transparent and have smooth surface when the amounts of the glycerin are 15.0 g and 20.0 g.

3.1.4 examination of triethanolamine amount

As shown in fig. 26, the amount of triethanolamine was increased and the viscosity of the system was increased. When the dosage is 0mL, the viscosity is low, the solution is in a solution state, and gel cannot be formed; when the dosage is 0.2mL, a thin gel can be formed, and the color is not transparent. When the dosage is 0.3mL or 0.4mL, the viscosity change of the system is small, the color and luster of the gel are transparent, and the viscosity is moderate.

3.1.5 propylene glycol dosage examination results

As can be seen from fig. 27, the gel viscosity does not change much with the increase of the amount of propylene glycol, the transparency of the gel is poor when the amount of propylene glycol is 1g or 3g, and the transparency and the spreading property are good when the amount of propylene glycol is 5g, 7g or 9g, the viscosity is moderate, and the moldability is good. Considering the experimental cost, 5g can be initially selected as the final dosage.

3.2 model building and analysis of variance results

The response surface analysis results are shown in table 17, and Design expert 8.0.6 software is adopted to analyze the table 18 data to obtain a quadratic polynomial regression model of the influence of 3 factors of the sanguinea root extract gel on the comprehensive evaluation score: y ═ 9.11+0.33A +0.59B + 0.019C-1.24 AB-1.71 AC-0.77 BC-2.10A ² －0.96B ² －1.35C ² . Obtaining a correlation coefficient R by calculation ² _Pred 0.9142, adjusting the decision coefficient R ² _Adj 0.9852, the model can account for changes in 98.52% response values. As can be seen from Table 18, the variation of the blood ginseng extract gel has a certain linear relationship with the selected factors of carbomer 940, glycerol and triethanolamine, and the gel under different conditions can be analyzed by using the model. The model P is 0.0001 < 0.0100, which shows that the model is very obvious, the mismatching item P is 0.0616 > 0.05 (is not obvious), which shows that the fitting degree of the experimental result and the model is good, the experimental error is small, and the relation between the comprehensive value of the sanguinarine extract gel and the factors of the amount of carbomer 940, the amount of glycerin and the amount of triethanolamine can be better reflected, so that the optimal preparation process of the sanguinarine extract gel can be presumed by using the mathematical model. The factor influencing the forming process of the sanguinea ginseng extract gel in the test can be presumed to be B & gt A & gt C according to the F value.

TABLE 17 response surface design and test

TABLE 18 response surface analysis of variance

Sources of variance	Sum of squares	Degree of freedom	Mean square	F value	P value	Significance of
							Model (model)	57.01	9	6.33	119.67	＜0.0001	High in significance
A	0.85	1	0.85	15.96	0.0052	Is remarkable in that
							B	2.75	1	2.75	51.59	0.0002	Is remarkable in that
C	2.812E-003	1	2.812E-003	0.053	0.8243
							AB	6.18	1	6.18	116.15	＜0.0001
AC	11.73	1	11.73	221.60	＜0.0001
							BC	2.37	1	2.73	44.80	0.0003
A ²	18.54	1	18.54	350.27	＜0.0001
							B ²	3.85	1	3.85	72.69	＜0.0001
C ²	7.69	1	7.69	145.18	＜0.0001
							Residual error	0.37	7	0.053
Missimilitude term	0.30	3	0.10	5.78	0.0616	Is not significant
							Pure error	0.069	4	0.017
Sum of	57.38	16

3.3 analysis of response surface results

According to a regression equation, sequentially fixing carbomer 940(A), glycerin (B) and triethanolamine (C) at an origin, drawing a 3D effect graph and a contour map of other two factors and a Y value, predicting the optimal prescription of the sanguinarine extract gel, and obtaining the influence of interaction of the factors on the comprehensive score of the sanguinarine extract gel (see fig. 28-30), wherein the circle in the contour map is an ellipse, so that the interaction is remarkable, and if the contour map is a circle, the interaction is not remarkable. In order to analyze the influence of two interactive factors on the sensory evaluation of the sanguin ginseng extract gel more intuitively, a Design-Expert 8.0.6 software is used for establishing a response surface according to a regression equation, and the inclination degree of the response surface reflects the influence degree of the interactive factors on a Y value, namely, the larger the inclination angle of the response surface is, the more the curved surface is, the larger the influence of the interactive influence on a comprehensive value is. From the 3D response surface curvature and contour plots, the terms AB, AC, BC interact significantly. Through analysis, the optimal formula proportion of the sanguinea root extract gel is 9400.4g of carbomer, 18.35g of glycerol and 0.29mL of triethanolamine, and the performance of the gel prepared by the formula is optimal.

3.4 addition of drugs

Accurately weighing 2.0g, 5.0g and 8.0g of XUERENSHEN extract respectively, adding appropriate amount of solvent, dissolving, slowly adding into blank gel matrix, and stirring to obtain medicated gel with drug loading of 2%, 5% and 8%. The optimal drug loading capacity is determined by observing the appearance and the spreadability of the drug. The results are shown in Table 19, and the optimum drug loading was determined to be 5% by comprehensive evaluation.

TABLE 19 drug Loading test results

Adding medicine quantity (g)	Appearance character	Spreadability
			2.0	The appearance is brownish red and glossy, but a small amount of bubbles are left	Has moderate viscosity and easy application
5.0	The appearance is bloody red, transparent and glossy, and the surface is smooth and fine	Moderate viscosity and easy application
			8.0	The appearance is dark red, transparent, the glossiness is general, and the surface is fine and smooth	Has slightly dilute viscosity and is easy to apply

3.5 optimal prescription verification

Weighing 0.4g of carbomer 940 according to a preferred optimal formula, scattering into 18.35g of glycerol, adding 50mL of deionized water, swelling for 24h, heating in a water bath at 85 ℃, stirring and dissolving, adding 0.1g of sodium hyaluronate, 0.15g of allantoin and 1g of azone, stirring for 1h to enable the mixture to be colloidal, taking down the mixture, and cooling to be used as a phase A for later use; weighing 5g of XUERENSHEN extract powder, dissolving with appropriate amount of solvent, adding 5g of propylene glycol and 0.1g of methyl hydroxybenzoate, and stirring to dissolve as phase B. Slowly adding phase B into phase A, stirring well, adding triethanolamine 0.29mL to adjust pH, adding deionized water to 100g, and stirring well to obtain the Xuezhen extract gel, wherein the prepared gel has fine and smooth feeling without particles, good transparency, moderate viscosity, easy spreading, pH of 6.30, and is a Xuezhong gel. The product diagram is shown in fig. 31.

4 conclusion and discussion

The gel is a novel external preparation, can be tightly adhered to the action part for a long time, and is comfortable to use. The gel can be added with single medicine extract and can be prepared into compound gel, namely the traditional compound extract is combined with the gel preparation technology, so that the gel is convenient to use, easy to accept and very wide in prospect. Glycerol also has moisture-keeping effect as plasticizer, and azone can improve curative effect as transdermal penetration enhancer. The optimal formula is obtained by analyzing by using Design Expert 8.0.6 software, wherein carbomer 940, glycerol and triethanolamine are preferably selected as response surface analysis software, the appearance, spreadability and viscosity are taken as comprehensive evaluation indexes: 9400.4g of carbomer, 18.35g of glycerol and 0.29mL of triethanolamine. The blood ginseng extract gel optimized by the response surface method has the advantages of simple preparation process, stable and feasible prescription and reliable method.

Claims

1. A preparation process of a sanguinea root extract gel is characterized by comprising the following steps: the method comprises the following steps:

(3) slowly adding phase B into phase A, stirring, adding 0.27-0.31 part triethanolamine to adjust pH, adding 95-105 parts deionized water, and stirring to obtain XUERENSHEN extract gel;

the preparation method of the blood ginseng extract powder in the step (2) comprises the following steps:

1) extracting radix Ginseng powder with 50-70% ethanol for 3 times (each for 1.5-2.5 hr), filtering the extractive solution, and recovering ethanol from the filtrate at 40-50 deg.C under reduced pressure in a rotary evaporator to obtain concentrated solution;

2) diluting the concentrated solution obtained in the step 1) with 0.8-1.5 times of water, extracting with 0.8-1.2 times of petroleum ether for 1-2 times, discarding the petroleum ether layer, continuously adding 0.8-1.2 times of ethyl acetate into the water layer, extracting for 5-7 times, discarding the water layer, concentrating the ethyl acetate extract under reduced pressure to recover ethyl acetate, and drying in a vacuum drying oven to obtain an ethyl acetate part extract;

3) pretreatment of macroporous adsorption resin: taking AB-8 type macroporous adsorbent resin, adding 2BV 95% ethanol, soaking for 24h to fully swell the resin, repeatedly washing with distilled water after filtering until no white turbidity and no ethanol smell exist, then respectively soaking with 2-5% hydrochloric acid solution and 2-5% NaOH solution for 2.5-3.5h, repeatedly washing with distilled water until the solution is neutral, finally preserving with distilled water, sealing and storing to obtain pretreated AB-8 type macroporous adsorbent resin;

4) preparation of a sample solution: taking the ethyl acetate part extract obtained in the step 2), adding distilled water for ultrasonic dissolution, and preparing a blood ginseng extract solution with the concentration of 5-5.5 mg/mL;

5) and (3) purification: taking 8-12g of the pretreated AB-8 type macroporous adsorption resin obtained in the step 3), carrying out wet column packing, slowly loading the column into a chromatographic column, taking 65-75mL4.7BV of the blood ginseng extract solution obtained in the step 4) with the pH value adjusted to 3.5-4.5, loading the sample at the volume flow of 0.8-1.2mL/min, carrying out impurity removal on the sample after adsorbing for 2.5-3.5h, eluting with 110-130mL of 65-75% ethanol of 8BV at the volume flow of 2.5-3.5mL/min, collecting the concentration of eluent, and drying to obtain the blood ginseng extract powder.

2. The preparation process of the sanguinarine extract gel according to claim 1, characterized in that: the method comprises the following steps:

(1) according to the weight parts, 0.4 part of carbomer 940 is scattered into 18.35 parts of glycerol, 50 parts of deionized water is added for swelling for 24 hours, the mixture is heated in a water bath at 85 ℃ and stirred for dissolving, 0.1 part of sodium hyaluronate, 0.15 part of allantoin and 1 part of azone are added, the mixture is stirred for 1 hour to form a colloid, and the colloid is cooled to room temperature after being taken down to obtain phase A;

3. The preparation process of the sanguinarine extract gel according to claim 1, characterized in that: the preparation method of the blood ginseng extract powder in the step (2) comprises the following steps:

1) weighing radix Ginseng Indici powder, extracting with 60% ethanol for 3 times, each for 2 hr, filtering the extractive solution, and recovering ethanol from the filtrate under reduced pressure at 45 deg.C in a rotary evaporator until no alcohol smell exists to obtain concentrated solution;

2) diluting the concentrated solution obtained in the step 1) by adding 1 time of water, extracting petroleum ether with the same volume for 1 time, removing a petroleum ether layer, continuously adding ethyl acetate with the same volume into a water layer for extracting for 6 times, removing a water layer, concentrating an ethyl acetate extract under reduced pressure, recovering ethyl acetate, and drying in a vacuum drying oven to obtain an ethyl acetate part extract;

3) pretreatment of macroporous adsorption resin: taking AB-8 type macroporous adsorption resin, adding 2BV 95% ethanol, soaking for 24h to fully swell the resin, repeatedly washing with distilled water after filtering until no white turbidity and no ethanol smell exist, then respectively soaking with 5% hydrochloric acid solution and 5% NaOH solution for 3h, repeatedly washing with distilled water until the solution is neutral, finally preserving with distilled water, sealing and storing to obtain pretreated AB-8 type macroporous adsorption resin;

4) preparation of a sample solution: taking the ethyl acetate part extract obtained in the step 2), adding a proper amount of distilled water, and ultrasonically dissolving to prepare a blood ginseng extract solution with the concentration of 5.38 mg/mL;

5) and (3) purification: precisely weighing 10.0g of the pretreated AB-8 type macroporous adsorption resin obtained in the step 3), filling the resin into a column by a wet method, slowly filling the resin into a chromatographic column, taking 70mL4.7BV of the blood ginseng extract solution obtained in the step 4) with the pH value being adjusted to be 4, sampling at the volume flow of 1mL/min, removing impurities by pure water after adsorbing for 3 hours, eluting at the volume flow of 3mL/min by using 120mL8BV 70% ethanol, collecting the eluent, and drying to obtain the blood ginseng extract powder.