CN116023423B - Ginsenoside Rk3 and preparation and application thereof in preparation of folliculitis medicines - Google Patents

Abstract

Ginsenoside Rk3 and the preparation and the application in preparing folliculitis medicines belong to the technical field of natural compound preparation. In order to solve the technical problems existing in the preparation of ginsenoside Rk3 in the prior art, the function of the ginsenoside Rk3 is researched, and the invention discloses a method for preparing high-purity ginsenoside Rk3. Compared with the prior art, the method provided by the invention reduces the separation and purification process by using macroporous resin and silica gel, reduces the production cost, does not pollute the environment, and is suitable for industrial production; the method adopts the cheap and easily available total saponins of the stems and leaves or fruits of the ginseng as the raw material, thereby promoting the development and utilization of the ginseng. The invention discovers that ginsenoside Rk3 has remarkable treatment effect on folliculitis.

Description

Ginsenoside Rk3 and preparation and application thereof in preparation of folliculitis medicines

Technical Field

The invention belongs to the technical field of natural compound preparation, and particularly relates to ginsenoside Rk3 and preparation and application thereof in preparation of folliculitis medicines.

Background

Ginseng (Panax ginseng C.A. Meyer) is the root of the perennial herb of Araliaceae, and is known from ancient times as "Baicaowang". Modern pharmacological researches have shown that the main active substance of ginseng is ginsenoside, and has the effects of regulating central nervous system and cardiovascular and cerebrovascular, improving digestive system, promoting metabolism, enhancing immunity, resisting aging, improving intelligence, etc.

Pharmacological studies show that the rare ginsenoside Rk3 has various pharmacological activities, such as anticancer, anti-inflammatory and immunity improving. However, the rare ginsenoside Rk3 content in the original ginseng is extremely low, and the chemical total synthesis cannot be realized by utilizing the existing scientific technology because the structure of the original ginseng is complex. Rare ginsenoside Rk3 can be produced by hydrolyzing common ginsenoside. The ginsenoside Rk3 is obtained by removing glucose at 21 st position C from common panaxatriol type saponin Rg1, converting into Rh1, and removing a molecule of water at 20 th position C from Rh 1. At present, the preparation method of rare ginsenoside Rk3 generally comprises the steps of firstly converting common saponins into ginsenoside Rk3 through hydrolysis such as acid, alkali, enzyme, microorganism and the like, and then obtaining high-purity ginsenoside Rk3 through conventional separation and purification methods such as macroporous resin, silica gel, preparative liquid chromatography and the like. However, the common hydrolysis method has the problems of complex reaction, more side reactions, difficult control of reaction conditions and low yield; however, the conventional separation and purification method has low extraction efficiency, and a large amount of ginsenoside Rk3 cannot be obtained. For example, CN201811301790.1 discloses a method for preparing ginsenoside Rk3 by using cordyceps seed liquid conversion: firstly preparing cordyceps sinensis seed liquid, then inoculating the cordyceps sinensis seed liquid to the sterilized raw ginseng medicinal material, culturing until mycelia grow fully, taking out the raw ginseng medicinal material with the fully grown mycelia, and drying to obtain a primary product containing ginseng rare saponin Rk3; extracting the primary product with ethanol under reflux, recovering solvent, volatilizing until no ethanol smell, suspending with water, extracting with ethyl acetate, and recovering solvent to obtain ethyl acetate layer extract; performing gradient elution by using a silica gel chromatographic column and dichloromethane-methanol with gradient concentration to obtain a part rich in ginsenoside Rk3; and then carrying out gradient elution by using gradient concentration methanol through an open ODS column to obtain a part rich in ginsenoside Rk3, and preparing a high-purity ginsenoside Rk3 monomer compound through a semi-prepared liquid phase of the part. CN201310107895.4 discloses a method for preparing ginsenoside Rk3 by steaming rhizome of pseudo-ginseng: extracting steamed Notoginseng radix rhizome with ethanol, mixing the extract with diatomite, granulating, oven drying, extracting with ethyl acetate to obtain concentrate, performing silica gel column chromatography, eluting with mixed solvent of dichloromethane and methanol to obtain ginsenoside Rk3 and Rh4 crude product; the crude product is prepared into a mixture of ginsenoside Rk3 and Rh4 through preparation liquid chromatography, and then the mixture of ginsenoside Rk3 and Rh4 is separated by high-speed countercurrent to obtain refined ginsenoside Rk3 and refined ginsenoside Rh4.

As can be seen from the above comparison, the following problems generally exist in the isolation of ginsenoside Rk3 at present: (1) The method combines acid-base or microbial conversion with purification modes such as silica gel, macroporous resin and the like, has complex process and long separation time, needs desalting treatment, is easy to cause resource waste and is not friendly to the environment; (2) The ginsenoside has a plurality of impurities after conversion, and the monomer Rk3 is separated by column chromatography, so that the separation cost is high and the environment is not friendly; is not suitable for industrialized mass production. Therefore, there is a need to explore a production method of high-purity ginsenoside Rk3, which is low in cost, environment-friendly and suitable for large-scale industrial production. It has been reported that rare ginsenosides can be obtained by degrading the total ginsenosides by steaming with amino acids. Amino acid is essential nutrient component of animal and plant, is basic material for constructing cell and repairing tissue of biological organism, and has certain biological activity, no toxic side effect and can be absorbed and degraded. Therefore, the selection of the proper amino acid as the catalyst has important significance for green, safe and efficient conversion of the rare ginsenoside.

Folliculitis is a disease produced by malasseziaMalassezia) Or staphylococcus aureus @ sStaphylococcus aureus) The acute suppurative inflammation caused by invasion of hair follicle and sebaceous glands is an initial miliaria of large follicular type, pustules are gradually formed, the pustules appear in batches and are not fused with each other, a small amount of pus blood can be discharged after the pustules are ruptured, no scar is left after healing, the pustules tend to relapse, and the pustules often last for weeks or months, and subjective pruritus or causalgia mainly invade the head and neck, and also see the four limbs, the axillary part, the pudendum and the like. Clinically, antibiotics are usually taken orally, and neomycin ointment, mupirocin ointment or iodine tincture are coated externally. However, no specific medicine exists at present, and antibiotics are widely used, so that the body generates medicine resistance, repeated attacks are repeated, the patient cannot recover for a long time, and great pain and inconvenience are brought to the patient. The antibacterial effect of traditional Chinese medicines has become a hotspot of the current antibacterial research due to the characteristics of high efficiency, low toxic and side effects and the like. The ginsenoside has strong activity in bacteriostasis and inhibition of bacterial biomembrane. The natural medicine is safe, reliable, cheap and easy to obtain,the method for searching and preventing folliculitis from natural Chinese herbal medicines has wide prospect.

Disclosure of Invention

In order to solve the technical problems of the prior art for preparing ginsenoside Rk3 and further research functions of the ginsenoside Rk3, the invention provides a preparation method of a ginsenoside Rk3 cyclodextrin inclusion compound, which comprises the following steps:

(1) Adding an aspartic acid aqueous solution with the pH of 1-5 into ginseng total saponins according to the feed liquid ratio of 1 g to 10 mL, uniformly stirring, carrying out water bath at 80-100 ℃ for 3-5 h, standing, cooling, centrifuging, and collecting supernatant;

(2) Adding cyclodextrin into the supernatant obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rk3 cyclodextrin clathrate; the adding amount of the cyclodextrin is 0.8-1.4 times of the total mass of the ginsenoside;

(3) Eluting the ginsenoside Rk3 cyclodextrin inclusion compound obtained in the step (2) by using absolute ethyl alcohol to obtain an ethanol eluent, concentrating, drying to obtain a crude product of the ginsenoside Rk3, dissolving the crude product of the ginsenoside Rk3 in the absolute ethyl alcohol at 60 ℃, adding 5% ethyl acetate, uniformly stirring, cooling and recrystallizing to obtain the ginsenoside Rk3.

Further defined, the ginseng total saponins in step (1) include ginseng stem and leaf total saponins, ginseng fruit total saponins, american ginseng stem and leaf total saponins, american ginseng fruit total saponins.

Further defined is that the aqueous aspartic acid solution of step (1) has a pH of 3.

Further defined, the water bath temperature of step (1) is 100 ℃.

Further defined, the water bath time of step (1) is 4 h.

Further defined, the adding amount of the cyclodextrin in the step (2) is 1.2 times of the total mass of the ginsenoside.

Further defined, step (2) the cyclodextrin includes an alpha cyclodextrin, a beta cyclodextrin, a gamma cyclodextrin.

The invention also provides the ginsenoside Rk3 obtained by the preparation method.

The invention also provides application of the ginsenoside Rk3 obtained by the preparation method in preparing medicines for treating folliculitis.

Further limited, the medicament is prepared by taking ginsenoside Rk3 as an active ingredient and matching auxiliary materials for medicaments.

Further defined, the medicament may act by oral or parenteral administration; the dosage forms of the medicine comprise tablets, capsules, powder, pills, granules, injections and patches.

The invention also provides a preparation method of the spray for treating folliculitis, wherein the spray is prepared by dissolving ginsenoside Rk3 in a mixed solution of Tween 80, propylene glycol and water, wherein the mass ratio of Tween 80, propylene glycol and water in the mixed solution is 1:1:18, and the concentration of the ginsenoside Rk3 in the mixed solution is 10 mg/mL.

The invention also provides the spray for treating folliculitis, which is prepared by the preparation method.

The invention has the beneficial effects that:

the invention takes the ginseng total saponins as raw materials, adopts a novel amino acid hydrolysis technology, and obtains the high-purity ginsenoside Rk3 by the characteristics that the ginsenoside Rk3 is included with cyclodextrin under a specific pH value condition and precipitated after the high-temperature degradation of the ginseng total saponins. The preparation method of the ginsenoside Rk3 reduces the process of separating and purifying by using macroporous resin and silica gel, reduces the production cost, has strong practical value, and is suitable for industrial production; the method is simple and efficient, does not pollute the environment, can realize industrialized obtainment of reaction substrates, can be purchased from the market, can be completely used for preparing ginsenoside Rk3, and adopts cheap and easily obtained total saponins of stems, leaves and fruits of ginseng as raw materials to promote development and utilization of ginseng plants. In addition, the ginsenoside Rk3 has remarkable treatment effect on folliculitis and has the advantage of small toxic and side effects.

Drawings

FIG. 1 is a graph showing the effect of 5 different amino acids on ginsenoside Rk3 content;

FIG. 2 is a graph showing the effect of pH of an amino acid solution on purity of ginsenoside Rk3;

FIG. 3 is a graph showing the effect of water bath temperature on purity of ginsenoside Rk3;

FIG. 4 is a graph showing the effect of water bath time on purity of ginsenoside Rk3;

FIG. 5 is a graph showing the effect of the mass ratio of beta-cyclodextrin to total saponins of ginseng stems and leaves on the yield of ginsenoside Rk3;

FIG. 6 is a HPLC chromatogram of ginsenoside Rk3 before and after hydrolysis and separation and purification of total saponins of stems and leaves of Ginseng radix in example 1; wherein a in fig. 6 is an HPLC chromatogram before total saponins of ginseng stem and leaf are hydrolyzed, b in fig. 6 is an HPLC chromatogram after total saponins of ginseng stem and leaf are hydrolyzed, and c in fig. 6 is an HPLC chromatogram of ginsenoside Rk3 after separation and purification;

FIG. 7 is a graph showing the results of experiments on the inhibition of ginsenoside Rk3 against Staphylococcus aureus and Malachillea; wherein a in fig. 7 is an inhibition graph of ginsenoside Rk3 on staphylococcus aureus, a is blank, b is positive control, c is ginsenoside Rk3; b in fig. 7 is a graph of inhibition of ginsenoside Rk3 on malassezia, a is blank, B is a positive control, c is ginsenoside Rk3;

FIG. 8 is a graph showing the effect of ginsenoside Rk3 on the level of the THP-1 cell inflammatory factor IL-1 beta protein mediated by Malachite bacteria;

FIG. 9 is a graph showing the effect of ginsenoside Rk3 on the level of THP-1 cell inflammatory factor IL-8 protein expression mediated by Malachite bacteria;

FIG. 10 is a graph showing the comparison of ERK protein expression levels in different groups and a WB graph; wherein, beta-actin in figure 10 is reference protein;

FIG. 11 is a graph showing comparison of JNK protein expression levels in different groups and a WB graph; wherein, beta-actin in figure 11 is reference protein;

FIG. 12 is a graph showing the comparison of p38 protein expression levels in different groups and a WB map; wherein, beta-actin in figure 12 is reference protein;

FIG. 13 is a graph showing comparison of the expression levels of extranuclear P-P65 and P65 proteins in different groups and a WB plot; wherein, beta-action in FIG. 13 is an internal reference protein;

FIG. 14 is a graph showing comparison of the expression levels of P-P65 and P65 proteins in nuclei in different groups and a WB plot; wherein, laminB in FIG. 14 is an internal reference protein.

Detailed Description

The present invention is further illustrated by the following examples and drawings, which are not intended to be limiting, but any modifications, equivalents, improvements, etc. within the spirit and principles of the present invention are intended to be included within the scope of the present invention.

The method for preparing ginsenoside Rk3 comprises the following steps:

(1) Weighing ginseng total saponins, adding an aspartic acid aqueous solution according to a feed liquid ratio of 1 g to 10 mL, stirring uniformly, carrying out water bath, standing and cooling, and centrifuging to collect supernatant;

(2) Adding cyclodextrin into the supernatant obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rk3 cyclodextrin clathrate;

(3) Eluting the ginsenoside Rk3 cyclodextrin inclusion compound obtained in the step (2) by using absolute ethyl alcohol to obtain an ethanol eluent, concentrating, drying to obtain a crude product of the ginsenoside Rk3, dissolving the crude product of the ginsenoside Rk3 in the absolute ethyl alcohol at 60 ℃, adding 5% ethyl acetate, uniformly stirring, cooling and recrystallizing to obtain the ginsenoside Rk3.

In order to obtain the ginsenoside Rk3 with higher purity, the invention optimizes the corresponding parameters in the method, wherein the total ginsenoside of the ginseng stems and leaves (the total content is about 80%) is selected in the optimization process, and the beta cyclodextrin is used for the ring paste selection, and the specific optimization process is as follows:

1. optimizing the type of amino acid in step (1):

five amino acids were selected, including two acidic amino acids aspartic acid and glutamic acid, and three basic amino acids arginine, histidine and lysine. The pH value of the amino acid aqueous solution is set to 3 for controlling the variable, the reaction water bath temperature is set to 100 ℃, the water bath time is set to 1 h, the adding amount of beta cyclodextrin is set to be the same as the mass of the total saponins of the ginseng stem and leaf, and the content of ginsenoside Rk3 in the crude product is detected by an HPLC method.

As shown in fig. 1, the conversion rate of 5 amino acids to rare ginsenoside Rk3 is obviously different (P < 0.05), and the conversion effects of arginine, lysine, histidine, glutamic acid and aspartic acid to rare ginsenoside Rk3 are sequentially enhanced; wherein, the conversion of the acidic amino acid to rare ginsenoside Rk3 is significantly higher than that of the basic amino acid (P < 0.05). Aspartic acid is the best conversion rate of rare ginsenoside Rk3. Aspartic acid was therefore ultimately selected as the best catalyst.

2. Optimizing the pH value of the aspartic acid aqueous solution in the step (1):

aspartic acid aqueous solutions (1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 and 6.0) with different pH values are prepared, the water bath temperature is set to 100 ℃ for a controlled variable, the water bath time is set to 1 h, the addition amount of beta cyclodextrin is set to be the same as the quality of total saponins of ginseng stems and leaves, and the content of ginsenoside Rk3 in the crude product is detected by an HPLC method.

As a result, as shown in FIG. 2, the pH of the aqueous aspartic acid solution in step (1) has a significant effect on the purity of the obtained crude ginsenoside Rk3. When the pH of the aspartic acid aqueous solution is 1-6, ginsenoside Rk3 can be obtained, but the content of the ginsenoside Rk3 is greatly different; when the pH of the aspartic acid aqueous solution is 1-5, the purity of the crude ginsenoside Rk3 is more than 40%; when the pH of the aspartic acid aqueous solution is 3, the purity of ginsenoside Rk3 is highest and is 93.2%. Thus, the pH of the aqueous aspartic acid solution in step (1) is selected to be 1-5, preferably 3.

3. The water bath temperature is optimized:

the water bath temperature was limited to 60 ℃, 70 ℃, 80 ℃,90 ℃, 100 ℃, 110 ℃, 120 ℃, the pH of the aqueous aspartic acid solution in step (1) was set to 3, the water bath time was set to 1 h, and the amount of beta-cyclodextrin added was set to be the same as the mass of total saponins of ginseng stem and leaf, respectively. And detecting the purity of ginsenoside Rk3 in the crude product by using an HPLC method.

As shown in FIG. 3, the water bath temperature has a significant effect on the purity of ginsenoside Rk3 in the resulting crude product. The purity of the ginsenoside Rk3 is gradually increased when the water bath temperature is increased from 60 ℃ to 90 ℃, and the purity of the ginsenoside Rk3 is basically stable and does not change obviously when the water bath temperature is increased from 90 ℃ to 100 ℃, wherein the purity of the ginsenoside Rk3 is the highest and is 95.8% when the water bath temperature is 100 ℃. Thus, the water bath temperature is selected to be 80℃to 100℃and preferably 100 ℃.

4. The water bath time is optimized:

the water bath times were set to 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, respectively, the pH of the aqueous aspartic acid solution in step (1) was set to 3 for the control variable, the water bath temperature was set to 100 ℃, and the amount of beta-cyclodextrin added was set to be the same as the mass of total saponins of ginseng stem and leaf. And detecting the purity of ginsenoside Rk3 in the crude product by using an HPLC method.

As shown in FIG. 4, the water bath time has a significant effect on the purity of ginsenoside Rk3 in the crude product obtained. The purity of ginsenoside Rk3 gradually increased when the water bath time was prolonged from 1 h to 4 h, and the purity of ginsenoside Rk3 was substantially stable and did not change significantly when the water bath time was prolonged from 4 h to 6 h, wherein the purity of ginsenoside Rk3 was highest at 94.2% when the water bath time was 4 h. Thus, a water bath time of 3 h-5 h, preferably 4 h, is selected.

5. The addition amount of cyclodextrin is optimized:

the addition amounts of beta cyclodextrin were set to 0.4, 0.6, 0.8, 1.0, 1.1, 1.2, 1.4 times the mass of total saponins of ginseng stem and leaf, respectively, the pH of the aqueous aspartic acid solution in step (1) was set to 3 for the control variable, the water bath temperature was set to 100 ℃, and the water bath time was set to 4 h. And detecting the yield of the crude ginsenoside Rk3 by using an HPLC method.

As shown in figure 5, the addition amount of beta cyclodextrin has a remarkable effect on the yield of the obtained crude ginsenoside Rk3. When the adding amount of beta cyclodextrin is increased from 0.4 times to 1.2 times of the total saponins of the ginseng stems and leaves, the yield of the ginsenoside Rk3 is gradually increased, when the adding amount of beta cyclodextrin is increased from 1.2 times to 1.4 times of the total saponins of the ginseng stems and leaves, the yield of the ginsenoside Rk3 is slowly increased, and when the adding amount of beta cyclodextrin reaches 1.2 times of the total saponins of the ginseng stems and leaves, the yield of the ginsenoside Rk3 is maximum and is 8.6%. Therefore, the addition amount of the beta cyclodextrin is selected to be 0.8-1.4 times, preferably 1.2 times of the total saponins of the ginseng stems and leaves.

Through the optimization process, the invention obtains the preparation method for obtaining the ginsenoside Rk3 with higher purity, which comprises the following steps:

(1) Adding aspartic acid water solution with pH of 1-5% according to feed liquid ratio of 1 g:10 mL, stirring, standing in water bath at 80-100deg.C for 3-5 h, cooling, centrifuging, and collecting supernatant;

(2) Adding cyclodextrin into the supernatant obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rk3 cyclodextrin clathrate; the adding amount of the cyclodextrin is 0.8-1.4 times of the total mass of the ginsenoside;

(3) Eluting the ginsenoside Rk3 cyclodextrin inclusion compound obtained in the step (2) by using absolute ethyl alcohol to obtain an ethanol eluent, concentrating, drying to obtain a crude product of the ginsenoside Rk3, dissolving the crude product of the ginsenoside Rk3 in the absolute ethyl alcohol at 60 ℃, adding 5% ethyl acetate, uniformly stirring, cooling and recrystallizing to obtain the ginsenoside Rk3.

The invention provides the following examples aiming at the optimized preparation method:

three parallel experiments were performed in each of the following examples

Example 1:

(1) Taking total saponins of ginseng stem and leaf, adding an aspartic acid water solution with pH of 5 according to a feed liquid ratio of 1 g to 10 mL by a UV method, stirring uniformly, carrying out water bath at 80 ℃ for 5 h, standing and cooling, and centrifugally collecting supernatant;

(2) Adding alpha cyclodextrin into the supernatant obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rk3 cyclodextrin clathrate; the addition amount of the alpha cyclodextrin is 1.4 times of the mass of the total saponins of the ginseng;

(3) Eluting the ginsenoside Rk3 cyclodextrin inclusion compound obtained in the step (2) by using absolute ethyl alcohol to obtain an ethanol eluent, concentrating, drying to obtain a crude product of the ginsenoside Rk3, dissolving the crude product of the ginsenoside Rk3 in the absolute ethyl alcohol at 60 ℃, adding 5% ethyl acetate, uniformly stirring, cooling and recrystallizing to obtain the ginsenoside Rk3. And (3) performing HPLC analysis on the three batches of Rk3 obtained after the recrystallization, wherein the purity is 92.3% -95.2%. In this example, the HPLC chromatogram of hydrolyzed total saponins of stem and leaf of Ginseng radix and the HPLC chromatogram of ginsenoside Rk3 after separation and purification are shown in FIG. 6.

Example 2:

(1) Taking total saponins of herba Herminii, adding aspartic acid water solution with pH of 1 according to the ratio of 1 g to 10 mL (UV method), stirring, standing in water bath at 90deg.C for 4 h, cooling, centrifuging, and collecting supernatant;

(2) Adding beta cyclodextrin into the supernatant obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rk3 cyclodextrin clathrate; the addition amount of the beta cyclodextrin is 1.0 times of the mass of the total saponins of the ginseng;

(3) Eluting the ginsenoside Rk3 cyclodextrin inclusion compound obtained in the step (2) by utilizing ethanol to obtain ethanol eluent, concentrating, drying to obtain a crude product of the ginsenoside Rk3, dissolving the crude product of the ginsenoside Rk3 in absolute ethanol at 60 ℃, adding 5% ethyl acetate, uniformly stirring, cooling and recrystallizing to obtain the ginsenoside Rk3. And (3) performing HPLC analysis on the three batches of Rk3 obtained after the recrystallization, wherein the purity is 94.2% -96.1%.

Example 3:

(1) Taking total saponins of American ginseng fruit, adding an aspartic acid water solution with pH of 2 according to a feed liquid ratio of 1 g to 10 mL (UV method), stirring uniformly, standing in a water bath of 3 h at 100 ℃, cooling, centrifuging, and collecting supernatant;

(2) Adding gamma cyclodextrin into the supernatant obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rk3 cyclodextrin clathrate; the addition amount of the gamma cyclodextrin is 1.2 times of the mass of the total saponins of the ginseng;

(3) Eluting the ginsenoside Rk3 cyclodextrin inclusion compound obtained in the step (2) by utilizing ethanol to obtain ethanol eluent, concentrating, drying to obtain a crude product of the ginsenoside Rk3, dissolving the crude product of the ginsenoside Rk3 in absolute ethanol at 60 ℃, adding 5% ethyl acetate, uniformly stirring, cooling and recrystallizing to obtain the ginsenoside Rk3. And (3) performing HPLC analysis on the three batches of Rk3 obtained after the recrystallization, wherein the purity is 94.5-97.1%.

Example 4:

(1) Taking total saponins of stems and leaves of American ginseng, adding an aspartic acid aqueous solution with pH of 3 according to a feed liquid ratio of 1 g to 10 mL (UV method), stirring uniformly, standing in a water bath of 3 h at 100 ℃, cooling, and centrifugally collecting supernatant;

(2) Adding gamma cyclodextrin into the supernatant obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rk3 cyclodextrin clathrate; the addition amount of the gamma cyclodextrin is 0.8 times of the mass of the total saponins of the ginseng;

(3) Eluting the ginsenoside Rk3 cyclodextrin inclusion compound obtained in the step (2) by using absolute ethyl alcohol to obtain an ethanol eluent, concentrating, drying to obtain a crude product of the ginsenoside Rk3, dissolving the crude product of the ginsenoside Rk3 in the absolute ethyl alcohol at 60 ℃, adding 5% ethyl acetate, uniformly stirring, cooling and recrystallizing to obtain the ginsenoside Rk3. And (3) performing HPLC analysis on the three batches of ginsenoside Rk3 obtained after recrystallization, wherein the purity is 94.1-98.1%.

Example 5: application of ginsenoside Rk3 in treatment of folliculitis

Antibacterial experiment of ginsenoside Rk3

Materials:

ginsenoside Rk3: obtained from example 4 with a purity of (97.5%);

staphylococcus aureus (ATCC 6538) and malassezia (ATCC 44344) were purchased from the beijing collection of microorganisms.

Nutrient broth medium was purchased from beijing obozhong biotechnology limited.

Ginsenoside Rk3 and gentamicin 10 mg are respectively weighed and dissolved in 10 mL water to prepare a sample solution with the concentration of 1 mg/mL. The sterilized round filter paper sheet (diameter 6 mm) is put into a sample solution to be soaked for 30 min, taken out and then put on weighing paper to be dried, and the weighing paper sheet is placed under an ultraviolet lamp. The staphylococcus aureus is absorbed by a pipetting gun to be 200 uL bacteria liquid and evenly coated on the surface of the culture medium; spreading the filter paper sheet on the surface of the culture medium in a quadrilateral shape by using tweezers; the petri dishes are placed in a biochemical constant temperature incubator at 37 ℃ for 24 h, the diameter of the inhibition zone is measured by a crisscross method, the diameter is measured for 3 times in parallel, and the average value is obtained.

The malassezia strain pathogenic bacteria are activated and cultured, a bacterial cake with the diameter of 6 mm is taken out from the edge of a fungus colony by a puncher, and the pathogenic bacterial cake is placed in the center of a PDA culture medium of 90 mm. Sterile filter paper sheets (phi=8 mm) immersed in the Rk3 solution were placed on PDA solid medium at 15 mm from the pathogenic bacteria cake, incubated at 26 ℃, 7 d, and the bacteriostatic distance (distance between the edge of pathogenic bacteria colony and the edge of sterile filter paper sheet) was measured to determine the bacteriostatic activity of each endophyte. 3 parallel controls were set up for each group of experiments, sterile water was used as a blank control, and gentamicin at equal concentrations was used as a positive control.

The calculation formula of the bacteriostasis rate is as follows: antibacterial ratio (%) = (control pathogen colony diameter-treated pathogen colony diameter)/control pathogen colony diameter x 100%

The results of the bacteriostasis experiments are shown in fig. 7, and the A in fig. 7 shows that the ginsenoside Rk3 has a strong inhibition effect on staphylococcus aureus, and the inhibition rate can reach 83.1%. As shown in the B in FIG. 7, ginsenoside Rk3 has a strong inhibition effect on malassezia, and the inhibition rate is up to 60.8%.

Application of ginsenoside Rk3 in preparation of folliculitis medicines

Experimental materials:

ginsenoside Rk3: obtained as in example 4 with a purity of (97.5%).

Human monocyte THP-1 cells: purchased from cell institute of academy of sciences of China.

Malassezia (ATCC 44344) was purchased from the beijing collection of microorganisms.

Reagent:

methanol; acetonitrile; fetal bovine serum, specialty grade of Ji Taiyi kemel, shanghai; MTT, shanghai wei aobio limited; RPMI1640 medium, jiangsu Kaiki Biotechnology Co., ltd; bulled beef granules, qingdao Niaoshui biotechnology Co., ltd; ground meat media, qingdao Nikki Biotechnology Co., ltd; human IL-1 beta kit, shanghai Ji Taiyi family Seai Co; human IL-8 ELISA kit, shanghai Ji Taiyi family Sai Bio Inc.; rabbit p-NF-kB antibody, signalway; rabbit-derived LaminB antibodies, boster; rabbit ERK antibodies, proteontech; rabbit p-ERK antibody, signalway; rabbit JNK antibody, proteontech; rabbit p-JNK antibody, signalway; rabbit p38 antibody, proteontech; rabbit P-P38 antibody, signalway; murine GAPDH antibodies, proteontech; FITC-goat anti-rabbit IgG, shanghai Weiao Biol.Co; SDS-PAGE gel configuration kit, shanghai WeiaobioLimited; 5 Xprotein loading buffer, shanghai WeiaobioLimited.

(1) Establishment of model of folliculitis

THP-1 cells were co-cultured with Malachite bacteria to stimulate monocytes to produce specific inflammatory responses, thereby establishing a folliculitis-related inflammatory model. The THP-1 cells are stimulated by living bacteria or heat-inactivated malassezia with different quantitative ratios to establish a folliculitis-related inflammation model, and the specific operation steps are as follows:

a. taking THP-1 cells in exponential growth phase, adjusting their concentration with new serum-free medium containing no antibiotics, and culturing with 5×10 ⁵ Density of/mL inoculated in sterile 96 well cell culture plates, 200 uL per well;

b. culturing Moraxella 3 d by centrifugation, washing with PBS buffer solution for 3 times, preparing bacterial suspension with PBS buffer solution, dividing the bacterial suspension into two parts, adding one part directly into 96-well plate in live bacteria form, inactivating the other part in hot water bath at 90deg.C for 30 min, adding the other part into the well plate in heat inactivated bacteria form, wherein the number ratio of bacteria to cells is 1:1, 10:1, and 100:1, each group of parallel, 3 wells, using PBS buffer solution without bacteria as blank, adding 5% CO at 37deg.C ₂ Incubating 24 h in an incubator;

c. after stimulation, cell supernatants from the well plates were collected by centrifugation.

(2) ELISA method for measuring cell inflammatory factors IL-1 beta and IL-8

THP-1 cells produce IL-1 beta and IL-8 specifically when they undergo an immune reaction, and therefore they are generally used as an index for screening inflammatory diseases. The THP-1 cells 4 h were pre-incubated with Rk3 at various concentrations, after which the cells 24 h were stimulated according to the stimulation conditions of the above-described inflammation model, and the cell culture supernatants were collected by centrifugation, and the changes in IL-1. Beta. And IL-8 content in the culture supernatants were immediately examined by ELISA.

As shown in fig. 8 and 9, malassezia induction caused THP-1 cells to produce inflammatory responses, and the secretion of extracellular inflammatory factors IL-1 beta and IL-8 was significantly increased (p < 0.01), i.e., proved successful in model establishment. When 100 uM ginsenoside Rk3 is used for pretreatment of cells, the secretion of inflammatory factors induced by malassezia is inhibited, and the inhibition rates of IL-1 beta and IL-8 are respectively as high as 90.4% and 94.5%; the results also show that ginsenoside Rk3 inhibits the secretion of extracellular inflammatory factors in a concentration-dependent manner.

(3) Western blot method for detecting MAPK and NF-kB signal channels and NF-kB nucleoplasm transfer proteins

When malassezia is incubated with THP-1 cells, downstream MAPK signaling pathways are stimulated and an immune inflammatory response is generated. At the same time, the downstream NF-kB signaling pathway is activated, thereby migrating from outside the nucleus to inside the nucleus and participating in the subsequent inflammatory response. Therefore, the influence of ginsenoside Rk3 on MAPK signal pathway, NF-kB signal pathway and NF-kB nuclear mass transfer in THP-1 cells induced by malassezia is detected in the experiment. The specific method comprises the following steps:

A. the cells were collected.

B. The method for extracting the total cell protein comprises the following steps:

and respectively adding an appropriate amount of IP lysate containing a mixed inhibitor (PMSF+phosphate inhibitor+protease inhibitor) into each sample, uniformly mixing, cracking for 10 min on ice, centrifuging for 10 min at 13000 rpm, and carefully sucking out the supernatant for later use.

C. The extraction method of the nuclear protein and the plasma protein comprises the following steps:

a. adding 1.5V mixed Buffer (Buffer A: buffer B=1:9) according to the cell pressure volume V in the sample, uniformly mixing, and reacting on ice for 30 min;

b.3000 Centrifuging at 4 ℃ for 10 min at rpm, wherein the supernatant is the cytoplasmic protein;

c. adding Buffer C with 1.2 times of cell pressure volume into the precipitate, mixing, reacting on ice for 45 min, and shaking vigorously for 15 s every 10 min;

d.12000 And (3) centrifuging at 4 ℃ for 30 min at rpm, wherein the supernatant is the nuclear protein.

(4) Western blotting

a. Adding a loading buffer solution (protein solution: loading buffer solution=4:1) into the extracted protein sample, and heating in a metal bath at 100 ℃ for 5 min to combine SDS with the protein;

b. the protein loading amount is set to be 10 ug, and the volume of a sample needing electrophoresis is calculated;

c. protein samples were separated by 10% SDS-PAGE electrophoresis, electrotransferred to PVDF membrane, and blocked with 5% BSA solution at 4 h;

d. incubation with primary antibody working solution overnight at 4 ℃, after 3 washes, incubation with secondary antibody working solution at 37 ℃ 1 h;

e. after washing 3 times, the color was developed by ECL color development, and the photograph was observed.

When malassezia is incubated with THP-1 cells, downstream MAPK signaling pathways are stimulated and an immune inflammatory response is generated. Therefore, the present experiment examined the effect of ginsenoside Rk3 on MAPK signaling pathway in malassezia-induced THP-1 cells. From FIGS. 10, 11 and 12, malassezia increased ERK, JNK and p38 protein phosphorylation of MAPK. When incubated with different concentrations of Rk3 (100 uM,50 uM and 10 uM), the levels of phosphorylation of these proteins were all found to be reduced and dose dependent. When 100 uM ginsenoside Rk3 acts on THP-1 cells, the phosphorylation levels of ERK, JNK and p38 proteins in the cells are inhibited, the inhibition rates of 75.8%, 57.5% and 53.2% of ginsenoside Rk3 on ERK and JNK proteins are respectively strong, and the phosphorylation degrees of JNK and p38 can be reduced to the level of an unused stimulation group. This suggests that ginsenoside Rk3 can inhibit malassezia-induced inflammatory responses by inhibiting the MAPK signaling pathway.

When malassezia induces THP-1 cells, the downstream NF-kB signaling pathway is activated, thereby transferring from extranuclear to nuclear and participating in subsequent inflammatory reactions. As shown in fig. 13 and 14, when high concentrations of ginsenoside Rk3 (100 uM) were used to act on THP-1 cells, intracellular P-P65 protein bands became concentrated and nuclear P-P65 protein bands became thin, indicating that nuclear penetration of phosphorylated P65 was significantly inhibited by the action of ginsenoside Rk3, and that the inhibition was dose-dependent, indicating that ginsenoside Rk3 could inhibit the occurrence of folliculitis by inhibiting nuclear translocation of NF-kB signaling pathway.

(5) Data analysis:

the SPSS 13.0 software is adopted, the data are expressed by mean plus or minus standard deviation (x+/-s), the results are all processed by t test, the comparison among groups adopts single factor analysis of variance, and the P at two sides is less than 0.05, so that the statistical significance is achieved.

(III) human body experiments

Ginsenoside Rk3: obtained from example 4 with a purity of (97.5%);

(1) Preparation of ginsenoside Rk3 spray

10 g ginsenoside Rk3, dissolved in 1L (5 % Tween 80,5% propylene glycol, 90% water) at the concentration of: 10 mg/mL, and filling the spray 30 mL/bottle.

(2) Administration of drugs

50 folliculitis patients were recruited as volunteers. Spraying on affected part, 2 times daily, and continuously filling in questionnaires according to cure condition of each symptom after 10 days, and then counting questionnaire results.

(3) Results

A score is given based on the treatment of each patient. 70% of the patients are completely cured, 25% of the symptoms are substantially eliminated, 4% of the patients are improved, and 1% of the patients are not affected. No side effects occur. The spray prepared from the ginsenoside Rk3 has obvious treatment effect on folliculitis and has no side effect.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (8)

1. A method for preparing ginsenoside Rk3, which is characterized by comprising the following steps:

(1) Adding an aspartic acid aqueous solution with the pH of 1-5 into ginseng total saponins according to the feed liquid ratio of 1 g to 10 mL, uniformly stirring, carrying out water bath at 80-100 ℃ for 3-5 h, standing, cooling, centrifuging, and collecting supernatant;

(2) Adding cyclodextrin into the supernatant obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rk3 cyclodextrin clathrate; the adding amount of the cyclodextrin is 0.8-1.4 times of the total mass of the ginsenoside;

(3) Eluting the ginsenoside Rk3 cyclodextrin inclusion compound obtained in the step (2) by using absolute ethyl alcohol to obtain an ethanol eluent, concentrating, drying to obtain a crude product of the ginsenoside Rk3, dissolving the crude product of the ginsenoside Rk3 in the absolute ethyl alcohol at 60 ℃, adding 5% ethyl acetate, uniformly stirring, cooling and recrystallizing to obtain the ginsenoside Rk3.

2. The method according to claim 1, wherein the ginseng total saponins in step (1) include ginseng stem and leaf total saponins, ginseng fruit total saponins, american ginseng stem and leaf total saponins, american ginseng fruit total saponins.

3. The method according to claim 1, wherein the aqueous aspartic acid solution in step (1) has a pH of 3.

4. The method of claim 1, wherein the water bath temperature in step (1) is 100 ℃ and the water bath time is 4 h.

5. The preparation method according to claim 1, wherein the cyclodextrin is added in an amount of 1.2 times the mass of total saponins of ginseng in step (2); the cyclodextrin is alpha cyclodextrin or beta cyclodextrin or gamma cyclodextrin.

6. An application method of ginsenoside Rk3, which is characterized in that the ginsenoside Rk3 obtained by the preparation method of any one of claims 1-5 is used for preparing medicines for treating folliculitis.

7. The method according to claim 6, wherein the medicament is prepared by using ginsenoside Rk3 as an active ingredient and adding auxiliary materials for medicaments.

8. The application of the spray containing the ginsenoside Rk3 in preparing the medicine for treating the folliculitis is characterized in that the spray is prepared by dissolving the ginsenoside Rk3 in a mixed solution of Tween 80, propylene glycol and water, wherein the mass ratio of the Tween 80 to the propylene glycol to the water in the mixed solution is 1:1:18, and the concentration of the ginsenoside Rk3 in the mixed solution is 10 mg/mL.

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