CN116120389B - Ginsenoside Rg5 and preparation and application thereof in preparing allergic rhinitis medicine - Google Patents

Ginsenoside Rg5 and preparation and application thereof in preparing allergic rhinitis medicine Download PDF

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CN116120389B
CN116120389B CN202310314680.3A CN202310314680A CN116120389B CN 116120389 B CN116120389 B CN 116120389B CN 202310314680 A CN202310314680 A CN 202310314680A CN 116120389 B CN116120389 B CN 116120389B
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ginsenoside
cyclodextrin
ginseng
total saponins
ethanol
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陈建波
李美佳
孙印石
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Institute Special Animal and Plant Sciences CAAS
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Abstract

Ginsenoside Rg5 and the application thereof in preparing allergic rhinitis medicines belong to the technical field of natural compound preparation. In order to solve the technical problems existing in the preparation of ginsenoside Rg5 in the prior art and further research on the functions of the ginsenoside Rg5, the invention discloses a method for preparing high-purity ginsenoside Rg5, which utilizes the characteristics that the ginsenoside Rg5 is included with cyclodextrin after the hydrolysis of total ginsenoside acid and is precipitated and separated out under specific acidic conditions. 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 total saponins of the stems, leaves or fruits of the ginseng are taken as raw materials, so that the development and the utilization of the ginseng are promoted. In addition, the ginsenoside Rg5 has remarkable treatment effect on allergic rhinitis and has the advantage of small toxic and side effects.

Description

Ginsenoside Rg5 and preparation and application thereof in preparing allergic rhinitis medicine
Technical Field
The invention belongs to the technical field of natural compound preparation, and particularly relates to ginsenoside Rg5 and preparation and application thereof in preparing medicines for treating allergic rhinitis.
Background
Ginsenoside Rg5 (ginsenoside Rg 5) is a tetracyclic triterpene saponin existing in Ginseng radix Rubri, and has very small content. Animal and human clinical trial studies show that Rg5 not only has remarkable effects in resisting cancer, improving lung inflammation, improving memory and the like, but also shows good safety (She Anqi. Research progress of ginsenoside Rg5. University of Shenyang medical science, 2020, 37,12 1140-300). Ginsenoside Rg5 is a rare saponin, has extremely low content in natural ginseng plants, and can be produced in a large amount after processing, wherein the mass percentage of the ginsenoside Rg5 is about 0.03-0.08%. Ginsenoside Rg5 can be produced by hydrolyzing ginsenoside. Common hydrolysis modes are acid, alkali, enzyme, microorganism and the like. Among them, acid hydrolysis is the most commonly used method for preparing ginsenoside Rg5, and researches show that the diols of ginsenoside Rb1, rb2, rc, rd are heated or partially hydrolyzed in acid environment to generate ginsenoside Rg3, and the ginsenoside Rg3 is further dehydrated at-OH at 20 position and-H at 22 position to form double bond to finally form ginsenoside Rg5. When the acid strength, hydrolysis temperature and hydrolysis time are not properly controlled, insufficient hydrolysis or excessive hydrolysis is liable to occur, and there is a problem that the yield is too low. The production technology is complex, the requirement is too high, the application and development of the ginsenoside Rg5 are limited, the resource waste is caused, the ginsenoside Rg5 with high purity is difficult to obtain, and the pharmacological activity research is also greatly limited.
For example: CN201710865039.3 discloses a method for preparing Rg5 from total saponins of ginseng. Reflux extracting the steamed ginseng plant material with solvent, subjecting the extractive solution to macroporous adsorbent resin column chromatography to obtain total saponins containing ginsenoside Rg5 and Rk1, subjecting the total saponins, steamed ginseng total saponins, notoginseng radix total saponins or steamed ginsenoside Rb1 to macroporous adsorbent resin column chromatography to obtain group saponins with total ginsenoside Rg5 and Rk1 content greater than 97%, and subjecting the group saponins to macroporous adsorbent resin column repeated column chromatography to obtain monomeric ginsenoside Rg5 and monomeric ginsenoside Rk1 with purity greater than 90%. CN201810333188.X discloses a preparation method of low-polarity rare ginsenoside Rg5/Rk1 and Rh3/Rk 2. Sequentially adding ethanol, sodium hydroxide and benzoyl peroxide into the stem and leaf extract of Araliaceae plant, introducing oxygen, heating the reaction liquid, concentrating the reaction liquid to paste by a rotary evaporator after the heating reaction is finished, adding distilled water until solid precipitate is released, filtering and collecting the solid precipitate, washing with water, drying to remove water, extracting with n-hexane to remove low-polarity substances, extracting with absolute ethanol, and evaporating the solvent to obtain rare ginsenoside. Ethyl acetate and n-hexane solvent were stirred into the silica gel column, ethyl acetate and n-hexane solution were washed for 3 retention volumes, the 2 nd and 3 rd retention volumes were collected Rh3 and Rk2, then a second ethyl acetate and n-hexane solution was used for 5:1 for 3 retention volumes, a total of 6 retention volumes were washed, and the 5 th and 6 th retention volumes were collected Rg5 and Rk1.
As can be seen from the comparison, the conventional separation and purification of ginsenoside Rg5 generally has the following problems: (1) The method combines acid conversion and macroporous resin purification, has complex process, long separation time, needs desalting treatment, easily causes resource waste and is not friendly to the environment; (2) The ginsenoside is converted mostly to obtain the whole Rg5, and impurities are contained, and the single Rg5 is separated by column chromatography such as silica gel, 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 Rg5, which is low in cost, environment-friendly and suitable for large-scale industrial production.
Allergic Rhinitis (AR) refers to a condition that a specific individual is stimulated by external antigens to release a large amount of histamine, which is a main inflammatory mediator mediated by IgE, so as to induce various immunocompetent cells, cytokines and other non-infectious inflammatory diseases of nasal mucosa, clinical symptoms mainly include sudden and repeated nasal itching, sneezing, nasal discharge and nasal obstruction, work, study and life of people are seriously affected, and the conventional antihistaminic medicines can relieve part of clinical symptoms, but have side effects of somnolence, hepatorenal toxicity and the like, and cannot solve essential problems. The related data show that the incidence of allergic rhinitis is wider, the incidence of allergic rhinitis is different in different areas throughout the world, the incidence of allergic rhinitis is up to 40% in some areas, and the industrialized degree of developed countries is relatively perfect and is influenced by industrialization, the incidence of allergic rhinitis is relatively higher, the allergic rhinitis gradually develops into a worldwide health problem, and the allergic rhinitis is widely paid attention to related scholars.
Drug therapy is one of the main treatment methods of allergic rhinitis at present. The commonly used western medicines for AR treatment comprise antihistamines, glucocorticoids, anti-leukotriene receptor medicines, mast cell membrane stabilizers and decongestion, but only can relieve partial clinical symptoms of patients and has large toxic and side effects. At present, a plurality of chemotherapeutic drugs are directly or indirectly derived from the purification of the Chinese herbal medicine components or are modified by taking the chemotherapeutic drugs as precursors to prepare the anti-allergic rhinitis drug with strong efficacy, small side effect, safety and low toxicity. Modern pharmacological studies have shown that astragalus has an effect of enhancing immune function against increases in capillary permeability caused by histamine and 5-hydroxytryptamine. Flos Magnoliae dispelling wind and relieving pain and freeing nose orifices, ancient people took Xin Yilie as a special drug for treating ear and nose, and modern researches also prove that volatile oil components contained in flos Magnoliae can effectively resist anaphylactic reaction, and it is easy to see that natural drugs are safe, reliable, cheap and easily available, and have wide prospects for preventing and treating allergic rhinitis from natural Chinese herbal medicines.
Disclosure of Invention
In order to solve the technical problems existing in the preparation of ginsenoside Rg5 in the prior art and further research functions of the ginsenoside Rg5, the invention provides a preparation method of the ginsenoside Rg5, which comprises the following steps:
(1) Adding 5-15% citric acid aqueous solution according to a feed liquid ratio of 1 g to 5 mL, uniformly stirring, and cooling in water bath at 80-100deg.C for 2-4 h at room temperature to obtain hydrolysate;
(2) Adding cyclodextrin into the hydrolysate obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rg5 cyclodextrin clathrate; the adding amount of the cyclodextrin is 0.8-1.0 times of the total mass of the ginsenoside;
(3) Eluting the ginsenoside Rg5 cyclodextrin inclusion compound obtained in the step (2) by using ethanol with the volume fraction of 95% -100%, obtaining ethanol eluent, concentrating, drying to obtain a ginsenoside Rg5 crude product, dissolving in ethanol with the volume fraction of 95% at 60 ℃, adding ethyl acetate to the ethanol with the volume fraction of 5%, stirring uniformly, cooling, and recrystallizing to obtain the refined ginsenoside Rg5.
The citric acid solution in the preparation method can be replaced by organic acid solution such as formic acid, acetic acid, acidic amino acid, malic acid, oxalic acid, lactic acid and the like with the same acidity or inorganic acid solution such as hydrochloric acid, sulfuric acid and the like with the same acidity.
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 citric acid solution of step (1) has a mass fraction of 10%.
Further defined, the water bath temperature of step (1) is 100 ℃.
Further defined, the water bath time of step (1) is 3 h.
Further defined, the cyclodextrin in the step (2) is added in an amount which is 1.0 times of the total mass of the ginseng saponins.
Further defined, step (2) the cyclodextrin includes an alpha cyclodextrin, a beta cyclodextrin, a gamma cyclodextrin.
The invention also provides the ginsenoside Rg5 obtained by the preparation method.
The invention also provides application of the ginsenoside Rg5 obtained by the preparation method in preparing medicines for treating allergic rhinitis.
Further limited, the medicament is prepared by taking ginsenoside Rg5 as an active ingredient and matching auxiliary materials which can be used for medicaments.
The invention has the beneficial effects that:
the invention adopts the ginseng total saponin as the raw material, and utilizes the characteristics that the ginsenoside Rg5 is included with cyclodextrin after acid hydrolysis and is precipitated and separated out under specific acidic conditions to obtain the high-purity ginsenoside Rg5. The preparation method of the ginsenoside Rg5 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 Rg5, 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 Rg5 has remarkable treatment effect on allergic rhinitis and has the advantage of small toxic and side effects.
Drawings
FIG. 1 is a graph showing the effect of mass fraction of aqueous citric acid solution on the purity of ginsenoside Rg5 in crude product in step (1);
FIG. 2 is a graph showing the effect of water bath temperature on the purity of ginsenoside Rg5 in crude product;
FIG. 3 is a graph showing the effect of water bath time on the purity of ginsenoside Rg5 in crude product;
FIG. 4 is a graph showing the effect of mass ratio of alpha cyclodextrin to total saponins of stem and leaf of Ginseng on the yield of ginsenoside Rg 5;
FIG. 5 is a HPLC chromatogram of ginsenoside Rg5 in the hydrolysate obtained after hydrolysis of total saponins of American ginseng fruit in example 3, and a HPLC chromatogram of purified ginsenoside Rg5 obtained; wherein A in FIG. 5 is HPLC chromatogram of ginsenoside Rg5 in the hydrolyzed solution obtained after hydrolysis, and B in FIG. 5 is HPLC chromatogram of refined ginsenoside Rg 5;
FIG. 6 is a graph showing the effect of ginsenoside Rg5 on inflammatory factor levels in mice; wherein, compared with the blank group, * p<0.05， ** p<0.01; compared with the model group # p<0.05， ## p<0.01；
FIG. 7 is a graph showing the effect of ginsenoside Rg5 on IgE, igG1 and IgG2a levels in mice; wherein, compared with the blank group, * p<0.05， ** p<0.01; compared with the model group # p<0.05， ## p<0.01；
FIG. 8 is a graph showing the effect of ginsenoside Rg5 on eosinophil and mast cell levels in mice; wherein, compared with the blank group, * p<0.05， ** p<0.01; compared with the model group # p<0.05， ## p<0.01；
FIG. 9 is a graph showing the evaluation of the effect of ginsenoside Rg5 capsule on treating allergic rhinitis; wherein, A in figure 9 is the overall effect evaluation diagram of the ginsenoside Rg5 capsule for treating allergic rhinitis, and B in figure 9 is the treatment effect diagram of the ginsenoside Rg5 capsule for treating each symptom of allergic rhinitis.
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 Rg5 comprises the following steps:
(1) Weighing ginseng total saponins, adding citric acid aqueous solution according to a feed liquid ratio of 1 g to 5 mL, stirring uniformly, and then carrying out water bath and cooling at room temperature to obtain hydrolysate;
(2) Adding cyclodextrin into the hydrolysate obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rg5 cyclodextrin clathrate;
(3) Eluting the ginsenoside Rg5 cyclodextrin inclusion compound obtained in the step (2) by using ethanol to obtain ethanol eluent, concentrating, drying to obtain a ginsenoside Rg5 crude product, dissolving the crude product in ethanol with the volume fraction of 95% at 60 ℃, adding ethyl acetate to the volume fraction of 5%, uniformly stirring, cooling, and recrystallizing to obtain the refined ginsenoside Rg5.
In order to obtain the ginsenoside Rg5 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 85%) is selected in the optimization process, and the alpha cyclodextrin is used for the ring paste selection, and the specific optimization process is as follows:
1. optimizing the mass fraction of the citric acid aqueous solution in the step (1):
different mass fractions of aqueous citric acid solutions (1%, 2%, 4%, 6%, 8%, 10%, 12%, 15%, 18%, 20%) were prepared, the water bath temperature was set to 100 ℃ for the control variable, the water bath time was set to 1 h, the addition amount of α -cyclodextrin was set to be the same as the mass of total saponins of ginseng stem and leaf, and the purity of crude ginsenoside Rg5 was detected by HPLC.
The results are shown in FIG. 1, and the mass fraction of the aqueous solution of citric acid in the step (1) has a significant effect on the purity of the obtained crude ginsenoside Rg5. When the mass fraction of the citric acid aqueous solution is 1% -20%, ginsenoside Rg5 can be obtained, but the purity of the ginsenoside Rg5 is greatly different; when the mass fraction of the aqueous solution of citric acid is 5% -15%, the purity of the ginsenoside Rg5 is higher, and when the mass fraction of the aqueous solution of citric acid is 10%, the purity of the obtained crude ginsenoside Rg5 is the highest and is 95%. Thus, the mass fraction of the aqueous citric acid solution in step (1) is selected to be 5% -15%, preferably 10%.
2. The water bath temperature is optimized:
the water bath temperature was limited to 60 ℃, 80 ℃, 90 ℃, 100 ℃, 110 ℃, 120 ℃ respectively, the mass fraction of the aqueous citric acid solution in step (1) was set to 10% as a control variable, the water bath time was set to 1 h, and the addition amount of the α -cyclodextrin was set to be the same as the mass of total saponins of ginseng stem and leaf. The purity of the crude ginsenoside Rg5 is detected by utilizing an HPLC method.
As shown in FIG. 2, the water bath temperature has a significant effect on the purity of the crude ginsenoside Rg5 obtained. When the water bath temperature is increased from 60 ℃ to 90 ℃, the purity of the ginsenoside Rg5 is gradually increased, and when the water bath temperature is increased from 90 ℃ to 100 ℃, the purity of the ginsenoside Rg5 is basically stable and the change is not obvious, wherein when the water bath temperature is 100 ℃, the purity of the ginsenoside Rg5 is the highest and is 95%. Thus, the water bath temperature is selected to be 80℃to 100℃and preferably 100 ℃.
3. The water bath time is optimized:
the water bath time was set to 0.5 h, 1 h, 2 h, 3 h,4 h, respectively, the mass fraction of the aqueous citric acid solution in step (1) was set to 10% for the control variable, the water bath temperature was set to 100 ℃, and the addition amount of the α -cyclodextrin was set to be the same as the mass of total saponins of ginseng stem and leaf. The purity of the crude ginsenoside Rg5 is detected by utilizing an HPLC method.
As shown in FIG. 3, the water bath time has a significant effect on the purity of the crude ginsenoside Rg5 obtained. When the water bath time is prolonged from 0.5 to h to 2 h, the purity of the ginsenoside Rg5 is gradually increased, when the water bath time is prolonged from 2 h to 3 h, the purity of the ginsenoside Rg5 is basically stable, and the change is not obvious, wherein when the water bath time is 3 h, the purity of the ginsenoside Rg5 is the highest and is 95%. Thus, a water bath time of 2 h-4 h, preferably 3 h, is selected.
4. The addition amount of cyclodextrin is optimized:
the addition amount of the alpha cyclodextrin is respectively set to be 0.4 times, 0.6 times, 0.8 times, 1.0 times, 1.1 times, 1.2 times and 1.4 times of the total saponins of the ginseng stems and leaves, the mass fraction of the aqueous solution of citric acid in the step (1) is set to be 10% for the control variable, the water bath temperature is set to be 100 ℃, and the water bath time is set to be 3 h. And detecting the yield of the crude ginsenoside Rg5 by using an HPLC method.
As shown in figure 4, the addition amount of the alpha cyclodextrin has a remarkable effect on the yield of the obtained crude ginsenoside Rg5. When the addition amount of the alpha cyclodextrin is increased from 0.4 times to 0.8 times of the total saponins of the ginseng stems and leaves, the yield of the ginsenoside Rg5 is gradually increased, when the addition amount of the alpha cyclodextrin is increased from 0.8 times to 1.0 times of the total saponins of the ginseng stems and leaves, the yield of the ginsenoside Rg5 is slowly increased, and when the addition amount of the alpha cyclodextrin reaches 1.0 times of the total saponins of the ginseng stems and leaves, the yield of the ginsenoside Rg5 is maximum and is 10%. Therefore, the addition amount of the alpha cyclodextrin is selected to be 0.8-1.0 times, preferably 1.0 times of the total saponins of the ginseng stems and leaves.
Through the optimization process, the preparation method for obtaining the ginsenoside Rg5 with higher purity is obtained, and specifically comprises the following steps:
(1) Adding 5-15% citric acid aqueous solution according to a feed liquid ratio of 1 g to 5 mL, uniformly stirring, and cooling in water bath at 80-100deg.C for 2-4 h at room temperature to obtain hydrolysate;
(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 Rg5 cyclodextrin clathrate; the adding amount of the cyclodextrin is 0.8-1.0 times of the total mass of the ginsenoside;
(3) Eluting the ginsenoside Rg5 cyclodextrin inclusion compound obtained in the step (2) by using ethanol with the volume fraction of 95% -100%, obtaining ethanol eluent, concentrating, drying to obtain a ginsenoside Rg5 crude product, dissolving in ethanol with the volume fraction of 95% at 60 ℃, adding ethyl acetate to the ethanol with the volume fraction of 5%, stirring uniformly, cooling, and recrystallizing to obtain the refined ginsenoside Rg5.
The invention provides the following examples aiming at the optimized preparation method:
example 1:
(1) Taking total saponins of ginseng stems and leaves, adding 5% citric acid aqueous solution according to the feed liquid ratio of 1 g to 5 mL by a UV method, uniformly stirring, and cooling in water bath 2 h at 80 ℃ at room temperature to obtain hydrolysate;
(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 Rg5 cyclodextrin clathrate; the addition amount of the alpha cyclodextrin is 0.8 times of the mass of the total saponins of the ginseng;
(3) Eluting the ginsenoside Rg5 cyclodextrin inclusion compound obtained in the step (2) by using ethanol with the volume fraction of 95%, obtaining ethanol eluent, concentrating, drying to obtain a ginsenoside Rg5 crude product, dissolving the crude product in ethanol with the volume fraction of 95% at 60 ℃, adding ethyl acetate to the ethanol with the volume fraction of 5%, uniformly stirring, cooling, and recrystallizing to obtain the refined ginsenoside Rg5. And (3) performing HPLC analysis on three batches of Rg5 obtained after the recrystallization, wherein the purity is 95.1% -98.1%.
Example 2:
(1) Taking ginseng fruit total saponins with the total content of about 85 percent (UV method), adding 15 percent citric acid aqueous solution according to the feed liquid ratio of 1 g to 5 mL, uniformly stirring, and cooling in water bath 4 h at 90 ℃ at room temperature to obtain hydrolysate;
(2) Adding beta cyclodextrin into the hydrolysate obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rg5 cyclodextrin clathrate; the addition amount of the beta cyclodextrin is 0.9 times of the mass of the total saponins of the ginseng;
(3) Eluting the ginsenoside Rg5 cyclodextrin inclusion compound obtained in the step (2) by using ethanol to obtain ethanol eluent, concentrating, drying to obtain a ginsenoside Rg5 crude product, dissolving the crude product in ethanol with the volume fraction of 95% at 60 ℃, adding ethyl acetate to the volume fraction of 5%, uniformly stirring, cooling, and recrystallizing to obtain the refined ginsenoside Rg5. And (3) performing HPLC analysis on three batches of Rg5 obtained after the recrystallization, wherein the purity is 96.1% -98.5%.
Example 3:
(1) Taking total saponins of American ginseng fruit, adding 10% citric acid aqueous solution according to the feed liquid ratio of 1 g to 5 mL, stirring uniformly, and cooling in water bath 3 h at 100 ℃ at room temperature to obtain hydrolysate;
(2) Adding beta cyclodextrin into the hydrolysate obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rg5 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 Rg5 cyclodextrin inclusion compound obtained in the step (2) by using ethanol to obtain ethanol eluent, concentrating, drying to obtain a ginsenoside Rg5 crude product, dissolving the crude product in ethanol with the volume fraction of 95% at 60 ℃, adding ethyl acetate to the volume fraction of 5%, uniformly stirring, cooling, and recrystallizing to obtain the refined ginsenoside Rg5. And (3) performing HPLC analysis on the three batches of Rg5 obtained after the recrystallization, wherein the purity is 96.6-98.1%. In this example, HPLC chromatogram of ginsenoside Rg5 in the hydrolysate obtained after hydrolysis of total saponins of American ginseng fruit is shown in FIG. 5.
Example 4:
(1) Taking total saponins of stems and leaves of American ginseng, adding 10% citric acid aqueous solution according to a feed liquid ratio of 1 g to 5 mL, stirring uniformly, and cooling in water bath 3 h at 100 ℃ at room temperature to obtain hydrolysate;
(2) Adding gamma cyclodextrin into the hydrolysate obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rg5 cyclodextrin clathrate; the addition amount of the gamma cyclodextrin is 1.0 times of the mass of the total saponins of the ginseng;
(3) Eluting the ginsenoside Rg5 cyclodextrin inclusion compound obtained in the step (2) by using ethanol with the volume fraction of 95%, obtaining ethanol eluent, concentrating, drying to obtain a ginsenoside Rg5 crude product, dissolving the crude product in ethanol with the volume fraction of 95% at 60 ℃, adding ethyl acetate to the ethanol with the volume fraction of 5%, uniformly stirring, cooling, and recrystallizing to obtain the refined ginsenoside Rg5. And (3) performing HPLC analysis on the three batches of Rg5 obtained after the recrystallization, wherein the purity is 96.2-98.4%.
Example 5: application of ginsenoside Rg5 in preparing medicine for treating allergic rhinitis
Experimental animals:
SPF-grade healthy BALB/c mice were purchased from Liaoning Long biotechnology Co., ltd, kept at 20-25℃and kept in the laboratory center of the national institute of agricultural sciences.
Reagent:
RPIM1640 medium; immunoglobulin E (IgE), immunoglobulin G1 (IgG 1), immunoglobulin G2a (IgG 2 a), interferon gamma (IFN-. Gamma.), interleukin 4 (IL-4), interleukin 12 (IL-12) and interleukin 13 (IL-13) ELISA kits were all purchased from Shanghai enzyme-linked biotechnology Co. OVA, alum and PBS working fluids were purchased from Shanghai Sigma-Aldrich; pancreatin and foetal calf serum were purchased from Shanghai Biyun biotechnology Co. Hematoxylin-eosin (HE) staining kit and flow cytometry kit were purchased from shanghai bi yunshan biotechnology limited.
Instrument:
microplate reader (iMark, bio-Rad company, usa); high-speed centrifuges (model QY-200-T8, shanghai Touo instruments, inc.), bench-top refrigerated high-capacity centrifuges, tissue microtomes (model RM2235, germany, leka), flow cytometry (FACSCalibur, product of BD Co., USA).
The experimental method of the mice comprises the following steps:
(1) Modeling Allergic Rhinitis (AR)
Mice were given peritoneal injections of 200. Mu.L (500 mg/L) of OVA solution sensitization solution on days 1,8 and 15 to induce systemic sensitization. On days 22-28, mice were given 20. Mu.L of nasal inhalation sensitization solution to induce AR. The behavior, morphology and feeding of the mice were observed from day 1 after the mice were modelled, and the mice were observed for nasal scratching, sneezing and runny nose after each challenge and scored. Wherein, scratching nose: the single claw gently flexes the nose for 1 minute, the double claw repeatedly flexes the nose for 2 minutes and the double claw rubs everywhere for 3 minutes; sneeze 1-3 times for 1 minute, 4-10 times for 2 minutes, and more than 11 times for 3 minutes; nasal discharge: nasal discharge flows to anterior nares 1 minute, nasal discharge exceeds anterior nares 2 minutes, nasal discharge is full of face 3 minutes; and if the total score exceeds 5, the molding is considered successful.
(2) Experimental grouping and administration method
The 60 mice were randomly divided into 6 groups: blank group, model group, positive drug group, ginsenoside Rg5 low dose experimental group, ginsenoside Rg5 medium dose experimental group and ginsenoside Rg5 high dose experimental group, each group comprises 10. Mice of the blank group were not treated, and mice of the model group, the positive drug group and the experimental group were constructed as AR mice according to the method in (1). After molding, low, medium and high dose experimental mice were orally gavaged 10, 20, 40 mg kg daily from day 16 to day 28 -1 Ginsenoside Rg5, 20 mg/kg of gastric loratadine is infused daily by positive drug group from 16 th day to 28 th day after modeling -1 The method comprises the steps of carrying out a first treatment on the surface of the Model group mice were perfused with 200 μl of physiological saline.
The ginsenoside Rg5 used in this example is the refined ginsenoside Rg5 obtained in example 2.
(3) Peripheral blood sampling and morphological observation of nasal mucosa tissue
On day 29, blood is taken from eyeballs of the mice, the blood is kept stand for 2-3 h, 850 Xg is centrifugated for 15 min at 4 ℃, the supernatant is transferred to a new tube, and the tube is put into a refrigerator at-80 ℃ for standby. After taking blood, the cervical vertebra of the mice is dislocation and lethal, the head is broken, the nasal mucosa of the respiratory area is rapidly opened, the spleen, the muscles and other accessory tissues are removed, then the mice are put into 10% formaldehyde for fixation, taken out after 1 week, conventionally tabletted, decalcified, trimmed, paraffin embedded, spread, copied, cut into pieces, and the nasal mucosa middle section mucosa HE is dyed, and the morphological observation of the nasal mucosa tissue is carried out.
(4) ELISA method for detecting peripheral blood inflammatory factor, OVA-IgE and OV-IgG level
Optical Density (OD) values were measured with a microplate reader at 450 nm and IFN-. Gamma., IL-4, IL-12, IL-13, igE, igG1 and IgG2a levels were calculated according to ELISA kit instructions. And detecting an OD value at a position of 450 nm by using an enzyme-labeled instrument, and finally accurately calculating the concentration of the target protein in the sample according to a standard curve.
(5) Detection of cellular levels by flow cytometry
After anesthetizing the mice, alveoli were lavaged with complete medium and bronchoalveolar lavage fluid (BALF) was collected. Centrifuging at 5000 rpm for 5 min, discarding supernatant, suspending cells with PBS, and concentrating at 2×10 6 Individual cells were incubated with Fc receptor blocker in 100 μl of buffer for 5 min to prevent specific binding. Eosinophil and mast cell levels were then determined according to flow cytometry kit instructions. Finally, the cellular level was measured with a flow cytometer.
(6) Statistical analysis method
SPSS 13.0 software was used. The data are expressed by mean plus or minus standard deviation (x+/-s), the results are all subjected to 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.
Experimental results:
(1) Influence of ginsenoside Rg5 on pathological conditions of mouse nose
By observing the morphology of nasal mucosa tissues of each group of mice, the following is found: the AR model group can be used for greatly shedding cilia structures, seriously edema tissues, severely congestion of nasal mucosa and eosinophil infiltration of the subcutaneous lamina propria of the mucosa; the high-dose experimental group of ginsenoside Rg5 can be used for obviously reducing the damage of epithelial cells, obviously reducing the phenomenon of tissue edema, relieving nasal mucosa congestion and obviously reducing eosinophil infiltration; in the medium dose experimental group, the damage of epithelial cells is partially reduced, and the congestion of nasal mucosa is reduced; the low dose experimental group can see severe edema of tissues, slightly reduced nasal mucosa congestion and eosinophil infiltration of the submucosa of the mucosa epithelium.
(2) Influence of ginsenoside Rg5 on mouse inflammatory factor level
Detection of IFN-gamma, IL-4, IL-12, IL-13 levels in peripheral blood of mice from each group revealed that: compared with the blank group, IFN-gamma and IL-12 in the mice of the model group are significantly reduced, the difference is statistically significant (P < 0.05), and the IL-4 and IL-13 levels are significantly increased, the difference is statistically significant (P < 0.05). The low, medium and high dose experimental groups showed increasing IFN-gamma and IL-12 levels and decreasing IL-4 and IL-13 levels in sequence compared to the model groups, and the extent of the effect exhibited concentration dependence. The high dose group levels were comparable to the positive control levels (see figure 6).
(3) Effect of ginsenoside Rg5 on mouse IgE, igG1 and IgG2a levels
Detection of IgE, igG1 and IgG2a levels in peripheral blood from mice of each group revealed that: compared with the blank group, the IgE and IgG1 levels in the mice of the model group are significantly increased, the IgG2a level is significantly reduced, and the differences are statistically significant (P < 0.05). The low, medium and high dose experimental groups showed a statistically significant difference (P < 0.05) in that IgE and IgG1 levels were sequentially decreased and IgG2a levels were sequentially increased, as compared to the model group, and the extent of the effect exhibited a concentration dependence (see FIG. 7).
(4) Influence of ginsenoside Rg5 on eosinophil and mast cell levels in mice
Detection of eosinophil and mast cell levels in each group of mice revealed that: compared with the blank group, the eosinophil and mast cell levels of the model group are significantly increased, and the differences are statistically significant (P < 0.05). The eosinophil and mast cell levels were sequentially reduced in the low, medium and high dose experimental groups compared to the model group, and the extent of the effect exhibited concentration dependence, with differences of statistical significance (P < 0.05) (see FIG. 8).
The above experimental results are combined to show that the ginsenoside Rg5 has therapeutic effect on allergic rhinitis.
(II) human body experiments
(1) Preparation of ginsenoside Rg5 capsule
100 g, ginsenoside Rg5 (purity is 98.5%) obtained in example 2, medicinal starch 1950 and g, mixing, encapsulating, and making into 4000 capsules, each with weight of 0.5g, each containing ginsenoside Rg5, 25 and mg.
(2) Administration of drugs
100 allergic rhinitis patients were collected as volunteers, whose major allergic symptoms were: nasal obstruction, runny nose, sneeze, itching nose, itching eyes, and the like. The administration mode of the patient is that 2 granules are orally taken each time, 2 times a day, and after 15 days of continuous administration, a questionnaire is filled in according to the improvement condition of each symptom, and then the questionnaire result is counted.
(3) Results
And giving a score according to the rhinitis improvement condition of each patient, and obtaining the overall improvement condition. 30% of patients were substantially or completely improved, 55% of patients were greatly improved, 13% of patients were improved, and only 2% of patients had no improvement in feedback (reflecting special or mid-drinking, night, etc. conditions of rhinitis) as shown in fig. 9. Meanwhile, except for 1 patient, slight ear swelling (special allergic constitution) occurred after taking, and the rest did not have any side effect. Of which 20 patients reflected a great improvement in sleep quality.
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 (7)

1. A method for preparing ginsenoside Rg5, which is characterized by comprising the following steps:
(1) Adding 5-15% citric acid aqueous solution according to a feed liquid ratio of 1 g to 5 mL, uniformly stirring, and cooling in water bath at 80-100deg.C for 2-4 h at room temperature to obtain hydrolysate;
(2) Adding cyclodextrin into the hydrolysate obtained in the step (1), stirring while adding, standing for 12 h, and centrifuging to collect precipitate to obtain ginsenoside Rg5 cyclodextrin clathrate; the adding amount of the cyclodextrin is 0.8-1.0 times of the total mass of the ginsenoside;
(3) Eluting the ginsenoside Rg5 cyclodextrin inclusion compound obtained in the step (2) by using ethanol with the volume fraction of 95% -100%, obtaining ethanol eluent, concentrating, drying to obtain a ginsenoside Rg5 crude product, dissolving in ethanol with the volume fraction of 95% at 60 ℃, adding ethyl acetate to the ethanol with the volume fraction of 5%, stirring uniformly, cooling, and recrystallizing to obtain the refined ginsenoside Rg5.
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 citric acid solution in step (1) has a mass fraction of 10%.
4. The method of claim 1, wherein the water bath temperature in step (1) is 100 ℃.
5. The method of claim 1, wherein the water bath time of step (1) is 3 h.
6. The preparation method according to claim 1, wherein the cyclodextrin is added in an amount of 1.0 times the mass of total saponins of ginseng in step (2).
7. The method of claim 1, wherein the cyclodextrin in step (2) is an alpha cyclodextrin or a beta cyclodextrin or a gamma cyclodextrin.
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