A saponin extraction method

Abstract

The application provides a saponin extraction method and relates to the technical field of natural product separation. The method comprises the following steps: extraction, water precipitation, resin adsorption, elution and decolorization treatment, active carbon decolorization and drying. The elution process comprises the following steps: first, washing with 0.1%-0.5% NaOH aqueous solution, then washing with water, finally, resolving with alcohol, and collecting the eluent of alcohol resolution; the volume ratio of the NaOH aqueous solution, water and alcohol is 1:7-8:2-3. The method extracts and separates active substances in the stems and leaves of Gypsophila oldhamiana Miq., and for the first time, saponin Rb1 and saponin Rd are separated and extracted from the stems and leaves of Gypsophila oldhamiana Miq., the total extraction content reaches 19.7%, and important basis is provided for the research on the active components of the stems and leaves of Gypsophila oldhamiana Miq.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural product separation, in particular to a saponin extraction method. BACKGROUND

[0002] Ginsenoside Rb1 is one of the most abundant saponins in ginseng, especially in American ginseng. It is the precursor of many other saponins, including ginsenoside Rd. Its main effects include improving learning and memory, anti-cerebral ischemia-reperfusion injury, sedation, cardiovascular protection, anti-fatigue, antioxidant and anti-aging, etc. Ginsenoside Rd content is usually lower than Rb1, but it is metabolized from Rb1 in vivo. In the cerebral ischemia model, Rd shows stronger protective effect than Rb1, and can more effectively reduce the infarction area, which is a drug for clinical treatment of acute cerebral infarction. In addition, ginsenoside Rd is also a potent natural anti-inflammatory agent, and has analgesic effect.

[0003] Ginsenoside Rb1 mainly exists in the roots and flower buds of ginseng, American ginseng and sanchi, and the leaves of gynostemma pentaphyllum; ginsenoside Rd mainly exists in the leaves, stems, flower buds and seeds of ginseng, American ginseng and sanchi, and the leaves of gynostemma pentaphyllum and the aboveground stem and leaf parts of pearl ginseng.

[0004] Campanumoea lancifolia (Roxb.), also known as long-leafed campanumoea, spider fruit, etc., is a perennial herb of Campanumoea lancifolia (Roxb.) in Campanulaceae. At present, the research on the stems and leaves of Campanumoea lancifolia is less, and most of the research is still focused on the roots and fruits of Campanumoea lancifolia. The stems and leaves are regarded as non-medicinal parts. The roots are mainly dug, and the stems and leaves are often discarded. However, the stems and leaves of Campanumoea lancifolia, as an important part of Campanumoea lancifolia, have a yield much higher than that of roots and fruits, and have important research prospects. SUMMARY

[0005] The purpose of the present application is to provide a saponin extraction method, which separates and extracts ginsenoside Rb1 and ginsenoside Rd from the stems and leaves of Campanumoea lancifolia for the first time, with a total extraction content of 19.7%, providing an important basis for the research on the active ingredients of the stems and leaves of Campanumoea lancifolia.

[0006] A saponin extraction method, comprising the following contents:

[0007] S100, crushing the plant stems and leaves and placing them in a multifunctional extraction tank, extracting three times with alcohol hot reflux, filtering, combining the filtrates and concentrating under reduced pressure to obtain an extract;

[0008] S200, placing the above extract in a settling tank, adding water according to a solid-liquid ratio of 1:2-5, stirring, dissolving, settling, filtering to obtain a filtrate; passing the filtrate through a macroporous adsorption resin column to elute and obtain an eluate;

[0009] In step S200, the elution process includes the following: first, flushing with 0.1%~0.5% NaOH aqueous solution, then flushing with water, and finally resolving with alcohol, collecting the alcohol resolution eluent, the flow rate of the elution process is 50 mL / min; the volume ratio of NaOH aqueous solution, water and alcohol is 1:7~8:2~3;

[0010] The alcohol includes one or both of methanol or ethanol, and the volume fraction of the alcohol is 50%~80%;

[0011] The macroporous adsorption resin column is a non-polar macroporous adsorption resin column, and a polystyrene type ball column can also be used.

[0012] S300, after the above eluent is concentrated, decolorization resin column is used for decolorization treatment to obtain a decolorization liquid, and the decolorization liquid is concentrated to obtain a paste;

[0013] S400, after the above paste is dissolved in alcohol, activated carbon is added for decolorization, filtration, concentration and drying to obtain an extract;

[0014] S500, the above extract is passed through a chromatographic silica gel column, chloroform-methanol-water is used as an eluent, thin layer chromatography is used to detect the flow portion, the flow portion is collected, and two crude products of compound 1 and compound 2 are obtained;

[0015] S600, the above two crude products are respectively passed through a C18 medium pressure column, methanol-water is used as an eluent for purification, the flow portion is collected, concentrated and dried, and after identification, ginsenoside Rb1 and ginsenoside Rd are obtained.

[0016] The present application uses red fruit ginseng stems and leaves as raw materials, and extracts saponins through alcohol hot reflux, further removes filter residues by water precipitation on the obtained filtrate, first adsorbs saponins onto resin, then washes off components (sugar, protein, starch, gum, etc.) that cannot be adsorbed by the resin with alkaline water and water, and then elutes active components from the resin with methanol or ethanol with a volume fraction of 60%~80%; then, through the combination of resin and activated carbon, decolorization is performed to obtain a red fruit ginseng extract; the red fruit ginseng extract is separated and purified by using a chromatographic silica gel column combined with thin layer chromatography to obtain pure products of compound 1 and compound 2, and through identification, compound 1 is ginsenoside Rb1 and compound 2 is ginsenoside Rd. The present application first extracts ginsenoside Rb1 and ginsenoside Rd from red fruit ginseng stems and leaves, and through high performance liquid chromatography, it is found that the content of ginsenoside Rb1 in the red fruit ginseng extract reaches 13.4%, the content of ginsenoside Rd reaches 6.31%, and the total content of saponins reaches 19.71%, which provides an important basis for the study of active components of red fruit ginseng stems and leaves, provides a new raw material source for the extraction of natural products of ginsenoside Rb1 and Rd, and improves the economic value of red fruit ginseng stems and leaves.

[0017] The inventors found that the yield of the red ginseng extract is related to the type of solvent used for extraction. In step S100, when a mixed alcohol of methanol and ethanol is selected, the yield of the red ginseng extract is significantly higher than that when methanol or ethanol alone is used as the extraction agent, and when the proportion of methanol in the mixed alcohol is higher than that of ethanol, the yield is the best. The inventors speculate that the pH of the methanol-ethanol mixed solution may be more matched with the stable pH of saponins, thereby reducing the hydrolysis of saponins under acidic or basic conditions. In addition, the molecule of methanol is smaller, and it is easier to quickly penetrate the cell membrane micropore. Fast penetration of methanol reduces extraction time and reduces the degradation of heat-sensitive components. When the proportion of methanol is high, fast extraction may dominate.

[0018] When mixed alcohol is used for extraction, the yield of compound 1 obtained in S500 is significantly higher than that when methanol or ethanol alone is used, and the yield of compound 2 obtained is lower than that when ethanol alone is used as the extraction agent. The inventors speculate that the mixed alcohol may form a microenvironment, significantly increasing the solubility of compound 1; or the optimal solubility parameter of compound 1 may be between methanol and ethanol, and the polarity range of the mixed alcohol is more matched with its solubility requirement. The mixed alcohol may destroy the specific interaction between compound 2 and ethanol, such as hydrogen bond or van der Waals force, resulting in a decrease in the extraction yield of compound 2 in the mixed alcohol extraction agent.

[0019] The D941 decolorizing resin used in the present application has high selective adsorption capacity for macromolecular pigments (such as chlorophyll, polyphenol polymer), but has weak adsorption for saponin components (such as Rb1, Rd), which can preferentially remove most of the pigments and reduce the processing load of the subsequent activated carbon. The resin decolorization conditions are mild, avoiding the hydrolysis or isomerization of saponins under high temperature or strong acid and alkali environment. Activated carbon (especially needle grade) has strong adsorption capacity for small molecular pigments, tannins, sugars and other residual impurities, which can make up for the insufficient removal of small molecular impurities by resin decolorization. The staged decolorization can significantly reduce the residual pigments in the final product. The present application realizes deep decolorization by selective adsorption in stages, while maximizing the retention of saponins.

[0020] Further, in step S100, the method of alcohol hot reflux extraction comprises: adding 9-11 times the amount of alcohol, hot refluxing for 1.5-2.5 h, and filtering to obtain a first filtrate; adding 7-9 times the amount of alcohol to the obtained filter residue, hot refluxing for 1.5-2.5 h, and filtering to obtain a second filtrate; adding 5-7 times the amount of alcohol to the obtained filter residue, hot refluxing for 1.5-2.5 h, and filtering to obtain a third filtrate, and combining the three obtained filtrates.

[0021] The present application adopts the way of gradient decreasing ethanol dosage to perform three times of hot reflux dynamic extraction on the stems and leaves of Hongguo Ginseng. The first extraction adopts 4-6 times of alcohol to fully dissolve most of saponins, and the subsequent alcohol dosage is gradually decreased to extract residual components, thereby avoiding excessive solvent used at one time to cause subsequent concentration burden, effectively saving the amount of ethanol, and being suitable for industrial large-scale extraction. Compared with single extraction, the multi-time extraction can reduce the mass transfer resistance and improve the diffusion efficiency of saponins from plant cells to solvent, thereby improving the total extraction rate of saponins.

[0022] Further, in S100, the alcohol includes methanol or ethanol, and the volume fraction of the alcohol is 50%-75%; the solid-liquid ratio of the stems and leaves of Hongguo Ginseng to alcohol is 1:5-11 during each extraction; and the temperature of the hot reflux is 65-85 DEG C.

[0023] Further, in S300, the decolorizing resin column is a D941 decolorizing resin column, the flow rate is 30-50 mL / min, and the eluent is ethanol with a volume fraction of 50%-80%.

[0024] Further, in S400, the method for decolorizing with activated carbon includes: adding alcohol to dilute the paste to 10-15 times of volume, adding needle activated carbon, heating and refluxing for 1.5-2.5 h, and then standing and filtering to obtain Hongguo Ginseng extract.

[0025] The alcohol includes methanol or ethanol, and the volume fraction is 30%-100%, and the mass ratio of the needle activated carbon to the extract is 1:8-12.

[0026] The method for decolorizing with activated carbon in the present application is performed by the combined way of alcohol dilution-activated carbon adsorption-heating boiling, which can significantly improve the product purity: the alcohol is diluted by 30%-100% to reduce the solution polarity, promote the hydrophobic pigments (such as polyphenols and caramel pigments) to be preferentially adsorbed by activated carbon, improve the solubility of saponin components, prevent saponins from being precipitated due to excessive concentration, and promote the release of impurities; and the removal of impurities with different polarity can be adapted. The activated carbon physically adsorbs pigment molecules through the large specific surface area and microporous structure, and the oxygen-containing functional groups on the surface of the activated carbon can also combine with the polar impurities to adsorb and remove the impurities. High-temperature boiling improves the molecular thermal motion speed, accelerates the diffusion of pigments to the interstices of activated carbon, and also decomposes part of colloidal substances to promote the release of pigments and improve the adsorption capacity.

[0027] Further, in S400, the filtration is performed by using a polypropylene filter membrane with a membrane pore size of 5-10 μm, a filtration pressure of 0.1-0.3 MPa, and a filtration temperature of 60-80 DEG C, and the filter cake is washed 3-5 times with 50%-70% ethanol.

[0028] The polypropylene filter membrane has a pore size of 5-10 mu m, can effectively intercept the needle active carbon particles and macromolecular polymer impurities, low pressure filtration can avoid filter membrane deformation, and reduce the penetration ability of active carbon particles, and heat filtration is beneficial to reduce liquid viscosity, improve flow rate, and prevent target component saponin from being precipitated at low temperature to block the filter membrane. The specific method of ethanol washing is: 70% ethanol washing 1-2 times, 60% ethanol washing 1-2 times, and 50% ethanol washing 1 time. The 70% ethanol washing can remove the fat-soluble impurities, and the 50% ethanol washing can wash the water-soluble impurities.

[0029] Further, in S500, the volume ratio of chloroform, methanol and water in the eluent is 14-16:1:0.1.

[0030] Further, in S600, the volume ratio of methanol and water in the eluent is 3.0-3.5:10, and thin layer chromatography is used to detect the flow fraction.

[0031] Further, in S500 and S600, the lower layer solution of chloroform-ethyl acetate-methanol-water after standing at 10 DEG C is used as the developing agent for thin layer chromatography detection, and the volume ratio of chloroform, ethyl acetate, methanol and water is 15:40:22:10, and the color developing agent includes 10% sulfuric acid ethanol solution or anisaldehyde-sulfuric acid reagent.

[0032] Advantages of the application

[0033] 1. The application separates and extracts saponin Rb1 and saponin Rd from the stems and leaves of red fruit ginseng, and the total extraction content reaches 19.7%, which provides an important basis for the study of the active components of the stems and leaves of red fruit ginseng, provides a new raw material source for the extraction of natural products of ginsenoside Rb1 and Rd, and improves the economic value of the stems and leaves of red fruit ginseng.

[0034] 2. The application uses methanol / ethanol hot reflux to extract saponin from the stems and leaves of red fruit ginseng, which is simple, efficient and suitable for industrial production and application.

[0035] The molecular formula of ginsenoside Rb1 is C 54 H 92 O 23 , the molecular weight is 1108.6, and the chemical name is 3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl]-20-O-[β-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl]-dammar-24-ene-3β,12β,20S-diol.

[0036] The molecular formula of ginsenoside Rd is C 48 H 82 O19 Molecular weight: 963.15, Chemical name: 3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl]-20-O-β-D-glucopyranosyl-dammar-24-ene-3β, 12β, 20S-triol. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Mass spectrum of ginsenoside Rb1 (compound 1) extracted by the present application;

[0038] Figure 2 NMR hydrogen spectrum of ginsenoside Rb1 (compound 1) extracted by the present application;

[0039] Figure 3 NMR carbon spectrum of ginsenoside Rb1 (compound 1) extracted by the present application;

[0040] Figure 4 Mass spectrum of ginsenoside Rd (compound 2) extracted by the present application;

[0041] Figure 5 NMR hydrogen spectrum of ginsenoside Rd (compound 2) extracted by the present application;

[0042] Figure 6 NMR carbon spectrum of ginsenoside Rd (compound 2) extracted by the present application;

[0043] Figure 7 High performance liquid chromatogram of red fruit ginseng extract of the present application;

[0044] Figure 8 High performance liquid chromatogram of red fruit ginseng extract of the present application; DETAILED DESCRIPTION

[0045] Instruments and materials: extraction tank, single-effect concentrator, storage tank, settling tank, decarburization tank, titanium rod filter, vacuum drying oven, red fruit ginseng stems and leaves from Yunnan.

[0046] Example 1

[0047] A saponin extraction method, comprising the following contents:

[0048] 1. Take fresh red fruit ginseng stems and leaves 3500g, add 70% methanol, add 35L for the first time, heat reflux for 2h; add 28L for the second time, heat reflux for 2h; add 21L for the third time, heat reflux for 2h. Combine the filtrate, concentrate the filtrate to a paste 1865g, heat measure at 60℃, the relative density of the paste is 1.21g / cm 3 .

[0049] 2. The extract was added with water to 5L, heated, dissolved, cooled to room temperature, filtered with 200 mesh, removed the residue, the filtrate was passed through a macroporous adsorption resin column, model HPD-100, quantity 2000 mL, flow rate 5 mL / min, after loading, stand for 30 min;

[0050] Elution: first with 0.2% sodium hydroxide solution 2000 ml, flow rate 50 mL / min; with tap water 15000 ml, flow rate 50 mL / min; analysis, with 75% methanol 5000 ml, flow rate 50 mL / min; eluent concentrated to 1000 ml.

[0051] 3. Decolorization: the above solution concentrated liquid was passed through a decolorizing resin column, model D941, quantity 1000 mL, 80% methanol wash, decolorization treatment. The decolorization liquid was concentrated to paste 20 g.

[0052] 4. After decolorization, the extract was added with 50% methanol 200 ml, activated carbon 2 g, heated to reflux for 2 h, filtered, concentrated and dried to obtain red ginseng stem and leaf extract 8.4 g, yield 0.24%.

[0053] 5. Separation: take fresh red ginseng stem and leaf extract 8.0 g, dissolved with methanol, and mix with 24 g of chromatographic silica gel.

[0054] Take 120 g of chromatographic silica gel to pack column, pack the mixed silica gel, use chloroform-methanol-water (15:1:0.1) as eluent, 250 ml as 1 flow, use thin layer chromatography to detect flow, use chloroform-ethyl acetate-methanol-water (15:40:22:10) 10 ℃ lower solution as developing agent, 10% sulfuric acid ethanol as color developing agent, detect, combine the flow containing same position and same color spots to obtain compound 1 crude product 1.24 g and compound 2 crude product 0.64 g.

[0055] Example 2

[0056] A saponin extraction method, comprising the following contents:

[0057] 1. Take fresh red ginseng stem and leaf 3500 g, add 70% ethanol, first add 35 L, heat reflux for 2 h; secondly add 28 L, heat reflux for 2 h; thirdly add 21 L, heat reflux for 2 h. Combine the filtrate, concentrate the filtrate to paste 1898 g, heat measurement at 60 ℃, the relative density of the paste is 1.18 g / cm3.

[0058] 2. The extract was added with water to 5L, heated, dissolved, cooled to room temperature, filtered with 200 mesh, removed the residue, the filtrate was passed through a macroporous adsorption resin column, model HPD-100, quantity 2000 mL, flow rate 5 mL / min, after loading, stand for 30 min;

[0059] Elution: first 2000 ml of 0.2% sodium hydroxide aqueous solution at a flow rate of 50 mL / min; 15000 ml of tap water at a flow rate of 50 mL / min; resolution, 5000 ml of 75% methanol at a flow rate of 50 mL / min; and eluent concentration to 1000 ml.

[0060] 3. Decolorization: the concentrated solution was passed through a decolorization resin column, model D941, 1000 ml, 80% methanol was used for washing, and the decolorization treatment was performed. The decolorization liquid was concentrated to 20 g of paste, and the relative density was 1.0 g / cm3 at 60°C.

[0061] 4. After decolorization, the extract was added with 200 ml of 50% methanol and 2 g of activated carbon, and heated to reflux for 2 h. Filtration, concentration and drying were performed to obtain 8.1 g of red ginseng stem and leaf extract, with a yield of 0.23%.

[0062] 5. Separation: 8 g of fresh red ginseng stem and leaf extract was dissolved in methanol, and 24 g of chromatographic silica gel was used for sample mixing.

[0063] 120 g of chromatographic silica gel was used for column loading, and the mixed silica gel was used for sample mixing. Chloroform-methanol-water (15:1:0.1) was used as eluent, 250 ml was used as 1 flow, and thin layer chromatography was used for detection. The lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) at 10°C was used as developing agent, and 10% sulfuric acid ethanol was used as color developing agent. After detection, the flow fractions containing the same position and color spots were combined to obtain 1.16 g of compound 1 crude product and 0.76 g of compound 2 crude product.

[0064] Example 3

[0065] A saponin extraction method, comprising the following contents:

[0066] 1. 3500 g of fresh red ginseng stem and leaf was added with 70% mixed alcohol, and the ratio of methanol and ethanol in the mixed alcohol was 3:1. 35 L was added for the first time, heated to reflux for 2 h; 28 L was added for the second time, heated to reflux for 2 h; and 21 L was added for the third time, heated to reflux for 2 h. The filtrate was combined and concentrated to 2143 g of paste, and the relative density of the paste was 1.20 g / cm3 at 60°C.

[0067] 2. The extract was added with water to 5 L, heated, dissolved, cooled to room temperature, filtered with a 200 mesh filter, and the filter residue was removed. The filtrate was passed through a macroporous adsorption resin column, model HPD-100, 2000 ml, and the flow rate was 5 ml / min. After the sample was loaded, it was left for 30 min.

[0068] Elution: first 2000 ml of 0.2% sodium hydroxide aqueous solution at a flow rate of 50 mL / min; 15000 ml of tap water at a flow rate of 50 mL / min; resolution, 5000 ml of 75% methanol at a flow rate of 50 mL / min; and eluent concentration to 1000 ml.

[0069] 3. Decolorization: the concentrated solution was passed through a decolorization resin column, model D941, 1000 ml, 80% methanol was used for washing, and the decolorization treatment was performed. The decolorization liquid was concentrated to 20 g of paste, and the relative density was 1.0 g / cm3 at 60°C.

[0070] 4. After decolorization, the extract was added with 200 ml of 50% methanol and 2 g of activated carbon, and heated to reflux for 2 h. Filtration, concentration and drying were performed to obtain 9.6 g of red ginseng stem and leaf extract, with a yield of 0.27%.

[0071] 5. Separation: 8 g of fresh red ginseng stem and leaf extract was dissolved in methanol, and 24 g of chromatographic silica gel was used for sample mixing.

[0072] 120 g of chromatographic silica gel was taken for column packing, and the mixed silica gel was packed. Chloroform-methanol-water (15:1:0.1) was used as the eluent, 250 ml was 1 flow, thin layer chromatography was used for detection, and the lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) at 10°C was used as the developing agent, 10% sulfuric acid ethanol was used as the color developing agent, and detection was performed. The flow fractions containing the same position and color spots were combined to obtain 1.6 g of compound 1 crude product and 0.72 g of compound 2 crude product.

[0073] Example 4

[0074] A saponin extraction method, comprising the following contents:

[0075] 1. 3500 g of fresh red ginseng stem and leaf was taken, 35 L of 70% mixed alcohol was added, the ratio of methanol and ethanol in the mixed alcohol was 1:1, and the hot reflux was performed for 2 h. 28 L of 70% mixed alcohol was added for the second time, and the hot reflux was performed for 2 h. 21 L of 70% mixed alcohol was added for the third time, and the hot reflux was performed for 2 h. The filtrate was combined and concentrated to 1989 g of paste, and the relative density of the paste was 1.23 g / cm3 at 60°C.

[0076] 2. The extract was added with water to 5 L, heated, dissolved, cooled to room temperature, filtered through a 200 mesh sieve, and the filter residue was removed. The filtrate was passed through a macroporous adsorption resin column, model HPD-100, 2000 ml, and the flow rate was 5 ml / min. After the sample was added, it was left for 30 min.

[0077] Elution: first 2000 ml of 0.2% sodium hydroxide aqueous solution at a flow rate of 50 mL / min; 15000 ml of tap water at a flow rate of 50 mL / min; resolution, 5000 ml of 75% methanol at a flow rate of 50 mL / min; and eluent concentration to 1000 ml.

[0078] 3. Decolorization: the concentrated solution was passed through a decolorization resin column, model D941, 1000 ml, 80% methanol was used for washing, and the decolorization treatment was performed. The decolorization liquid was concentrated to 20 g of paste, and the relative density was 1.0 g / cm3 at 60°C.

[0079] 4. After decolorization, the extract was added with 200 ml of 50% methanol and 2 g of activated carbon, and heated to reflux for 2 h. Filtration, concentration and drying were performed to obtain 9.1 g of red ginseng stem and leaf extract, with a yield of 0.26%.

[0080] 5. Separation: 8 g of fresh red ginseng stem and leaf extract was dissolved in methanol, and 24 g of chromatographic silica gel was used for sample mixing.

[0081] 120 g of chromatographic silica gel was used for column loading, and the mixed silica gel was used for sample mixing. Chloroform-methanol-water (15:1:0.1) was used as the eluent, 250 ml was used as 1 flow, and thin layer chromatography was used for detection. The lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) at 10°C was used as the developing agent, and 10% sulfuric acid ethanol was used as the color developing agent. After detection, the flow fractions containing the same position and color spots were combined to obtain 1.4 g of compound 1 crude product and 0.72 g of compound 2 crude product.

[0082] Example 5

[0083] A saponin extraction method, comprising the following contents:

[0084] 1. 3500 g of fresh red ginseng stem and leaf was added with 70% mixed alcohol, and the ratio of methanol and ethanol in the mixed alcohol was 1:3. 35 L was added for the first time, heated to reflux for 2 h; 28 L was added for the second time, heated to reflux for 2 h; and 21 L was added for the third time, heated to reflux for 2 h. The filtrate was combined and concentrated to 1933 g of paste, and the relative density of the paste was 1.21 g / cm3 at 60°C.

[0085] 2. The extract was added with water to 5 L, heated, dissolved, cooled to room temperature, filtered with a 200 mesh screen, and the filter residue was removed. The filtrate was passed through a macroporous adsorption resin column, model HPD-100, 2000 ml, and the flow rate was 5 ml / min. After the sample was loaded, it was left for 30 min.

[0086] Elution: first 2000 ml of 0.2% sodium hydroxide aqueous solution at a flow rate of 50 mL / min; 15000 ml of tap water at a flow rate of 50 mL / min; resolution, 5000 ml of 75% methanol at a flow rate of 50 mL / min; and eluent concentration to 1000 ml.

[0087] 3. Decolorization: the concentrated solution was passed through a decolorization resin column, model D941, 1000 ml, 80% methanol was used for washing, and the decolorization treatment was performed. The decolorization liquid was concentrated to 20 g of paste, and the relative density was 1.0 g / cm3 at 60°C.

[0088] 4. After decolorization, the extract was added with 200 ml of 50% methanol and 2 g of activated carbon, and heated to reflux for 2 h. Filtration, concentration and drying were performed to obtain 8.9 g of red ginseng stem and leaf extract, with a yield of 0.26%.

[0089] 5. Separation: 8 g of fresh red ginseng stem and leaf extract was dissolved in methanol, and 24 g of chromatographic silica gel was used for sample mixing.

[0090] 120 g of chromatographic silica gel was taken for column loading, and the mixed silica gel was loaded. Chloroform-methanol-water (15:1:0.1) was used as eluent, 250 ml was 1 flow, thin layer chromatography was used for detection, and the lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) at 10°C was used as developing agent, 10% sulfuric acid ethanol was used as color developing agent, and detection was performed. The flow containing the same position and color spots was combined to obtain 1.52 g of compound 1 crude product and 0.68 g of compound 2 crude product.

[0091] Example 6

[0092] A saponin extraction method, comprising the following contents:

[0093] 1. 3500 g of fresh red ginseng stem and leaf was added with 50% methanol, 31.5 L was added for the first time, heated to reflux for 1.5 h; 31.5 L was added for the second time, heated to reflux for 2.5 h; and 24.5 L was added for the third time, heated to reflux for 1.5 h. The filtrate was combined and concentrated to 1845 g of paste, the relative density of the paste was 1.21 g / cm3 at 60°C.

[0094] 2. The extract was added with water to 3.8 L, heated, dissolved, cooled to room temperature, filtered with 200 mesh, the filter residue was removed, and the filtrate was passed through a macroporous adsorption resin column, model HPD-100, 2000 ml, flow rate 5 ml / min, and the sample was loaded for 30 min.

[0095] Elution: first 2000 ml of 0.1% sodium hydroxide aqueous solution at a flow rate of 50 mL / min; 14000 ml of tap water at a flow rate of 50 mL / min; resolution, 4000 ml of 60% methanol at a flow rate of 50 mL / min; and eluent concentration to 900 ml.

[0096] 3. Decolorization: the concentrated solution was passed through a decolorization resin column, model D941, 1000 ml, 80% methanol was used for washing, and the decolorization treatment was performed. The decolorization liquid was concentrated to 20 g of paste, and the relative density was 1.0 g / cm3 at 60°C.

[0097] 4. After decolorization, the extract was added with 200 ml of 70% methanol and 2 g of activated carbon, and heated to reflux for 2 h. Filtration, concentration and drying were performed to obtain 8.2 g of red ginseng stem and leaf extract, with a yield of 0.23%.

[0098] 5. Separation: 8.0 g of fresh red ginseng stem and leaf extract was dissolved in methanol, and 24 g of chromatographic silica gel was used for sample mixing.

[0099] 120 g of chromatographic silica gel was used for column loading, and the mixed silica gel was loaded. Chloroform-methanol-water (15:1:0.1) was used as the eluent, 250 ml was used as 1 flow, and thin layer chromatography was used for detection. The lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) at 10°C was used as the developing agent, and 10% sulfuric acid ethanol was used as the color developing agent. After detection, the flow fractions containing the same position and color spots were combined to obtain 1.21 g of compound 1 crude product and 0.65 g of compound 2 crude product.

[0100] Example 7

[0101] A saponin extraction method, comprising the following contents:

[0102] 1. 3500 g of fresh red ginseng stem and leaf was added with 38.5 L of 75% methanol, heated to reflux for 2.5 h; 24.5 L of 75% methanol was added for the second time, heated to reflux for 1.5 h; and 17.5 L of 75% methanol was added for the third time, heated to reflux for 2.5 h. The filtrate was combined and concentrated to 1888 g of paste, and the relative density of the paste was 1.21 g / cm3 at 60°C.

[0103] 2. The extract was added with water to 9.4 L, heated, dissolved, and cooled to room temperature. Filtration was performed with a 200 mesh filter to remove the filter residue, and the filtrate was passed through a macroporous adsorption resin column, model HPD-100, 2000 ml, at a flow rate of 5 mL / min. After the sample was loaded, it was left to stand for 30 min.

[0104] Elution: first 2000 ml of 0.3% sodium hydroxide aqueous solution at a flow rate of 50 mL / min; 15000 ml of tap water at a flow rate of 50 mL / min; resolution, 5000 ml of 75% methanol at a flow rate of 50 mL / min; and eluent concentration to 1000 ml.

[0105] 3. Decolorization: the concentrated solution was passed through a decolorization resin column, type D941, 1000 ml, 70% methanol washing, and decolorization treatment. The decolorization liquid was concentrated to 20 g of paste, and the relative density was 1.0 g / cm3 at 60°C.

[0106] 4. After decolorization, the extract was added with 30% methanol 200 ml, 2 g of activated carbon, heated and refluxed for 2 h, filtered, concentrated and dried to obtain 8.3 g of red ginseng stem and leaf extract, with a yield of 0.24%.

[0107] 5. Separation: 8.0 g of fresh red ginseng stem and leaf extract was taken and dissolved in methanol, and 24 g of chromatographic silica gel was used for sample mixing.

[0108] 120 g of chromatographic silica gel was taken and packed, and the mixed silica gel was packed, and chloroform-methanol-water (15:1:0.1) was used as eluent, 250 ml was 1 flow, and thin layer chromatography was used for detection, and chloroform-ethyl acetate-methanol-water (15:40:22:10) 10°C was used as developing agent, and 10% sulfuric acid ethanol was used as color developing agent, and detection was performed, and the flow containing the same position and color spots was combined to obtain 1.26 g of compound 1 crude product and 0.61 g of compound 2 crude product.

[0109] Example 8

[0110] A saponin extraction method, comprising the following contents:

[0111] 1. 3500 g of fresh red ginseng stem and leaf was taken, 31.5 L of 50% ethanol was added for the first time, heated and refluxed for 1.5 h; 30.5 L of 50% ethanol was added for the second time, heated and refluxed for 2.5 h; and 24.5 L of 50% ethanol was added for the third time, heated and refluxed for 1.5 h. The filtrate was combined and concentrated to 1890 g of paste, and the relative density of the paste was 1.18 g / cm3 at 60°C.

[0112] 2. The extract was added with water to 7.5 L, heated and dissolved, cooled to room temperature, filtered with a 200 mesh screen, and the filter residue was removed. The filtrate was passed through a macroporous adsorption resin column, type HPD-100, 2000 ml, and the flow rate was 5 mL / min. After the sample was added, it was left for 30 min.

[0113] Elution: first 2000 ml of 0.4% sodium hydroxide aqueous solution at a flow rate of 50 mL / min; 14000 ml of tap water at a flow rate of 50 mL / min; resolution, 4000 ml of 50% methanol at a flow rate of 50 mL / min; and eluent concentration to 900 ml.

[0114] 3. Decolorization: the concentrated solution was passed through a decolorization resin column, model D941, 1000 ml, 80% ethanol elution, and decolorization treatment. The decolorization liquid was concentrated to 20 g of paste, and the relative density was 1.0 g / cm3 at 60°C.

[0115] 4. After decolorization, the extract was added with 200 ml of 80% methanol and 2 g of activated carbon, heated and refluxed for 1 h, filtered, concentrated and dried to obtain 8.2 g of red ginseng stem and leaf extract, with a yield of 0.23%.

[0116] 5. Separation: 8 g of fresh red ginseng stem and leaf extract was dissolved in methanol, and 24 g of chromatographic silica gel was used for sample mixing.

[0117] 120 g of chromatographic silica gel was used for column packing, and the mixed silica gel was used for sample packing. Chloroform-methanol-water (15:1:0.1) was used as eluent, 250 ml was 1 flow, thin layer chromatography was used for flow detection, chloroform-ethyl acetate-methanol-water (15:40:22:10) was used as developing agent, 10% sulfuric acid ethanol was used as color developing agent, and detection was performed. The flow fractions containing the same position and color spots were combined to obtain 1.2 g of compound 1 crude product and 0.73 g of compound 2 crude product.

[0118] Example 9

[0119] A saponin extraction method, comprising the following contents:

[0120] 1. 3500 g of fresh red ginseng stem and leaf was added with 38.5 L of 75% ethanol for the first time, heated and refluxed for 2.5 h; 24.5 L of 75% ethanol was added for the second time, heated and refluxed for 1.5 h; and 17.5 L of 75% ethanol was added for the third time, heated and refluxed for 2.5 h. The filtrate was combined and concentrated to 1902 g of paste, and the relative density of the paste was 1.17 g / cm3 at 60°C.

[0121] 2. The extract was added with water to 8.3 L, heated and dissolved, cooled to room temperature, filtered with a 200 mesh filter, and the filter residue was removed. The filtrate was passed through a macroporous adsorption resin column, model HPD-100, 2000 ml, and the flow rate was 5 ml / min. After the sample was loaded, it was left for 30 min.

[0122] Elution: first 2000 ml of 0.5% sodium hydroxide aqueous solution at a flow rate of 50 mL / min; 16000 ml of tap water at a flow rate of 50 mL / min; resolution, 6000 ml of 80% methanol at a flow rate of 50 mL / min; and eluent concentration to 1000 ml.

[0123] 3. Decolorization: the concentrated solution was passed through a decolorization resin column, model D941, 1000 ml, 60% methanol was used for washing, and the decolorization treatment was performed. The decolorization liquid was concentrated to 20 g of paste, and the relative density was 1.0 g / cm3 at 60°C.

[0124] 4. After decolorization, the extract was added with 200 ml of 60% methanol and 2 g of activated carbon, and heated to reflux for 2 h. After filtration, drying, 8.2 g of red ginseng stem and leaf extract was obtained, and the yield was 0.23%.

[0125] 5. Separation: 8 g of fresh red ginseng stem and leaf extract was taken, dissolved with methanol, and mixed with 24 g of chromatographic silica gel.

[0126] 120 g of chromatographic silica gel was taken and packed, and the mixed silica gel was packed. Chloroform-methanol-water (15:1:0.1) was used as the eluent, 250 ml was taken as 1 flow, and thin layer chromatography was used for detection. The lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) at 10°C was used as the developing agent, and 10% sulfuric acid ethanol was used as the color developing agent. After detection, the flow containing the same position and color spots was combined, and 1.18 g of compound 1 crude product and 0.75 g of compound 2 crude product were obtained.

[0127] Example 10

[0128] A saponin extraction method, comprising the following contents:

[0129] 1. 3500 g of fresh red ginseng stem and leaf was taken, and 38.5 L of 50% mixed alcohol was added, in which the ratio of methanol and ethanol was 3:2. The mixture was heated to reflux for 2.5 h. 24.5 L of 50% mixed alcohol was added, and the mixture was heated to reflux for 1.5 h. 17.5 L of 50% mixed alcohol was added, and the mixture was heated to reflux for 2.5 h. The filtrate was combined and concentrated to 2143 g of paste, and the relative density of the paste was 1.20 g / cm3 at 60°C.

[0130] 2. The extract was added with water to 6.5 L, heated, dissolved, and cooled to room temperature. After 200 mesh filtration, the filter residue was removed, and the filtrate was passed through a macroporous adsorption resin column, model HPD-100, 2000 ml, and the flow rate was 5 ml / min. After the sample was added, it was left for 30 min.

[0131] Elution: first 2000 ml of 0.2% sodium hydroxide aqueous solution at a flow rate of 50 mL / min; 14000 ml of tap water at a flow rate of 50 mL / min; resolution, 6000 ml of 60% methanol at a flow rate of 50 mL / min; and eluent concentration to 1000 ml.

[0132] 3. Decolorization: the concentrated solution was passed through a decolorization resin column, model D941, 1000 ml, 60% methanol was used for washing, and the decolorization treatment was performed. The decolorization liquid was concentrated to 20 g of paste, and the relative density was 1.0 g / cm3 at 60°C.

[0133] 4. After decolorization, the extract was added with 30% methanol 200 ml, 2 g of activated carbon, heated and refluxed for 2 h, filtered, concentrated and dried to obtain 9.4 g of red ginseng stem and leaf extract, with a yield of 0.268%.

[0134] 5. Separation: 8 g of fresh red ginseng stem and leaf extract was dissolved in methanol, and 24 g of chromatographic silica gel was used for sample mixing.

[0135] 120 g of chromatographic silica gel was taken and packed, and the mixed silica gel was used for sample mixing. Chloroform-methanol-water (15:1:0.1) was used as eluent, 250 ml was 1 flow, thin layer chromatography was used for detection, the lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) at 10°C was used as developing agent, 10% sulfuric acid ethanol was used as color developing agent, and detection was performed. The flow fractions containing the same position and color spots were combined to obtain 1.6 g of compound 1 crude product and 0.7 g of compound 2 crude product.

[0136] Example 11

[0137] A saponin extraction method, comprising the following contents:

[0138] 1. 3500 g of fresh red ginseng stem and leaf was taken, 75% mixed alcohol was added, the ratio of methanol and ethanol in the mixed alcohol was 1:2, 31.5 L was added for the first time, heated and refluxed for 1.5 h; 30.5 L was added for the second time, heated and refluxed for 2.5 h; 24.5 L was added for the third time, heated and refluxed for 1.5 h. The filtrate was combined and concentrated to 1942 g of paste, the relative density of the paste was 1.2 g / cm3 at 60°C.

[0139] 2. The extract was added with water to 5 L, heated, dissolved, cooled to room temperature, filtered through a 200 mesh screen, the filter residue was removed, and the filtrate was passed through a macroporous adsorption resin column, model HPD-100, 2000 ml, flow rate 5 ml / min, and after the sample was added, it was left for 30 min.

[0140] Elution: first 2000 ml of 0.2% sodium hydroxide aqueous solution at a flow rate of 50 mL / min; then 16000 ml of tap water at a flow rate of 50 mL / min; and finally 4000 ml of 75% methanol at a flow rate of 50 mL / min; the eluent was concentrated to 1000 ml.

[0141] 3. Decolorization: the concentrated solution was passed through a decolorization resin column, type D941, 1000 ml, 50% methanol was used for washing, and the solution was treated by decolorization. The decolorization solution was concentrated to 20 g of paste, which was measured at 60°C, and the relative density was 1.0 g / cm3.

[0142] 4. After decolorization, the extract was added with 200 ml of 80% methanol and 2 g of activated carbon, and heated to reflux for 2 h. After filtration, drying, 8.8 g of red ginseng stem and leaf extract was obtained, with a yield of 0.26%.

[0143] 5. Separation: 8 g of fresh red ginseng stem and leaf extract was dissolved in methanol, and mixed with 24 g of chromatographic silica gel.

[0144] 120 g of chromatographic silica gel was taken and packed into a column, and the mixed silica gel was packed. Chloroform-methanol-water (15:1:0.1) was used as the eluent, 250 ml was taken as one fraction, and thin layer chromatography was used for detection. The lower layer solution of chloroform-ethyl acetate-methanol-water (15:40:22:10) at 10°C was used as the developing agent, and 10% sulfuric acid ethanol was used as the color developing agent. After detection, the fractions containing the same position and color spots were combined, and 1.5 g of compound 1 crude product and 0.7 g of compound 2 crude product were obtained.

[0145] The effects of different extraction solvents on the yield of red ginseng extract and the yield of compound 1 and compound 2 crude product are shown in Table 1.

[0146] Table 1 Effects of different extraction solvents on the yield of red ginseng extract and the yield of compound 1 and compound 2 crude product

[0147]

[0148] As can be seen from Table 1, the yield of red ginseng extract is significantly improved when using a mixture of methanol and ethanol as the extraction agent, and the yield is the highest when the proportion of methanol in the mixture is large. The yield of compound 1 crude product is significantly higher when using a mixture of methanol and ethanol as the extraction agent than when using methanol or ethanol alone as the extraction agent. The yield of compound 2 crude product is significantly higher than when using methanol alone as the extraction agent, but lower than when using ethanol alone as the extraction agent.

[0149] Purification of compound 1 and compound 2 crude product:

[0150] Purification of compound 1:

[0151] The crude compound 1 3.1 g was dissolved in 30 ml of water, and was passed through a C18 medium pressure column, and was eluted with 30% methanol water, and was collected in 100 ml as one fraction, and was detected by thin layer chromatography, and the lower layer solution placed at 10°C was used as developing agent, and 10% sulfuric acid ethanol was used as color developing agent, and the fractions containing the same position and color spots and having high concentration were combined, and were concentrated and dried to obtain 2.46 g of pure compound 1, which was identified as ginsenoside Rb1. The mass spectrum of compound 1 is shown in Figure 1 Figure 2 Figure 3 The nuclear magnetic resonance spectrum of compound 1 was compared with the standard nuclear magnetic resonance spectrum of ginsenoside Rb1, and it was confirmed that compound 1 was ginsenoside Rb1.

[0152] Purification of compound 2:

[0153] The crude compound 2 1.6 g was dissolved in 30 ml of water, and was passed through a C18 medium pressure column, and was eluted with 35% methanol water, and was collected in 100 ml as one fraction, and was detected by thin layer chromatography, and the lower layer solution placed at 10°C was used as developing agent, and 10% sulfuric acid ethanol was used as color developing agent, and the fractions containing the same position and color spots and having high concentration were combined, and were concentrated and dried to obtain 1.1 g of pure compound 2, which was identified as ginsenoside Rd by mass spectrum. The mass spectrum of compound 2 is shown in Figure 4 Figure 5 Figure 6 The nuclear magnetic resonance spectrum of compound 2 was compared with the standard nuclear magnetic resonance spectrum of ginsenoside Rd, and it was confirmed that compound 2 was ginsenoside Rd.

[0154] Determination of the content of Hongguoshen leaf extract: Determined according to high performance liquid chromatography (general rule 0512).

[0155] Chromatographic conditions and system suitability test:

[0156] Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, and water was used as mobile phase B, and gradient elution was performed according to the conditions in Table 2; the flow rate was 1.5 ml per minute; the detection wavelength was 203 nm; and the column temperature was 25°C.

[0157] Table 2 Gradient elution conditions

[0158]

[0159] Preparation of the control extract solution

[0160] ​​​​Take ginsenoside Rb1, ginsenoside Rd control product appropriate amount, precision weighing, add 70% methanol solution and dilution to 0.2mg per 1ml solution, namely.

[0161] Preparation of test sample solution:

[0162] Take 25mg of the product, precision weighing, placed in 10ml flask, add 70% methanol solution and dilution to the mark, shake, namely.

[0163] Determination method:

[0164] Respectively, precision pipette control extract solution and test sample solution 10ul, inject liquid chromatograph, determination, namely.

[0165] As shown in Figure 7 The retention time of 36~37min, ginsenoside Rb1, in the retention time of 39~40min, ginsenoside Rd.

[0166] As shown in Figure 8 The content of ginsenoside Rb1 in Hongguoshen extract reaches 13.4%, ginsenoside Rd reaches 6.31%, the total content of saponin reaches 19.71%.

Claims

1. A method of saponin extraction, characterized by, Includes the following: S100. The plant stems and leaves are crushed and placed in a multi-functional extraction tank. The extraction is performed three times by hot reflux with alcohol. The mixture is filtered, and the filtrates are combined and concentrated under reduced pressure to obtain an extract. The plant is ginseng. S200. Place the above extract in a settling tank, add water at a solid-liquid ratio of 1:2~5, stir to dissolve, settle, and filter to obtain filtrate. The filtrate was passed through a macroporous adsorption resin column for elution to obtain the eluent. In step S200, the elution process includes the following: first rinsing with 0.1%~0.5% NaOH aqueous solution, then rinsing with water, and finally eluting with alcohol, and collecting the eluent eluted from the alcohol. The elution process was carried out at a flow rate of 50 mL / min; The volume ratio of NaOH aqueous solution, water, and alcohol is 1:7~8:2~3; The alcohol is selected from one or both of methanol and ethanol, and the volume fraction of the alcohol is 50% to 80%. S300. After concentrating the above eluent, it is decolorized by passing it through a decolorizing resin column to obtain a decolorized solution, and the decolorized solution is concentrated to obtain a paste. S400. Dissolve the above paste in alcohol, add activated charcoal for decolorization, filter, concentrate and dry to obtain the extract. S500. The above extract was packed into a silica gel column for chromatography, using chloroform-methanol-water as the eluent. The fractions were detected by thin-layer chromatography and collected to obtain two crude products, compound 1 and compound 2. S600. The two crude products were purified by passing them through a C18 medium-pressure column with methanol-water as the eluent. The fractions were collected, concentrated, dried, and identified to confirm the presence of ginsenoside Rb1 and ginsenoside Rd.

2. The saponin extraction method according to claim 1, characterized in that, In step S100, the method of alcoholic hot reflux extraction includes: using 9 to 11 times the amount of alcohol, hot refluxing for 1.5 to 2.5 hours, filtering to obtain the first filtrate; adding 7 to 9 times the amount of alcohol to the obtained filter residue, hot refluxing for 1.5 to 2.5 hours, filtering to obtain the second filtrate; adding 5 to 7 times the amount of alcohol to the obtained filter residue, hot refluxing for 1.5 to 2.5 hours, filtering to obtain the third filtrate, and combining the three filtrates.

3. The saponin extraction method according to claim 2, characterized in that, In S100, the alcohol is selected from methanol or ethanol, and the volume fraction of the alcohol is 50%~75%; during each extraction, the solid-liquid ratio of plant stems and leaves to alcohol is 1:5~11; the temperature of the hot reflux is 65~85℃.

4. The saponin extraction method according to claim 1, characterized in that, In S300, the decolorizing resin column is a D941 decolorizing resin column, the flow rate is 30~50 mL / min, and the eluent is ethanol with a volume fraction of 50%~80%.

5. The saponin extraction method according to claim 1, characterized in that, In S400, the method for decolorizing activated carbon includes: diluting the paste with alcohol to 10-15 times its volume, adding needle-grade activated carbon, heating under reflux for 1.5-2.5 hours, allowing to stand and filtering to obtain the extract; wherein the alcohol is selected from methanol or ethanol, with a volume fraction of 30%-100%, and the mass ratio of needle-grade activated carbon to the extract is 1:8-12.

6. The saponin extraction method according to claim 1, characterized in that, In the S400, filtration is carried out using a polypropylene membrane with a pore size of 5~10μm, a filtration pressure of 0.1~0.3MPa, a filtration temperature of 60~80℃, and the filter cake is washed with 50%~70% ethanol 3~5 times.

7. The saponin extraction method according to claim 1, characterized in that, In S500, the volume ratio of chloroform, methanol, and water in the eluent is 14~16:1:0.

1.

8. The saponin extraction method according to claim 1, characterized in that, In S600, the volume ratio of methanol to water in the eluent is 3.0~3.5:10, and thin-layer chromatography is used to detect the fractions.

9. The saponin extraction method according to claim 1, characterized in that, In both S500 and S600, the thin-layer chromatography detection uses the lower layer solution of chloroform-ethyl acetate-methanol-water after standing at 10℃ as the developing solvent, with a volume ratio of chloroform, ethyl acetate, methanol, and water of 15:40:22:

10. The colorimetric reagent is selected from 10% sulfuric acid ethanol solution or anisaldehyde-sulfuric acid reagent.

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