A method for extracting saponin components from American ginseng stems and leaves and its application

Abstract

The present invention relates to the field of biomedicine, and in particular to a method for extracting saponin components from American ginseng stems and leaves and an application thereof. The method uses American ginseng stems and leaves as raw materials, and adopts a method of preparing with longan pulp, removing pigments with a Soxhlet extractor, and extracting total saponins to obtain total saponins with a purity of more than 95%, and then uses three-phase high-speed countercurrent chromatography for separation and purification to obtain protopanaxandiol saponins: the ratio of American ginsenoside Rb1, American ginsenoside Rb2, and American ginsenoside Rb3 is 5:2:15; C-17 side chain isomerization type saponins: the ratio of American ginsenoside L1, American ginsenoside L2, and American ginsenoside L3 is 3:5:8. In the present invention, the L-type American ginseng total saponins are prepared in American ginseng stems and leaves for the first time, and in addition, the saponin components obtained by the present invention play an important role in treating liver cancer.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a method for extracting saponin components from American ginseng stems and leaves and applications thereof. Background Art

[0002] American ginseng (Panax quinquefolius L.) is a perennial herbaceous plant of the genus Panax in the Araliaceae family. The root of American ginseng is cylindrical or fusiform; the leaves are broadly ovate or obovate with coarse serrations on the edges; the inflorescence is umbellate, green, and bell-shaped; the petals are green-white, and the anthers are ovate or rectangular; the berries are oblate and bright red when ripe, with a flowering period of July-August and a fruiting period of September. American ginseng was first discovered in the Atlantic coastal jungles of the Montreal area, and was named after its place of origin. It belongs to the same genus as ginseng but is a different species.

[0003] American ginseng is native to southern Canada and northern United States in North America, and is now distributed in high-altitude mountainous areas in Jilin, Beijing, Shandong, Shaanxi, Shanxi, Fujian, Yunnan and other southern provinces. American ginseng prefers a shady and humid environment and avoids strong light and high temperature. It is suitable to be planted in slightly acidic to neutral sandy loam with thick soil, good drainage, loose and fertile soil. Propagation is mainly based on seed propagation.

[0004] In recent years, ginsenosides have become the focus of research in the field of anti-cancer. Ginsenosides have a good effect on liver cancer cells such as human SMMC-7721 cells, SK-HEP-1 cells and Bel-7404 cells, as well as lung adenocarcinoma and esophageal cancer. The chemical composition and efficacy of American ginseng and ginseng are basically the same, and the planting area of ​​American ginseng in my country is gradually increasing. The stems and leaves of American ginseng are a huge above-ground resource of American ginseng. In the development and utilization of American ginseng, most people only pursue the utilization of American ginseng roots and ignore the development of stems and leaves, resulting in a large waste of resources. Studies have shown that the total saponin content of American ginseng stems and leaves is significantly higher than that of American ginseng roots and ginseng. In addition, the main ingredient of Xinyue Capsules on the market is also American ginseng stem and leaf saponins, all of which indicate that the stems and leaves of American ginseng have great development and research value.

[0005] At present, the commonly used extraction and preparation methods include decoction, water extraction and alcohol precipitation or alcohol extraction and water precipitation, ultrasound, macroporous resin adsorption, etc. The extraction rates of these methods are limited and it is difficult to meet large demands. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art, the present invention provides a method for extracting saponin components from American ginseng stems and leaves.

[0007] The present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a method for extracting saponin components from American ginseng stems and leaves, comprising the following steps:

[0009] (1) In order to increase the content of B and L type rare saponins, the harvested American ginseng stems and leaves were used as raw materials, the American ginseng stems and leaves and longan pulp were steamed for 5-8 h, the longan pulp was removed, and the American ginseng stems and leaves were dried to obtain the American ginseng stem and leaf steamed product;

[0010] (2) Removing pigments: Steamed American ginseng stems and leaves are taken, crushed through a 100-300 mesh sieve, divided into several 300 mesh bags, placed in a Soxhlet extractor, and 500 ml of a mixed solution of ammonia water and ethyl acetate in a volume ratio of 0.5:99.5 is added, heated and refluxed for 1.5 hours, and the mixed solution of ammonia water and ethyl acetate is discarded. The residue is evaporated to remove the ammonia water and ethyl acetate, and the depigmented American ginseng stems and leaves are obtained;

[0011] (3) Extracting total saponins: The depigmented American ginseng stems and leaves obtained in step (2) were placed back into a Soxhlet extractor, 1000 ml of ammonia-methanol mixed solution with a volume ratio of 0.5:99.5 was added, and the mixture was heated under reflux for extraction for 12 h. The ammonia-methanol mixed solution was recovered until dry, and 300 ml of water saturated with n-butanol was added to the residue to dissolve it, and the residue was transferred to a separatory funnel, and extracted with n-butanol saturated with water by shaking for at least 4 times, and the n-butanol solution was combined;

[0012] (4) Removing impurities: Studies have shown that saponin components can be extracted with ammonia methanol. Saponin components are dissolved in n-butanol and extracted and purified with n-butanol to remove water-soluble polar impurities such as polysaccharides and starch. Therefore, the n-butanol solution obtained in step (3) is washed with 1% hydrochloric acid solution, the hydrochloric acid solution is discarded, and then washed with water saturated with n-butanol until neutral, the washing liquid is discarded, and the n-butanol is recovered to dryness to obtain a total saponin extract of American ginseng stems and leaves with a purity of more than 95%;

[0013] (5) Three-phase high-speed countercurrent chromatography separation and purification: Conventional high-speed countercurrent chromatography is a two-phase system (liquid-liquid system), which is not suitable for the separation and purification of highly polar components such as saponins. The addition of MWCNTs / OMCS can form a three-phase system, which effectively amplifies the differences in the distribution coefficients and distribution times of different saponin components in the system, thereby achieving separation.

[0014] Therefore, the present invention uses an aqueous phase dispersed with O-carboxymethyl chitosan functionalized multi-walled carbon nanotubes as the stationary phase, mobile phase A is water-saturated 5:1 carbon tetrachloride-acetone, mobile phase B is water-saturated 10:1 n-butanol-methanol, and the gradient flow rate is set to 0 min, 95% A; 60 min, 65% A; 120 min, 35% A; the column volume of the high-speed countercurrent chromatograph is 500 ml, the sample amount is 50 mg, the rotation speed is 900 rpm, the flow rate is 2 mL / min, and the detector is an evaporative light detector;

[0015] Collect the eluate from 30min to 60min, evaporate the solvent to dryness, and obtain protopanaxadiol saponins: ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3;

[0016] The eluate was collected at 90 min to 120 min, and the solvent was evaporated to dryness to obtain C-17 side chain isomeric saponins: American ginsenoside L1, American ginsenoside L2, and American ginsenoside L3.

[0017] Furthermore, in step (1), 100 g of American ginseng stems and leaves and 100 g of longan pulp are prepared; and in step (2), 100 g of steamed American ginseng stems and leaves are prepared.

[0018] Furthermore, in step (5), the ratio of protopanaxadiol saponins: ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3 is 5:2:15.

[0019] Furthermore, in step (5), the ratio of C-17 side chain isomeric saponin: American ginsenoside L1, American ginsenoside L2, and American ginsenoside L3 is 3:5:8.

[0020] In a second aspect, the present invention provides a use of protopanaxadiol saponins prepared by the above-mentioned extraction method in the preparation of drugs for treating liver cancer.

[0021] In a third aspect, the present invention provides a use of a C-17 side chain isomeric saponin prepared by the above extraction method in the preparation of a drug for treating liver cancer.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1. The present invention uses American ginseng stems and leaves as raw materials, and adopts a method of processing with longan pulp, removing pigments by a Soxhlet extractor, and extracting total saponins to obtain total saponins with a purity of more than 95%, and then uses three-phase high-speed countercurrent chromatography for separation and purification to obtain protopanaxadiol saponins: American ginsenoside Rb1, American ginsenoside Rb2, and American ginsenoside Rb3 in a ratio of 5:2:15; C-17 side chain isomerization saponins: American ginsenoside L1, American ginsenoside L2, and American ginsenoside L3 in a ratio of 3:5:8, wherein the L-type American ginseng total saponins are prepared from American ginseng stems and leaves for the first time in the present invention, and the saponin components obtained in the present invention play an important role in the treatment of liver cancer.

[0024] 2. The present invention is the first to introduce an aqueous phase dispersed with O-carboxymethyl chitosan functionalized multi-walled carbon nanotubes as the stationary phase to form three phases, which effectively amplifies the differences in the distribution coefficients and distribution times of different saponin components in the system, thereby achieving separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below in conjunction with the accompanying drawings.

[0026] Figure 1 It is the standard curve diagram in Example 1 of the present invention;

[0027] Figure 2 Ginsenoside Rb of the present invention 1 , Rb 2 , Rb 3 Liquid phase spectrum of reference substance;

[0028] Figure 3 Ginsenoside Rb of the present invention 1 , Rb 2 , Rb 3 Liquid phase spectrum of the test sample;

[0029] Figure 4 Ginsenoside L of the present invention 1 , L 2 ,,L 3 Liquid phase spectrum of reference substance;

[0030] Figure 5 Ginsenoside L of the present invention 1 , L 2 ,,L 3 Liquid phase spectrum of the test sample;

[0031] Figure 6 The chemical structure diagram of O-carboxymethyl chitosan of the present invention. DETAILED DESCRIPTION

[0032] The following is a further description of the extraction method and application of saponin components in American ginseng stems and leaves through specific examples, but is not limited to the present invention.

[0033] Example 1: Preparation of saponin components from American ginseng stems and leaves - origin screening.

[0034] (1) Using American ginseng stems and leaves harvested from different origins as raw materials; see Table 1 below;

[0035] (2) Steam 100 g of American ginseng stems and leaves and 100 g of longan pulp for 5-8 h, remove the longan pulp, and dry the American ginseng stems and leaves to obtain the American ginseng stem and leaf steamed product;

[0036] (3) Removing pigments: Take 100 g of the steamed American ginseng stems and leaves, crush them through a 100-mesh sieve, divide them into several 300-mesh bags, put them in a Soxhlet extractor, add 500 ml of a mixed solution of ammonia water and ethyl acetate in a volume ratio of 0.5:99.5, heat and reflux for 1.5 hours, discard the mixed solution of ammonia water and ethyl acetate, evaporate the residue to remove the pigments of American ginseng stems and leaves;

[0037] (4) Extraction of total saponins: After the depigmentation of the American ginseng stems and leaves in step (3), place them back in the Soxhlet extractor, add 1000 ml of ammonia-methanol mixed solution with a volume ratio of 1.5:98.5, heat and reflux for extraction for 12 hours, and recover the ammonia-methanol mixed solution until dry. Add 300 ml of water saturated with n-butanol to the residue to dissolve it, transfer it to a separatory funnel, and shake and extract it 4 times with n-butanol saturated with water, 300 ml each time, and combine the n-butanol solutions;

[0038] (5) Removing impurities: The n-butanol solution in step (4) is washed three times with 1% hydrochloric acid solution, 300 ml each time, the hydrochloric acid solution is discarded, and then washed with water saturated with n-butanol until neutral, the washing liquid is discarded, and the n-butanol is recovered to dryness to obtain a total saponin extract of American ginseng stems and leaves with a purity of more than 95%;

[0039] The detection method is as follows:

[0040] Preparation of reference solution: Take an appropriate amount of ginsenoside Re reference substance, weigh it accurately, and add methanol to make a solution containing 0.5 mg per 1 ml.

[0041] Preparation of standard curve

[0042] Accurately pipette 30μl, 60μl, 90μl, 120μl, 150μl and 200μl of the reference solution into stoppered test tubes respectively, evaporate the solvent at low temperature, add 0.5ml of 1% vanillin perchloric acid test solution, mix thoroughly in a 60℃ constant temperature water bath, heat for 15 minutes, immediately cool with ice water for 2 minutes, add 5ml of 77% sulfuric acid solution and shake well; use the corresponding reagent as blank, measure the absorbance at a wavelength of 540nm according to the UV-Vis spectrophotometry (General Rule 0401), and draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0043] Determination method

[0044] Take about 4g of dried American ginseng stems and leaves, crush them (pass through a No. 4 sieve), weigh them accurately, place them in a stoppered conical flask, add 50ml of water-saturated n-butanol, heat and reflux for 60 minutes, filter, evaporate to dryness, add an appropriate amount of methanol to dissolve, place them in a 25ml volumetric flask and dilute to the scale with methanol, shake well, accurately measure 70μl, and follow the method under the standard curve preparation item, starting from "place in a stoppered test tube", determine the absorbance, and read the ginsenoside Rb in the test solution from the standard curve. 2 The amount of the calculation result is multiplied by 0.84, and the standard curve is shown in Figure 1 .

[0045] Table 1 Comparison of total saponin extraction rates from American ginseng stems and leaves from different origins

[0046]

[0047] Result analysis: The total saponin content in the stems and leaves of American ginseng from different origins is significantly different. The total saponin content extracted from the stems and leaves of American ginseng from Weihai is the highest, so this study used American ginseng from Weihai for extraction and preparation.

[0048] Example 2: Preparation of saponin components from American ginseng stems and leaves - screening of processing methods.

[0049] 1. Unprocessed

[0050] (1) The raw materials are American ginseng stems and leaves harvested from October to November in Weihai;

[0051] (2) Removing pigments: Take 100 g of the steamed American ginseng stems and leaves, crush them through a 100-mesh sieve, divide them into several 300-mesh bags, put them in a Soxhlet extractor, add 500 ml of a mixed solution of ammonia water and ethyl acetate in a volume ratio of 0.5:99.5, heat and reflux for 1.5 hours, discard the mixed solution of ammonia water and ethyl acetate, evaporate the residue to remove the pigments of American ginseng stems and leaves;

[0052] (3) Extraction of total saponins: After the pigment is removed from the stems and leaves of American ginseng in step (3), place them back in the Soxhlet extractor, add 1000 ml of ammonia-methanol mixed solution with a volume ratio of 1.5:98.5, heat and reflux for extraction for 12 hours, and recover the ammonia-methanol mixed solution until dry. Add 300 ml of water saturated with n-butanol to the residue to dissolve it, transfer it to a separatory funnel, and shake and extract it 4 times with n-butanol saturated with water, 300 ml each time, and combine the n-butanol solutions;

[0053] (4) Removing impurities: The n-butanol solution in step (3) is washed three times with 1% hydrochloric acid solution, 300 ml each time, the hydrochloric acid solution is discarded, and the solution is then washed with water saturated with n-butanol until neutral, the washing liquid is discarded, and the n-butanol is recovered to dryness to obtain a total saponin extract of American ginseng stems and leaves with a purity of more than 95%.

[0054] 2. Preparation of Longan Meat

[0055] (1) The raw materials are American ginseng stems and leaves harvested from October to November in Weihai;

[0056] (2) Steam 100 g of American ginseng stems and leaves and 100 g of longan pulp for 5-8 h, remove the longan pulp, and dry the American ginseng stems and leaves to obtain the American ginseng stem and leaf steamed product;

[0057] (3) Removing pigments: Take 100 g of the steamed American ginseng stems and leaves, crush them through a 100-mesh sieve, divide them into several 300-mesh bags, put them in a Soxhlet extractor, add 500 ml of a mixed solution of ammonia water and ethyl acetate in a volume ratio of 0.5:99.5, heat and reflux for 1.5 hours, discard the mixed solution of ammonia water and ethyl acetate, evaporate the residue to remove the pigments of American ginseng stems and leaves;

[0058] (4) Extraction of total saponins: After the depigmentation of the American ginseng stems and leaves in step (3), place them back in the Soxhlet extractor, add 1000 ml of ammonia-methanol mixed solution with a volume ratio of 1.5:98.5, heat and reflux for extraction for 12 hours, and recover the ammonia-methanol mixed solution until dry. Add 300 ml of water saturated with n-butanol to the residue to dissolve it, transfer it to a separatory funnel, and shake and extract it 4 times with n-butanol saturated with water, 300 ml each time, and combine the n-butanol solutions;

[0059] (5) Removing impurities: The n-butanol solution in step (4) is washed three times with 1% hydrochloric acid solution, 300 ml each time, the hydrochloric acid solution is discarded, and then washed with water saturated with n-butanol until neutral, the washing liquid is discarded, and the n-butanol is recovered to dryness to obtain a total saponin extract of American ginseng stems and leaves with a purity of more than 95%.

[0060] The detection method was determined according to the total saponin detection method in Example 1.

[0061] Table 2 is a comparison of extraction rates of different processing methods

[0062]

[0063] Result analysis: There was little difference in the total saponin content between unprocessed and processed American ginseng stems and leaves. The total saponin content extracted from the American ginseng stems and leaves processed with longan pulp was slightly higher. Therefore, this study added longan pulp to process American ginseng for extraction and preparation.

[0064] Example 3

[0065] (1) The raw materials are American ginseng stems and leaves harvested from October to November in Weihai;

[0066] (2) Steam 100 g of American ginseng stems and leaves and 100 g of longan pulp for 5-8 h, remove the longan pulp, and dry the American ginseng stems and leaves to obtain the American ginseng stem and leaf steamed product;

[0067] (3) Removing pigments: Take 100 g of the steamed American ginseng stems and leaves, crush them through a 100-mesh sieve, divide them into several 300-mesh bags, put them in a Soxhlet extractor, add 500 ml of a mixed solution of ammonia water and ethyl acetate in a volume ratio of 0.5:99.5, heat and reflux for 1.5 hours, discard the mixed solution of ammonia water and ethyl acetate, evaporate the residue to remove the pigments of American ginseng stems and leaves;

[0068] (4) Extraction of total saponins: After the depigmentation of the American ginseng stems and leaves in step (3), place them back in the Soxhlet extractor, add 1000 ml of ammonia-methanol mixed solution with a volume ratio of 1.5:98.5, heat and reflux for extraction for 12 hours, and recover the ammonia-methanol mixed solution until dry. Add 300 ml of water saturated with n-butanol to the residue to dissolve it, transfer it to a separatory funnel, and shake and extract it 4 times with n-butanol saturated with water, 300 ml each time, and combine the n-butanol solutions;

[0069] (5) Removing impurities: The n-butanol solution in step (4) is washed three times with 1% hydrochloric acid solution, 300 ml each time, the hydrochloric acid solution is discarded, and then washed with water saturated with n-butanol until neutral, the washing liquid is discarded, and the n-butanol is recovered to dryness to obtain a total saponin extract of American ginseng stems and leaves with a purity of more than 95%;

[0070] (6) Three-phase high-speed countercurrent chromatography separation and purification: The stationary phase was an aqueous phase dispersed with O-carboxymethyl chitosan functionalized multi-walled carbon nanotubes, the mobile phase A was water-saturated 5:1 carbon tetrachloride-acetone, the mobile phase B was water-saturated 10:1 n-butanol-methanol, the gradient flow rate was set to 0 min, 95% A; 60 min, 65% A; 120 min, 35% A; the column volume of the high-speed countercurrent chromatography was 500 ml, the sample load was 50 mg, the rotation speed was 900 rpm, the flow rate was 2 mL / min, and the detector was an evaporative light detector;

[0071] Collect the eluate from 30min to 60min, evaporate the solvent to dryness, and obtain protopanaxadiol saponins: ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3;

[0072] The eluate was collected at 90 min to 120 min, and the solvent was evaporated to dryness to obtain C-17 side chain isomeric saponins: American ginsenoside L1, American ginsenoside L2, and American ginsenoside L3.

[0073] Example 4

[0074] (1) The raw materials are American ginseng stems and leaves harvested from October to November in Weihai;

[0075] (2) Steam 100 g of American ginseng stems and leaves and 100 g of longan pulp for 8 h, remove the longan pulp, and dry the American ginseng stems and leaves to obtain the steamed American ginseng stems and leaves product;

[0076] (3) Removing pigments: Take 100 g of the steamed American ginseng stems and leaves, crush them through an 80-mesh sieve, divide them into several 300-mesh bags, put them in a Soxhlet extractor, add 500 ml of a mixed solution of ammonia water and ethyl acetate in a volume ratio of 1.5:98.5, heat and reflux for 1 hour, discard the mixed solution of ammonia water and ethyl acetate, evaporate the residue to remove the pigments of the American ginseng stems and leaves;

[0077] (4) Extraction of total saponins: After the depigmentation of the American ginseng stems and leaves in step (3), place them back in the Soxhlet extractor, add 1000 ml of ammonia-methanol mixed solution with a volume ratio of 0.5:99.5, heat and reflux for extraction for 12 hours, and recover the ammonia-methanol mixed solution until dry. Add 300 ml of water saturated with n-butanol to the residue to dissolve it, transfer it to a separatory funnel, and shake and extract it 4 times with n-butanol saturated with water, 300 ml each time, and combine the n-butanol solutions;

[0078] (5) Removing impurities: The n-butanol solution in step (4) is washed three times with 1% hydrochloric acid solution, 300 ml each time, the hydrochloric acid solution is discarded, and then washed with water saturated with n-butanol until neutral, the washing liquid is discarded, and the n-butanol is recovered to dryness to obtain a total saponin extract of American ginseng stems and leaves with a purity of more than 95%;

[0079] (6) Three-phase high-speed countercurrent chromatography separation and purification: The stationary phase was an aqueous phase dispersed with O-carboxymethyl chitosan functionalized multi-walled carbon nanotubes, the mobile phase A was water-saturated 5:1 carbon tetrachloride-acetone, the mobile phase B was water-saturated 10:1 n-butanol-methanol, the gradient flow rate was set to 0 min, 95% A; 60 min, 65% A; 120 min, 35% A; the column volume of the high-speed countercurrent chromatography was 500 ml, the sample load was 50 mg, the rotation speed was 900 rpm, the flow rate was 2 mL / min, and the detector was an evaporative light detector;

[0080] Collect the eluate from 30min to 60min, evaporate the solvent to dryness, and obtain protopanaxadiol saponins: ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3;

[0081] The eluate was collected at 90 min to 120 min, and the solvent was evaporated to dryness to obtain C-17 side chain isomeric saponins: American ginsenoside L1, American ginsenoside L2, and American ginsenoside L3.

[0082] Example 5

[0083] (1) The raw materials are American ginseng stems and leaves harvested from October to November in Weihai;

[0084] (2) Steam 100 g of American ginseng stems and leaves and 100 g of longan pulp for 7 h, remove the longan pulp, and dry the American ginseng stems and leaves to obtain the steamed American ginseng stems and leaves product;

[0085] (3) Removing pigments: Take 100 g of the steamed American ginseng stems and leaves, crush them through a 120-mesh sieve, divide them into several 300-mesh bags, put them in a Soxhlet extractor, add 500 ml of a mixed solution of ammonia water and ethyl acetate in a volume ratio of 1.5:98.5, heat and reflux for 1 hour, discard the mixed solution of ammonia water and ethyl acetate, evaporate the residue to remove the pigments of American ginseng stems and leaves;

[0086] (4) Extraction of total saponins: After the depigmentation of the American ginseng stems and leaves in step (3), place them back in the Soxhlet extractor, add 1000 ml of ammonia-methanol mixed solution with a volume ratio of 2:98, heat and reflux for extraction for 12 hours, and recover the ammonia-methanol mixed solution until dry. Add 300 ml of water saturated with n-butanol to the residue to dissolve it, transfer it to a separatory funnel, and shake and extract it 4 times with n-butanol saturated with water, 300 ml each time, and combine the n-butanol solutions;

[0087] (5) Removing impurities: The n-butanol solution in step (4) is washed three times with 1% hydrochloric acid solution, 300 ml each time, the hydrochloric acid solution is discarded, and then washed with water saturated with n-butanol until neutral, the washing liquid is discarded, and the n-butanol is recovered to dryness to obtain a total saponin extract of American ginseng stems and leaves with a purity of more than 95%;

[0088] (6) Three-phase high-speed countercurrent chromatography separation and purification: The stationary phase was an aqueous phase dispersed with O-carboxymethyl chitosan functionalized multi-walled carbon nanotubes, the mobile phase A was water-saturated 5:1 carbon tetrachloride-acetone, the mobile phase B was water-saturated 10:1 n-butanol-methanol, the gradient flow rate was set to 0 min, 95% A; 60 min, 65% A; 120 min, 35% A; the column volume of the high-speed countercurrent chromatography was 500 ml, the sample load was 50 mg, the rotation speed was 900 rpm, the flow rate was 2 mL / min, and the detector was an evaporative light detector;

[0089] Collect the eluate from 30min to 60min, evaporate the solvent to dryness, and obtain protopanaxadiol saponins: ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3;

[0090] The eluate was collected at 90 min to 120 min, and the solvent was evaporated to dryness to obtain C-17 side chain isomeric saponins: American ginsenoside L1, American ginsenoside L2, and American ginsenoside L3.

[0091] Example 6: Determination of saponin components in American ginseng stems and leaves.

[0092] (1) Determination method of R-type saponin

[0093] Chromatographic conditions and system suitability test

[0094] Octadecylsilane bonded silica gel was used as filler; acetonitrile-0.01% phosphoric acid solution (25:85) was used as mobile phase; the detection wavelength was 203 nm. The theoretical plate number calculated based on the ginsenoside Rb2 peak should be no less than 1500.

[0095] Preparation of reference solution

[0096] Take appropriate amounts of ginsenoside Rb1 reference substance, ginsenoside Rb2 reference substance, and ginsenoside Rb3 reference substance, weigh them accurately, and add methanol to prepare solutions containing 0.25 mg of ginsenoside Rb1, 0.5 mg of ginsenoside Rb2, and 0.20 mg of ginsenoside Rb3 per 1 ml.

[0097] Preparation of test solution

[0098] Take about 0.5g of the powder of this product, weigh it accurately, put it in a Soxhlet extractor, add 30ml of chloroform, heat and reflux for 1 hour, discard the chloroform solution, evaporate the chloroform of the residue, add 30ml of methanol, heat and reflux for 3 hours, evaporate the extract to dryness at low temperature, add 10ml of water to dissolve it, add petroleum ether (30-60℃) to extract 3 times, 20ml each time, discard the ether solution, pass the aqueous solution through a D101 macroporous adsorption resin column (inner diameter 1.5cm, column length 15cm), and elute with 50mL of water, 40% methanol, and 80% methanol in turn, collect 30ml of 80% methanol eluate, evaporate to dryness, and dissolve the residue in 1ml of ethanol to obtain the product.

[0099] Determination method

[0100] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and measure, and the result is shown in Table 1. Figure 2-Figure 3 .

[0101] (2) Determination method of L-type saponin

[0102] Chromatographic conditions and system suitability test

[0103] Octadecylsilane bonded silica gel was used as filler; acetonitrile-0.05% formic acid solution (25:85) was used as mobile phase; the detection wavelength was 203 nm. The theoretical plate number calculated based on the ginsenoside L1 peak should not be less than 5000.

[0104] Preparation of reference solution

[0105] Take appropriate amounts of ginsenoside L1 reference substance, ginsenoside L2 reference substance, and ginsenoside L3 reference substance, weigh them accurately, and add methanol to make solutions containing 0.10 mg of ginsenoside L1, 0.25 mg of ginsenoside L2, and 0.20 mg of ginsenoside L3 per 1 ml.

[0106] Preparation of test solution

[0107] Take about 0.5 g of the powder of this product, weigh it accurately, and dissolve it in 1 ml of methanol.

[0108] Determination method

[0109] Accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and measure, and the result is shown in Table 1. Figure 4-Figure 5 .

[0110] Example 7: Application of saponin components in American ginseng stems and leaves in the treatment of liver cancer.

[0111] 1. Experimental Materials

[0112] 1.1 Experimental animals

[0113] Mice, female, weighing 20±2g, were obtained from Shandong Ailek Biotechnology Co., Ltd. The temperature of the animal room was 20±2℃, the relative humidity was 65%-75%, and the feed was standard ordinary feed. During the experiment, the animals had free access to water and food.

[0114] 1.2 Drugs and reagents

[0115] American ginseng saponin extract; cyclophosphamide; medical alcohol, normal saline, high-glucose culture medium

[0116] 1.3 Main instruments

[0117] (1) -80℃ ultra-low temperature refrigerator (SANYO, Japan);

[0118] (2) HFsafe-1200LC biological safety cabinet (Shanghai Lishen Scientific Instrument Co., Ltd.);

[0119] (3) Centrifuge (BIOBASE);

[0120] (4) Carbon dioxide incubator (Shanghai Haozhuang Instrument Co., Ltd.);

[0121] (5) DMI3000b inverted fluorescence microscope (Thermo Scientific);

[0122] (6) 1 / 10,000 electronic balance (Sartorius Scientific Instruments Co., Ltd.)

[0123] 1.4 Hepatocellular carcinoma cell lines

[0124] H22 liver cancer tumor line (Nanjing Kebai Biotechnology Co., Ltd.).

[0125] 2. Experimental Methods

[0126] 2.1 Recovery of liver cancer cell lines

[0127] Take out the frozen tumor cell line from the -80℃ ultra-low temperature refrigerator and thaw quickly in a 37℃ water bath. Transfer the frozen cells to a centrifuge tube in a biosafety cabinet, centrifuge at 3000r for 5min, discard the supernatant, add culture medium to the centrifuge tube, blow off the cells, transfer to a culture bottle, replenish the culture medium, blow them evenly, observe the cell morphology under a microscope, and incubate at 37℃ and 5% CO 2 Culture in an incubator.

[0128] 2.2 Cell culture via intraperitoneal cavity

[0129] Take out the well-growing liver cancer cells from the incubator and blow them with a pipette in a biosafety cabinet to make them

[0130] The cells were evenly dispersed in the culture medium, transferred to a centrifuge tube, centrifuged at 3000r for 5min, the supernatant was discarded, physiological saline was added, shaken evenly, and centrifuged again, and repeated 3 times. The cell concentration was adjusted to 2×10 7 The suspension was inoculated into the peritoneal cavity of Kunming mice at a rate of 0.3 mL / mouse. The cells were passaged intraperitoneally for 3 times to allow the liver cancer cells to adapt to the animal's internal environment and grow stably.

[0131] 2.3 Model establishment

[0132] Ascites was extracted from the peritoneal cavity of mice inoculated with H22 tumor strain. The ascites was light milky white and diluted with normal saline. After thorough mixing, the number of live cells was counted on a hemocytometer and the cell concentration was adjusted to 1×10 7 The suspension was inoculated into the right armpit of Kunming mice at a rate of 0.2 mL / mouse.

[0133] 2.4 Grouping and Dosing

[0134] The animals were divided into a blank control group (A), a tumor-bearing control group (B), a cyclophosphamide group (C), a low-dose group (D), a medium-dose group (E), and a high-dose group (F). 24 hours after inoculation, the mice in each group were weighed and a certain amount of drugs were administered.

[0135] Group A: 0.9% saline, 0.1 mL / 10 g;

[0136] Group B: 0.9% saline, 0.1 mL / 10 g;

[0137] Group C: 30 mg / kg, 0.1 mL / 10 g;

[0138] Group D: American ginsenoside extract, 5 mg / kg, 0.1 mL / 10 g;

[0139] Group E: American ginsenoside extract, 12 mg / kg, 0.1 mL / 10 g;

[0140] Group F: American ginseng saponin extract, 30 mg / kg, 0.1 mL / 10 g.

[0141] Except for the positive drug cyclophosphamide, which is administered by intraperitoneal injection,

[0142] The other groups were all given the drug by gavage for 20 consecutive days.

[0143] 2.5 Observation indicators

[0144] 2.5.1 General items

[0145] After the experiment started, the living conditions of the mice were observed, including their coat color, diet, drinking water, activity, and mental state.

[0146] Keep detailed records of the patient's condition and tumor growth.

[0147] 2.5.2 Calculation of tumor inhibition rate

[0148] 24 hours after the last administration, all mice were killed by cervical dislocation, and the intact tumor was peeled off and weighed to calculate the tumor inhibition rate. Tumor inhibition rate = (average tumor weight of tumor-bearing control group - average tumor weight of drug-treated group) / average tumor weight of control group × 100%.

[0149] 3. Experimental results

[0150] 3.1 Survival status of mice

[0151] After administration, the mice in the blank group ate, drank water and moved well, and no abnormal phenomena occurred. The mice in the tumor-bearing control group began to show mental depression, reduced eating and drinking, and the tumor volume in the right armpit continued to increase; the mice in the C group were more active, and the tumor volume grew slowly; the low-dose group drank water, ate, and moved better than the model group mice, and the tumor volume first grew slowly and then grew rapidly; the mice in the medium-dose group drank water, ate, and moved better than the model group mice, and the tumor volume grew slowly; the mice in the high-dose group were more depressed, and their eating and drinking conditions were poor, and the tumor volume first grew rapidly and then grew slowly.

[0152] 3.2 Tumor inhibitory effect

[0153] All doses of American ginseng saponin extract can inhibit tumor growth, and low and medium doses can significantly inhibit tumor growth, with obvious anti-tumor effects, and the tumor inhibition rates are 53.03% and 62.44% respectively. Therefore, American ginseng saponin extract has a good anti-tumor effect.

[0154] Table 2 Tumor inhibition rate of each group of drugs

[0155] ;

[0156] In this experiment, all doses of American ginseng saponin extract showed different degrees of anti-tumor effects, among which the medium-dose group had the strongest anti-tumor effect, with a tumor inhibition rate of 62.44%.

Claims

1. A method for extracting saponin components from American ginseng stems and leaves, characterized in that: The following steps are involved: (1) Using the harvested American ginseng stems and leaves as raw materials, the American ginseng stems and leaves and longan pulp are steamed for 5-8 hours, the longan pulp is removed, and the American ginseng stems and leaves are dried to obtain the American ginseng stem and leaf steamed product; (2) Removing pigments: Steamed American ginseng stems and leaves are taken, crushed through a 100-300 mesh sieve, divided into several 300 mesh bags, placed in a Soxhlet extractor, and 500 ml of a mixed solution of ammonia water and ethyl acetate in a volume ratio of 0.5:99.5 is added, heated and refluxed for 1.5 hours, and the mixed solution of ammonia water and ethyl acetate is discarded. The residue is evaporated to remove the ammonia water and ethyl acetate, and the depigmented American ginseng stems and leaves are obtained; (3) Extracting total saponins: The depigmented American ginseng stems and leaves obtained in step (2) were placed back into a Soxhlet extractor, 1000 ml of ammonia-methanol mixed solution with a volume ratio of 0.5:99.5 was added, and the mixture was heated under reflux for extraction for 12 h. The ammonia-methanol mixed solution was recovered until dry, and 300 ml of water saturated with n-butanol was added to the residue to dissolve it, and the residue was transferred to a separatory funnel, and extracted with n-butanol saturated with water by shaking for at least 4 times, and the n-butanol solution was combined; (4) Removing impurities: washing the n-butanol solution obtained in step (3) with a 1% hydrochloric acid solution, discarding the hydrochloric acid solution, and then washing with water saturated with n-butanol until neutral, discarding the water washing solution, and recovering the n-butanol to dryness to obtain a total saponin extract of American ginseng stems and leaves with a purity of more than 95%; (5) Three-phase high-speed countercurrent chromatography separation and purification: The stationary phase was an aqueous phase dispersed with O-carboxymethyl chitosan functionalized multi-walled carbon nanotubes, the mobile phase A was water-saturated 5:1 carbon tetrachloride-acetone, the mobile phase B was water-saturated 10:1 n-butanol-methanol, the gradient flow rate was set to 0 min, 95% A; 60 min, 65% A; 120 min, 35% A; the column volume of the high-speed countercurrent chromatography was 500 ml, the sample load was 50 mg, the rotation speed was 900 rpm, the flow rate was 2 mL / min, and the detector was an evaporative light detector; Collect the eluate from 30min to 60min, evaporate the solvent to dryness, and obtain protopanaxadiol saponins: ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3; The eluate was collected at 90 min to 120 min, and the solvent was evaporated to dryness to obtain C-17 side chain isomeric saponins: American ginsenoside L1, American ginsenoside L2, and American ginsenoside L3.

2. The extraction method according to claim 1, characterized in that: Step (1) comprises 100 g of American ginseng stems and leaves and 100 g of longan pulp; and step (2) comprises 100 g of steamed American ginseng stems and leaves.

3. The extraction method according to claim 1, characterized in that: In step (5), the ratio of protopanaxadiol saponins: ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rb3 is 5:2:

15.

4. The extraction method according to claim 1, characterized in that: In step (5), the ratio of C-17 side chain isomeric saponin: American ginsenoside L1, American ginsenoside L2, and American ginsenoside L3 is 3:5:

8.

5. Use of protopanaxadiol saponin prepared by the extraction method according to claim 1 in the preparation of drugs for treating liver cancer.

6. Use of the C-17 side chain isomeric saponin prepared by the extraction method of claim 1 in the preparation of a drug for treating liver cancer.

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