A method for separating and purifying chemical components in Gynostemma pentaphyllum

Abstract

The present invention belongs to the technical field of natural chemical component extraction, relates to the extraction of chemical components of traditional Chinese medicine, and specifically relates to a method for separating and purifying chemical components in Gynostemma pentaphyllum. Gynostemma pentaphyllum is soaked in an ethanol aqueous solution, and then heated under reflux extraction to obtain a total extract of Gynostemma pentaphyllum, which is then separated and purified using gradient countercurrent chromatography; in the gradient countercurrent chromatography, the upper phase of n-butanol-water is mobile phase A, the upper phase of ethyl acetate-water is mobile phase B, and the lower phase of ethyl acetate-water is stationary phase; the injection amount of the gradient countercurrent chromatography is 70-100 mg; the separated and purified compounds are quercetin-3-O-neohesperidin, gypenosyl saponin LVI, ginsenoside Rb3, and gypenosyl saponin XLVI.

Description

Technical Field

[0001] The invention belongs to the technical field of natural chemical component extraction, relates to the extraction of chemical components of traditional Chinese medicine, and particularly relates to a method for separating and purifying chemical components in Gynostemma pentaphyllum. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Saponins are an important class of natural compounds found in nature. Studies have shown that saponins are the main active ingredients of many Chinese medicinal herbs, such as ginseng, Panax notoginseng, Gynostemma pentaphyllum, Licorice root, Acanthopanax senticosus, Bupleurum chinense, Sanguisorba officinalis, Pulsatilla chinensis, and Polygala tenuifolia. Their main pharmacological effects include anti-inflammatory, antibacterial, anti-tumor, hypoglycemic, liver protection, and immune regulation. Saponins are mainly divided into triterpenoid saponins and steroidal saponins. According to the inventors' research, there is less research on chemical components such as saponins in Gynostemma pentaphyllum compared to Chinese medicinal herbs such as ginseng and Panax notoginseng. Therefore, there is a need to separate and purify the chemical components, including saponins, in Gynostemma pentaphyllum. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention aims to provide a method for separating and purifying the chemical components in Gynostemma pentaphyllum, which can separate quercetin-3-O-neohesperidin, gypenosyl saponin LVI, ginsenoside Rb3, and gypenosyl saponin XLVI from Gynostemma pentaphyllum.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] A method for separating and purifying chemical components in Gynostemma pentaphyllum comprises soaking Gynostemma pentaphyllum in an ethanol-water solution, heating and refluxing to obtain a Gynostemma pentaphyllum total extract, and separating and purifying the Gynostemma pentaphyllum total extract by gradient countercurrent chromatography.

[0007] In gradient countercurrent chromatography, the upper phase of n-butanol-water is mobile phase A, the upper phase of ethyl acetate-water is mobile phase B, and the lower phase of ethyl acetate-water is the stationary phase;

[0008] The gradient conditions in the gradient countercurrent chromatography are: 0-20 min, 10% A; 50-100 min, 20% A; 110-140 min, 30% A; 160-240 min, 60% A; or, 0-20 min, 10% A; 50-100 min, 20% A; 130 min, 30% A; 140 min, 35% A; 160-240 min, 60% A;

[0009] The injection volume for gradient countercurrent chromatography was 70–100 mg;

[0010] The isolated and purified compounds are quercetin-3-O-neohesperidin, gypenosapside LVI, ginsenoside Rb3, and gypenosapside XLVI.

[0011] The present invention adopts countercurrent chromatography technology, and realizes the separation and purification of quercetin-3-O-neohesperidin, gypenosapogenin LVI, ginsenoside Rb3 and gypenosapogenin XLVI in Gynostemma pentaphyllum by selecting a mobile phase, setting gradient conditions and adjusting the injection volume.

[0012] Among them, the study showed that when the gradient conditions are 0-20 min, 10% A; 50-100 min, 20% A; 130 min, 30% A; 140 min, 35% A; 160-240 min, 60% A, the elution time can be shortened and the efficiency of separating and purifying quercetin-3-O-neohesperidin, gypenosyl saponin LVI, ginsenoside Rb3, and gypenosyl saponin XLVI from Gynostemma pentaphyllum can be improved.

[0013] Studies have shown that after the mobile phase, stationary phase and gradient conditions are determined, the injection volume will also affect the separation and purification of quercetin-3-O-neohesperidin, gypenoside LVI, ginsenoside Rb3, and gypenoside XLVI. When the injection volume is 70-100 mg, the separation and purification of quercetin-3-O-neohesperidin, gypenoside LVI, ginsenoside Rb3, and gypenoside XLVI can be completely guaranteed. However, when the injection volume is outside the range of 70-100 mg, especially at 150 mg, the separation and purification of quercetin-3-O-neohesperidin, gypenoside LVI, ginsenoside Rb3, and gypenoside XLVI cannot be completely achieved. When the injection volume is between 70 and 100 mg, the higher the injection volume, the more corresponding compounds are obtained. Therefore, when the injection volume is between 95 and 100 mg, the separation and purification effect of quercetin-3-O-neohesperidin, gypenosyl saponin LVI, ginsenoside Rb3, and gypenosyl saponin XLVI is better.

[0014] Further research found that the combined components of kaempferol-3-O-robinoside and kaempferol-3-O-neohesperidin can also be separated by gradient countercurrent chromatography. However, kaempferol-3-O-robinoside and kaempferol-3-O-neohesperidin are isomers, and it is difficult to achieve the separation and purification of kaempferol-3-O-robinoside and kaempferol-3-O-neohesperidin using only gradient countercurrent chromatography. Therefore, in some embodiments, the components of kaempferol-3-O-robinoside and kaempferol-3-O-neohesperidin separated by gradient countercurrent chromatography are used as raw materials, and cyclic countercurrent chromatography separation is further performed to achieve the separation and purification of kaempferol-3-O-robinoside and kaempferol-3-O-neohesperidin.

[0015] The beneficial effects of the present invention are:

[0016] The present invention establishes a suitable linear gradient elution countercurrent chromatography for the gynostemma pentaphyllum total extract, constructs a solvent system of n-butanol / ethyl acetate / water, optimizes the gradient elution conditions and the injection volume, thereby achieving the separation and purification of quercetin-3-O-neohesperidin, gypenosylceraside LVI, ginsenoside Rb3, and gypenosylceraside XLVI in the gynostemma pentaphyllum total extract. In view of the problem that kaempferol-3-O-acacia glycoside and kaempferol-3-O-neohesperidin are difficult to separate using linear gradient elution countercurrent chromatography, the present invention combines a circulating countercurrent mode to achieve complete separation of kaempferol-3-O-acacia glycoside and kaempferol-3-O-neohesperidin, thereby completing the separation and purification of quercetin-3-O-neohesperidin, kaempferol-3-O-acacia sugar, kaempferol-3-O-neohesperidin, gypenosapogenin LVI, ginsenoside Rb3 and gypenosapogenin XLVI in the total extract of Gynostemma pentaphyllum. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 The chromatogram is a chromatogram of gradient countercurrent chromatography separation performed under the initial gradient condition (gradient condition 1) and an injection volume of 70 mg in an embodiment of the present invention;

[0019] Figure 2 The chromatogram is a chromatogram of gradient countercurrent chromatography separation performed under gradient condition 2 and an injection volume of 70 mg according to an embodiment of the present invention;

[0020] Figure 3 The chromatogram is a chromatogram of gradient countercurrent chromatography separation performed under gradient condition 3 and an injection volume of 70 mg according to an embodiment of the present invention;

[0021] Figure 4 This is a chromatogram of gradient countercurrent chromatography separation performed under gradient condition 3 and an injection volume of 100 mg in an embodiment of the present invention;

[0022] Figure 5 This is a chromatogram of gradient countercurrent chromatography separation performed under gradient condition 3 and an injection volume of 150 mg in an embodiment of the present invention;

[0023] Figure 6 This is a chromatogram of the cyclic countercurrent chromatography separation of compounds 2 and 3 in an embodiment of the present invention;

[0024] Figure 7Figure 1 is an HPLC detection diagram of different components in the separation process of an embodiment of the present invention, wherein a is a total extract of Gynostemma pentaphyllum, b is a component of compounds 2 and 3 separated by gradient countercurrent chromatography, c is a component of compound 1 separated by gradient countercurrent chromatography, d is a component of compound 2 separated by circulating countercurrent chromatography, e is a component of compound 3 separated by circulating countercurrent chromatography, f is a component of compound 4 separated by gradient countercurrent chromatography, g is a component of compound 5 separated by gradient countercurrent chromatography, and h is a component of compound 6 separated by gradient countercurrent chromatography. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0026] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0027] In order to separate and purify chemical components including saponins in Gynostemma pentaphyllum, the present invention provides a method for separating and purifying chemical components in Gynostemma pentaphyllum.

[0028] A typical embodiment of the present invention provides a method for separating and purifying chemical components from Gynostemma pentaphyllum, comprising soaking Gynostemma pentaphyllum in an ethanol aqueous solution, heating and refluxing to obtain a Gynostemma pentaphyllum total extract, and separating and purifying the Gynostemma pentaphyllum total extract using gradient countercurrent chromatography;

[0029] In gradient countercurrent chromatography, the upper phase of n-butanol-water is mobile phase A, the upper phase of ethyl acetate-water is mobile phase B, and the lower phase of ethyl acetate-water is the stationary phase;

[0030] The gradient conditions in the gradient countercurrent chromatography are: 0-20 min, 10% A; 50-100 min, 20% A; 110-140 min, 30% A; 160-240 min, 60% A; or, 0-20 min, 10% A; 50-100 min, 20% A; 130 min, 30% A; 140 min, 35% A; 160-240 min, 60% A;

[0031] The injection volume for gradient countercurrent chromatography was 70–100 mg;

[0032] The isolated and purified compounds are quercetin-3-O-neohesperidin, gypenosapside LVI, ginsenoside Rb3, and gypenosapside XLVI.

[0033] In some embodiments, the gradient conditions in the gradient countercurrent chromatography are: 0-20 min, 10% A; 50-100 min, 20% A; 130 min, 30% A; 140 min, 35% A; 160-240 min, 60% A. % A is the volume fraction of mobile phase A in the mobile phase.

[0034] In some embodiments, in gradient countercurrent chromatography, mobile phase A is n-butanol saturated with water.

[0035] In some embodiments, in gradient countercurrent chromatography, mobile phase B is ethyl acetate saturated with water.

[0036] In some embodiments, the stationary phase in gradient countercurrent chromatography is saturated ethyl acetate in water.

[0037] In some embodiments, in gradient countercurrent chromatography, the injection amount is 95-100 mg.

[0038] In some embodiments, in gradient countercurrent chromatography, the flow rate of mobile phase B is 4-6 mL / min.

[0039] In some embodiments, in gradient countercurrent chromatography, an evaporative light detector is used for detection.

[0040] In some embodiments, the ethanol aqueous solution is an ethanol aqueous solution with a volume fraction of 78-82%.

[0041] In some embodiments, the solid-to-liquid ratio of Gynostemma pentaphyllum to ethanol aqueous solution is 250-350:1, g:L.

[0042] In some embodiments, the components of kaempferol-3-O-sophoroside and kaempferol-3-O-neohesperidin separated by gradient countercurrent chromatography are used as raw materials to continue cyclic countercurrent chromatography separation.

[0043] In one or more embodiments, in the circulating countercurrent chromatography, the upper phase of n-butanol / ethyl acetate / water is used as the stationary phase, and the lower phase of n-butanol / ethyl acetate / water is used as the mobile phase.

[0044] Specifically, in the circulating countercurrent chromatography, the volume ratio of n-butanol, ethyl acetate and water is 1.4:8.6:10 to 1.6:8.4:10.

[0045] In one or more embodiments, in the circulating countercurrent chromatography, the flow rate of the mobile phase is 4 to 6 mL / min.

[0046] In one or more embodiments, the cyclic countercurrent chromatography is performed with cyclic separation at least 4 times, so as to completely separate kaempferol-3-O-sophoroside from kaempferol-3-O-neohesperidin.

[0047] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0048] Example

[0049] Material

[0050] Gynostemma pentaphyllum was purchased from Bozhou Medicinal Materials Market in Anhui Province and identified as Gynostemma pentaphyllum (Thunb.) Makino of the Cucurbitaceae family by researcher Wang Daijie from Shandong Provincial Analysis and Testing Center.

[0051] Ethyl acetate, analytical grade, Tianjin Kermiou Chemical Reagent Co., Ltd.; methanol, analytical grade, Tianjin Kermiou Chemical Reagent Co., Ltd.; n-butanol, analytical grade, Tianjin Kermiou Chemical Reagent Co., Ltd.; acetonitrile, chromatographic grade, Tianjin Concord Technology Co., Ltd.

[0052] Extraction of compounds from Gynostemma pentaphyllum:

[0053] Weigh 600 g of Gynostemma pentaphyllum in a reflux flask, add 2 L of 80% ethanol solution, and soak overnight. The next day, open the reflux extraction device, heat reflux for 2 hours, extract three times in total, filter and combine the extracts, and concentrate by rotary evaporation to obtain 58.6 g of Gynostemma pentaphyllum total extract.

[0054] Solvent system determination:

[0055] Prepare 10mL of solvent according to different solvent systems, shake it thoroughly and evenly, and let it stand for stratification. After stratification, take 2mL of the upper and lower phases and put them in the same test tube, add about 1mg of sample, shake it vigorously to dissolve, and let it stand. After the upper and lower phases are completely separated, take about 0.8mL of solution from each phase into a test tube, place the test tube in a centrifugal concentrator to concentrate, and allow the solvent to evaporate completely. After it is completely evaporated, add equal amounts of methanol solution to the upper and lower phases for dissolution. Pass the sample solution through a 0.22μm nylon filter membrane and place it in a liquid phase bottle. Use HPLC to determine the peak area of each target peak in the upper phase and lower phase. The peak area of each target peak in the upper phase is recorded as A1, and the peak area of each target peak in the lower phase is recorded as A2. Calculate the distribution coefficient K of the sample in different solvent systems. D .

[0056] The distribution coefficient K of the sample D Calculated as follows:

[0057] K D =A1 / A2

[0058] Solvent system and sample solution preparation:

[0059] (1) Preparation of gradient countercurrent chromatography solvent system:

[0060] A certain amount of n-butanol and water were added to a separatory funnel. The separatory funnel was shaken vigorously and then allowed to stand. After separation, the upper phase was n-butanol saturated with water, which served as mobile phase A. A certain amount of ethyl acetate and water were added to a separatory funnel. The separatory funnel was shaken vigorously and then allowed to stand. After separation, the upper phase was ethyl acetate saturated with water, which served as mobile phase B, and the lower phase was water saturated with ethyl acetate, which served as the stationary phase.

[0061] (2) Preparation of sample solution:

[0062] Accurately weigh 40 mg of sample, take 5 mL of mobile phase and stationary phase respectively and dissolve them in a test tube, shake vigorously to fully dissolve them and set aside.

[0063] Countercurrent chromatography procedure:

[0064] (1) Gradient countercurrent chromatography technology

[0065] This experiment used an ethyl acetate-n-butanol-water solvent system. The n-butanol-water upper phase was mobile phase A, the ethyl acetate-water lower phase was the stationary phase, and the upper phase was mobile phase B. The temperature was set at 25°C. After the HSCCC was fully pumped with the stationary phase at a flow rate of 20 mL / min, the "FWD-OUT" operating mode was selected, the speed was 800 rpm, and the mobile phase B was pumped in at a flow rate of 5.0 mL / min. The volume of the stationary phase lost was recorded when the solvent system reached dynamic equilibrium in the CCC column. The oil-free air pump, evaporative light detector, and online recording software were turned on, and the evaporative light gain value was set to 6. When the solvent system reached dynamic equilibrium in the CCC column, the volume of the stationary phase lost was recorded, and the pre-prepared sample solution was injected into the injection valve. The flow rate and speed were maintained unchanged, and high-speed countercurrent chromatography separation was initiated.

[0066] (2) Circulating countercurrent chromatography technology

[0067] According to the ultraviolet absorption graph of high-speed countercurrent chromatography, one tube was collected every 2 minutes, the collected components were detected by HPLC and the collected elution solutions with the same components were combined, concentrated by rotary evaporation and re-dissolved with methanol, filtered with a 0.22 μm organic filter membrane and then tested for purity by HPLC.

[0068] Segmented collection and detection of countercurrent chromatography separations:

[0069] High-speed countercurrent chromatography separation experiments were performed under optimal conditions, with one tube (total volume of 10 mL) collected every 2 minutes. The collected components were detected by HPLC, and the elution solutions with the same components were combined and concentrated by rotary evaporation. The powdered samples were obtained after freeze-drying, and their purity was detected by HPLC.

[0070] HPLC detection of chemical components in Gynostemma pentaphyllum:

[0071] HPLC analysis conditions for Gynostemma pentaphyllum extract and countercurrent chromatography fractions were as follows: SYMMETRY-C18 column (250 mm × 4.6 mm, 5.0 μm); mobile phase: acetonitrile (A)-water (B): 0 min, 15% A; 5 min, 22% A; 10 min, 22% A; 15 min, 40% A; 20 min, 45% A; 30 min, 95% A; 35 min, 95% A. The flow rate was 1.0 mL / min, the injection volume was 10 μL, and the detector wavelength was 203 nm.

[0072] Results and Discussion

[0073] Choice of solvent system:

[0074] The 1 / KD values of Gynostemma pentaphyllum extract in different solvent systems of ethyl acetate-n-butanol-water are shown in Table 1.

[0075] Table 1 Partition coefficients of Gynostemma pentaphyllum extracts in different solvent systems

[0076]

[0077] In the solvent system (ethyl acetate / n-butanol / water, v / v), as the proportion of n-butanol gradually increases, the elution ability of each target component gradually increases, and the 1 / KD value gradually decreases. As shown in the table, compounds 1, 2, and 3 can be eluted when the proportion of n-butanol is 20%, but the α values of compounds 2 and 3 are small and cannot be completely separated. When the proportion of n-butanol is 40%, compounds 5 and 6 are eluted in sequence according to polarity. Compound 4 requires an n-butanol concentration of 60% to elute. Based on the results of the determination of the target compound distribution coefficient, this experiment selected the gradient countercurrent separation mode. The upper phase of the solvent system composed of n-butanol-water is fluidity A, the lower phase of the solvent system composed of ethyl acetate-water is stationary, and the upper phase is fluidity B. The n-butanol proportion varies from 10% to 60%.

[0078] Gradient countercurrent chromatography separation:

[0079] The stationary phase was pumped into the HSCCC at a flow rate of 20.0 mL / min. The HSCCC was operated in the "FWD-OUT" mode at 800 rpm, at a temperature of 25°C, and mobile phase B was pumped in at a flow rate of 5 mL / min. After reaching equilibrium, the stationary phase retention was calculated to be 71.5%. The gradient conditions and injection volume were continuously adjusted and optimized, and experimental verification was performed. The experimental adjustment and optimization process is as follows: Figure 1-5 As shown, the gradient conditions and injection volume optimization tests are as follows:

[0080] The initial gradient conditions were set as 0-20 min, 10% A; 50 min, 20% A; 70 min, 22% A; 110 min, 24% A; 140 min, 30% A; 180 min, 36% A; 190-240 min, 60% A, with a flow rate of 5.0 mL / min and an injection volume of 70 mg. Figure 1 As shown, all target compounds were eluted, while compounds 1, 2, 3 and compounds 5 and 6 were not completely separated, and the elution time of compound 4 was longer, which was 2.8h. Figure 2 As shown in Figure 2, gradient elution condition 2 reduces the elution capacity of the first half based on the initial gradient. Compound 1 is completely separated, but compounds 2 and 3 are still not separated. Compounds 5 and 6 are completely separated in about 2.5 hours, and the elution time is extended to 3.2 hours. Figure 3 As shown, gradient elution condition 3 keeps the elution gradient unchanged in the first half, increases the change in n-butanol concentration after 2.5 h, accelerates the elution of compound 4, and shortens the elution time to 2.8 h.

[0081] Increasing the injection volume can produce more target compounds. The injection volume of 150 mg was tested under the optimized gradient condition 3. Figure 5 As can be seen from the figure, when the injection volume is increased to 150 mg, the separation effect of each target component is not ideal, and the loss of fixed components during elution is serious. Under solvent gradient condition 3, the injection volume is adjusted to 100 mg. Figure 4 As shown, good separation of compounds 2 and 3 and 1, 4, 5, and 6 was achieved.

[0082] Taking into account the loss of stationary phase and the separation effect, the gradient condition 3 was determined as the optimal condition for gradient countercurrent elution of Gynostemma pentaphyllum: 0-20 min, 10% A; 50-100 min, 20% A; 130 min, 30% A; 140 min, 35% A; 160-240 min, 60% A, with a flow rate of 5.0 mL / min and an injection volume of 100 mg.

[0083] Circulating countercurrent chromatography:

[0084] Using the gradient countercurrent elution mode, compounds 2 and 3 were not completely separated. The two compounds were further separated using a circular countercurrent. The solvent system was n-butanol / ethyl acetate / water (1.5:8.5:10, v / v), the upper phase was the stationary phase, and the lower phase was the mobile phase. The stationary phase was pumped into the HSCCC at a flow rate of 20.0 mL / min. The HSCCC was set to "FWD-IN" operation mode and a speed of 800 rpm. The temperature was 25°C and the mobile phase was pumped in at a flow rate of 5.0 mL / min. After reaching dynamic equilibrium, the stationary phase retention rate was calculated to be 56.7%. As shown in Figure 6, the compound was eluted at 60 minutes, and the eluent was pumped into the CCC for further separation. After four cycles of separation, compounds 2 and 3 were completely separated.

[0085] HPLC analysis of Gynostemma pentaphyllum extract Figure 7 shown.

[0086] Structural identification of isolated substances:

[0087] Each component 1 H and 13 The C(NMR) data were compared with those reported previously, and the structures of the compounds isolated from the processed black ginseng were determined to be: quercetin-3-O-neohesperidin (1), kaempferol-3-O-sophoroside (2), kaempferol-3-O-neohesperidin (3), gypenosyl saponin LVI (4), ginsenoside Rb3 (5), and gypenosyl saponin XLVI (6).

[0088] Its structural analysis data is as follows:

[0089] Quercetin-3-O-neohesperidin (1): ESI-MS m / z 611.1[M+H] + , molecular formula is C 27 H 30 O 16 . 1 H NMR (500MHz, DMSO) δ7.61(dd,J=8.4,1.7Hz,1H,H-6'),7.53(d,J=1.8Hz,1H,H-2'),6.83(d,J=8.5Hz,1H,H-5'),6.39(d,J=1.6H z,1H,H-8),6.19(d,J=1.6Hz,1H,H-6),5.65(d,J=7.7Hz,1H,Glc-1),5.29(d,J=5.5Hz,1H,Rha-1),0.77(d,J=6.1Hz,3H,Rha-6). 13C NMR(125MHz,DMSO)δ177.3(C-4),164.1(C-7),161.3(C-5),156.3(C-2),156.1(C-9),148.4(C-4') ,144.9(C-3'),132.9(C-3),121.7(C-6'),121.2(C-1'),116.0(C-2'),115.1(C-5'),104.0(C-10) ,100.5(Rha-1),98.7(Glc-1),98.4(C-6),93.5(C-8),77.5(Glc-2),77.3(Glc-3),77.3(Glc-5),7 1.9(Rha-4),70.6(Rha-2),70.6(Rha-3),70.3(Glc-4),68.3(Rha-5),61.0(Glc-6),17.2(Rha-6).

[0090] The chemical structure of quercetin-3-O-neohesperidin (1) is shown below:

[0091]

[0092] Kaempferol-3-O-sophora bioside (2): ESI-MS m / z 629.5 [M+Cl] - , molecular formula is C 27 H 30 O 15 . 1 HNMR(500MHz,DMSO)δ8.05(d,J=8.9Hz,2H,H-2',6'),6.86(d,J=8.9Hz,2H,H-3',5'),6.43(d,J=2.1Hz,1H,H-8),6 .20(d,J=2.1Hz,1H,H-6),5.32(d,J=7.7Hz,1H,Gal-1),5.20(d,J=4.5Hz,1H,Rha-1),1.06(d,J=6.2Hz,3H,Rha-6). 13C NMR(125MHz,DMSO)δ177.0(C-4),164.1(C-7),161.2(C-5),160.0(C-4'),156.6(C-2),156.4 (C-9),133.3(C-3),131.0(C-2',6'),120.8(C-1'),115.1(C-3',5'),104.0(C-10),102.0(Ga l-1),100.1(Rha-1),98.7(C-6),93.7(C-8),73.5(Gul-5),73.0(Gul-3),71.9(Rha-4),71.1( Rha-2),70.6(gul-2),70.4(Rha-3),68.3(Rha-5),68.0(gul-4),65.3(gul-6),19.9(Rha-6).

[0093] The chemical structure of kaempferol-3-O-sophoroside (2) is shown below:

[0094]

[0095] Kaempferol-3-O-neohesperidin (3): ESI-MS m / z[M+Na] + , molecular formula is C 27 H 30 O 15 . 1 H NMR (500MHz, DMSO) δ8.03(d,J=8.8Hz,2H,H-2',6'),6.88(d,J=8.8Hz,2H,H-3',5'),6.43(d,J=1.9Hz,1H,H-8),6. 20(d,J=1.9Hz,1H,H-6),5.66(d,J=7.4Hz,1H,Glc-1),5.28(d,J=5.6Hz,1H,Rha-1),0.76(d,J=6.1Hz,3H,Rha-6). 13C NMR(125MHz,DMSO)δ177.4(C-4),164.1(C-7),161.3(C-5),159.9(C-4'),156.3(C-2),156.1(C- 9),132.7(C-3),130.8(C-2',6'),120.9(C-1),115.1(C-3',5'),104.0(C-10),100.6(Rha-1),9 8.7(C-8),98.3(glc-1),93.7(C-6),17.3(Rha-6),77.5(glc-2),77.5(glc-3),77.3(glc-5),71 .8(Rha-4),70.6(Rha-2),70.6(Rha-3),70.2(glc-4),68.3(Rha-5),60.8(glc-6),17.3(Rha-6).

[0096] The chemical structure of kaempferol-3-O-neohesperidin (3) is shown below:

[0097]

[0098] Gynostemma pentaphyllum saponin LVI(4): ESI-MS m / z 1095.6[M+H] + , molecular formula is C 53 H 90 O 23 . 13C NMR(101MHz,MeOD)δ:132.2(C-25),126.1(C-24),105.6(GlcⅢ-1),104.8(GlcⅠ-1),104.3(GlcⅡ -1),98.1(Xyl-1),96.7(C-3),84.9(C-20),80.6(GlcⅠ-2),78.6(GlcⅢ-3),78.6(GlcⅡ-3),78.3 (GlcⅠ-3),78.1(GlcⅠ-5),78.0(GlcⅡ-5),77.5(Xyl-3),76.7(GlcⅢ-5),76.1(GlcⅡ-2),75.3(Gl cⅢ-2),74.8(Xyl-2),72.0(GlcⅠ-4),71.5(C-12),71.4(GlcⅢ-4),71.2(GlcⅡ-4),71.2(Xyl-4), 70.1(GlcⅢ-6),68.1(C-12),66.8(Xyl-5),63.2(GlcⅠ-6),62.4(GlcⅡ-6),57.2(C-5),52.9(C-1 7),52.4(C-14),51.0(C-9),49.7(C-13),41.9(C-4),41.0(C-8),38.8(C-10),36.7(C-10),36. 7(C-22),35.7(C-7),31.5(C-11),31.0(C-15),28.7(C-29),27.3(C-16),25.9(C-26),23.8(C- 23),22.4(C-21),19.3(C-6),19.3(C-27),18.0(C-28),17.8(C-19),17.4(C-30),16.3(C-18).

[0099] The chemical structure of gypenoside LVI (4) is shown below:

[0100]

[0101] Ginsenoside Rb3(5): ESI-MS m / z 1113.5[M+Cl] - , molecular formula is C 53 H 90 O 22 . 13C NMR(101MHz,MeOD)δ:132.2(C-25),126.1(C-24),105.6(Xyl-1),105.4(GlcⅠ-1),104.5(Glc Ⅱ-1),98.1(GlcⅢ-1),91.3(C-3),85.6(C-20),81.1(C-2),78.6(GlcⅡ-5),78.5(GlcⅡ-3),78. 3(GlcⅢ-3),77.9(Xyl-3),77.7(GlcⅠ-5),77.5(GlcⅠ-3),76.7(GlcⅢ-5),76.3(GlcⅡ-2),75.3 (GlcⅢ-2),74.8(Xyl-2),71.9(GlcⅡ-4),71.7(C-12),71.6(GlcⅠ-4),71.5(GlcⅢ-4),71.2(Xy l-4),70.1(GlcⅢ-6),66.8(Xyl-5),63.1(GlcⅡ-6),62.9(GlcⅠ-6),57.6(C-5),52.9(C-17),5 2.4(C-14),51.1(C-9),49.7(C-13),41.0(C-8),40.6(C-4),40.2(C-1),37.9(C-10),36.8(C -22),35.9(C-7),31.5(C-15),30.9(C-11),28.4(C-28),27.3(C-16),25.9(C-26),23.8(C-2 3),22.4(C-21),19.2(C-6),18.0(C-27),17.4(C-30),16.7(C-29),16.7(C-19),16.3(C-18).

[0102] The chemical structure of ginsenoside Rb3(5) is shown below:

[0103]

[0104] Gynostemma pentaphyllum saponin XLVI(6): ESI-MS m / z 963.5[M+H] + , molecular formula is C 48 H 82 O 19 . 13C NMR(101MHz,MeOD)δ:132.3(C-25),125.9(C-24),104.8(GlcⅠ-1),104.4(GlcⅡ-1),98 .3(GlcⅢ-1),96.7(C-3),84.9(C-20),80.6(GlcⅠ-2),78.6(GlcⅡ-3),78.3(GlcⅢ-3),7 8.2(GlcⅠ-3),78.1(GlcⅢ-5),78.2(GlcⅡ-5),77.9(GlcⅠ-5),76.2(GlcⅡ-2),75.4(Glc Ⅲ-2),72.1(GlcⅠ-4),71.8(C-12),71.2(GlcⅡ-4),71.2(GlcⅢ-4),68.1(C-2),63.2(Gl cⅠ-6),62.6(GlcⅢ-6),62.4(GlcⅡ-6),57.2(C-5),53.1(C-17),52.5(C-14),51.0(C-9 ),49.8(C-13),47.9(C-1),41.9(C-4),41.0(C-8),38.8(C-10),36.7(C-22),35.7(C- 7),31.6(C-11),31.2(C-15),28.7(C-29),27.2(C-16),25.9(C-26),24.2(C-23),22. 8(C-21),19.3(C-6),17.9(C-27),17.8(C-28),17.8(C-19),17.2(C-30),16.2(C-18).

[0105] The chemical structure of gypenosanol XLVI (6) is shown below:

[0106]

[0107] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for separating and purifying chemical components in Gynostemma pentaphyllum, characterized in that: The Gynostemma pentaphyllum is soaked in an ethanol-water solution, and then heated under reflux for extraction to obtain a total extract of Gynostemma pentaphyllum, and the total extract of Gynostemma pentaphyllum is separated and purified by gradient countercurrent chromatography; In gradient countercurrent chromatography, the upper phase of n-butanol-water is mobile phase A, the upper phase of ethyl acetate-water is mobile phase B, and the lower phase of ethyl acetate-water is the stationary phase; The gradient conditions in the gradient countercurrent chromatography are: 0-20 min, 10% A; 50-100 min, 20% A; 110-140 min, 30% A; 160-240 min, 60% A; or, 0-20 min, 10% A; 50-100 min, 20% A; 130 min, 30% A; 140 min, 35% A; 160-240 min, 60% A; The injection volume for gradient countercurrent chromatography was 70–100 mg; The isolated and purified compounds were quercetin-3-O-neohesperidin, gypenosyl saponin LVI, ginsenoside Rb3, and gypenosyl saponin XLVI; In gradient countercurrent chromatography, mobile phase A is n-butanol saturated with water; In gradient countercurrent chromatography, mobile phase B is ethyl acetate saturated with water; In gradient countercurrent chromatography, the stationary phase is water saturated with ethyl acetate; In gradient countercurrent chromatography, the flow rate of mobile phase B is 4–6 mL / min; In gradient countercurrent chromatography, evaporative light detection was used for detection.

2. The method for separating and purifying chemical components in Gynostemma pentaphyllum as claimed in claim 1, wherein: The gradient conditions in the gradient countercurrent chromatography were: 0-20 min, 10% A; 50-100 min, 20% A; 130 min, 30% A; 140 min, 35% A; 160-240min, 60%A.

3. The method for separating and purifying chemical components in Gynostemma pentaphyllum as claimed in claim 1, wherein: In gradient countercurrent chromatography, the injection volume is 95-100 mg.

4. The method for separating and purifying chemical components in Gynostemma pentaphyllum as claimed in claim 1, wherein: The ethanol aqueous solution is an ethanol aqueous solution with a volume fraction of 78 to 82%; Alternatively, the solid-to-liquid ratio of Gynostemma pentaphyllum to the ethanol aqueous solution is 250-350:1, g:L.

5. The method for separating and purifying chemical components in Gynostemma pentaphyllum as claimed in claim 1, wherein: The components of kaempferol-3-O-sophoroside and kaempferol-3-O-neohesperidin separated by gradient countercurrent chromatography are used as raw materials to continue the cyclic countercurrent chromatography separation; In cyclic countercurrent chromatography, the upper phase of n-butanol / ethyl acetate / water was used as the stationary phase, and the lower phase of n-butanol / ethyl acetate / water was used as the mobile phase; In the circulating countercurrent chromatography, the volume ratio of n-butanol, ethyl acetate and water is 1.4:8.6:10 to 1.6:8.4:

10.

6. The method for separating and purifying chemical components in Gynostemma pentaphyllum as claimed in claim 5, wherein: In circulating countercurrent chromatography, the mobile phase flow rate is 4 to 6 mL / min.

7. The method for separating and purifying chemical components in Gynostemma pentaphyllum as claimed in claim 5, wherein: In cyclic countercurrent chromatography, the separation is cyclically repeated at least four times.

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