Gynostemma pentaphylla-derived triterpenoid saponin compounds and application thereof in preparation of anti-fatigue products

Abstract

The invention discloses a triterpenoid saponin compound derived from gynostemma pentaphylla and application of the triterpenoid saponin compound in preparation of an anti-fatigue product. The triterpenoid saponin compound provided by the invention is selected from one of the following compounds 1 to 29. The invention further provides application of the triterpenoid saponin compound shown in the general formula I in preparation of anti-fatigue products. Experiments show that the triterpenoid saponin compound derived from the gynostemma pentaphylla has remarkable anti-fatigue activity and can be used for preparing anti-fatigue products.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to triterpenoid saponin compounds derived from Gynostemma pentaphyllum and applications thereof in the preparation of anti-fatigue products. Background Art

[0002] The Chinese medicine Gynostemma pentaphyllum is the whole herb of many plants of the genus Gynostemma pentaphyllum in the Cucurbitaceae family, such as Gynostemmapentaphyllum (Thunb.) Makino, G. longipes CYWu ex CYWu et S.K. Chen, G. compressum, G. yixingense and G. burmanicum. Gynostemma pentaphyllum is a commonly used Chinese medicine and herbal tea in my country. It is included in the "Medicinal Materials and Pieces Not Included in Formula Preparations" section of the 2015 edition of the "Chinese Pharmacopoeia". It is also included in the "List of Chinese Medicinal Materials that Can Be Used in Health Foods" catalog by the former Ministry of Health of my country. Existing chemical composition studies have shown that Gynostemma pentaphyllum contains triterpenoid saponins, flavonoids, polysaccharides and other chemical components, among which dammarane-type tetracyclic triterpenoid saponins are the main active ingredients of Gynostemma pentaphyllum, also known as Gynostemma pentaphyllum saponins. So far, nearly 500 triterpenoid saponin compounds have been isolated and identified from Gynostemma pentaphyllum. Related pharmacological studies have shown that Gynostemma pentaphyllum has a wide range of pharmacological activities, such as lowering lipids, enhancing immunity, delaying aging, and improving brain function.

[0003] However, Gynostemma pentaphyllum is widely distributed, and there are natural hybrids among them. The saponin components of Gynostemma pentaphyllum produced in different regions vary greatly. For example, Tae Young KIM et al. applied for a patent for the medical use of Gynostemma pentaphyllum stem and leaf extract for anti-fatigue and its preparation method (US20190201468A1). The main components of the extract are Gynostemma pentaphyllum saponin L and Gynostemma pentaphyllum saponin LI, which are characterized by hydroxyl substitution at the C-2 and C-12 positions; Yang Junli et al. applied for and obtained the patent CN113135978A of "A Gynostemma pentaphyllum active ingredient dammarane-type triterpenoid saponin and its separation and application", and the structural feature of the active ingredient is that the side chain at the C-17 position is cyclized into a five-membered ring.

[0004] The present invention systematically collects Gynostemma pentaphyllum from major producing areas across the country, and studies its chemical composition and anti-fatigue effect. It is found that although the saponin components in Gynostemma pentaphyllum from different regions are all triterpenoid saponins, their anti-fatigue effects are significantly different. Summary of the invention

[0005] The present invention aims to provide a triterpenoid saponin compound derived from Gynostemma pentaphyllum, which has specific structural characteristics and shows significant anti-fatigue activity.

[0006] In the first aspect, the triterpenoid saponin compounds provided by the present invention are selected from one or more of the following compounds 1 to 29:

[0007]

[0008] In a second aspect, the present invention also provides a method for extracting and separating the above-mentioned triterpenoid saponin compounds, comprising the following steps: extracting Gynostemma pentaphyllum with water and precipitating with alcohol, concentrating the supernatant, and obtaining one or more of Compounds 1 to 29 by separation and purification.

[0009] Specifically, the extraction and separation method of the above triterpenoid saponin compounds comprises the following steps:

[0010] (1) extracting Gynostemma pentaphyllum with water, concentrating it, using 90% ethanol to precipitate it, taking the supernatant and concentrating it, taking the supernatant and concentrating it, performing D101 macroporous adsorption resin column chromatography, and then using an ethanol-water solvent system for gradient elution (elution procedure: 20%, 70%, 85% ethanol aqueous solution by volume), concentrating and drying, and obtaining three components Fr.D1 to Fr.D3;

[0011] (2) Fr.D1 obtained in step (1) was subjected to SP825 macroporous adsorption resin column chromatography, and gradient elution was performed using an ethanol-water solvent system (elution procedure: volume fraction of 30%, 40%, 50%, 60%, 70% ethanol-water solution), and concentrated and dried to obtain five components Fr.D1A to Fr.D1E;

[0012] (3) subjecting the component Fr.D1B obtained in step (2) to MCI resin column chromatography, using a methanol-water solvent system for gradient elution (elution procedure: 40%, 50%, 60%, 70%, 80%, 90% methanol-water solution by volume fraction), and combining after UHPLC-CAD identification to obtain 6 components Fr.D1B1 to Fr.D1B6;

[0013] (4) Fr.D1B3 obtained in step (3) was separated using a reverse phase C18 chromatographic column and gradient eluted using a methanol-water solvent system (elution procedure: volume fractions of 40%, 50%, 60%, 70%, 80%, 90%, and 100% methanol aqueous solution) to obtain 7 components Fr.D1B3a to Fr.D1B3g; among them, Fr.D1B3d was separated and purified by preparative liquid phase to obtain compound 28, Fr.D1B3e was separated and purified by preparative liquid phase to obtain compound 9, compound 10, compound 15, compound 16, and compound 29, and Fr.D1B3f was separated and purified by preparative liquid phase to obtain compound 5 and compound 27;

[0014] (5) Fr.D1B4 obtained in step (3) was separated using a reverse phase C18 column and gradient eluted using a methanol-water solvent system (elution procedure: volume fractions of 40%, 50%, 60%, 70%, and 80% methanol-water solution), and then identified and combined by UHPLC-CAD to obtain five components Fr.D1B4a-Fr.D1B4e; among them, Fr.D1B4c was separated and purified by preparative liquid phase to obtain compounds 21 and 24, and Fr.D1B4d was separated and purified by preparative liquid phase to obtain compounds 25 and 26;

[0015] (6) Fr.D3 obtained in step (1) was subjected to silica gel column chromatography, and gradient elution was performed using a chloroform-methanol-water solvent system (elution procedure: volume ratio of 95:5:0.5, 90:10:1, 85:15:1.5, 80:20:6, 75:25:7.5, 70:30:9, 100:0:0), and the mixture was combined after TLC identification to obtain Fr.D3-1 to Fr.D3-22; the components Fr.D3-10 to Fr.D3-23 were separated. 13, after merging, separated by reverse phase C18 column, gradient eluted by methanol-water solvent system (elution procedure: volume fraction of 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 100% methanol aqueous solution), and combined after UHPLC-CAD analysis to obtain 9 components Fr.D3-10a~Fr.D3-10h; among them, Fr.D3-10g was separated and purified by preparative liquid phase to obtain compound 14 and compound 23;

[0016] (7) Fr.D3-14 obtained in step (6) was subjected to reverse phase C18 column chromatography, and gradient elution was performed using a methanol-water solvent system (elution procedure was: volume fractions of 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 100% methanol aqueous solution). After UHPLC-CAD identification and merging, 27 components Fr.D3-14-1 to Fr.D3-14-27 were obtained; among them, Fr.D3-14-8 was separated and purified by preparative liquid phase to obtain compound 12, compound 13, compound 17, compound 18, compound 19, compound 20, and compound 22;

[0017] (8) Fr.D3-15 obtained in step (6) was separated and purified by reverse phase C18 column chromatography, and gradient elution was performed using a methanol-water solvent system (elution procedure: volume fraction of 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 100% methanol aqueous solution), and 21 components Fr.D3-15a to Fr.D3-15u were obtained after UHPLC-CAD identification and merging; among them, Fr.D3-15i was separated and purified by preparative liquid phase to obtain compound 6 and compound 8; Fr.D3-15j was separated and purified by preparative liquid phase to obtain compound 7;

[0018] (9) Fr.D2 obtained in step (1) was separated and purified by silica gel column chromatography using a chloroform-methanol-water solvent system gradient elution (elution procedure: volume ratio of 95:5:0.5, 90:10:1, 85:15:1.5, 80:20:6, 75:25:7.5, 70:30:9, 100:0:0), and after TLC identification and merging, 22 components Fr.D2-1 to Fr.D2-22 were obtained; among which, Fr.D2- 8 was separated by a reverse phase C18 column and gradient eluted using a methanol-water solvent system (elution procedure: volume fraction of 50%, 60%, 70%, 80%, 90%, 100% methanol-water solution), and 18 components Fr.D2-8a to Fr.D2-8r were obtained after UHPLC-CAD identification and merging; among them, Fr.D2-8j was separated and purified by preparative liquid phase to obtain compound 11; Fr.D2-8n was separated and purified by preparative liquid phase to obtain compound 4;

[0019] (10) Fr.D2-14 obtained in step (9) was separated by a reverse phase C18 column, and gradient eluted using a methanol-water solvent system (elution procedure: volume fraction of 50%, 60%, 70%, 75%, 80%, 85%, 90%, 100% methanol aqueous solution), and 8 components Fr.D2-14a to Fr.D2-14h were obtained after UHPLC-CAD identification and merging; among them, Fr.D2-14h was separated and purified by preparative liquid phase to obtain compound 3;

[0020] (11) Fr.D2-18 obtained in step (9) was separated by a reverse phase C18 column and gradient eluted using a methanol-water solvent system (elution procedure: volume fraction of 50%, 60%, 70%, 80%, 90%, 100% methanol-water solution), and six components F were obtained after being combined and identified by UHPLC-CAD. r .D2-18a~F r .D2-18f; among them, F r .D2-18e was separated and purified by preparative liquid phase to obtain compound 1 and compound 2.

[0021] In a third aspect, the present invention also provides the use of triterpenoid saponin compounds in the preparation of anti-fatigue products;

[0022] The structure of the triterpenoid saponin compound is shown in general formula I:

[0023]

[0024] in,

[0025] R3 is -OH, -O-Glc,

[0026] R 12 is -OH or oxo (=O);

[0027] R 20 is -OH, -O-Glc,

[0028] R 22 for Or cyclize with the C-12 hydroxyl group to form a seven-membered ring

[0029] The abbreviations in the structural formula are defined as follows:

[0030] Glc: glucose; Rha: rhamnose; Xyl: xylose; Ac: acetyl.

[0031] In the application, the products include anti-physical fatigue products, anti-mental fatigue products and muscle-building products.

[0032] In the application, the product is a food, a dietary supplement or a medicine.

[0033] In the application, the dosage form of the drug is granules, oral liquid, capsule, tablet, effervescent tablet, powder injection, water injection or injection;

[0034] In the application, the medicine includes a single dosage form or a compound dosage form.

[0035] The application of the Gynostemma pentaphyllum extract in the preparation of anti-fatigue products is achieved through at least one of the following indicators:

[0036] 1) Increase the weight of the quadriceps and gastrocnemius muscles;

[0037] 2) Promote ATP production in myotube cells;

[0038] 3) Antagonize FXR signaling.

[0039] In a fourth aspect, the present invention further provides an anti-fatigue composition, comprising a triterpenoid saponin compound, wherein the triterpenoid saponin compound is selected from one or more of the above-mentioned compounds 1 to 29.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. Compounds 1 to 29 provided by the present invention have novel structural characteristics and significant anti-fatigue activity.

[0042] 2. The triterpenoid saponin compounds derived from Gynostemma pentaphyllum and having the structure shown in general formula I provided by the present invention have significant anti-fatigue activity and can be used as lead compounds to develop related anti-fatigue products. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the evaluation result of the myotube ATP production activity of triterpenoid saponin monomer compounds.

[0044] Figure 2 These are the evaluation results of the triterpenoid saponin monomer compounds' activity in promoting myotube mitochondrial ATP production.

[0045] Figure 3 These are the evaluation results of the FXR antagonistic activity of triterpenoid saponin monomer compounds.

[0046] Figure 4 The dose-effect relationship of saponin monomer compounds with FXR antagonistic activity.

[0047] Figure 5 This is the treadmill performance of mice after compound 31 intervention.

[0048] Figure 6 This is the exhaustive swimming performance of mice after compound 31 intervention.

[0049] Figure 7 This is the pulling force of the limbs of mice after the intervention of compound 31.

[0050] Figure 8 is the ratio of hind limb muscle weight to body weight of mice after compound 31 intervention. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0052] Example 1. Extraction of triterpenoid saponins

[0053] Here are the steps:

[0054] (1) Take 10 kg of Gynostemma pentaphyllum (produced in Guilin, Guangxi), decoct and extract three times with water, concentrate and precipitate with 90% ethanol (the percentage used is volume concentration, the same below), discard the precipitate, concentrate the supernatant until there is no alcohol taste, use D-101 macroporous adsorption resin for column chromatography, elute the unprecipitated oligosaccharides and flavonoids with water, and then elute with 20%, 70%, and 85% ethanol respectively, and concentrate and dry to obtain three components Fr.D1 to Fr.D3.

[0055] (2) Fr.D1 (200 g) was subjected to SP825 macroporous adsorption resin column chromatography, with ethanol and water as eluents, eluted with 30%, 40%, 50%, 60%, and 70% ethanol, respectively, and the fractions were collected and concentrated and dried to obtain five fractions Fr.D1A to Fr.D1E.

[0056] (3) Component Fr.D1B (24.2 g) was subjected to MCI resin column chromatography and eluted with 40%, 50%, 60%, 70%, 80%, and 90% methanol aqueous solution by volume. After UHPLC-CAD identification, it was combined into 6 components Fr.D1B1 to Fr.D1B6.

[0057] (4) Fr.D1B3 (4.1 g) was separated by reverse phase C18 column and eluted with methanol aqueous solution with volume fraction of 40%, 50%, 60%, 70%, 80%, 90% and 100%, and 7 components Fr.D1B3a to Fr.D1B3g were obtained. Among them, Fr.D1B3d was separated and purified by preparative liquid phase to obtain compound 28 (8 mg); Fr.D1B3e was separated and purified by preparative liquid phase to obtain compounds 9 (31.2 mg), 10 (52.9 mg), 15 (60.7 mg), 16 (19.8 mg) and 29 (3.7 mg); Fr.D1B3f was separated and purified by preparative liquid phase to obtain compounds 5 (4.9 mg) and 27 (13 mg).

[0058] (5) Fr.D1B4 (1.5 g) was separated by a reverse phase C18 column, eluted with 40%, 50%, 60%, 70%, and 80% methanol aqueous solution, and identified and combined by UHPLC-CAD to obtain five components Fr.D1B4a-Fr.D1B4e. Among them, Fr.D1B4c was separated and purified by preparative liquid phase to obtain compounds 21 (5.6 mg) and 24 (16.1 mg); Fr.D1B4d was separated and purified by preparative liquid phase to obtain compounds 25 (6.8 mg) and 26 (21.8 mg).

[0059] (6) Fr.D3 (40 g) was separated by silica gel column chromatography and eluted with chloroform-methanol-water (95:5:0.5-70:30:9) (elution procedure: volume ratios of 95:5:0.5, 90:10:1, 85:15:1.5, 80:20:6, 75:25:7.5, 70:30:9, 100:0:0). After TLC identification and merging, Fr.D3-1 to Fr.D3-22 were obtained. The components Fr.D3-10 to Fr.D3-13 were combined and separated using a reverse phase C18 column, eluted with 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 100% methanol aqueous solution, and combined after UHPLC-CAD analysis to obtain a total of 9 components Fr.D3-10a to Fr.D3-10h. Among them, Fr.D3-10g was separated and purified by preparative liquid phase to obtain compounds 14 (8 mg) and 23 (7.5 mg).

[0060] (7) Fr.D3-14 (1.8 g) was subjected to reverse phase C18 column chromatography, eluted with methanol aqueous solution with volume fractions of 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 100%, and 27 components Fr.D3-14-1 to Fr.D3-14-27 were obtained after UHPLC-CAD identification and merging. Among them, Fr.D3-14-8 was separated and purified by preparative liquid phase to obtain compounds 12 (8.2 mg), 13 (25 mg), 17 (9 mg), 18 (3 mg), 19 (7 mg), 20 (5.9 mg), and 22 (2.5 mg).

[0061] (8) Fr.D3-15 was separated and purified by reverse phase C18 column, eluted with methanol aqueous solution with volume fractions of 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 100%, and then identified and combined by UHPLC-CAD to obtain 21 components Fr.D3-15a to Fr.D3-15u. Among them, Fr.D3-15i was separated and purified by preparative liquid phase to obtain compounds 6 (120 mg) and 8 (100 mg); Fr.D3-15j was separated and purified by preparative liquid phase to obtain compound 7 (12.6 mg).

[0062] (9) Fr.D2 (110 g) was separated and purified by silica gel column chromatography, eluted with chloroform-methanol-water (95:5:0.5-70:30:9) (elution procedure: volume ratio of 95:5:0.5, 90:10:1, 85:15:1.5, 80:20:6, 75:25:7.5, 70:30:9, 100:0:0), and 22 components Fr.D2-1 to Fr.D2-22 were obtained after TLC identification and merging. Among them, Fr.D2-8 was separated by reverse phase C18 column, eluted with a methanol-water solution with a volume fraction of 50%, 60%, 70%, 80%, 90%, and 100%, and 18 components were obtained after UHPLC-CAD identification and merging. Among them, Fr.D2-8j was separated and purified by preparative liquid phase to obtain compound 11 (4.5 mg); Fr.D2-8n was separated and purified by preparative liquid phase to obtain compound 4 (4.8 mg).

[0063] (10) Fr.D2-14 (2.1 g) was separated by reverse phase C18 column chromatography, and gradient eluted with methanol aqueous solution with volume fractions of 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 100%. After UHPLC-CAD identification and merging, 8 components Fr.D2-14a to Fr.D2-14h were obtained. Among them, Fr.D2-14h was separated and purified by preparative liquid phase to obtain compound 3 (6 mg).

[0064] (11) Fr.D2-18 (2.3 g) was separated by reverse phase C18 column, and eluted with 50%, 60%, 70%, 80%, 90%, 100% methanol-water gradient, and identified and combined by UHPLC-CAD to obtain 6 components Fr.D2-18a to Fr.D2-18f. Among them, Fr.D2-18e was separated and purified by preparative liquid phase to obtain compounds 1 (462 mg) and 2 (92.6 mg).

[0065] Structural characterization of compounds 1 to 29

[0066] The new compound obtained in Example 1 was subjected to high-resolution mass spectrometry, nuclear magnetic resonance analysis and acid hydrolysis experiment to determine the structure of compound 1-29.

[0067] The acid hydrolysis experiment method is as follows: the monomer compound obtained from the Gynostemma pentaphyllum extract (1.0 mg each) is dissolved in 6 mol / L trifluoroacetic acid (1 mL), heated in a water bath at 90°C for 2 h, cooled to room temperature, extracted three times with chloroform, and the aqueous layer is concentrated to obtain the sugar-containing residue.

[0068] Dissolve the obtained sugar residues and monosaccharide reference substance with anhydrous pyridine (200 μL), add L-cysteine ​​methyl ester hydrochloride (1 mg), and heat in a 60°C water bath for 1 h. Then add 10 μL of o-toluene isothiocyanate and heat in a 60°C water bath for 1 h. After the reaction is completed, cool to room temperature, filter the supernatant, and analyze using UHPLC-CAD. UHPLC-CAD analysis conditions: Waters ACQUITY TM UPLC HSS T3 column (100×2.1 mm, 1.8 μm); mobile phase: 0.1% formic acid water (A)-acetonitrile (B); elution program: 0-8 min, 20%-30% B; flow rate: 0.6 mL / min; column temperature 40°C; nebulization temperature 35°C; filtering 1 s.

[0069] By comparing the retention time of the derivatized product with that of the monosaccharide reference substance, the absolute configuration of each sugar moiety was determined, including D-glucose (retention time: 4.5 min), D-xylose (retention time: 4.9 min), and L-rhamnose (retention time: 6.3 min). The details are as follows:

[0070] Compound 1: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -6.00 (concentration c 0.089, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 1; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1253.6572 [MH] - (C 60 H 101 O 27 , calculated value: 1253.6530). Compound 1 was identified as (3β,12β,20S)-trihydroxydammarane-24-ene-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-β-D-glucopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0071] Compound 2: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -10.52 (concentration c 0.15, solvent is methanol); hydrogen spectrum 1 H and C spectra 13C NMR data are shown in Table 1; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1237.6639 [MH] - (C 60 H 101 O 26 , calculated value: 1237.6581). Compound 2 was identified as (3β,12β,20S)-trihydroxydammarane-24-ene-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-α-L-rhamnopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0072] Compound 3: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 +2.00 (concentration c 0.08, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 1; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1133.6354 [MH] - (C 56 H 93 O 23 , calculated value: 1133.6180). Compound 3 was identified as (3β,12β,20S)-trihydroxydammarane-24-ene-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-(2-O-acetyl)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0073] Compound 4: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 +23.33 (concentration c 0.08, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 1; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 987.9188 [MH] - (C 50 H 83 O 19, calculated value: 987.5529). Compound 4 was identified as (3β,12β,20S)-trihydroxydammarane-24-ene-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-(6-O-acetyl)-β-D-glucopyranoside.

[0074] Compound 5: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 +2.3 (concentration c 0.087, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 2; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1107.5935 [MH]-(C 54 H 91 O 23 , calculated value: 1107.5951). Compound 5 was identified as (3β,12β,20S,26)-tetrahydroxydammarane-24-ene-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0075] Compound 6: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 +14.00 (concentration c 0.08, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 2; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1133.6005 [MH] - (C 56 H 93 O 23 , calculated value: 1133.6180). Compound 6 was identified as (3β,12β,20S)-trihydroxydammarane-24-ene-3-O-[6-O-acetyl-β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0076] Compound 7: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5+14.00 (concentration c 0.08, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 2; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1133.6047 [MH] - (C 56 H 93 O 23 , calculated value: 1133.6180). Compound 7 was identified as (3β,12β,20S)-trihydroxydammarane-24-ene-3-O-[3-O-acetyl-β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0077] Compound 8: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -9.0 (concentration c 0.08, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 2; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1265.6510 [MH] - (C 61 H 101 O 27 , calculated value: 1265.6530). Compound 8 was identified as (3β,12β,20S)-trihydroxydammarane-24-ene-3-O-[6-O-acetyl-β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-β-D-xylopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0078] Compound 9: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -6.9 (concentration c 0.146, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 3; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1269.6469 [MH] - (C 60 H 101 O 28, calculated value: 1269.6479). Compound 9 was identified as (3β,12β,20S,25)-tetrahydroxydammarane-23-ene-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-β-D-glucopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0079] Compound 10: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 +8.7 (concentration c 0.092, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 3; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1107.5942 [MH]-(C 59 H 99 O 27 , calculated value: 1107.5951). Compound 10 was identified as (3β,12β,20S,25)-tetrahydroxydammarane-23-ene-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0080] Compound 11: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 +1.0 (concentration c 0.075, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 3; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1003.9702 [MH] - (C 50 H 83 O 20 , calculated value: 1003.5478). Compound 11 was identified as (3β,12β,20S)-trihydroxy-25-peroxyhydroperdamaran-23-ene-3-O-[β-D-pyranose glucose (1→2)-β-D-pyranose glucose syl]-20-O-(6-O-acetyl)-β-D-pyranose glucoside.

[0081] Compound 12: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D5 +18.0 (concentration c 0.1, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 4; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 945.5383 [MH] - (C 48 H 81 O 18 , calculated value: 945.5423). Compound 12 was identified as (3β,12β,20S,25)-tetrahydroxydammarane-23-ene-3-O-β-D-glucopyranosyl-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0082] Compound 13: White amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 +8.0 (concentration c 0.1, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 4; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 961.5368 [MH] - (C 48 H 81 O 19 , calculated value: 961.5372). Compound 13 was identified as (3β,12β,20S)-trihydroxy-25-peroxydamaran-23-ene-3-O-β-D-glucopyranosyl-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0083] Compound 14: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -7.00 (concentration c 0.09, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 4; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 931.5378 [MH] - (C 47 H 79 O 18, calculated value: 931.5266). Compound 14 was identified as (3β,12β,20S)-trihydroxy-25-peroxydamaran-23-ene-20-O-β-D-xylopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0084] Compound 15: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -14.00 (concentration c 0.086, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 5; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1239.6238 [MH] - (C 59 H 99 O 27 , calculated value: 1239.6374). Compound 15 was identified as (3β,12β,20R,24S)-tetrahydroxydammarane-25-ene-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-β-D-xylopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0085] Compound 16: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -9.3 (concentration c 0.097, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 5; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1107.5933 [MH] - (C 54 H 91 O 23 , calculated value: 1107.5951). Compound 16 was identified as (3β,12β,20S,24S)-tetrahydroxydammarane-25-ene-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0086] Compound 17: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D5 -1.0 (concentration c 0.08, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 5; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 945.5446 [MH] - (C 48 H 81 O 18 , calculated value: 945.5423). Compound 17 was identified as (3β,12β,20S,24S)-tetrahydroxydammarane-25-ene-3-O-β-D-glucopyranosyl-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0087] Compound 18: White amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -2.0 (concentration c 0.08, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 6; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 945.5411 [MH] - (C 48 H 81 O 18 , calculated value: 945.5423). Compound 18 was identified as (3β,12β,20S,24R)-tetrahydroxydammarane-25-ene-3-O-β-D-glucopyranosyl-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0088] Compound 19: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 +5.00 (concentration c 0.07, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 6; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 961.5332 [MH] - (C 48 H 81 O 19, calculated value: 961.5372). Compound 19 was identified as (3β,12β,20S)-trihydroxy-24(S)-peroxyhydrodammarane-25-ene-3-O-β-D-glucopyranosyl-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0089] Compound 20: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -5.00 (concentration c 0.02, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 6; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 961.5469 [MH] - (C 48 H 81 O 19 , calculated value: 961.5372). Compound 20 was identified as (3β,12β,20S)-trihydroxy-24(R)-peroxyhydrodammarane-25-ene-3-O-β-D-glucopyranosyl-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0090] Compound 21: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -14.6 (concentration c 0.089, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 7; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1237.6202 [MH] - (C 59 H 97 O 27 , calculated value: 1237.6217). Compound 21 was identified as (3β,12β,20S)-trihydroxydammarane-25-ene-24-one-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-β-D-xylopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0091] Compound 22: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D5 +5.00 (concentration c 0.05, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 7; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 943.5236 [MH] - (C 48 H 79 O 18 , calculated value: 943.5266). Compound 22 was identified as (3β,12β,20S)-trihydroxydammarane-25-ene-24-one-3-O-β-D-glucopyranosyl-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0092] Compound 23: White amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -7.00 (concentration c 0.088, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 7; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 913.5160 [MH] - (C 47 H 77 O 17 , calculated value: 913.5161). Compound 23 was identified as (3β,12β,20S)-trihydroxydammarane-25-ene-24-one-20-O-β-D-xylopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0093] Compound 24: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -13.00 (concentration c 0.115, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 8; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1221.6262 [MH] - (C 59 H 97 O 26, calculated value: 1221.6268). Compound 24 was identified as (3β,20S)-dihydroxydammarane-12-one-24-ene-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-β-D-xylopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0094] Compound 25: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -13.00 (concentration c 0.145, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 8; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1235.6409 [MH]-(C 60 H 99 O 26 , calculated value: 1235.6425). Compound 25 was identified as (3β,20S)-dihydroxydammarane-12-one-24-ene-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-α-L-rhamnopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0095] Compound 26: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -6.9 (concentration c 0.146, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 8; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1089.5289 [MH] - (C 54 H 89 O 22 , calculated value: 1089.5845). Compound 26 was identified as (3β,20S)-dihydroxydammarane-12-one-24-ene-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0096] Compound 27: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2D 5 -11.4 (concentration c 0.088, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 9; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1241.6525 [MH] - (C 59 H 101 O 27 , calculated value: 1241.6530). Compound 27 was identified as (3β,12β,20S,25)-tetrahydroxydammarane-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-β-D-xylopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0097] Compound 28: White amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -15.1 (concentration c 0.073, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 9; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1225.6198 [MH] - (C 58 H 97 O 27 , calculated value: 1225.6217). Compound 28 was identified as (3β,12β,20S)-trihydroxy-25-keto-26-nordamarian-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-β-D-xylopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0098] Compound 29: white amorphous powder, easily soluble in pyridine, methanol and acetonitrile, purple-red when detected by 10% sulfuric acid ethanol solution; optical rotation value [α] 2 D 5 -16.0 (concentration c 0.050, solvent is methanol); hydrogen spectrum 1 H and C spectra 13 C NMR data are shown in Table 9; High resolution mass spectrometry HR-ESI-MS: molecular ion peak m / z 1221.6113 [MH] - (C 59 H 97 O 26, calculated value; 1221.6268). Compound 29 was identified as (3β,20R)-dihydroxy-24-ene-12β,23R-epoxydammarane-3-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranosyl]-20-O-β-D-xylopyranosyl (1→3)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside.

[0099] Compounds 30-42 are all known compounds. Their structures were determined by comparing with the NMR data reported in the literature and were identified as: Gynostemma pentaphyllum saponin LXXXVII (30, m / z 1223.6473 [MH] - ), Gynostemma pentaphyllum saponin V (31, m / z1091.6028[MH] - ), Gynostemma pentaphyllum saponin XCIV (32, m / z 1265.6536 [MH] - )、Gynostemma pentaphyllum saponin LXXXVIII(33,m / z 1061.5909[MH] - ), Gynostemma pentaphyllum saponin X (34, m / z 929.7080 [MH] - ), Gynostemma pentaphyllum saponin LXXXVI (35, m / z 899.5311 [MH] - ), Gynostemma pentaphyllum saponin XCVIII (36, m / z 1239.6371 [MH] - )、(3β,12β,20S)-trihydroxy-25-peroxydamaran-23-ene-3-O-[β-D-pyranoglucopyranosyl (1→2)-β-D-pyranoglucopyranosyl]-20-O-β-D-pyranoglucopyranoside (37, m / z 977.5319 [MH] - )、(3β,12β,20R)-trihydroxy-25-peroxydamaran-25-ene-3-O-β-D-pyranoglucopyranosyl(1→2)-β-D-pyranoglucopyranoside(38,m / z 815.8386[MH] - )、Gynostemma pentaphyllum saponin XC(39,m / z 1239.6388[MH] - )、(3β,12β,20S)-trihydroxy-24(S)-peroxyhydrodammarane-25-ene-3-O-β-D-pyranoglucopyranosyl(1→2)-β-D-pyranoglucopyranoside(40,m / z 815.4744[MH] - )、Gynostemma pentaphyllum saponin CI (41, m / z 1239.6224 [MH] - ) and gypenoside LXXXIX (42, m / z 1221.6138 [MH] - ), the carbon spectrum data of the above compounds are shown in Table 10.

[0100] Table 1 Hydrogen spectrum (600MHz) and carbon spectrum (150MHz) data of compounds 1-4 in deuterated pyridine

[0101]

[0102] Table 2 Hydrogen spectrum (600MHz) and carbon spectrum (150MHz) data of compounds 5-8 in deuterated pyridine

[0103]

[0104] Table 3 Proton spectrum (600 MHz) and carbon spectrum (150 MHz) data of compounds 9-11 in deuterated pyridine

[0105]

[0106] Table 4 Hydrogen spectrum (600 MHz) and carbon spectrum (150 MHz) data of compounds 12-14 in deuterated pyridine

[0107]

[0108] Table 5 Hydrogen spectrum (600 MHz) and carbon spectrum (150 MHz) data of compounds 15-17 in deuterated pyridine

[0109]

[0110] Table 6 Hydrogen spectrum (600 MHz) and carbon spectrum (150 MHz) data of compounds 18-20 in deuterated pyridine

[0111]

[0112] Table 7 Hydrogen spectrum (600 MHz) and carbon spectrum (150 MHz) data of compounds 21-23 in deuterated pyridine

[0113]

[0114] Table 8 Hydrogen spectrum (600 MHz) and carbon spectrum (150 MHz) data of compounds 24-26 in deuterated pyridine

[0115]

[0116] Table 9 Hydrogen spectrum (600 MHz) and carbon spectrum (150 MHz) data of compounds 27-29 in deuterated pyridine

[0117]

[0118] Table 10 Carbon spectrum (150 MHz) data of compounds 30-42 in deuterated pyridine

[0119] No. 30 31 32 33 34 35 36 37 38 39 40 41 42 1 39.2 39.4 39.2 39.2 39.1 39.4 39.2 39.2 39.1 39.2 39.2 39.2 39.2 2 26.8 26.9 26.8 26.8 26.8 28.7 26.8 26.8 26.8 26.8 26.8 26.8 26.5 3 89.0 89.2 89.4 88.8 88.8 78.1 89.0 89.0 88.9 89.0 89.0 89.0 88.6 4 39.7 39.9 39.7 39.7 39.7 39.6 39.7 39.7 39.7 39.8 39.7 39.7 39.5 5 56.4 56.6 56.5 56.4 56.4 56.4 56.4 56.4 56.3 56.4 56.4 56.4 56.2 6 18.6 18.7 18.5 18.6 18.7 18.8 18.6 18.5 18.5 18.6 18.5 18.6 18.2 7 35.2 35.3 35.2 35.1 35.1 35.2 35.2 35.1 35.2 35.1 35.2 35.1 34.9 8 40.1 40.2 40.1 40.1 40.0 40.1 40.1 39.8 40.0 40.1 40.0 40.0 39.5 9 50.3 50.3 50.3 50.3 50.2 50.4 50.3 50.1 50.4 50.2 50.4 50.2 50.4 10 36.9 37.1 37.0 37.0 37.0 37.4 36.9 36.9 36.9 36.9 37.0 36.9 36.8 11 30.9 30.9 30.9 30.9 30.7 30.7 30.9 31.0 32.2 30.9 32.4 31.0 29.8 12 70.1 70.6 70.1 70.1 70.4 70.2 70.1 70.5 70.9 70.5 71.0 70.3 79.4 13 49.5 49.5 49.5 49.5 49.4 49.5 49.5 49.6 49.4 49.6 48.6 49.5 49.5 14 51.4 51.6 51.5 51.4 51.5 51.4 51.4 51.5 51.1 51.6 51.7 51.6 51.1 15 30.7 30.9 30.7 30.7 30.7 30.9 30.7 30.7 31.4 30.7 31.4 30.7 32.3 16 26.8 26.9 26.7 26.8 26.7 26.8 26.7 26.4 26.4 26.5 26.9 26.8 26.5 17 51.9 52.0 51.4 51.4 51.8 51.5 51.4 52.2 51.8 51.5 54.8 51.5 46.4 18 16.1 16.2 16.1 16.1 16.0 16.1 16.1 16.0 15.9 16.1 15.9 16.0 15.3 19 16.3 16.4 16.3 16.3 16.3 16.4 16.3 17.2 17.2 16.3 16.4 16.3 16.2 20 83.6 83.6 83.4 83.4 83.4 83.5 83.6 83.2 73.7 83.5 72.9 83.6 81.8 21 22.0 22.4 22.0 22.0 22.2 22.0 22.1 23.3 22.6 23.1 27.3 22.4 27.8 22 36.2 36.3 36.2 36.2 36.1 36.2 36.0 40.1 46.5 39.7 32.2 32.6 52.1 23 23.0 23.3 23.0 23.0 23.2 23.0 22.6 126.6 127.0 122.8 26.6 30.3 72.1 24 126.1 126.0 126.1 126.1 126.0 126.1 125.6 138.1 138.0 142.4 90.4 76.0 129.0 25 131.1 131.1 131.1 131.1 131.1 131.1 136.4 81.3 81.3 70.0 146.5 149.9 131.0 26 25.8 25.9 25.8 25.8 25.8 25.8 68.2 25.4 25.3 30.6 113.2 110.4 25.4 27 17.9 18.0 17.9 17.9 17.9 17.9 14.1 25.2 25.3 30.6 17.5 18.2 18.7 28 28.1 28.3 28.2 28.2 28.2 28.3 28.1 28.2 28.1 28.1 28.2 28.1 27.8 29 16.6 16.8 16.6 16.8 16.8 16.4 16.6 16.7 16.7 16.6 16.6 16.6 16.3 30 17.5 17.5 17.5 17.5 17.3 17.5 17.5 16.3 16.4 17.3 17.1 17.4 16.7 3-O- Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc 1' 105.1 105.1 105.1 107.0 107.0 105.1 105.2 105.2 105.1 105.2 105.1 104.8 2' 83.4 83.5 83.4 75.8 75.8 83.4 83.5 83.5 83.4 83.5 83.1 83.2 3' 78.5 78.1 78.1 78.8 78.8 78.5 78.3 78.3 78.4 78.3 78.4 78.1 4' 71.7 72.0 71.7 72.0 71.9 72.1 71.7 71.7 71.7 71.7 71.7 77.4 5' 78.3 78.1 75.0 78.5 78.4 78.4 78.2 78.2 78.3 78.2 78.2 77.8 6' 62.9 63.0 64.4 63.2 63.1 62.7 62.9 62.9 62.7 62.9 62.7 62.4 2'-O- Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc 1” 106.1 106.1 106.1 106.2 106.1 106.1 106.1 106.1 106.1 105.8 2” 77.2 76.7 76.8 77.2 77.2 77.2 77.3 77.2 77.2 76.9 3” 78.0 78.5 78.0 78.3 78.4 78.4 78.4 78.4 78.4 78.0 4” 71.7 72.0 71.2 71.8 71.7 71.7 71.9 71.7 71.8 72.7

[0120] The following is the performance test of triterpenoid saponins.

[0121] Test experiment 1: Promoting the production of total ATP and mitochondrial ATP in myotube cells

[0122] Skeletal muscle ATP depletion and mitochondrial dysfunction are important signs of physical fatigue. Regulation of skeletal muscle ATP production and mitochondrial function is of great significance for improving the body's fatigue state, enhancing exercise endurance and improving muscle atrophy. Therefore, we used the myotube and its mitochondrial ATP production activity screening model to evaluate the effect of triterpenoid saponins in Gynostemma pentaphyllum on promoting total ATP production in myotube cells and mitochondrial ATP, thereby reflecting its anti-fatigue activity.

[0123] 1 Experimental reagents, materials and instruments

[0124] Positive drug Acadesine (AICAR, batch S1515) was purchased from Beyotime; Oligomycin (O24530) was purchased from ACMEC BIO. The screened monomer compounds were obtained by separation and purification in Example 1. High-glucose DMEM medium, DMEM / F12 medium, OPTI-MEM medium and fetal bovine serum were all purchased from Gibco; penicillin-streptomycin mixture, 0.25% EDTA-trypsin, and horse serum were purchased from Solebol; Lipofectamine 2000 transfection reagent was purchased from ThermoFisher, USA; CellTiter-Lumi TM The luminescence cell viability detection kit (C0059L) was purchased from Biotech. The instrument used in the screening was a multifunctional microplate reader (SYNERGY-LX, purchased from Biotek, USA).

[0125] 2 Experimental methods

[0126] C2C12 cells were cultured in 96-well plates and grown in complete medium (DMEM high glucose medium + 10% fetal bovine serum) until fully confluent. The medium was then replaced with differentiation medium (DMEM / F12 medium containing 1% glucose + 2% horse serum) to induce myoblast differentiation and fusion to form multinucleated myotubes. After 4 days of differentiation, the test substance and positive drug AICAR were added. The final concentration of the test substance was 20 μM and the concentration of the positive drug was 100 μM. The cell ATP content was detected 24 hours after the addition of the drug to the differentiated myotubes. The operation steps were as described in CellTiter-Lumi TMThe instructions of the luminescence cell viability detection kit recommend that the cells be rinsed with PBS and then 100 μL / well CellTiter-Lumi TM Steady Plus II luminescence detection reagent, shake at 200 rpm for 2 minutes at room temperature, then incubate for 10 minutes. After the luminescence signal stabilizes, chemiluminescence detection is performed using a multifunctional microplate reader with chemiluminescence function.

[0127] Furthermore, the mitochondrial-dependent ATP content was determined by incubation in galactose medium with or without oligomycin (mitochondrial ATP synthase inhibitor). After 21 h of drug culture, the differentiated myotubes were switched to galactose medium with or without oligomycin (mitochondrial ATP synthase inhibitor) for incubation for 3 h, and the ATP content was determined. Mitochondrial ATP production was calculated based on the following formula:

[0128]

[0129] Compounds that increased total ATP or decreased glycolytic ATP and increased mitochondrial ATP by more than 7.5% compared to the negative control group were considered to have the effect of activating mitochondrial ATP production.

[0130] The experimental data were statistically analyzed. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001.

[0131] 3 Experimental results

[0132] like Figure 1 The results showed that compared with the blank control group, the screened triterpenoid saponin compounds all exhibited certain activity in promoting ATP production in myotube cells, among which compounds 1, 6, 8, 13, 30, 31, 38 and 40 had significant activity in promoting ATP production in myotube cells, and were all more active than the positive drug AICAR (an AMPK agonist).

[0133] In addition, the generation of ATP in cells is mainly mediated by two processes: glycolysis in the cytoplasm and oxidative phosphorylation in the mitochondria. The energy generated by mitochondrial energy transfer mediated by oxidative phosphorylation accounts for about 60%-80% of the total ATP produced by cells. Improving mitochondrial energy production can affect the body's heat production, exercise endurance and muscle strength. Further experimental results showed that the total ATP content of myotube cells intervened by active saponin compounds 1, 6, 13, 31, 38 and 40 increased, and the total ATP content decreased after the addition of oligomycin to inhibit its mitochondrial oxidative phosphorylation, and dropped to the same level as the control group after drug addition (such as Figure 2The results show that the mechanism of the active Gynostemma pentaphyllum saponin monomer in promoting the ATP production of myotube cells is to promote mitochondrial oxidative phosphorylation rather than glycolysis. At the same time, we used the MitoATP index to measure the ability of active monomers to promote the production of ATP from myotube mitochondria. The active saponin compounds 1, 6, 13, 31, 38 and 40 found in the previous experiments all have the effect of promoting the production of ATP from myotube mitochondria, and compound 6 has the strongest activity in the production of ATP from myotube mitochondria.

[0134] In summary, multiple saponin compounds have the ability to promote the production of ATP derived from myotube mitochondria, that is, triterpenoid compounds derived from Gynostemma pentaphyllum have different degrees of anti-fatigue activity.

[0135] Test experiment 2: FXR antagonistic activity test

[0136] Farnesoid X receptor (FXR) is a member of the nuclear hormone receptor family that is mainly present in the liver and intestines and regulates the synthesis, transport, intestinal reabsorption and excretion of bile acids. Stimulation of bile acid receptors such as TGR5 has been reported to have anti-fatigue, exercise endurance and muscle-building effects. Gynostemma pentaphyllum extract has been found to upregulate bile acid anabolism by antagonizing intestinal FXR, thereby stimulating bile acid receptors to exert anti-fatigue effects. We used a luciferase dual reporter gene assay-based FXR antagonist activity screening model to evaluate whether triterpenoid saponins in Gynostemma pentaphyllum also have FXR antagonist activity.

[0137] 1 Experimental reagents, materials and instruments

[0138] Ursodeoxycholic acid (UDCA, S25093) was purchased from Yuanye Bio; GW4064 (FXR agonist, batch G872312) was purchased from McLean; the screened monomer compounds were obtained by separation and purification in Example 1. Dual luciferase reporter gene detection kit (E1910) was purchased from Promega, USA; Lipofectamine 2000 transfection reagent was purchased from ThermoFisher, USA; 293T cells, empty plasmids, and Renilla luciferase plasmids were donated by Xing Yaling's research group at the Institute of Infectious Diseases and Epidemiology, Academy of Military Medical Sciences; pCMV-Script-hFXR plasmids and pGL4.11-hSHP-Luciferase plasmids were donated by Xie Cen's research group at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Multifunctional microplate reader (SYNERGY-LX) was purchased from Biotek, USA.

[0139] 2 Experimental methods

[0140] 293T cells were cultured in 96-well plates, and liposomes were used for transient transfection when the confluence reached 70%. The operation steps were carried out according to the instructions of the Lipofectamine2000 kit. In the experimental group, 100ng / well pCMV-Script-hFXR plasmid, 100ng / well pGL4.11-hSHP-Luc plasmid, and 5ng / well TK plasmid were gently mixed with 25μL OPTI-MEM serum-free medium to prepare a plasmid mixture. In addition, 0.5μL / well Lipofectamine 2000 was gently mixed with 25μL / well OPTI-MEM serum-free medium to prepare a Lipofectamine 2000 dilution, and allowed to stand for 5min. The plasmid mixture was gently mixed with the Lipofectamine 2000 dilution, and after standing for 20min, 50μL / well of the compound solution was added to the cells and gently shaken. 6 hours after transfection, the medium was changed to complete medium containing antibiotics. 24 hours later, the test substance and positive drug UDCA were added. The final concentration of the test substance was 10 μM and the concentration of the positive drug was 20 μM.

[0141] Dual luciferase reporter gene activity was detected 24 hours after cell drug addition, and the operation steps were carried out according to the instructions of the LuciferaseReporter Assay System kit. After rinsing the cells with PBS, 20μL 1×PLB lysis buffer was added, and the cells were shaken at 200rpm for 15 minutes at room temperature. 50μL / well LAR II reagent was added to the lysate, and after gentle mixing, the firefly luciferase was detected using a multifunctional microplate reader with chemiluminescence function. After the detection was completed, 50μL / well Stop&Glo reagent was added, and after gentle mixing, the sea cucumber luciferase was detected using a multifunctional microplate reader. The ratio of the two test values ​​was used as the final result for subsequent statistical analysis. Compared with the DMSO group, # P<0.05, ## P<0.01, ### P<0.001; compared with the GW4064 group, *P<0.05, **P<0.01, ***P<0.001.

[0142] 3 Experimental results

[0143] like Figure 3 The results showed that the monomer compounds 1, 6, 13, 16, 26, 31, 37 and 38 had strong FXR antagonistic activity. Among them, compounds 31, 37 and 38 showed stronger antagonistic activity than the positive drug UDCA (20 μM) at a concentration of 10 μM; further experiments showed that the above three compounds can antagonize FXR receptors according to the dose-effect relationship, and the IC of compound 31 is 50The IC value of compound 37 was 19.43 μM. 50 The IC value of compound 38 was 10.61 μM. 50 is 10.85μM. For specific results, see Figure 4 .

[0144] In summary, multiple saponin compounds have FXR antagonist activity, that is, triterpenoid compounds derived from Gynostemma pentaphyllum have different degrees of anti-fatigue effects.

[0145] Test experiment 3: Exhaustive treadmill exercise experiment in mice

[0146] The anti-fatigue effect of compound 31 was verified by exhaustive treadmill exercise test in mice.

[0147] 1 Animal grouping and dosage

[0148] A total of 5 groups of mice were set up, with 8 mice in each group, including a blank control group (Veh), a group treated with 300 mg / kg of chemical type A Gynostemma pentaphyllum extract (Gyp), and groups treated with low, medium, and high doses (10, 30, and 90 mg / kg) of compound 31 (GVL, GVM, and GVH).

[0149] 2 Animal experimental methods

[0150] After 7 days of adaptive feeding, mice were gavaged and administered for 2 weeks. The first behavioral test was performed 1 hour after the last gavage. The mice moved at a constant speed of 20 m / min, and the total distance they traveled to reach exhaustion was recorded. The exhaustion state was considered when the mice failed to continue to move for three consecutive times and stayed on the electric shock plate for 10 seconds and refused to move.

[0151] 3 Experimental results

[0152] like Figure 5 The results showed that compound 31 at different doses could significantly increase the treadmill performance of mice, indicating that the compound has an anti-fatigue effect. (*, **, *** represent that the difference between this group and the normal control group in the one-way ANOVA is statistically significant, p<0.05, p<0.01, p<0.001 respectively)

[0153] Test experiment 4: Exhaustive swimming test of mice

[0154] The anti-fatigue effect of compound 31 was verified by the mouse exhaustive swimming test.

[0155] 1 Animal grouping and dosage

[0156] The specific animal grouping and drug dosage were the same as those in test experiment 3.

[0157] 2 Animal experimental methods

[0158] After 7 days of adaptive feeding, mice were given drugs by gavage. The drugs were given for 2 weeks. One hour after the last gavage, the weight-bearing swimming experiment was started. The mouse pool was 30 cm deep and the water temperature was 25±1°C. A weight of 5% of the mouse's body weight was tied to the mouse's tail to make it swim to exhaustion. If the mouse could not float to the surface for 7 consecutive seconds, it was considered to have reached the state of exhaustion, and the swimming time was recorded.

[0159] 3 Experimental results

[0160] like Figure 6 The results showed that compound 31 can significantly increase the exhaustive swimming performance of mice in a dose-dependent manner, indicating that the compound has an anti-fatigue effect. (*, **, *** represent that the difference between this group and the normal control group in the one-way ANOVA is statistically significant, p<0.05, p<0.01, p<0.001 respectively)

[0161] Test experiment 5: Mouse limb tension test

[0162] The anti-fatigue effect of compound 31 was verified by the mouse limb tension test.

[0163] 1 Animal grouping and dosage

[0164] The specific animal grouping and drug dosage were the same as those in test experiment 3.

[0165] 2 Animal experimental methods

[0166] After 7 days of adaptive feeding, mice were gavaged and administered for 2 weeks. The limb pulling test was performed half an hour after the last gavage. The mice were placed on a wire mesh. After the limbs of the mice were all grasped stably, the 1 / 3 of the tail of the mice was controlled and the mice were dragged backward with a pulling force parallel to the wire mesh. The maximum pulling force was recorded after the limbs of the mice were all out of the wire mesh. This experiment was repeated three times, and the data obtained from the three times were statistically analyzed together.

[0167] 3 Experimental results

[0168] like Figure 7 The results showed that compound 31 significantly increased the pulling force of the four limbs of mice after intervention, indicating that the compound has the effect of enhancing physical fitness. (*, **, *** represent that the difference between this group and the normal control group in the one-way ANOVA is statistically significant, p<0.05, p<0.01, p<0.001 respectively)

[0169] Test experiment 6: Detection of the weight ratio of quadriceps and gastrocnemius muscles in mice

[0170] The muscle-enhancing effect of compound 31 was verified by detecting the body weight ratio of the quadriceps and gastrocnemius muscles of mice.

[0171] 1 Animal grouping and dosage

[0172] The specific animal grouping and drug dosage were the same as those in test experiment 3.

[0173] 2 Animal experimental methods

[0174] The swimming exhaustion test was the same as that in test experiment 4. The mice were anesthetized and bled after reaching exhaustion for half an hour, and the quadriceps femoris (QFM) and gastrocnemius (Gastro.) of mice in different groups were collected, and their weights were measured and divided by body weight to calculate their proportion to body weight.

[0175] 3 Experimental results

[0176] like Figure 8 The results showed that compound 31 can significantly increase the weight ratio of quadriceps and gastrocnemius muscles of mice after two weeks of administration, and each dose has a dose-effect relationship, indicating that compound 31 has the effect of increasing muscle and enhancing physical fitness. (*, **, *** represent that the difference between this group and the normal control group in the one-way ANOVA is statistically significant, p<0.05, p<0.01, p<0.001 respectively)

[0177] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A triterpenoid saponin compound selected from one or more of the following compounds 1 to 29:

2. The method for extracting and separating triterpenoid saponins according to claim 1, comprising the following steps: extracting Gynostemma pentaphyllum with water and precipitating with alcohol, concentrating the supernatant, and obtaining triterpenoid saponins with different structures by separation and purification.

3. The extraction and separation method according to claim 2, characterized in that: The steps include: (1) extracting Gynostemma pentaphyllum with water, concentrating it, and using 90% ethanol to precipitate it. After the supernatant is concentrated, it is subjected to D101 macroporous adsorption resin column chromatography, and then gradient eluted using an ethanol-water solvent system, concentrated and dried to obtain three components Fr.D1 to Fr.D3; (2) Fr.D1 obtained in step (1) was subjected to SP825 macroporous adsorption resin column chromatography, gradient elution was performed using an ethanol-water solvent system, and the mixture was concentrated and dried to obtain five components Fr.D1A to Fr.D1E; (3) subjecting the component Fr.D1B obtained in step (2) to MCI resin column chromatography, using a methanol-water solvent system for gradient elution, and combining after UHPLC-CAD identification to obtain 6 components Fr.D1B1 to Fr.D1B6; (4) Fr.D1B3 obtained in step (3) was separated using a reverse phase C18 chromatographic column and gradient eluted using a methanol-water solvent system to obtain 7 components Fr.D1B3a to Fr.D1B3g; among them, Fr.D1B3d was separated and purified by preparative liquid phase to obtain compound 28, Fr.D1B3e was separated and purified by preparative liquid phase to obtain compound 9, compound 10, compound 15, compound 16 and compound 29, and Fr.D1B3f was separated and purified by preparative liquid phase to obtain compound 5 and compound 27; (5) Fr.D1B4 obtained in step (3) was separated using a reverse phase C18 column, gradient eluted using a methanol-water solvent system, and identified and combined by UHPLC-CAD to obtain five components Fr.D1B4a-Fr.D1B4e; among them, Fr.D1B4c was separated and purified by preparative liquid phase to obtain compounds 21 and 24, and Fr.D1B4d was separated and purified by preparative liquid phase to obtain compounds 25 and 26; (6) Fr.D3 obtained in step (1) was subjected to silica gel column chromatography, and gradient elution was performed using a chloroform-methanol-water solvent system. After TLC identification, the components Fr.D3-1 to Fr.D3-22 were obtained. Fr.D3-10 to Fr.D3-13 were combined and separated using a reverse phase C18 column, and gradient eluted using a methanol-water solvent system. After UHPLC-CAD analysis, the components Fr.D3-10a to Fr.D3-10h were obtained. Among them, Fr.D3-10g was separated and purified by preparative liquid phase separation to obtain compound 14 and compound 23. (7) Fr.D3-14 obtained in step (6) was subjected to reverse phase C18 column chromatography, and gradient elution was performed using a methanol-water solvent system. After identification and merging by UHPLC-CAD, 27 components Fr.D3-14-1 to Fr.D3-14-27 were obtained; among them, Fr.D3-14-8 was separated and purified by preparative liquid phase to obtain compound 12, compound 13, compound 17, compound 18, compound 19, compound 20 and compound 22; (8) Fr.D3-15 obtained in step (6) was separated and purified by a reverse phase C18 column, and gradient elution was performed using a methanol-water solvent system. After identification and merging by UHPLC-CAD, 21 components Fr.D3-15a to Fr.D3-15u were obtained; among them, Fr.D3-15i was separated and purified by preparative liquid phase to obtain compound 6 and compound 8; Fr.D3-15j was separated and purified by preparative liquid phase to obtain compound 7; (9) Fr.D2 obtained in step (1) was separated and purified by silica gel column chromatography, using a chloroform-methanol-water solvent system for gradient elution, and after TLC identification and merging, 22 components Fr.D2-1 to Fr.D2-22 were obtained; among them, Fr.D2-8 was separated by a reverse phase C18 column, using a methanol-water solvent system for gradient elution, and after UHPLC-CAD identification and merging, 18 components Fr.D2-8a to Fr.D2-8r were obtained; among them, Fr.D2-8j was separated and purified by preparative liquid phase to obtain compound 11; Fr.D2-8n was separated and purified by preparative liquid phase to obtain compound 4; (10) Fr.D2-14 obtained in step (9) was separated by a reverse phase C18 column, and gradient eluted using a methanol-water solvent system. After UHPLC-CAD identification and merging, 8 components Fr.D2-14a to Fr.D2-14h were obtained; among them, Fr.D2-14h was separated and purified by preparative liquid phase to obtain compound 3; (11) Fr.D2-18 obtained in step (9) was separated by a reverse phase C18 column and gradient eluted using a methanol-water solvent system. After identification and merging by UHPLC-CAD, six components Fr.D2-18a to Fr.D2-18f were obtained; among them, Fr.D2-18e was separated and purified by preparative liquid phase to obtain compounds 1 and 2.

4. Application of triterpenoid saponin compounds in the preparation of anti-fatigue products; The structure of the triterpenoid saponin compound is shown in general formula I: in, R3 is R 12 is -OH or =O; R 20 for R 22 for Or cyclize with the C-12 hydroxyl group to form a seven-membered ring The abbreviations in the structural formula are defined as follows: Glc: glucose; Rha: rhamnose; Xyl: xylose; Ac: acetyl.

5. The use according to claim 4, characterized in that The products include anti-physical fatigue products, anti-mental fatigue products and muscle-building products.

6. Use according to claim 5, characterized in that The product is a food, a dietary supplement or a medicine.

7. The product according to claim 6, characterized in that The dosage form of the drug is granules, oral liquid, capsule, tablet, effervescent tablet, powder injection, water injection or injection; The medicine includes a single dosage form or a compound dosage form.

8. An anti-fatigue composition comprising the triterpenoid saponin compound according to claim 1.

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