Gynostemma pentaphyllum-derived triterpenoid saponin compound and use thereof in preparation of Anti-fatigue products

Abstract

Provided are a Gynostemma pentaphyllum-derived triterpenoid saponin compound and a use thereof in the preparation of anti-fatigue products. The provided triterpenoid saponin compound is selected from one of compounds 1-29. Also provided are a use of a triterpenoid saponin compound as represented by general formula I in the preparation of anti-fatigue products. Experiments show that the Gynostemma pentaphyllum-derived triterpenoid saponin compound has significant anti-fatigue activity and can be used for preparing anti-fatigue products.

Description

Triterpenoid saponins derived from Gynostemma pentaphyllum and their application in the preparation of anti-fatigue products Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to triterpenoid saponins derived from Gynostemma pentaphyllum and their application in the preparation of anti-fatigue products. Background Technology

[0002] The traditional Chinese medicine Gynostemma pentaphyllum refers to the whole herb of various plants in the genus Gynostemma of the Cucurbitaceae family, such as Gynostemma pentaphyllum (Thunb.) Makino, G. longipes CYWu ex CYWu et SKChen, G. compressum, G. yixingense, and G. burmanicum. Chemical studies have shown that Gynostemma pentaphyllum contains various chemical components, including triterpenoid saponins, flavonoids, and polysaccharides. Dammarane-type tetracyclic triterpenoid saponins are the main active ingredients and are also known as Gynostemma pentaphyllum saponins. To date, nearly 500 triterpenoid saponin compounds have been isolated and identified from Gynostemma pentaphyllum. Related pharmacological studies have shown that Gynostemma pentaphyllum possesses a wide range of pharmacological activities, including lowering lipids, enhancing immunity, delaying aging, and improving brain function.

[0003] However, Gynostemma pentaphyllum is widely distributed, and natural hybridization occurs among its members, resulting in significant differences in saponin composition among Gynostemma pentaphyllum from different regions. For example, Tae Young Kim et al. applied for a patent (US20190201468A1) for the medicinal use of Gynostemma pentaphyllum stem and leaf extract for anti-fatigue purposes and its preparation method. The main components of this extract are Gynostemma pentaphyllum saponin L and Gynostemma pentaphyllum saponin LI, characterized by hydroxyl substitution at both C-2 and C-12 positions. Yang Junli et al. applied for and obtained a patent (CN113135978A) for "A Dammarane-type Triterpenoid Saponin, an Active Ingredient of Gynostemma pentaphyllum, and its Separation and Application," which features a structural characteristic of a five-membered ring synthesized from the C-17 side chain.

[0004] This invention systematically collected Gynostemma pentaphyllum from major producing areas across the country and studied its chemical composition and anti-fatigue effects. It was found that although the saponin components in Gynostemma pentaphyllum from different regions are all triterpenoid saponins, their anti-fatigue effects vary significantly. Summary of the Invention

[0005] The purpose of this invention is to provide a triterpenoid saponin compound derived from Gynostemma pentaphyllum, which has specific structural features and exhibits significant anti-fatigue activity.

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

[0007] Secondly, the present invention also provides a method for extracting and separating the above-mentioned triterpenoid saponins, comprising the following steps: extracting Gynostemma pentaphyllum with water and precipitating it with alcohol, concentrating the supernatant, and purifying it by separation to obtain one or more of compounds 1 to 29.

[0008] Specifically, the extraction and separation method for the above-mentioned triterpenoid saponins includes the following steps:

[0009] (1) Extract Gynostemma pentaphyllum with water, concentrate it, precipitate it with 90% ethanol, concentrate the supernatant, and then perform D101 macroporous adsorption resin column chromatography. Then, use an ethanol-water solvent system for gradient elution (elution program: 20%, 70%, and 85% ethanol aqueous solution by volume), concentrate and dry to obtain three components Fr.D1~Fr.D3;

[0010] (2) Take the Fr.D1 obtained in step (1) and perform SP825 macroporous adsorption resin column chromatography. Use an ethanol-water solvent system for gradient elution (elution program: volume fraction of 30%, 40%, 50%, 60%, 70% ethanol aqueous solution), concentrate and dry to obtain 5 components Fr.D1A~Fr.D1E;

[0011] (3) Take the fraction Fr.D1B obtained in step (2) and perform MCI resin column chromatography. Use a methanol-water solvent system for gradient elution (elution program: 40%, 50%, 60%, 70%, 80%, 90% methanol aqueous solution by volume fraction). After identification by UHPLC-CAD, combine to obtain 6 fractions Fr.D1B1~Fr.D1B6;

[0012] (4) Take Fr.D1B3 obtained in step (3), separate it using a reversed-phase C18 column, and perform gradient elution using a methanol-water solvent system (elution program: 40%, 50%, 60%, 70%, 80%, 90%, 100% methanol aqueous solution by volume fraction) to obtain 7 components Fr.D1B3a~Fr.D1B3g; among them, Fr.D1B3d was purified by preparative liquid chromatography to obtain compound 28, Fr.D1B3e was purified by preparative liquid chromatography to obtain compound 9, compound 10, compound 15, compound 16 and compound 29, and Fr.D1B3f was purified by preparative liquid chromatography to obtain compound 5 and compound 27;

[0013] (5) Take Fr.D1B4 obtained in step (3), separate it using a reversed-phase C18 column, and perform gradient elution using a methanol-water solvent system (elution program: 40%, 50%, 60%, 70%, 80% methanol aqueous solution by volume fraction). After identification and merging by UHPLC-CAD, five components Fr.D1B4a~Fr.D1B4e are obtained; among them, Fr.D1B4c is purified by preparative liquid chromatography to obtain compound 21 and compound 24, and Fr.D1B4d is purified by preparative liquid chromatography to obtain compound 25 and compound 26.

[0014] (6) Take the Fr.D3 obtained in step (1) and perform silica gel column chromatography separation. Use a chloroform-methanol-water solvent system for gradient elution (elution program: volume ratio 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 detection and merging, obtain Fr.D3-1 to Fr.D3-22; separate the components Fr.D3-10 to Fr.D3- 13. After merging, the fractions were separated using a reversed-phase C18 column and eluted using a methanol-water solvent system with gradient elution (elution program: 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 100% methanol-water solution). After analysis by UHPLC-CAD, the fractions were merged, yielding a total of 9 fractions Fr.D3-10a to Fr.D3-10h. Among them, Fr.D3-10g was purified by preparative liquid chromatography to obtain compounds 14 and 23.

[0015] (7) The Fr.D3-14 obtained in step (6) was subjected to reversed-phase C18 column chromatography and gradient elution was performed using a methanol-water solvent system (elution program: 50%, 60%, 70%, 75%, 80%, 85%, 90%, 100% methanol aqueous solution by volume fraction). 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 purified by preparative liquid chromatography to obtain compounds 12, 13, 17, 18, 19, 20 and 22.

[0016] (8) The Fr.D3-15 obtained in step (6) was separated and purified by reversed-phase C18 column chromatography. Gradient elution was performed using a methanol-water solvent system (elution program: 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 100% methanol aqueous solution by volume fraction). After identification and merging by UHPLC-CAD, 21 components Fr.D3-15a~Fr.D3-15u were obtained; among them, Fr.D3-15i was purified by preparative liquid chromatography to obtain compound 6 and compound 8; Fr.D3-15j was purified by preparative liquid chromatography to obtain compound 7.

[0017] (9) Take the Fr.D2 obtained in step (1) and purify it by silica gel column chromatography. Use a chloroform-methanol-water solvent system for gradient elution (elution program: volume ratio 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, 22 fractions Fr.D2-1 to Fr.D2-22 are obtained; among which, Fr.D2- 8. Separation was performed using a reversed-phase C18 column with gradient elution using a methanol-water solvent system (elution program: 50%, 60%, 70%, 80%, 90%, 100% methanol-water solution by volume). After identification and merging by UHPLC-CAD, 18 fractions Fr.D2-8a to Fr.D2-8r were obtained; among them, Fr.D2-8j was purified by preparative liquid chromatography to obtain compound 11; Fr.D2-8n was purified by preparative liquid chromatography to obtain compound 4.

[0018] (10) The Fr.D2-14 obtained in step (9) was separated by reversed-phase C18 column and gradient elution was performed using a methanol-water solvent system (elution program: 50%, 60%, 70%, 75%, 80%, 85%, 90%, 100% methanol aqueous solution by volume fraction). After identification and merging by UHPLC-CAD, eight components Fr.D2-14a to Fr.D2-14h were obtained; among them, Fr.D2-14h was purified by preparative liquid chromatography to obtain compound 3.

[0019] (11) The Fr.D2-18 obtained in step (9) was separated by reversed-phase C18 column and gradient eluted using a methanol-water solvent system (elution program: 50%, 60%, 70%, 80%, 90%, 100% methanol aqueous solution by volume fraction). After identification and merging by UHPLC-CAD, six components Fr.D2-18a to Fr.D2-18f were obtained; among them, Fr.D2-18e was purified by preparative liquid chromatography to obtain compound 1 and compound 2.

[0020] Thirdly, the present invention also provides the application of triterpenoid saponin compounds in the preparation of anti-fatigue products;

[0021] The structure of the triterpenoid saponin compound is shown in general formula I:

[0022] in,

[0023] R3 is -OH, -O-Glc.

[0024] R 12 For or oxygenation (=O);

[0025] R 20 -OH, -O-Glc,

[0026] R 22 for It may cyclize with the hydroxyl group at C-12 to form a seven-membered ring.

[0027] The abbreviations appearing in the structural formula are defined as follows:

[0028] Glc: glucose; Rha: rhamnose; Xyl: xylose; Ac: acetyl.

[0029] In the application, the products include anti-fatigue products, anti-mental fatigue products, and muscle-building products.

[0030] In the application described, the product is a food, dietary supplement, or medicine.

[0031] In the aforementioned applications, the dosage form of the drug is granules, oral liquid, capsules, tablets, effervescent tablets, powder for injection, water for injection, or injection.

[0032] In the application described, the drug includes single-drug formulations or compound formulations.

[0033] The Gynostemma pentaphyllum extract is used in anti-fatigue products by achieving at least one of the following indicators:

[0034] 1) Increase the weight of the quadriceps and gastrocnemius muscles;

[0035] 2) Promotes ATP production in myotube cells;

[0036] 3) Antagonize FXR signals.

[0037] Fourthly, the present invention also provides an anti-fatigue composition comprising triterpenoid saponins selected from one or more of compounds 1 to 29 described above. Attached Figure Description

[0038] Figure 1 shows the evaluation results of the myotube ATP-generating activity of triterpenoid saponin monomers.

[0039] Figure 2 shows the evaluation results of the activity of triterpenoid saponin monomers in promoting myotubular mitochondrial ATP production.

[0040] Figure 3 shows the FXR antagonistic activity evaluation results of triterpenoid saponin monomer compounds.

[0041] Figure 4 shows the dose-effect relationship of FXR antagonistic active saponin monomer compounds.

[0042] Figure 5 shows the treadmill exercise performance of mice after intervention with compound 31.

[0043] Figure 6 shows the performance of mice in exhaustive swimming after intervention with compound 31.

[0044] Figure 7 shows the limb tension in mice after intervention with compound 31.

[0045] Figure 8 shows the ratio of hind limb muscle weight to body weight in mice after intervention with compound 31. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0047] Example 1: Extraction of triterpenoid saponins

[0048] The steps are as follows:

[0049] (1) Take 10 kg of Gynostemma pentaphyllum (originating from Guilin, Guangxi), decoct it three times with water, concentrate it and precipitate it with 90% ethanol (the percentage used is volume concentration, the same below), discard the precipitate, concentrate the supernatant until there is no alcohol taste, and then perform column chromatography using D-101 macroporous adsorption resin. Elute the oligosaccharides and flavonoids that have not been precipitated with water, and then elute with 20%, 70% and 85% ethanol respectively. After concentration and drying, three components Fr.D1~Fr.D3 are obtained.

[0050] (2) Take Fr.D1 (200g) for SP825 macroporous adsorption resin column chromatography, using ethanol and water as eluents, and elute with 30%, 40%, 50%, 60% and 70% ethanol respectively. Collect the fractions, concentrate and dry to obtain 5 fractions Fr.D1A~Fr.D1E.

[0051] (3) The fraction Fr.D1B (24.2g) was subjected to MCI resin column chromatography and eluted with methanol aqueous solution of volume fractions of 40%, 50%, 60%, 70%, 80%, and 90%. After identification by UHPLC-CAD, it was merged into 6 fractions Fr.D1B1 to Fr.D1B6.

[0052] (4) Fr.D1B3 (4.1 g) was separated by reversed-phase C18 column and eluted with methanol-water solutions of 40%, 50%, 60%, 70%, 80%, 90%, and 100% (v / v) to obtain seven fractions Fr.D1B3a to Fr.D1B3g. Among them, Fr.D1B3d was purified by preparative liquid chromatography to obtain compound 28 (8 mg); Fr.D1B3e was purified by preparative liquid chromatography 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 purified by preparative liquid chromatography to obtain compounds 5 (4.9 mg) and 27 (13 mg).

[0053] (5) Fr.D1B4 (1.5 g) was separated using a reversed-phase C18 column and eluted with methanol-water solutions of 40%, 50%, 60%, 70%, and 80% (v / v). After identification and merging by UHPLC-CAD, five fractions Fr.D1B4a to Fr.D1B4e were obtained. Among them, Fr.D1B4c was purified by preparative liquid chromatography to obtain compounds 21 (5.6 mg) and 24 (16.1 mg); Fr.D1B4d was purified by preparative liquid chromatography to obtain compounds 25 (6.8 mg) and 26 (21.8 mg).

[0054] (6) Take Fr.D3 (40g) for silica gel column chromatography separation, and elute with chloroform-methanol-water (95:5:0.5~70:30:9) (elution program: volume ratio 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 detection and merging, Fr.D3-1~Fr.D3-22 were obtained. Fractions Fr.D3-10 to Fr.D3-13 were combined and separated using a reversed-phase C18 column, eluted with methanol-water solutions of 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 100% (v / v). After analysis by UHPLC-CAD, the fractions were combined to obtain a total of nine fractions Fr.D3-10a to Fr.D3-10h. Among them, Fr.D3-10g was purified by preparative liquid chromatography to obtain compounds 14 (8 mg) and 23 (7.5 mg).

[0055] (7) Fr.D3-14 (1.8 g) was subjected to reversed-phase C18 column chromatography, eluted with methanol-water solutions of 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 100% (v / v). After identification and merging by UHPLC-CAD, 27 fractions Fr.D3-14-1 to Fr.D3-14-27 were obtained. Among them, Fr.D3-14-8 was purified by preparative liquid chromatography 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).

[0056] (8) Fr.D3-15 was separated and purified by reversed-phase C18 column elution with methanol aqueous solution of 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 100% (v / v). After identification and merging by UHPLC-CAD, 21 fractions Fr.D3-15a to Fr.D3-15u were obtained. Among them, Fr.D3-15i was purified by preparative liquid chromatography to obtain compounds 6 (120 mg) and 8 (100 mg); Fr.D3-15j was purified by preparative liquid chromatography to obtain compound 7 (12.6 mg).

[0057] (9) Fr.D2 (110g) was separated and purified by silica gel column chromatography. Elution was performed using chloroform-methanol-water (95:5:0.5~70:30:9) (elution program: v / v ratio 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, 22 fractions Fr.D2-1~Fr.D2-22 were obtained. Fr.D2-8 was separated by reversed-phase C18 column chromatography with gradient elution of 50%, 60%, 70%, 80%, 90%, and 100% methanol-water solutions. After UHPLC-CAD identification and merging, 18 fractions were obtained. Among them, Fr.D2-8j was purified by preparative liquid chromatography to obtain compound 11 (4.5 mg); Fr.D2-8n was purified by preparative liquid chromatography to obtain compound 4 (4.8 mg).

[0058] (10) Fr.D2-14 (2.1 g) was separated by reversed-phase C18 column elution with a gradient of 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 100% methanol-water solutions. After identification and merging by UHPLC-CAD, eight fractions Fr.D2-14a to Fr.D2-14h were obtained. Among them, Fr.D2-14h was purified by preparative liquid chromatography to obtain compound 3 (6 mg).

[0059] (11) Fr.D2-18 (2.3 g) was separated by reversed-phase C18 column elution with a gradient of 50%, 60%, 70%, 80%, 90%, and 100% methanol-water solutions. After identification and merging by UHPLC-CAD, six fractions Fr.D2-18a to Fr.D2-18f were obtained. Among them, Fr.D2-18e was purified by preparative liquid chromatography to obtain compounds 1 (462 mg) and 2 (92.6 mg).

[0060] Structural characterization of compounds 1 to 29

[0061] The structures of compounds 1-29 were determined by high-resolution mass spectrometry, nuclear magnetic resonance analysis and acid hydrolysis experiments on the new compounds obtained in Example 1.

[0062] The acid hydrolysis experiment was conducted as follows: 1.0 mg of each monomeric compound obtained from the Gynostemma pentaphyllum extract was dissolved in 1 mL of 6 mol / L trifluoroacetic acid. The solution was heated in a water bath at 90 °C for 2 h, then cooled to room temperature. The solution was extracted three times with chloroform, and the aqueous layer was concentrated to obtain the sugar residues.

[0063] The obtained sugar residues and monosaccharide reference standards were dissolved in anhydrous pyridine (200 μL), and L-cysteine ​​methyl ester hydrochloride (1 mg) was added. The mixture was heated in a water bath at 60 °C for 1 h. Then, 10 μL of o-toluene isothiocyanate was added, and the mixture was heated in a water bath at 60 °C for 1 h. After the reaction was complete, the mixture was cooled to room temperature, the supernatant was filtered, and the solution was analyzed using UHPLC-CAD. UHPLC-CAD analytical conditions: Waters ACQUITY TM UPLC HSS T3 column (100×2.1mm, 1.8μm); mobile phase: 0.1% formic acid water (A)-acetonitrile (B); elution program: 0-8min, 20%-30% B; flow rate: 0.6mL / min; column temperature: 40℃; nebulization temperature: 35℃; filtering: 1s.

[0064] The absolute configurations of each glycosyl group were determined by comparing the retention times of the derivatized products with those of the monosaccharide reference standard, specifically D-glucose (retention time: 4.5 min), D-xylose (retention time: 4.9 min), and L-rhamnose (retention time: 6.3 min). Details are as follows:

[0065] Compound 1: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation value... (Concentration c 0.089, solvent: methanol); 1H NMR spectrum 1 H and carbon spectrum 13The C10 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.

[0066] Compound 2: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -10.52 (concentration c 0.15, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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-rhamnosyl(1→3)-α-L-rhamnosyl(1→6)-β-D-glucopyranoside.

[0067] Compound 3: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D+2.00 (concentration c 0.08, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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-rhamnosyl(1→6)-β-D-glucopyranoside.

[0068] Compound 4: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D+23.33 (concentration c 0.08, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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.

[0069] Compound 5: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D+2.3 (concentration c 0.087, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 C10 NMR data are shown in Table 2; High-resolution mass spectrometry (HR-ESI-MS): Molecular ion peak m / z 1107.5935 [MH]-(C10N M) 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-rhamnosyl(1→6)-β-D-glucopyranoside.

[0070] Compound 6: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D+14.00 (concentration c 0.08, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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 23Calculated 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.

[0071] Compound 7: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D+14.00 (concentration c 0.08, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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.

[0072] Compound 8: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -9.0 (concentration c 0.08, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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.

[0073] Compound 9: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -6.9 (concentration c 0.146, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13The C10 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.

[0074] Compound 10: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D+8.7 (concentration c 0.092, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 C10 NMR data are shown in Table 3; High-resolution mass spectrometry (HR-ESI-MS): Molecular ion peak m / z 1107.5942 [MH]-(C10N M) 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-rhamnosyl(1→6)-β-D-glucopyranoside.

[0075] Compound 11: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D+1.0 (concentration c 0.075, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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-peroxyhydrodammarane-23-ene-3-O-[β-D-glucopyranose(1→2)-β-D-glucopyranosyl]-20-O-(6-O-acetyl)-β-D-glucopyranoside.

[0076] Compound 12: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D+18.0 (concentration c 0.1, solvent methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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-rhamnosylpyranosyl(1→6)-β-D-glucopyranoside.

[0077] Compound 13: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D+8.0 (concentration c 0.1, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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-peroxy-dammarane-23-ene-3-O-β-D-glucopyranosyl-20-O-α-L-rhamnosylpyranosyl(1→6)-β-D-glucopyranoside.

[0078] Compound 14: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -7.00 (concentration c 0.09, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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-peroxy-dammarane-23-ene-20-O-β-D-xylanosylpyranosyl(1→3)-α-L-rhamnopyranosyl(1→6)-β-D-glucopyranoside.

[0079] Compound 15: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -14.00 (concentration c 0.086, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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.

[0080] Compound 16: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -9.3 (concentration c 0.097, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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-rhamnosyl(1→6)-β-D-glucopyranoside.

[0081] Compound 17: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D-1.0 (concentration c 0.08, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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 18Calculated value: 945.5423). Compound 17 was identified as (3β,12β,20S,24S)-tetrahydroxydammarane-25-ene-3-O-β-D-glucopyranosyl-20-O-α-L-rhamnosylpyranosyl(1→6)-β-D-glucopyranoside.

[0082] Compound 18: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D-2.0 (concentration c 0.08, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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-rhamnosylpyranosyl(1→6)-β-D-glucopyranoside.

[0083] Compound 19: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D+5.00 (concentration c 0.07, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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)-peroxy-dammarane-25-ene-3-O-β-D-glucopyranosyl-20-O-α-L-rhamnosylpyranosyl(1→6)-β-D-glucopyranoside.

[0084] Compound 20: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -5.00 (concentration c 0.02, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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 O19 Calculated value: 961.5372). Compound 20 was identified as (3β,12β,20S)-trihydroxy-24(R)-peroxy-dammarane-25-ene-3-O-β-D-glucopyranosyl-20-O-α-L-rhamnosylpyranosyl(1→6)-β-D-glucopyranoside.

[0085] Compound 21: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -14.6 (concentration c 0.089, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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-en-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.

[0086] Compound 22: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D+5.00 (concentration c 0.05, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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-en-24-one-3-O-β-D-glucopyranosyl-20-O-α-L-rhamnosylpyranosyl(1→6)-β-D-glucopyranoside.

[0087] Compound 23: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -7.00 (concentration c 0.088, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13The C10 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-en-24-one-20-O-β-D-xylanopyranosyl(1→3)-α-L-rhamnopyranosyl(1→6)-β-D-glucopyranoside.

[0088] Compound 24: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -13.00 (concentration c 0.115, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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.

[0089] Compound 25: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -13.00 (concentration c 0.145, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 C10 NMR data are shown in Table 8; High-resolution mass spectrometry (HR-ESI-MS): Molecular ion peak m / z 1235.6409 [MH]-(C10) 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-rhamnosyl(1→3)-α-L-rhamnosyl(1→6)-β-D-glucopyranoside.

[0090] Compound 26: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -6.9 (concentration c 0.146, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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-rhamnosyl(1→6)-β-D-glucopyranoside.

[0091] Compound 27: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -11.4 (concentration c 0.088, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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.

[0092] Compound 28: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -15.1 (concentration c 0.073, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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 27Calculated value: 1225.6217). Compound 28 was identified as (3β,12β,20S)-trihydroxy-25-one-26-nordammarane-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.

[0093] Compound 29: White amorphous powder, readily soluble in pyridine, methanol, and acetonitrile; shows a purple-red color upon detection in 10% sulfuric acid ethanol solution; optical rotation [α]25D -16.0 (concentration c 0.050, solvent: methanol); 1H NMR spectrum. 1 H and carbon spectrum 13 The C10 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.

[0094] Compounds 30-42 are all known compounds, and their structures were determined by comparison with NMR data reported in the literature. They were identified as: Gynostemma pentaphyllum saponin LXXXVII (30, m / z 1223.6473 [MH]). - ), Gynostemma pentaphyllum saponin V (31, m / z 1091.6028 [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-peroxy-dammarane-23-ene-3-O-[β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl]-20-O-β-D-glucopyranoside (37, m / z 977.5319 [MH]) - (3β,12β,20R)-trihydroxy-25-peroxy-dammarane-25-ene-3-O-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside (38, m / z 815.8386 [MH]) - ), Gynostemma pentaphyllum saponin XC (39, m / z 1239.6388 [MH]) - (3β,12β,20S)-trihydroxy-24(S)-peroxy-dammarane-25-ene-3-O-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside (40, m / z 815.4744 [MH]) - ), Gynostemma pentaphyllum saponin CI (41, m / z 1239.6224 [MH]) - ) and Gynostemma pentaphyllum saponin LXXXIX (42, m / z 1221.6138 [MH]) - The carbon spectral data of the above compounds are shown in Table 10.

[0095] Table 1. 1H NMR (600 MHz) and 1C NMR (150 MHz) data of compounds 1-4 in deuterated pyridine.

[0096] Table 2. 1H NMR (600 MHz) and 1C NMR (150 MHz) data for compounds 5-8 in deuterated pyridine.

[0097] Table 3. 1H NMR (600 MHz) and 1C NMR (150 MHz) data for compounds 9-11 in deuterated pyridine.

[0098] Table 4. 1H NMR (600 MHz) and 1C NMR (150 MHz) data of compounds 12-14 in deuterated pyridine.

[0099] Table 5. 1H NMR (600 MHz) and 1C NMR (150 MHz) data of compounds 15-17 in deuterated pyridine.

[0100] Table 6. 1H (600MHz) and 1C (150MHz) spectral data of compounds 18-20 in deuterated pyridine.

[0101] Table 7. 1H NMR (600 MHz) and 1C NMR (150 MHz) data for compounds 21-23 in deuterated pyridine.

[0102] Table 8. 1H NMR (600 MHz) and 1C NMR (150 MHz) data for compounds 24-26 in deuterated pyridine.

[0103] Table 9. 1H NMR (600 MHz) and 1C NMR (150 MHz) data for compounds 27-29 in deuterated pyridine.

[0104] Table 10. Carbon spectral data (150 MHz) of compounds 30-42 in deuterated pyridine

[0105] The following are performance tests of triterpenoid saponins.

[0106] Experiment 1: The role of total ATP production in myotube cells and its mitochondrial origin.

[0107] Skeletal muscle ATP depletion and mitochondrial dysfunction are important markers of physical fatigue. Regulating ATP production and mitochondrial function in skeletal muscle is crucial for improving fatigue levels, enhancing exercise endurance, and mitigating muscle atrophy. Therefore, we used a screening model of myotube and mitochondrial ATP production activity to evaluate the effects of triterpenoid saponins from Gynostemma pentaphyllum on promoting total ATP production in myotube cells and mitochondrial-derived ATP, thereby demonstrating their anti-fatigue activity.

[0108] 1. Experimental reagents, materials and instruments

[0109] The positive control agent Acadesine (AICAR, batch S1515) was purchased from Beyotime; oligomycin (Oligomycin, O24530) was purchased from ACMEC BIO. The screened monomeric compounds were obtained through the separation and purification process described 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 Solarbio; Lipofectamine 2000 transfection reagent was purchased from Thermo Fisher Scientific; CellTiter-Lumi... TM The chemiluminescence cell viability assay kit (C0059L) was purchased from Beyotime. A multi-functional microplate reader (SYNERGY-LX, purchased from Biotec, USA) was used for screening.

[0110] 2 Experimental Methods

[0111] C2C12 cells were cultured in 96-well plates and grown in complete medium (DMEM high-glucose medium + 10% fetal bovine serum) until complete confluence. The medium was then replaced with differentiation medium (DMEM / F12 medium containing 1% glucose + 2% horse serum) to induce myoblast differentiation and fusion into multinucleated myotubes. Four days after differentiation, the test substance and the positive control AICAR were added. The final concentration of the test substance was 20 μM, and the positive control AICAR concentration was 100 μM. ATP levels in the differentiated myotubes were measured 24 hours after drug administration, following the procedures outlined in CellTiter-Lumi. TM The instructions for the chemiluminescence cell viability assay kit recommend the following steps: After rinsing cells with PBS, add 100 μL / well CellTiter-Lumi. TM Steady Plus II chemiluminescence assay reagent: after lysis by shaking at 200 rpm for 2 min at room temperature, incubate for 10 min, and wait for the luminescence signal to stabilize before performing chemiluminescence detection using a multi-functional microplate reader with chemiluminescence function.

[0112] Furthermore, mitochondrial-dependent ATP levels were determined by incubation in galactose-containing / non-oligomycin (mitochondrial ATP synthase inhibitor) media. After 21 hours of drug-treated culture in differentiated muscle tubes, the tubes were transferred to galactose-containing / non-oligomycin (mitochondrial ATP synthase inhibitor) media and incubated for 3 hours. ATP levels were then measured, and mitochondrial ATP production was calculated based on the following formula:

[0113] Compared with the negative control group, compounds that increased total ATP or decreased glycolytic ATP and increased mitochondrial ATP by more than 7.5% were considered to have the effect of activating mitochondrial ATP production.

[0114] Statistical analysis of the experimental data showed that, compared with the control group, *P<0.05, **P<0.01, ***P<0.001.

[0115] 3 Experimental Results

[0116] As shown in Figure 1, compared with the blank control group, the screened triterpenoid saponin compounds all showed certain ATP-promoting activities in myotube cells. Among them, compounds 1, 6, 8, 13, 30, 31, 38 and 40 had significant ATP-promoting activities in myotube cells, and all of them had stronger activities than the positive control drug AICAR (an AMPK agonist).

[0117] Furthermore, intracellular ATP production is mainly mediated by two processes: glycolysis in the cytoplasm and oxidative phosphorylation in the mitochondria. Oxidative phosphorylation-mediated mitochondrial energy transfer accounts for approximately 60%-80% of the total ATP produced by the cell. Increasing mitochondrial energy production can affect the body's thermogenesis, exercise endurance, and muscle strength. Further experimental results showed that the total ATP content in myotube cells treated with active saponins 1, 6, 13, 31, 38, and 40 increased. After adding oligomycin to inhibit mitochondrial oxidative phosphorylation, the total ATP content decreased, reaching the same level as the control group after drug treatment (as shown in Figure 2). This indicates that the mechanism by which active Gynostemma pentaphyllum saponins promote ATP production in myotube cells is by promoting mitochondrial oxidative phosphorylation rather than promoting glycolysis. Simultaneously, we used the MitoATP index to measure the promoting ability of active monomers on myotube mitochondrial ATP production. The active saponins 1, 6, 13, 31, 38, and 40, found in previous experiments, all promoted myotube mitochondrial ATP production, with compound 6 exhibiting the strongest activity.

[0118] In summary, several saponin compounds have the ability to promote the production of ATP from myotubular mitochondria, meaning that triterpenoid compounds from Gynostemma pentaphyllum have different degrees of anti-fatigue activity.

[0119] Test Experiment 2: FXR Antagonistic Activity Test

[0120] Farnesol X receptors (FXRs) are members of the nuclear hormone receptor family, primarily found in the liver and intestines, and regulate the synthesis, transport, intestinal reabsorption, and excretion of bile acids. Arousal of bile acid receptors such as TGR5 has been reported to have anti-fatigue, exercise endurance-enhancing, and muscle-building effects. Gynostemma pentaphyllum extract has been found to upregulate bile acid synthesis and metabolism by antagonizing intestinal FXRs, thereby activating bile acid receptors to exert an anti-fatigue effect. We used an FXR antagonism activity screening model based on a luciferase dual reporter gene assay to evaluate whether triterpenoid saponins in Gynostemma pentaphyllum also possess FXR antagonistic activity.

[0121] 1. Experimental reagents, materials and instruments

[0122] Ursodeoxycholic acid (UDCA, S25093) was purchased from Yuanye Biotechnology; GW4064 (FXR agonist, batch G872312) was purchased from Maclean's; the screened monomeric compounds were obtained through the separation and purification process described in Example 1. The dual-luciferase reporter gene assay kit (E1910) was purchased from Promega, USA; Lipofectamine 2000 transfection reagent was purchased from Thermo Fisher Scientific, USA; 293T cells, empty vector plasmids, and Renilla luciferase plasmids were kindly provided by the research group of Professor Xing Yaling at the Institute of Infectious Disease Epidemiology, Academy of Military Medical Sciences; pCMV-Script-hFXR plasmid and pGL4.11-hSHP-Luciferase plasmid were kindly provided by the research group of Professor Xie Cen at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. A multi-functional microplate reader (SYNERGY-LX) was purchased from Biotec, USA.

[0123] 2 Experimental Methods

[0124] 293T cells were cultured in 96-well plates. When confluence reached 70%, transient transfection was performed using liposomes, following the instructions of the Lipofectamine 2000 kit. In the experimental group, 100 ng / well of pCMV-Script-hFXR plasmid, 100 ng / well of pGL4.11-hSHP-Luc plasmid, and 5 ng / well of TK plasmid were gently mixed with 25 μL of OPTI-MEM serum-free medium to prepare a plasmid mixture. Separately, 0.5 μL / well of Lipofectamine 2000 was gently mixed with 25 μL / well of OPTI-MEM serum-free medium to prepare a Lipofectamine 2000 dilution, and incubated for 5 min. The plasmid mixture was then gently mixed with the Lipofectamine 2000 dilution, and after incubation for 20 min, 50 μL / well of the mixture was added to the cells and gently shaken. Six hours after transfection, the medium was changed to a complete culture medium containing antibiotics. Twenty-four hours later, the test substance and positive control drug UDCA were added. The final concentration of the test substance was 10 μM and the concentration of the positive control drug was 20 μM.

[0125] 24 hours after drug administration, dual-luciferase reporter gene activity was detected in cells, following the instructions of the Luciferase Reporter Assay System kit. Cells were washed with PBS and then lysed with 20 μL of 1×PLB lysis buffer at room temperature and shaken at 200 rpm for 15 min. 50 μL / well LAR II reagent was added to the lysate, gently mixed, and then analyzed using a multi-mode microplate reader with chemiluminescence function for firefly luciferase detection. After the initial detection, 50 μL / well Stop&Glo reagent was added, gently mixed, and then analyzed using a multi-mode microplate reader for sea cucumber luciferase detection. The ratio of the two detection 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 GW4064 group, *P<0.05, **P<0.01, ***P<0.001.

[0126] 3 Experimental Results

[0127] As shown in Figure 3, monomeric compounds 1, 6, 13, 16, 26, 31, 37, and 38 exhibit strong FXR antagonistic activity. Among them, compounds 31, 37, and 38 showed stronger antagonistic activity than the positive control UDCA (20 μM) at a concentration of 10 μM. Further experiments showed that all three compounds antagonized the FXR receptor according to a dose-response relationship, with compound 31 showing the highest IC50 value. 50 The IC50 value of compound 37 was 19.43 μM. 50 The IC50 value of compound 38 was 10.61 μM. 50 The value was 10.85 μM, and the specific results are shown in Figure 4.

[0128] In summary, several saponin compounds exhibit FXR antagonistic activity, meaning that triterpenoid compounds derived from Gynostemma pentaphyllum have varying degrees of antibody fatigue effects.

[0129] Test Experiment 3: Mouse Exhaustion Treadmill Exercise Experiment

[0130] The anti-fatigue effect of compound 31 was verified through a mouse treadmill exercise experiment at exhaustion.

[0131] 1. Animal grouping and dosage

[0132] Five groups of mice were set up, with eight mice in each group: a blank control group (Veh), a chemical type A Gynostemma pentaphyllum extract group (Gyp) with 300 mg / kg, and low, medium and high doses (10, 30 and 90 mg / kg) of compound 31 (GVL, GVM and GVH).

[0133] 2. Animal Experiment Methods

[0134] Mice were acclimatized for 7 days before being administered the drug via gavage for 2 weeks. The first behavioral test was conducted 1 hour after the last gavage. Mice were exercised at a constant speed of 20 m / min, and the total distance traveled to reach exhaustion was recorded. Exhaustion was defined as a mouse failing to continue moving three times consecutively and remaining on the electric shock pad for 10 seconds.

[0135] 3 Experimental Results

[0136] As shown in Figure 5, different doses of compound 31 significantly increased the treadmill exercise performance of mice, indicating that the compound has an anti-fatigue effect. (*, **, and *** represent statistically significant differences between this group and the normal control group in one-way ANOVA, respectively, p<0.05, p<0.01, and p<0.001.)

[0137] Test Experiment 4: Mouse Exhaustion Swimming Experiment

[0138] The anti-fatigue effect of compound 31 was verified by a mouse exhaustive swimming experiment.

[0139] 1. Animal grouping and dosage

[0140] The specific animal grouping and drug dosage are the same as in test experiment 3.

[0141] 2. Animal Experiment Methods

[0142] Mice were acclimatized for 7 days before being administered the drug via gavage for 2 weeks. One hour after the last gavage, the mice were subjected to a weighted swimming experiment. The pool was 30 cm deep and the water temperature was 25 ± 1 ℃. A weight equal to 5% of the mouse's body weight was tied to the mouse's tail, and the mouse was made to swim until exhaustion. Exhaustion was defined as the mouse not being able to surface for 7 consecutive seconds. The swimming time was recorded.

[0143] 3 Experimental Results

[0144] As shown in Figure 6, compound 31 significantly increased the exhaustive swimming performance of mice in a dose-dependent manner, indicating that the compound has an anti-exhaustion effect. (*, **, and *** represent statistically significant differences between this group and the normal control group in one-way ANOVA, respectively, p<0.05, p<0.01, and p<0.001.)

[0145] Test Experiment 5: Pulling Test of Mouse Limbs

[0146] The anti-fatigue effect of compound 31 was verified by a mouse limb tensile test.

[0147] 1. Animal grouping and dosage

[0148] Specifically the same as the animal grouping and administration dosage in Test Experiment 3.

[0149] 2 Animal experiment methods

[0150] After 7 days of adaptive feeding, the mice were given drugs by gavage for 2 weeks. After half an hour of the last gavage, a four - limb pulling force experiment was conducted. The mice were placed on a wire mesh. After the four limbs of the mice were firmly grasped, the posterior 1 / 3 of the mouse's tail was controlled, and the mouse was dragged backward with a pulling force parallel to the wire mesh. After all four limbs of the mouse left the wire mesh, the maximum pulling force was recorded. This experiment was repeated three times, and the data obtained three times were analyzed statistically together.

[0151] 3 Experimental results

[0152] As shown in Figure 7, after the intervention of Compound 31, the four - limb pulling force of the mice was significantly increased, indicating that this compound has the effect of enhancing physical fitness. (*, **, *** represent that in one - way ANOVA, the difference between this group and the normal control is statistically significant, which are p < 0.05, p < 0.01, p < 0.001 respectively)

[0153] Test Experiment 6, Detection of the weight ratio of quadriceps femoris and gastrocnemius muscle to body weight in mice

[0154] The muscle - building effect of Compound 31 was verified by detecting the weight ratio of quadriceps femoris and gastrocnemius muscle to body weight in mice.

[0155] 1 Animal grouping and administration dosage

[0156] Specifically the same as the animal grouping and administration dosage in Test Experiment 3.

[0157] 2 Animal experiment methods

[0158] The swimming exhaustion test was the same as Test Experiment 4. After half an hour of exhaustion, the mice were anesthetized and sacrificed by blood collection. The quadriceps femoris muscle (QFM) and gastrocnemius muscle (Gastro.) of mice in different groups were collected, weighed and divided by the body weight to calculate the proportion of body weight they accounted for.

[0159] 3 Experimental results

[0160] As shown in Figure 8, after two weeks of administration of Compound 31, the weight - to - body - weight ratio of the quadriceps femoris and gastrocnemius muscles of the mice was significantly increased, and each dosage had a dose - effect relationship, indicating that Compound 31 has the effects of muscle - building and enhancing physical fitness. (*, **, *** represent that in one - way ANOVA, the difference between this group and the normal control is statistically significant, which are p < 0.05, p < 0.01, p < 0.001 respectively)

[0161] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0162] Cross-referencing of related applications:

[0163] This application claims priority to Chinese patent application No. 202510029035.6, filed on January 8, 2025, the entire contents of which are incorporated herein by reference.

[0164] Industrial applications

[0165] This invention has the following technical advantages:

[0166] 1. Compounds 1 to 29 provided by the present invention have novel structural features and significant anti-fatigue activity.

[0167] 2. The triterpenoid saponin compounds derived from Gynostemma pentaphyllum and having the structure shown in Formula I provided by this invention have significant anti-fatigue activity and can be used as lead compounds to develop related anti-fatigue products.

Claims

1. Triterpenoid saponins, selected from one or more of compounds 1 to 29 below:

2. The method for extraction and separation of triterpenoid saponins according to claim 1 includes the following steps: extracting Gynostemma pentaphyllum with water and precipitating it with alcohol, concentrating the supernatant, and obtaining triterpenoid saponins with different structures through separation and purification.

3. The extraction and separation method according to claim 2, characterized in that, Includes the following steps: (1) Extract Gynostemma pentaphyllum with water, concentrate it, precipitate it with 90% ethanol, take the supernatant, concentrate it, and then perform D101 macroporous adsorption resin column chromatography. Then, use an ethanol-water solvent system for gradient elution, concentrate and dry to obtain three components Fr.D1~Fr.D3. (2) Take the Fr.D1 obtained in step (1) and perform SP825 macroporous adsorption resin column chromatography. Use an ethanol-water solvent system for gradient elution, concentrate and dry to obtain 5 components Fr.D1A~Fr.D1E; (3) Take the fraction Fr.D1B obtained in step (2) and perform MCI resin column chromatography. Use a methanol-water solvent system for gradient elution. After identification by UHPLC-CAD, combine to obtain 6 fractions Fr.D1B1~Fr.D1B6. (4) Take Fr.D1B3 obtained in step (3), separate it using a reversed-phase C18 column, and perform gradient elution using a methanol-water solvent system to obtain 7 components Fr.D1B3a~Fr.D1B3g; among them, Fr.D1B3d was purified by preparative liquid chromatography to obtain compound 28, Fr.D1B3e was purified by preparative liquid chromatography to obtain compound 9, compound 10, compound 15, compound 16 and compound 29, and Fr.D1B3f was purified by preparative liquid chromatography to obtain compound 5 and compound 27; (5) Take Fr.D1B4 obtained in step (3), separate it using a reversed-phase C18 column, perform gradient elution using a methanol-water solvent system, and identify and combine it by UHPLC-CAD to obtain 5 components Fr.D1B4a~Fr.D1B4e; among them, Fr.D1B4c was purified by preparative liquid chromatography to obtain compound 21 and compound 24, and Fr.D1B4d was purified by preparative liquid chromatography to obtain compound 25 and compound 26; (6) Take Fr.D3 obtained in step (1) and perform silica gel column chromatography separation. Use a chloroform-methanol-water solvent system for gradient elution. After TLC detection and merging, Fr.D3-1 to Fr.D3-22 are obtained. Fr.D3-10 to Fr.D3-13 are merged and separated using a reversed-phase C18 column. Use a methanol-water solvent system for gradient elution. After UHPLC-CAD analysis, they are merged to obtain a total of 9 components Fr.D3-10a to Fr.D3-10h. Among them, Fr.D3-10g is purified by preparative liquid chromatography to obtain compound 14 and compound 23. (7) The Fr.D3-14 obtained in step (6) was subjected to reversed-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 purified by preparative liquid chromatography to obtain compound 12, compound 13, compound 17, compound 18, compound 19, compound 20 and compound 22. (8) The Fr.D3-15 obtained in step (6) was separated and purified by reversed-phase C18 column chromatography. Gradient elution was performed using a methanol-water solvent system. After identification and merging by UHPLC-CAD, 21 components Fr.D3-15a~Fr.D3-15u were obtained. Among them, Fr.D3-15i was purified by preparative liquid chromatography to obtain compound 6 and compound 8; Fr.D3-15j was purified by preparative liquid chromatography to obtain compound 7. (9) Take Fr.D2 obtained in step (1), separate and purify it by silica gel column chromatography, use a chloroform-methanol-water solvent system for gradient elution, and after TLC detection and merging, obtain 22 components Fr.D2-1 to Fr.D2-22; among them, Fr.D2-8 is separated by reversed-phase C18 column chromatography, uses a methanol-water solvent system for gradient elution, and after UHPLC-CAD detection and merging, obtain 18 components Fr.D2-8a to Fr.D2-8r; among them, Fr.D2-8j is purified by preparative liquid chromatography to obtain compound 11; Fr.D2-8n is purified by preparative liquid chromatography to obtain compound 4; (10) The Fr.D2-14 obtained in step (9) was separated by reversed-phase C18 column and gradient elution was performed using a methanol-water solvent system. After identification and merging by UHPLC-CAD, eight components Fr.D2-14a to Fr.D2-14h were obtained; among them, Fr.D2-14h was purified by preparative liquid chromatography to obtain compound 3. (11) The Fr.D2-18 obtained in step (9) was separated by reversed-phase C18 column, and gradient elution was performed using a methanol-water solvent system. After identification and merging by UHPLC-CAD, six components Fr.D2-18a~Fr.D2-18f were obtained; among them, Fr.D2-18e was purified by preparative liquid chromatography to obtain compound 1 and compound 2.

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

5. The application according to claim 4, characterized in that, The products include products for combating physical fatigue, products for combating mental fatigue, and products for building muscle.

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

7. The product according to claim 6, characterized in that, The dosage form of the drug is granules, oral liquid, capsules, tablets, effervescent tablets, powder for injection, water for injection, or injection. The drugs include single-drug formulations or compound formulations.

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

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