Gynostemma pentaphyllum extract and use thereof in preparation of Anti-fatigue product

Abstract

A Gynostemma pentaphyllum extract and the use thereof in the preparation of an anti-fatigue product. The Gynostemma pentaphyllum extract comprises one or more of the following compounds: (1) gypenoside LXXXV II; (2) (3β,12β,20S)-trihydroxydammar-24-en-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; (3) gypenoside V; (4) (3β,12β,20S)-trihydroxydammar-24-en-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; (5) gypenoside LXXXVIII; (6) gypenoside X; (7) (3β,12β,20R)-trihydroxy-25-hydroperoxydammar-25-en-3-O-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside; and (8) (3β,12β,20S)-trihydroxy-24S-hydroperoxydammar-25-en-3-O-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside; and (9) gypenoside LXXXVI. The provided Gynostemma pentaphyllum extract has a significant anti-fatigue effect.

Description

A Gynostemma pentaphyllum extract and its application in the preparation of anti-fatigue products Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a Gynostemma pentaphyllum extract and its application in the preparation of anti-fatigue products. Background Technology

[0002] The Chinese medicinal herb 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.

[0003] Chemical analysis has shown that Gynostemma pentaphyllum contains various chemical components, including triterpenoid saponins, flavonoids, and polysaccharides. Dammarane-type tetracyclic triterpenoid saponins are the main active ingredients of Gynostemma pentaphyllum, also known as gypenosides. 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, improving brain function, and combating fatigue. However, due to its wide distribution and natural hybridization, the saponin composition of Gynostemma pentaphyllum varies greatly between different regions. For example, TaeYoung KIM et al. applied for a patent (US20190201468A1) for the medicinal use of Gynostemma pentaphyllum stem and leaf extract for anti-fatigue and its preparation method. The main components of the 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 were granted a patent CN113135978A for "A Dammarane-type Triterpenoid Saponin of Gynostemma pentaphyllum Active Ingredient and Its Separation and Application". The active ingredient is characterized by a five-membered ring synthesized by homocyclic cyclization of the side chain at C-17.

[0004] We systematically collected Gynostemma pentaphyllum samples from major producing areas across China and studied its chemical composition and anti-fatigue effects. We found that although the saponin components in Gynostemma pentaphyllum from different regions were all triterpenoid saponins, their anti-fatigue effects varied significantly. Further analysis revealed that Gynostemma pentaphyllum from different regions contains triterpenoid saponins with different structural types (which we call different chemical types of Gynostemma pentaphyllum). This structural difference leads to variations in their anti-fatigue activities; that is, different chemical types of Gynostemma pentaphyllum exhibit different degrees of anti-fatigue activity. Summary of the Invention

[0005] The purpose of this invention is to provide a novel Gynostemma pentaphyllum extract, the compounds of which have a specific chemical structure that is different from existing known chemical Gynostemma pentaphyllum extracts, and which have significant anti-fatigue effects and can be used to prepare anti-fatigue products.

[0006] In a first aspect, the present invention provides a Gynostemma pentaphyllum extract containing one or more of compounds 1 to 9:

[0007] The chemical names of compounds 1 through 9 are as follows:

[0008] Compound 1: Gynostemma pentaphyllum saponin LXXXVⅡ;

[0009] Compound 2: (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-rhamnosyl(1→6)-β-D-glucopyranoside;

[0010] Compound 3: Gynostemma pentaphyllum saponin V;

[0011] Compound 4: (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;

[0012] Compound 5: Gynostemma pentaphyllum saponin LXXXVⅢ;

[0013] Compound 6: Gynostemma pentaphyllum saponin X;

[0014] Compound 7: (3β,12β,20R)-trihydroxy-25-peroxyhydro-dammarane-25-ene-3-O-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside;

[0015] Compound 8: (3β,12β,20S)-trihydroxy-24S-peroxy-dammarane-25-ene-3-O-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside;

[0016] Compound 9: Gynostemma pentaphyllum saponin LXXXVI.

[0017] Secondly, the present invention also provides an extraction method for the Gynostemma pentaphyllum extract (extraction method one), comprising the following steps: extracting Gynostemma pentaphyllum with alcohol, filtering, combining the filtrates, concentrating, and drying to obtain the Gynostemma pentaphyllum extract.

[0018] In extraction method one, the Gynostemma pentaphyllum can be directly soaked in alcohol for extraction, or it can be pulverized and then extracted with alcohol; the latter yields better extraction results. The alcohol extraction uses 20-70% ethanol by volume, preferably 50% ethanol; the amount of alcohol used is 8-12 times the volume of the Gynostemma pentaphyllum, preferably 10 times. The extraction conditions are: heating the material under reflux and extracting several times (e.g., 3 times), each time for 2 hours.

[0019] Thirdly, the present invention also provides another extraction method for the Gynostemma pentaphyllum extract (extraction method two), comprising the following steps: extracting Gynostemma pentaphyllum with water, filtering, concentrating, and allowing to stand; performing macroporous adsorption resin column chromatography on the supernatant, concentrating the eluent, and drying to obtain the Gynostemma pentaphyllum extract. The water extraction conditions are as follows: adding 12 times the volume of water to the Gynostemma pentaphyllum, decocting and extracting three times, each time for 1 hour. The chromatography includes: performing column chromatography on the supernatant using D101 macroporous adsorption resin, then eluting with 10% ethanol / water solution, discarding the eluent, and then eluting with 80% ethanol / water solution, collecting the eluent.

[0020] Both extraction methods can yield Gynostemma pentaphyllum extracts with significant anti-fatigue activity, with the extract obtained by extraction method two having relatively high saponin purity.

[0021] Fourthly, the present invention also provides the application of the above-mentioned Gynostemma pentaphyllum extract in the preparation of anti-fatigue products.

[0022] In this invention, the Gynostemma pentaphyllum extract is used in anti-fatigue products by achieving at least one of the following indicators:

[0023] 1) Reduce serum lactate levels after fatigue;

[0024] 2) Increase the ratio of quadriceps muscle weight to body weight;

[0025] 3) Reshape the area distribution of quadriceps muscle fibers and promote muscle fiber expansion;

[0026] 4) Promotes ATP production in myotube cells;

[0027] 5) Decrease 5-HT and 5-HIAA, increase DA, and decrease the overall 5-HT / DA ratio.

[0028] Fifthly, the present invention also provides an anti-fatigue product comprising the above-mentioned Gynostemma pentaphyllum extract.

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

[0030] In this invention, the products include food, dietary supplements, medicines, feed additives, and veterinary drugs.

[0031] In this invention, the dosage form of the drug is granules, oral liquid, capsules, tablets, effervescent tablets, powder for injection, water for injection, or injection; the drug includes single-ingredient dosage forms or compound dosage forms. Attached Figure Description

[0032] Figure 1 shows the base ion chromatogram (BPI) of liquid chromatography-mass spectrometry (LC-MS) of different chemical types of Gynostemma pentaphyllum extracts in negative ion mode.

[0033] Figure 2 shows the HPLC-CAD chromatograms of extracts of different chemical types of Gynostemma pentaphyllum.

[0034] Figure 3 shows the structural formulas of characteristic compounds in the chemical type A Gynostemma pentaphyllum extract.

[0035] Figure 4 shows the treadmill exercise performance of mice after administration of different chemical types of Gynostemma pentaphyllum extracts.

[0036] Figure 5 shows the performance of mice in exhaustive swimming after administration of different chemical types of Gynostemma pentaphyllum extracts.

[0037] Figure 6 shows the results of the limb tension test in mice after administration of different chemical types of Gynostemma pentaphyllum extracts.

[0038] Figure 7 shows the serum lactate levels in exhausted mice after administration of different chemical types of Gynostemma pentaphyllum extracts.

[0039] Figure 8 shows the treadmill exercise performance of mice after administration of chemical type A Gynostemma pentaphyllum, total ginsenosides, and chemical type B Gynostemma pentaphyllum extract (containing Gynostemma pentaphyllum saponin L).

[0040] Figure 9 shows the ratio of quadriceps muscle weight to body weight in mice after administration of chemical type A Gynostemma pentaphyllum extract.

[0041] Figure 10 shows the distribution of muscle fiber area in the quadriceps femoris muscle of mice after administration of chemical type A Gynostemma pentaphyllum extract.

[0042] Figure 11 shows the ATP content of C2C12 myoblasts differentiating into myotubes after intervention with chemical type A Gynostemma pentaphyllum extract.

[0043] Figure 12 shows the neurotransmitter levels in the hypothalamus of mice after intervention with chemical type A Gynostemma pentaphyllum extract. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0045] The chemical components in Gynostemma pentaphyllum extract were analyzed using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-QTOF-MS), as follows:

[0046] The mobile phase was 0.1% formic acid aqueous solution (A)-acetonitrile (B). The elution program was as follows: 0-2 min, 10%-25% B; 2-7 min, 25%-32% B; 7-10 min, 32%-33% B; 10-12 min, 33%-35% B; 12-22 min, 35%-46% B; 22-27 min, 46%-56% B; 27-28 min, 56%-95% B; 28-30 min, 95% B; 30-31 min, 95%-10% B; 31-33 min, 10% B. The flow rate was 0.5 mL / min, the column temperature was 40 °C, and the injection volume was 1 μL. Electrospray negative ion mode; capillary voltage 2.5kV; desolvation gas nitrogen, flow rate 1000L·h⁻¹; desolvation temperature 600℃; cone voltage 40V, ion source temperature 120℃, scanning range m / z 100~1500; trap voltage 6eV for low energy scanning, trap voltage 45~55eV for high energy scanning in negative ion mode; accurate mass number using Leucineek ephalin as calibration solution.

[0047] Screening test

[0048] Dried whole herb samples of Gynostemma pentaphyllum were collected from three different regions and labeled as chemical type A (originating from Guilin City, Guangxi Province), chemical type B (originating from Zhangzhou City, Fujian Province), and chemical type C (originating from Ankang City, Shaanxi Province).

[0049] Sample chemical type A, sample chemical type B, and sample chemical type C were each divided into two groups, and two extraction methods were used for each group to obtain a total of 6 extracts, which were labeled as chemical type AI, chemical type AⅡ, chemical type BI, chemical type BⅡ, chemical type CI, and chemical type CⅡ, respectively.

[0050] Extraction Method 1: Crush 5 kg of dried whole Gynostemma pentaphyllum into coarse powder, soak in 50% ethanol for 1 hour, and extract by reflux with 10 times the amount of 50% ethanol three times, 2 hours each time. Filter and combine the filtrates. Concentrate the filtrate and dry under pressure to obtain Gynostemma pentaphyllum extract (saponin purity I).

[0051] Extraction Method 2: Take 10 kg of dried whole Gynostemma pentaphyllum herb, decoct it three times with 12 times the amount of water, each time for 1 hour, filter, concentrate under reduced pressure, let stand, and perform D101 macroporous adsorption resin chromatography, eluting successively with 10% ethanol / water solution and 80% ethanol / water solution, collect the 80% ethanol eluent, concentrate under reduced pressure and dry to obtain Gynostemma pentaphyllum extract (saponin purity II).

[0052] Analysis results:

[0053] 1. Structural characterization

[0054] (1) Analysis using UHPLC-QTOF-MS revealed that the triterpenoid saponin composition of Gynostemma pentaphyllum extracts with the same chemical type but different saponin purities was basically consistent. Specifically, the triterpenoid saponin composition of Gynostemma pentaphyllum extracts of chemical types AI and AⅡ, BI and BⅡ, and CI and CⅡ was consistent, but the saponin purity differed. This indicates that the two extraction methods had no substantial impact on the triterpenoid saponin composition of the extracts.

[0055] (2) Structural characteristics of chemical type A Gynostemma pentaphyllum extract: A total of 23 compounds were detected and identified from chemical type A Gynostemma pentaphyllum extract (see Table 1). Their structural characteristics are as follows: hydroxyl substitution at C-12; sugar chain substitution at C-3 and C-20, the sugar chain at C-3 consists of 1 to 2 glucose units, and the sugar group at C-20 is diverse, consisting of 1 to 3 sugar groups, including glucose, rhamnose and xylose; the side chain at C-17 is a straight chain; among them, the ion chromatogram of chemical type AI Gynostemma pentaphyllum extract (saponin purity I) in negative ion mode is shown in Figure 1.

[0056] Table 1. Chemical components of triterpenoid saponins in extracts of chemical types AI and AⅡ of Gynostemma pentaphyllum.

[0057] *: Identified by reference standard.

[0058] Furthermore, based on the compound structures in Table 1, it can be seen that the main components of the chemical type AI and chemical type AⅡ Gynostemma pentaphyllum extracts have the core of protopanaxadiol, but the sugar chain substituents connected to the core are completely different from the known ginsenoside structures in terms of the types and number of monosaccharides, and there is a substantial structural difference between the two.

[0059] (3) Structural characteristics of chemical type B Gynostemma pentaphyllum extract: A total of 15 compounds were detected and identified from chemical type B Gynostemma pentaphyllum extract. Their structural characteristics are as follows: hydroxyl groups are attached at C-2 and C-12, and glycosyl groups are attached at C-3 and C-20. The glycosyl groups are diverse, consisting of 1 to 2 monosaccharides, including glucose and xylose. The side chain at C-17 is a straight chain. The ion chromatogram of chemical type B Gynostemma pentaphyllum extract (saponin purity I) in negative ion mode is shown in Figure 1.

[0060] The comparison shows that the component characteristics of chemical type B Gynostemma pentaphyllum extract are similar to those of the main components in US20190201468A1, both containing gypenosides L, but the chemical composition and structural characteristics are completely different from those of chemical type A Gynostemma pentaphyllum extract.

[0061] (4) Structural characteristics of chemical type C Gynostemma pentaphyllum extract: A total of 15 compounds were detected and identified from chemical type C Gynostemma pentaphyllum extract. Their structural characteristics are as follows: no hydroxyl substitution at C-12; sugar chain substitution at C-3, with diverse sugar types, consisting of 1 to 3 monosaccharides, including arabinose, rhamnose, xylose and glucose; polycyclic aromatic hydrocarbons forming a five-membered ring at C-17; carbonyl substitution visible at C-19; among them, the ion chromatogram of chemical type CI Gynostemma pentaphyllum extract (saponin purity I) in negative ion mode is shown in Figure 1.

[0062] The comparison shows that the component characteristics of chemical type C Gynostemma pentaphyllum extract are similar to those of the active ingredients in CN113135978A, but the component composition and structural characteristics are completely different from those of chemical type A Gynostemma pentaphyllum extract.

[0063] As can be seen from the above comparison, the chemical type A Gynostemma pentaphyllum extract obtained by the present invention is different from the existing publicly disclosed Gynostemma pentaphyllum extract and is a novel Gynostemma pentaphyllum extract.

[0064] 2. Comparative analysis of the relative content of chemical components

[0065] Since the response level of liquid chromatography-mass spectrometry (LC-MS) is related to the properties of the compound, rather than just its content, this invention uses a universal detector (HPLC-CAD) to compare the content of chemical components in different chemical types of Gynostemma pentaphyllum extracts. The method is as follows:

[0066] Phenomenex column (150 mm × 4.6 mm, 2.6 μm); mobile phase: 0.1% formic acid water (A) - acetonitrile (B); elution program: 0-8 min, 15%-30% B; 8-18 min, 30% B; 18-25 min, 30%-35% B; 25-35 min, 35% B; 35-36 min, 35%-40% B; 36-53 min, 40%-47% B; 53-54 min, 47%-95% B; 54-69 min, 95% B; flow rate: 1 mL / min; column temperature: 30 °C; nebulization temperature: 35 °C; filtering: 3.6 s.

[0067] The chromatograms of chemical type A Gynostemma pentaphyllum extract, chemical type B Gynostemma pentaphyllum extract, and chemical type C Gynostemma pentaphyllum extract are shown in Figure 2.

[0068] The characteristic compound structural formulas in the chemical type A Gynostemma pentaphyllum extract are shown in Figure 3. As can be seen from the comparison, the chemical composition of chemical type A Gynostemma pentaphyllum extract is significantly different from that of chemical type B and chemical type C Gynostemma pentaphyllum extracts.

[0069] 3. Analysis of anti-fatigue effects

[0070] To compare the anti-fatigue effects of different chemical types of Gynostemma pentaphyllum extracts, six types of Gynostemma pentaphyllum extracts (chemical type AI, chemical type AⅡ, chemical type BI, chemical type BⅡ, chemical type CI, and chemical type CⅡ) were weighed and prepared into stock solutions of different concentrations for subsequent pharmacological tests.

[0071] Test Experiment 1

[0072] The anti-fatigue effects of different chemical types of Gynostemma pentaphyllum extracts were investigated using a mouse exhaustive treadmill exercise experiment.

[0073] 1. Animal grouping and dosage setting

[0074] Eight groups of mice were set up, with eight mice in each group: blank control group (physiological saline), positive control group (caffeine 10 mg / kg, single administration 1 hour before behavioral test), chemical type AI 300 mg / kg group (AI-300 group), chemical type AⅡ 100 mg / kg group (AⅡ-100 group), chemical type AⅡ 300 mg / kg group (AⅡ-300 group), chemical type BI 300 mg / kg group (BI-300 group), chemical type BⅡ 100 mg / kg group (BⅡ-100 group), chemical type BⅡ 300 mg / kg group (BⅡ-300 group), chemical type CI 300 mg / kg group (CI-300 group), and chemical type CⅡ 300 mg / kg group (CⅡ-300 group).

[0075] 2. Animal experimental methods

[0076] Mice were acclimatized for 7 days before being administered the drug via gavage for 2 weeks. One hour after the last gavage, a treadmill exhaustion test was conducted on the mice. The mice 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 exercising three times consecutively and remaining on the electric shock pad for 10 seconds.

[0077] 3. Experimental Results

[0078] As shown in Figure 4, two weeks after administration, both chemical type AI and chemical type AⅡ significantly increased the treadmill exercise performance of mice, while other chemical types of Gynostemma pentaphyllum extracts had no significant effect. This indicates that chemical type A Gynostemma pentaphyllum extract has a good anti-fatigue effect. (*, **, *** represent statistically significant differences between this group and the normal control in one-way ANOVA, p<0.05, p<0.01, and p<0.001, respectively).

[0079] Test Experiment 2

[0080] The anti-fatigue effects of different chemical types of Gynostemma pentaphyllum extracts were investigated using a mouse exhaustive swimming test.

[0081] 1. Animal grouping and dosage setting

[0082] The specific animal groupings are the same as in test experiment 1.

[0083] 2. Animal experimental methods

[0084] After 7 days of acclimatization, mice were given oral medication. After 2 weeks of medication, 1 hour after the last oral administration, mice were subjected to a weighted swimming test. The pool was 30 cm deep and the water temperature was 25 ± 1 ℃. A weight of 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 float to the surface for 7 consecutive seconds. The swimming time was recorded.

[0085] 3. Experimental Results

[0086] As shown in Figure 5, two weeks of administration of chemical type AI and chemical type AⅡ significantly increased the exhaustive swimming performance of mice, while other chemical types of Gynostemma pentaphyllum extracts had no significant effect. This indicates that chemical type A Gynostemma pentaphyllum extract has a good anti-exhaustion effect. (*, **, *** represent statistically significant differences between this group and the normal control in one-way ANOVA, p<0.05, p<0.01, and p<0.001, respectively).

[0087] Test Experiment 3

[0088] The effect of different chemical types of Gynostemma pentaphyllum extracts on the limb pull strength of mice was investigated using a mouse limb pull strength test.

[0089] 1. Animal grouping and dosage setting

[0090] The specific animal groupings are the same as in test experiment 1.

[0091] 2. Animal experimental methods

[0092] The mice were subjected to a limb pulling test half an hour before the exhaustion test. The mice were placed on a wire mesh, and once they had a stable grip on all four limbs, the rear third of their tails was controlled, and the mice were pulled backward with a force parallel to the wire mesh. The maximum pulling force was recorded after all four limbs had detached from the wire mesh. This experiment was repeated three times, and the data from all three tests were statistically analyzed.

[0093] 3. Experimental Results

[0094] As shown in Figure 6, two weeks after administration, chemical type AI and chemical type AII significantly increased the limb pulling force of mice, while other chemical types of Gynostemma pentaphyllum extracts had no significant effect. This indicates that chemical type A Gynostemma pentaphyllum extract has the effect of enhancing muscle strength. (*, **, and *** represent statistically significant differences between this group and the normal control in one-way ANOVA, respectively, p<0.05, p<0.01, and p<0.001).

[0095] Test Experiment 4

[0096] The effects of different chemical types of Gynostemma pentaphyllum extracts on serum lactate levels in swimming exhausted mice were investigated.

[0097] 1. Animal grouping and dosage setting

[0098] The specific animal groupings are the same as in test experiment 2.

[0099] 2. Serum collection and lactate detection methods

[0100] Mice were anesthetized and euthanized half an hour after reaching exhaustion. Blood was collected from the mice, and serum was used for lactate level detection, following the instructions of the lactate detection kit. Enzyme working solution and 3 mmol / L standard working solution were prepared using enzyme stock solution, enzyme diluent, and standard powder. The test sample was mixed with the enzyme working solution and chromogenic reagent and reacted at 37°C for 10 min. After adding the stop solution and mixing, the absorbance was measured at 530 nm. The lactate content (mmol / L) was calculated as follows: (A...) 测定 -A 空白 / A 标准 -A 空白 )×C 标准 Calculate serum lactate levels by multiplying the dilution factor N.

[0101] 3. Experimental Results

[0102] As shown in Figure 7, administration of chemical type AI and chemical type AⅡ for two weeks significantly reduced serum lactate levels in exhausted mice, indicating that chemical type A Gynostemma pentaphyllum extract has the effect of reducing the accumulation of metabolites, that is, chemical type A Gynostemma pentaphyllum extract has a significant anti-fatigue effect. (*, **, *** represent statistically significant differences between this group and the normal control in one-way ANOVA, p<0.05, p<0.01, and p<0.001, respectively).

[0103] Test Experiment 5

[0104] The triterpenoid saponins in chemical types AI and AⅡ share the same parent nucleus as ginsenosides (protopanaxadiol type), but exhibit certain structural differences. To further demonstrate this, the present invention employed a mouse exhaustive treadmill exercise experiment to examine and compare the anti-fatigue effects of chemical type A Gynostemma pentaphyllum extract, total ginsenosides, and chemical type B Gynostemma pentaphyllum extract (containing Gynostemma pentaphyllum saponin L).

[0105] 1. Animal grouping and dosage setting

[0106] Six groups of mice were set up, with eight mice in each group: blank control group (physiological saline), positive control group (caffeine 10 mg / kg, single administration 1 hour before behavioral test), chemical AI 300 mg / kg group (AI-300), chemical AⅡ 300 mg / kg group (AⅡ-300), total ginsenosides 300 mg / kg group (PS-300), and chemical BII 300 mg / kg group (BII-300).

[0107] 2. Animal experimental methods

[0108] Mice were acclimatized for 7 days before being administered the drug via gavage for 2 weeks. One hour after the last gavage, a treadmill exhaustion test was conducted on the mice. The mice 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 exercising three times consecutively and remaining on the electric shock pad for 10 seconds.

[0109] 3. Experimental Results

[0110] As shown in Figure 8, two weeks after administration, chemical type A Gynostemma pentaphyllum extract and chemical type BI (containing gypenosides L) significantly increased the treadmill exercise performance of mice, while ginsenosides had no significant effect. Furthermore, chemical type A Gynostemma pentaphyllum extract was more effective than chemical type B Gynostemma pentaphyllum extract containing gypenosides L. This indicates that the saponin components of chemical type A Gynostemma pentaphyllum have a strong anti-fatigue effect. (*, **, *** represent statistically significant differences between this group and the normal control in one-way ANOVA, p<0.05, p<0.01, and p<0.001, respectively).

[0111] In the aforementioned tests 1-5, this invention observed and compared the anti-fatigue effects of different chemical types of Gynostemma pentaphyllum extracts through experiments such as mouse exhaustion treadmill exercise and mouse exhaustion swimming. The experimental results showed that chemical type A Gynostemma pentaphyllum extracts with different saponin purities all exhibited good anti-fatigue effects, while other chemical types of Gynostemma pentaphyllum extracts and total ginseng saponins did not show significant anti-fatigue effects. This provides a theoretical basis for further developing chemical type A Gynostemma pentaphyllum extract as an anti-fatigue product.

[0112] Test Experiment 6

[0113] The effect of chemical type A Gynostemma pentaphyllum extract on the quadriceps muscle weight-to-body weight ratio in mice exhausted from swimming was investigated.

[0114] 1. Animal experiments and quadriceps femoris muscle collection

[0115] The specific animal grouping, dosage settings, and animal experimental methods are the same as in Test Experiment 1.

[0116] Swimming-exhausted mice were anesthetized and euthanized half an hour after reaching exhaustion. Quadriceps femoris muscles were collected from mice in three groups: control group, chemical AI group, and chemical AⅡ group. The weight was measured and divided by the corresponding mouse body weight.

[0117] 2. Experimental Results

[0118] As shown in Figure 9, chemical type AⅡ significantly increased the quadriceps femoris muscle weight-to-body weight ratio in mice, indicating that chemical type A Gynostemma pentaphyllum extract has a skeletal muscle growth-promoting effect, meaning that chemical type A Gynostemma pentaphyllum total saponins have a significant muscle-building effect. (*, **, *** represent statistically significant differences between this group and the normal control in one-way ANOVA, p<0.05, p<0.01, and p<0.001, respectively.)

[0119] Test Experiment 7

[0120] Immunofluorescence staining was used to investigate the effect of chemical type AⅡ Gynostemma pentaphyllum extract on the area distribution of skeletal muscle fibers in the quadriceps femoris muscle of mice.

[0121] 1. Animal experiments and quadriceps femoris muscle sample testing

[0122] The specific methods for animal grouping and sample collection are the same as in test experiment 6.

[0123] The quadriceps femoris muscle tissue was fixed in 4% paraformaldehyde and embedded in paraffin. A cross-section (5 μm) was cut from the middle of the quadriceps femoris muscle and incubated overnight at 4°C with anti-laminus antibody (PA1-16730, ThermoFisher, USA). After washing in PBS containing 0.1% Tween-20, the sections were incubated with Alexa Flour 555 secondary antibody, rinsed, mounted, and examined under a microscope. The cross-sectional area of ​​the muscle fibers was calculated and statistically analyzed using ImageJ software.

[0124] 2. Experimental Results

[0125] Figure 10 shows that the chemical type A II Gynostemma pentaphyllum extract significantly remodeled the area distribution of muscle fibers in the quadriceps femoris muscle of mice and promoted muscle fiber enlargement, indicating that the chemical type A Gynostemma pentaphyllum extract has a skeletal muscle growth-promoting effect, that is, the chemical type A Gynostemma pentaphyllum extract has a significant muscle-building effect. (*, **, *** represent statistically significant differences between this group and the normal control in the independent samples t-test, respectively p<0.05, p<0.01, and p<0.001)

[0126] Test Experiment 8

[0127] The effect of chemical type A Gynostemma pentaphyllum extract on ATP production in myoblast differentiation of C2C12 mice was investigated using cellular ATP content detection.

[0128] 1. Cell culture and detection

[0129] 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, chemotypes AII (AII-10, AII-20, and AII-40 groups) and a positive control (Veh group) were added, with a final concentration of 20 μM for both. 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 lysing by shaking at 200 rpm for 2 minutes at room temperature, incubate for 10 minutes and wait for the luminescence signal to stabilize before performing chemiluminescence detection using a multi-functional microplate reader with chemiluminescence function.

[0130] 2. Experimental Results

[0131] As shown in Figure 11, the chemical type A Gynostemma pentaphyllum extract dose-dependently promoted ATP production in the myotubes of C2C12 mouse myoblasts, indicating that the chemical type A Gynostemma pentaphyllum extract has a skeletal muscle energy production-promoting effect, that is, the chemical type A Gynostemma pentaphyllum extract has a significant energy metabolism regulatory effect. (*, **, *** represent statistically significant differences between this group and the normal control in one-way ANOVA, p<0.05, p<0.01, and p<0.001, respectively)

[0132] Test Experiment 9

[0133] Monoamine neurotransmitters are associated with human emotions, motivation, and satisfaction, playing a crucial role in regulating mental activity. Changes in their brain levels and homeostasis imbalances are significant factors contributing to central fatigue. This study used ELISA to investigate the effects of chemically derived Gynostemma pentaphyllum extracts (purity I and purity II) on monoamine neurotransmitter levels in the hypothalamus of fatigued mice.

[0134] 1. Animal grouping and dosage setting

[0135] The specific animal groupings are the same as in test experiment 5.

[0136] Mice were anesthetized and euthanized half an hour after reaching exhaustion. Blood was collected from the hypothalamus of three groups of mice: the control group, the chemical AI 300 mg / kg group (AI-300 group), and the chemical AII 300 mg / kg group (AII-300 group). The levels of 5-HT, 5-HIAA, and DA in the hypothalamus tissue of mice were measured using ELISA (kits purchased from Nanjing Jiancheng Bioengineering Institute).

[0137] 2. Experimental Results

[0138] Figure 12 shows that chemically derived Gynostemma pentaphyllum extracts (purity I and purity II) can downregulate hypothalamic 5-HT and 5-HIAA levels and upregulate DA levels. Overall, by downregulating the hypothalamic 5-HT / DA ratio, they alleviate fatigue-induced neurotransmitter imbalances, suggesting that chemically derived Gynostemma pentaphyllum saponins have a central fatigue-improving effect, i.e., total saponins of chemically derived Gynostemma pentaphyllum have a significant effect on improving mental fatigue. (*, **, 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).

[0139] 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.

[0140] Cross-referencing of related applications:

[0141] This application claims priority to Chinese patent application No. 202510029039.4, filed on January 8, 2025, the entire contents of which are incorporated herein by reference.

[0142] Industrial applications

[0143] This invention has the following technical advantages:

[0144] 1. The Gynostemma pentaphyllum extract provided by this invention contains a variety of compounds with specific structures. These structural features are different from the chemical composition features of other chemical types of Gynostemma pentaphyllum extracts, and thus possess novelty.

[0145] 2. The Gynostemma pentaphyllum extract provided by this invention has a specific chemical structure, and therefore exhibits significantly superior anti-fatigue effects compared to other known chemical types of Gynostemma pentaphyllum extracts. Experiments using mouse exhaustive treadmill exercise, mouse exhaustive swimming models, and mouse limb pulling tests have verified that the anti-fatigue activity of the Gynostemma pentaphyllum extract provided by this invention includes anti-physical fatigue, mental fatigue, and muscle-building effects.

[0146] 3. The Gynostemma pentaphyllum extract provided by this invention has a wide range of applications and can be made into anti-fatigue products, such as food, dietary supplements, pharmaceuticals, feed additives and veterinary drugs, with good application prospects.

Claims

1. A Gynostemma pentaphyllum extract, comprising one or more of compounds 1 to 9:

2. The extraction method of Gynostemma pentaphyllum extract according to claim 1 includes the following steps: pulverizing Gynostemma pentaphyllum, extracting with alcohol, filtering, combining the filtrates, concentrating, and drying to obtain Gynostemma pentaphyllum extract.

3. The extraction method of Gynostemma pentaphyllum extract according to claim 1 includes the following steps: extracting Gynostemma pentaphyllum with water, filtering, concentrating, and allowing to stand; performing macroporous adsorption resin column chromatography on the supernatant, concentrating the eluent, and drying to obtain Gynostemma pentaphyllum extract.

4. The use of the Gynostemma pentaphyllum extract according to claim 1 in the preparation of anti-fatigue products.

5. The application according to claim 4, characterized in that, The Gynostemma pentaphyllum extract is used in anti-fatigue products by achieving at least one of the following indicators: 1) Reduce serum lactate levels after fatigue; 2) Increase the ratio of quadriceps muscle weight to body weight; 3) Reshape the area distribution of quadriceps muscle fibers and promote muscle fiber expansion; 4) Promotes ATP production in myotube cells; 5) Decrease 5-HT and 5-HIAA, increase DA, and decrease the overall 5-HT / DA ratio.

6. An anti-fatigue product comprising the Gynostemma pentaphyllum extract as described in claim 1.

7. The product according to claim 6, characterized in that, The products include products for combating physical fatigue, products for combating mental fatigue, and products for building muscle.

8. The product according to claim 6 or 7, characterized in that, The products mentioned are food, dietary supplements, medicines, feed additives, and veterinary drugs.

9. The product according to claim 8, 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.

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