Gynostemma pentaphylla extract and application thereof in preparation of anti-fatigue products

Abstract

The invention discloses a fiveleaf gynostemma herb extract and application of the fiveleaf gynostemma herb extract in preparation of an anti-fatigue product. The gynostemma pentaphyllum extract provided by the invention comprises one or more of the following compounds: (1) gynostemma pentaphyllum saponin LXXXV II; (2) (3beta, 12beta, 20S)-trihydroxy dammarane-24-ene-3-O-[beta-D-glucopyranosyl (1-> 2)-beta-D-glucopyranosyl]-20-O-beta-D-glucopyranosyl (1-> 3)-alpha-L-rhamnopyranosyl (1-> 6)-beta-D-glucopyranosyl; (3) gypenoside V; (4) (3beta, 12beta, 20S)-trihydroxy dammarane-24-ene-3-O-[6-O-acetyl-beta-D-glucopyranosyl (1-> 2)-beta-D-glucopyranosyl]-20-O-beta-D-xylopyranosyl (1-> 3)-alpha-L-rhamnopyranosyl (1-> 6)-beta-D-glucopyranoside, and (5) (3beta, 12beta, 20S)-trihydroxy dammarane-24-ene-3-O-[6-O-acetyl-beta-D- (5) gynostemma pentaphyllum saponin LXXXVIII; (6) preparing gypenoside X; (7) (3beta, 12beta, 20R)-trihydroxy-25-hydrogen peroxide group dammarane-25-ene-3-O-beta-D-glucopyranosyl (1-> 2)-beta-D-glucopyranoside, and (7) (3beta, 12beta, 20R)-trihydroxy-25-hydrogen peroxide group dammarane-25-ene-3- (8) (3beta, 12beta, 20S)-trihydroxy-24S-hydrogen peroxide group dammarane-25-ene-3-O-beta-D-glucopyranosyl (1-> 2)-beta-D-glucopyranoside, and (3) (3beta, 12beta, 20S)-trihydroxy-24S-hydrogen peroxide group dammarane-25-ene-3- And (9) the gynostemma pentaphyllum saponin LXXXVI is prepared. The fiveleaf gynostemma herb extract provided by the invention has a remarkable anti-fatigue effect.

Description

Technical Field

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

[0002] Existing chemical composition studies have shown that Gynostemma pentaphyllum contains various chemical components such as triterpenoid saponins, flavonoids, and polysaccharides. Among them, dammarane-type tetracyclic triterpenoid saponins are the main active components of Gynostemma pentaphyllum, also known as Gynostemma pentaphyllum saponins. Up to now, nearly 500 triterpenoid saponin compounds have been isolated and identified from Gynostemma pentaphyllum. Relevant pharmacological studies have shown that Gynostemma pentaphyllum has a wide range of pharmacological activities such as lipid-lowering, immune enhancement, anti-aging, improvement of brain function, and anti-fatigue.

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

[0004] We systematically collected Gynostemma pentaphyllum from the main producing areas across the country and studied its chemical composition and anti-fatigue effect. It was found that although the saponin components in Gynostemma pentaphyllum from different regions are all triterpenoid saponins, their anti-fatigue effects are significantly different. Further analysis found that Gynostemma pentaphyllum from different regions contains different structural types of triterpenoid saponins (we call them different chemical types of Gynostemma pentaphyllum). The difference in this structure makes their anti-fatigue activities different, that is, different chemical types of Gynostemma pentaphyllum have different degrees of anti-fatigue activities. Summary of the Invention

[0005] The purpose of the present invention is to provide a new Gynostemma pentaphyllum extract, the compounds contained therein have a specific chemical structure, which is different from the existing known chemical type Gynostemma pentaphyllum extracts, and has a significant anti-fatigue effect, and can be used in the preparation of anti-fatigue products.

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

[0007] The chemical names of the above compounds 1 to 9 are as follows in sequence: Compound 1: Gynostemma pentaphyllum saponin LXXXVⅡ; Compound 2: (3 β ,12 β ,20 S )-trihydroxydammar-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; Compound 3: Gypenoside V; Compound 4: (3 β ,12 β ,20 S )-trihydroxydammar-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; Compound 5: Gypenoside LXXXVIII; Compound 6: Gypenoside X; Compound 7: (3 β ,12 β ,20 R )-trihydroxy-25-hydroperoxydammar-25-ene-3- O - β -D-glucopyranosyl(1→2)- β -D-glucopyranoside; Compound 8: (3 β ,12 β ,20 S )-trihydroxy-24 S -hydroperoxydammar-25-ene-3- O - β -D-glucopyranosyl(1→2)- β -D-glucopyranoside; Compound 9: Gypenoside LXXXVI.

[0008] Second aspect, the present invention also provides an extraction method (Extraction Method 1) of the gynostemma pentaphyllum extract, comprising the following steps: subjecting gynostemma pentaphyllum to alcohol extraction, filtering, combining the filtrates, concentrating, and drying to obtain the gynostemma pentaphyllum extract.

[0009] In Extraction Method 1, the gynostemma pentaphyllum can be directly soaked in alcohol for extraction, or can be pulverized before alcohol extraction, with the latter having a better extraction effect. The alcohol extraction uses ethanol with a volume fraction of 20 - 70%, preferably ethanol with a volume fraction of 50%; the dosage of the alcohol is 8 - 12 times the volume of the gynostemma pentaphyllum, preferably 10 times. The conditions for alcohol extraction are: heating the material under reflux and extracting several times (such as 3 times), 2 h each time.

[0010] Third aspect, the present invention also provides another extraction method (Extraction Method 2) of the gynostemma pentaphyllum extract, comprising the following steps: subjecting gynostemma pentaphyllum to water extraction, filtering, concentrating, and standing; performing macroporous adsorption resin column chromatography on the supernatant, concentrating the eluate, and drying to obtain the gynostemma pentaphyllum extract. Among them, the conditions for water extraction are: adding water with a volume 12 times that of the gynostemma pentaphyllum and decocting for extraction 3 times, 1 h each time. The chromatography includes: performing column chromatography on the supernatant using D101 macroporous adsorption resin, then eluting with a 10% ethanol / aqueous solution, discarding the eluate, and then eluting with an 80% ethanol / aqueous solution and collecting the eluate.

[0011] Both of the above - mentioned extraction methods can obtain a gynostemma pentaphyllum extract with significantly anti - fatigue activity, and the extract obtained by Extraction Method 2 has a relatively high purity of saponins.

[0012] Fourth aspect, the present invention also provides the application of the above - mentioned gynostemma pentaphyllum extract in the preparation of anti - fatigue products.

[0013] In the present invention, the application of the gynostemma pentaphyllum extract in the preparation of anti - fatigue products is realized through at least one of the following indicators: 1) Reducing the serum lactic acid level after fatigue; 2) Increasing the weight - to - body - weight ratio of the quadriceps femoris; 3) Remodeling the area distribution of the quadriceps femoris muscle fibers and promoting the swelling of muscle fibers; 4) Promoting ATP production in myotube cells; 5) Down - regulating 5 - HT and 5 - HIAA, up - regulating DA, and overall down - regulating the 5 - HT / DA ratio.

[0014] Fifth aspect, the present invention also provides an anti - fatigue product, which comprises the above - mentioned gynostemma pentaphyllum extract.

[0015] In the present invention, the product includes anti - physical - fatigue products, anti - mental - fatigue products, and muscle - building products.

[0016] In the present invention, the product includes foods, dietary supplements, drugs, feed additives, and veterinary drugs.

[0017] In the present invention, the dosage form of the drug is granule, oral liquid, capsule, tablet, effervescent tablet, powder injection, aqueous injection or injection; the drug includes a single-agent dosage form or a compound dosage form.

[0018] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: 1. The gynostemma pentaphyllum extract provided by the present invention contains a variety of compounds with specific structures, and this structural feature is different from the chemical component characteristics of other chemical-type gynostemma pentaphyllum extracts, having novelty.

[0019] 2. Due to the specific chemical structure, the gynostemma pentaphyllum extract provided by the present invention has a significantly better anti-fatigue effect than other known chemical-type gynostemma pentaphyllum extracts. Through experiments such as the exhaustive treadmill exercise of mice, the exhaustive swimming model of mice, and the limb pulling force of mice, the anti-fatigue activity of the gynostemma pentaphyllum extract provided by the present invention includes anti-physical fatigue, anti-mental fatigue and muscle-building effects.

[0020] 3. The gynostemma pentaphyllum extract provided by the present invention has a wide range of uses and can be made into anti-fatigue products, such as foods, dietary supplements, drugs, feed additives and veterinary drugs, with good application prospects.

[0021] The additional aspects and advantages of the present invention will be partially given in the following description, partially become obvious from the following description, or be understood through the practice of the present invention. Description of the Drawings

[0022] Figure 1 It is the base peak ion chromatogram (BPI) of different chemical-type gynostemma pentaphyllum extracts in the negative ion mode.

[0023] Figure 2 It is the HPLC-CAD chromatogram of different chemical-type gynostemma pentaphyllum extracts.

[0024] Figure 3 It is the structural formula of the characteristic compound in the chemical-type A gynostemma pentaphyllum extract.

[0025] Figure 4 It is the treadmill exercise performance of mice after administration of different chemical-type gynostemma pentaphyllum extracts.

[0026] Figure 5 It is the exhaustive swimming exercise performance of mice after administration of different chemical-type gynostemma pentaphyllum extracts.

[0027] Figure 6 It is the result of the limb pulling force experiment of mice after administration of different chemical-type gynostemma pentaphyllum extracts.

[0028] Figure 7 It is the serum lactate level of exhausted mice after administration of different chemical-type gynostemma pentaphyllum extracts.

[0029] Figure 8 The treadmill exercise performance of mice after administration of Gynostemma pentaphyllum of chemical type A, total ginsenosides, and Gynostemma pentaphyllum extract of chemical type B (containing gypenoside L).

[0030] Figure 9 The weight-to-body weight ratio of the quadriceps femoris of mice after administration of Gynostemma pentaphyllum extract of chemical type A.

[0031] Figure 10 The distribution of muscle fiber area in the quadriceps femoris of mice after administration of Gynostemma pentaphyllum extract of chemical type A.

[0032] Figure 11 The ATP content of myotubes differentiated from C2C12 myoblasts after intervention with Gynostemma pentaphyllum extract of chemical type A.

[0033] Figure 12 The levels of neurotransmitters in the hypothalamus of mice after intervention with Gynostemma pentaphyllum extract of chemical type A. Detailed implementation methods

[0034] The chemical components in Gynostemma pentaphyllum extract were analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-QTOF-MS). The method is as follows: 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 was 2.5 kV; desolvation gas was nitrogen with a flow rate of 1000 L·h-1; desolvation temperature was 600 °C; cone voltage was 40 V, ion source temperature was 120 °C, scanning range m / z 100~1500; when performing low-energy scanning, the trap voltage was 6 eV, and when performing high-energy scanning, in the negative ion mode: trap voltage 45~55 eV; accurate mass number was calibrated with leucine enkephalin as the calibration solution.

[0035] Screening test Dry whole herb samples of Gynostemma pentaphyllum from 3 different regions were collected and labeled as sample of chemical type A (produced in Guilin City, Guangxi Zhuang Autonomous Region), sample of chemical type B (produced in Zhangzhou City, Fujian Province), and sample of chemical type C (produced in Ankang City, Shaanxi Province).

[0036] The samples of chemical type A, chemical type B, and chemical type C were each divided into 2 groups, and two extraction methods were used respectively, resulting in 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.

[0037] Extraction method 1: Grind 5 kg of dried whole Gynostemma pentaphyllum into coarse powder, soak it with 50% ethanol for 1 hour, extract it by heating under reflux with 10 times the amount of 50% ethanol for 3 times, 2 hours each time, filter, and combine the filtrates. Concentrate the filtrates and dry them under pressure to obtain Gynostemma pentaphyllum extract (saponin purity Ⅰ).

[0038] Extraction method 2: Take 10 kg of dried whole Gynostemma pentaphyllum, decoct it with 12 times the amount of water for extraction three times, 1 hour each time, filter, concentrate it under reduced pressure, let it stand, subject the supernatant to D101 macroporous adsorption resin chromatography, elute it successively with 10% ethanol / aqueous solution and 80% ethanol / aqueous solution, collect the 80% ethanol elution part, concentrate it under reduced pressure and dry it to obtain Gynostemma pentaphyllum extract (saponin purity Ⅱ).

[0039] Analysis results: 1. Structure characterization (1) Through the analysis by UHPLC-QTOF-MS technology, it was found that the compositions of triterpenoid saponin components in Gynostemma pentaphyllum extracts with the same chemical type and different saponin purities were basically the same, that is, the compositions of triterpenoid saponin components in chemical type AI and chemical type AⅡ, chemical type BI and chemical type BⅡ, and chemical type CI and chemical type CⅡ Gynostemma pentaphyllum extracts were the same, but the saponin purities were different. This shows that the two extraction methods have no substantial impact on the triterpenoid saponin components in the extracts.

[0040] (2) Structure 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). Its structure characteristics are: there is a hydroxyl substitution at C-12; there are sugar chain substitutions at C-3 and C-20. The sugar chain at C-3 is composed of 1-2 glucose groups, and the sugar group type at C-20 is diverse, composed of 1-3 sugar groups, including glucose, rhamnose, and xylose; the side chain at C-17 is a straight chain; among them, the ion flow diagram of chemical type AI Gynostemma pentaphyllum extract (saponin purity Ⅰ) in the negative ion mode is as Figure 1 shown.

[0041] Table 1 Chemical composition table of triterpenoid saponins in chemical type AI and chemical type AⅡ Gynostemma pentaphyllum extracts

[0042] *: Identified by reference substance.

[0043] Meanwhile, according to the compound structures in Table 1, it can also be known that the main components in the chemically typed AI and AⅡ Gynostemma pentaphyllum extracts have the parent nucleus of protopanaxadiol, but the sugar chain substituents connected to the parent nucleus are completely different from the known ginsenoside structures in terms of the types and quantities of monosaccharides, and there are substantial structural differences between the two.

[0044] (3) Structural characteristics of the chemically typed B Gynostemma pentaphyllum extract: A total of 15 compounds were detected and identified from the chemically typed B Gynostemma pentaphyllum extract. Its structural characteristics are as follows: Hydroxyl substituents are connected at C-2 and C-12 positions, glycosyl substituents are connected at C-3 and C-20 positions, and the glycosyl types are diverse, consisting of 1 to 2 monosaccharides, including glucose and xylose, and the side chain at C-17 is a straight chain; among them, the ion flow diagram of the chemically typed BI Gynostemma pentaphyllum extract (saponin purity Ⅰ) in the negative ion mode is as Figure 1 shown.

[0045] By comparison, it can be seen that the component characteristics in the chemically typed B Gynostemma pentaphyllum extract are similar to the main components in US20190201468A1, both containing Gynostemma pentaphyllum saponin L, but are completely different from the chemical composition and structural characteristics of the chemically typed A Gynostemma pentaphyllum extract.

[0046] (4) Structural characteristics of the chemically typed C Gynostemma pentaphyllum extract: A total of 15 compounds were detected and identified from the chemically typed C Gynostemma pentaphyllum extract. Its structural characteristics are as follows: There is no hydroxyl substituent at C-12 position; a sugar chain substituent is connected at C-3 position, and the glycosyl types are diverse, consisting of 1 to 3 monosaccharides, including arabinose, rhamnose, xylose and glucose; the side chain at C-17 is polycyclized into a five-membered ring; a carbonyl substituent can be seen at C-19 position; among them, the ion flow diagram of the chemically typed CI Gynostemma pentaphyllum extract (saponin purity Ⅰ) in the negative ion mode is as Figure 1 shown.

[0047] By comparison, it can be seen that the component characteristics in the chemically typed C Gynostemma pentaphyllum extract are similar to the active components in CN113135978A, but are completely different from the component composition and structural characteristics of the chemically typed A Gynostemma pentaphyllum extract.

[0048] From the above comparison, it can be seen that the chemically typed A Gynostemma pentaphyllum extract obtained in the present invention is different from the publicly available Gynostemma pentaphyllum extracts and is a new type of Gynostemma pentaphyllum extract.

[0049] 2. Comparative analysis of the relative contents of chemical components Since the response level of liquid chromatography-mass spectrometry is related to the properties of compounds rather than just the content, the present invention uses a universal detector (high performance liquid chromatography - charged aerosol detector HPLC-CAD) to compare the contents of chemical components in different chemically typed Gynostemma pentaphyllum extracts. The method is as follows: Phenomenex chromatographic column (150 mm×4.6 mm, 2.6 μm); mobile phase: 0.1% formic acid in 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; filter 3.6 s.

[0050] The chromatograms of gynostemma pentaphyllum extracts of chemical type A, chemical type B, and chemical type C are as Figure 2 shown.

[0051] The structural formula of the characteristic compounds in the gynostemma pentaphyllum extract of chemical type A is as Figure 3 shown. By comparison, it can be seen that the chemical compositions of the gynostemma pentaphyllum extract of chemical type A are significantly different from those of the gynostemma pentaphyllum extracts of chemical type B and chemical type C.

[0052] 3. Analysis of anti - fatigue effect In order to compare the anti - fatigue effects of gynostemma pentaphyllum extracts of different chemical types, six kinds of gynostemma pentaphyllum extracts of chemical type AI, chemical type AⅡ, chemical type BI, chemical type BⅡ, chemical type CI, and chemical type CⅡ were weighed respectively and made into stock solutions with different concentrations for subsequent pharmacological experiments.

[0053] Test experiment 1 The mouse exhaustive treadmill exercise experiment was used to investigate the anti - fatigue effects of gynostemma pentaphyllum extracts of different chemical types.

[0054] 1. Animal grouping and dose setting A total of 8 groups of mice were set up, with 8 mice in each group, namely blank control group (normal saline), positive control group (caffeine 10 mg / kg, administered once 1 h before behavioral testing), 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), chemical type CⅡ 300 mg / kg group (CⅡ - 300 group).

[0055] 2. Animal experiment method After 7 days of adaptive feeding, the mice were given drugs by gavage for 2 weeks. One hour after the last gavage, a treadmill exhaustion exercise test was conducted on the mice. The mice exercised at a constant speed of 20 m / min, and the total distance they traveled until exhaustion was recorded. The mice were considered to be in a state of exhaustion when they failed three consecutive attempts to continue exercising and stayed on the electric shock plate for 10 s, refusing to move.

[0056] 3. Experimental results As Figure 4 shown, after two weeks of drug administration, both Chemical Type AI and Chemical Type AⅡ could significantly increase the treadmill exercise performance of the mice, while other chemical types of Gynostemma pentaphyllum extracts had no significant effect. This indicates that the Gynostemma pentaphyllum extract of Chemical Type A has a good anti-fatigue effect. (*, **, *** represent that in one-way ANOVA, the difference in this group compared with the normal control is statistically significant, p < 0.05, p < 0.01, p < 0.001 respectively).

[0057] Test Experiment 2 A mouse exhaustive swimming experiment was used to investigate the anti-fatigue effects of different chemical types of Gynostemma pentaphyllum extracts.

[0058] 1. Animal grouping and dose setting Specifically, it was the same as the animal grouping in Test Experiment 1.

[0059] 2. Animal experiment method After 7 days of adaptive feeding, the mice were given drugs by gavage for 2 weeks. One hour after the last gavage, a weighted swimming experiment was conducted on the mice. The water depth in the mouse pool was 30 cm, and the water temperature was 25 ± 1°C. A weight equal to 5% of the mouse's body weight was tied to the mouse's tail to make it swim until exhaustion. The mouse was considered to reach the state of exhaustion when it could not surface for 7 consecutive seconds, and the swimming time was recorded.

[0060] 3. Experimental results As Figure 5 shown, after two weeks of administration of Chemical Type AI and Chemical Type AⅡ, the exhaustive swimming performance of the mice could be significantly increased, while other chemical types of Gynostemma pentaphyllum extracts had no significant effect. This indicates that the Gynostemma pentaphyllum extract of Chemical Type A has a good anti-fatigue effect. (*, **, *** represent that in one-way ANOVA, the difference in this group compared with the normal control is statistically significant, p < 0.05, p < 0.01, p < 0.001 respectively).

[0061] Test Experiment 3 A mouse four-limb pulling force measurement experiment was used to investigate the effects of different chemical types of Gynostemma pentaphyllum extracts on the four-limb pulling force of mice.

[0062] 1. Animal grouping and dose setting Same as the animal grouping in Test Experiment 1 specifically.

[0063] 2. Animal experiment method The above mice were subjected to a four - limb pulling force experiment half an hour before the exhaustion experiment. 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. The maximum pulling force was recorded after all four limbs of the mouse left the wire mesh. This experiment was repeated three times, and the data obtained three times were analyzed statistically together.

[0064] 3. Experimental results As Figure 6 shown, two weeks after administration, Chemical AI and Chemical AⅡ could significantly increase the four - limb pulling force of mice, while other chemical types of Gynostemma pentaphyllum extracts had no significant effect. This indicates that the chemical type A Gynostemma pentaphyllum extract has the effect of enhancing muscle strength. (*, **, *** represent that in one - way ANOVA, compared with the normal control group, the differences in this group are statistically significant, p < 0.05, p < 0.01, p < 0.001 respectively).

[0065] Test Experiment 4 Investigate the effects of different chemical types of Gynostemma pentaphyllum extracts on the lactic acid level in the serum of swimming - exhausted mice.

[0066] 1. Animal grouping and dose setting Same as the animal grouping in Test Experiment 2 specifically.

[0067] 2. Serum collection and lactic acid detection method The above mice were anesthetized and sacrificed half an hour after exhaustion, and blood was collected to obtain serum for lactic acid level detection. The operation steps were carried out according to the instructions of the lactic acid detection kit. An enzyme working solution and a 3 mmol / L standard working solution were prepared from the enzyme stock solution, enzyme diluent and standard powder. After the sample to be tested was mixed with the enzyme working solution and the color reagent, it was accurately reacted at 37 °C for 10 min. After adding the stop solution and mixing, the absorbance was measured at a wavelength of 530 nm, and the lactic acid content (mmol / L) = (A 测定 - A 空白 / A 标准 - A 空白 ) × C 标准 × dilution factor N was used to calculate the serum lactic acid level.

[0068] 3. Experimental results As Figure 7It is shown that two weeks of administration of Chemical AI and Chemical AⅡ can significantly reduce the serum lactic acid level in exhausted mice, indicating that the Chemical A gynostemma pentaphyllum extract has the effect of reducing the accumulation of metabolites, that is, the Chemical A gynostemma pentaphyllum extract has an obvious anti-fatigue effect. (*, **, *** represent that in one-way ANOVA, the difference in this group compared with the normal control is statistically significant, p < 0.05, p < 0.01, p < 0.001 respectively).

[0069] Test Experiment 5 The aglycone of the triterpenoid saponins in Chemical AI and Chemical AⅡ is the protopanaxadiol type triterpenoid saponin. Although it is the same as the aglycone of ginsenoside, there are certain structural differences. To further prove, the present invention uses the exhausted treadmill exercise experiment of mice to investigate and compare the anti-fatigue effects of the Chemical A gynostemma pentaphyllum extract, total ginsenosides, and the Chemical B gynostemma pentaphyllum extract (containing gypenoside L).

[0070] 1. Animal grouping and dose setting A total of 6 groups of mice were set up, with 8 mice in each group, namely the blank control group (normal saline), the positive control group (caffeine 10 mg / kg, administered once 1 h before the behavioral test), the Chemical AI 300 mg / kg group (AI-300), the Chemical AⅡ 300 mg / kg group (AⅡ-300), the total ginsenosides 300 mg / kg group (PS-300), and the Chemical BII 300 mg / kg group (BII-300).

[0071] 2. Animal experiment method After 7 days of adaptive feeding, the mice were given intragastric administration for 2 weeks. 1 h after the last intragastric administration, the treadmill exhaustion exercise experiment of the mice was carried out. The mice exercised at a constant speed of 20 m / min, and the total distance traveled until the exhausted state was recorded. The mice were regarded as being in the exhausted state when they failed three consecutive attempts to continue exercising and stayed on the electric shock plate for 10 s and refused to move.

[0072] 3. Experimental results As Figure 8 It is shown that after two weeks of administration, the Chemical A gynostemma pentaphyllum extract and Chemical BI (containing gypenoside L) can significantly increase the treadmill exercise performance of mice, while ginsenoside has no significant effect, and the effect of the Chemical A gynostemma pentaphyllum extract is better than that of the Chemical B gynostemma pentaphyllum extract containing gypenoside L. This indicates that the saponin components of the Chemical A gynostemma pentaphyllum have a strong anti-fatigue effect. (*, **, *** represent that in one-way ANOVA, the difference in this group compared with the normal control is statistically significant, p < 0.05, p < 0.01, p < 0.001 respectively) In the above Test Experiments 1 - 5, the present invention observed and compared the anti - fatigue effects of different chemical - type Gynostemma pentaphyllum extracts through experiments such as the exhaustive treadmill exercise experiment and the exhaustive swimming experiment on mice. The experimental results showed that the chemical - type A Gynostemma pentaphyllum extracts with different saponin purities all had good anti - fatigue effects, while the other chemical - type Gynostemma pentaphyllum extracts and total ginsenosides did not show obvious anti - fatigue effects, providing a theoretical basis for the further development of chemical - type A Gynostemma pentaphyllum extracts as anti - fatigue products.

[0073] Test Experiment 6 Investigate the effect of chemical - type A Gynostemma pentaphyllum extract on the ratio of the weight of the quadriceps femoris to body weight in swimming - exhausted mice.

[0074] 1. Animal experiment and collection of quadriceps femoris Specifically, it is the same as the animal grouping, dose setting, and animal experiment method in Test Experiment 1.

[0075] The swimming - exhausted mice were anesthetized and sacrificed by blood collection half an hour after exhaustion. The quadriceps femoris of the mice in the control group, chemical - type AI group, and chemical - type AⅡ group were collected, and the weight was measured and divided by the corresponding mouse body weight.

[0076] 2. Experimental results As Figure 9 shown, chemical - type AⅡ can significantly increase the ratio of the weight of the quadriceps femoris to body weight in mice, indicating that the chemical - type A Gynostemma pentaphyllum extract has the effect of promoting skeletal muscle growth, that is, the total saponins of chemical - type A Gynostemma pentaphyllum have an obvious muscle - building effect. (*, **, *** represent that in one - way ANOVA, the difference between this group and the normal control is statistically significant, with p < 0.05, p < 0.01, p < 0.001 respectively) Test Experiment 7 Adopt immunofluorescence staining experiment to investigate the effect of chemical - type AⅡ Gynostemma pentaphyllum extract on the distribution of skeletal muscle fiber area in the quadriceps femoris of mice.

[0077] 1. Animal experiment and detection of quadriceps femoris samples Specifically, it is the same as the animal grouping and sample collection method in Test Experiment 6.

[0078] The above - mentioned quadriceps femoris tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Cross - sections (5 μm) were cut from the middle of the quadriceps femoris and incubated overnight at 4℃ with anti - laminin antibody (PA1 - 16730, ThermoFisher, USA). After washing the sections in PBS containing 0.1% Tween - 20, they were incubated with Alexa Flour 555 secondary antibody, rinsed, and mounted. Microscopic detection was performed. The cross - sectional area of muscle fibers was calculated and statistically analyzed using ImageJ software.

[0079] 2. Experimental results AsFigure 10 It was shown that the chemical type A gynostemma pentaphyllum extract could significantly reshape the distribution of the muscle fiber area of the quadriceps femoris in mice and promote the enlargement of muscle fibers, indicating that the chemical type A gynostemma pentaphyllum extract has the effect of promoting skeletal muscle growth, that is, the chemical type A gynostemma pentaphyllum extract has an obvious muscle-building effect. (*, **, *** represent that in independent samples t In the t-test, compared with the normal control, the differences were statistically significant, being p < 0.05, p < 0.01, and p < 0.001 respectively) Test experiment 8 Cell ATP content detection was used to investigate the effect of the chemical type A gynostemma pentaphyllum extract on the ATP production of differentiated myotubes of C2C12 mouse myoblasts.

[0080] 1. Cell culture and detection C2C12 cells were cultured in 96-well plates. After growing to full confluence in complete medium (DMEM high-glucose medium + 10% fetal bovine serum), the medium was changed to differentiation medium (DMEM / F12 medium containing 1% glucose + 2% horse serum) for culture to induce the differentiation and fusion of myoblasts to form multinucleated myotubes. After 4 days of differentiation, chemical type AⅡ (groups AⅡ-10, AⅡ-20, AⅡ-40) and the positive drug (Veh group) were added, and the final concentration of the test substances was 20 μM. Cell ATP content detection was performed 24 h after adding the drugs to the differentiated myotubes, and the operation steps were carried out according to the instructions of the CellTiter-Lumi™ luminescence assay kit for cell viability detection. After rinsing the cells with PBS, 100 μL / well of CellTiter-Lumi™ Steady Plus Ⅱ luminescence detection reagent was added, and the cells were lysed by shaking at 200 rpm at room temperature for 2 min and then incubated for 10 min. After waiting for the luminescence signal to stabilize, chemiluminescence detection was performed using a multifunctional microplate reader with chemiluminescence function.

[0081] 2. Experimental results As Figure 11 It was shown that the chemical type A gynostemma pentaphyllum extract could dose-dependently promote the ATP production of differentiated myotubes of C2C12 mouse myoblasts. Thus, it was indicated that the chemical type A gynostemma pentaphyllum extract has the effect of promoting skeletal muscle energy production, that is, the chemical type A gynostemma pentaphyllum extract has an obvious energy metabolism regulation effect. (*, **, *** represent that in one-way ANOVA, compared with the normal control, the differences were statistically significant, being p < 0.05, p < 0.01, and p < 0.001 respectively) Test experiment 9 Monoamine neurotransmitters are related to human emotions, motivation, and satisfaction, and play an important role in the regulation of mental activities in the body. Changes in their levels in the brain and homeostasis imbalance are important factors leading to central fatigue. ELISA was used to detect the effects of chemically typed A Gynostemma pentaphyllum extracts (purity I and purity II) on the levels of monoamine neurotransmitters in the hypothalamus of fatigued mice.

[0082] 1. Animal grouping and dose setting Specifically, it is the same as the animal grouping in Test Experiment 5.

[0083] After the above mice reached exhaustion, they were anesthetized and sacrificed by blood collection half an hour later. The hypothalamus brain regions of the mice in the control group, chemically typed AI 300 mg / kg group (AI-300 group), and chemically typed AII 300 mg / kg group (AII-300 group) were collected. The contents of 5-HT, 5-HIAA, and DA in the hypothalamus tissues of the mice were measured by the Elisa method (the kit was purchased from Nanjing Jiancheng Bioengineering Institute).

[0084] 2. Experimental results Figure 12 It was shown that the chemically typed A Gynostemma pentaphyllum extracts (purity I and purity II) could down-regulate the levels of 5-HT and 5-HIAA in the hypothalamus and up-regulate the level of DA. Overall, by down-regulating the 5-HT / DA ratio in the hypothalamus, it relieved the neurotransmitter disorder induced by fatigue, suggesting that the saponin components of chemically typed A Gynostemma pentaphyllum have the effect of improving central fatigue, that is, the total saponins of chemically typed A Gynostemma pentaphyllum have an obvious effect on improving mental fatigue. (*, **, *** represent that in one-way ANOVA, the difference between this group and the normal control is statistically significant, p<0.05, p<0.01, p<0.001 respectively) Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

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

2. The method for extracting the Gynostemma pentaphyllum extract according to claim 1, comprising the steps of: crushing the Gynostemma pentaphyllum, extracting with alcohol, filtering, combining the filtrate, concentrating, and drying to obtain the Gynostemma pentaphyllum extract.

3. The method for extracting the Gynostemma pentaphyllum extract according to claim 1, comprising the following steps: extracting the Gynostemma pentaphyllum with water, filtering, concentrating, and standing; subjecting the supernatant to macroporous adsorption resin column chromatography, concentrating the eluate, and drying to obtain the Gynostemma pentaphyllum extract.

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

5. The use according to claim 4, characterized in that: The application of the Gynostemma pentaphyllum extract in the preparation of anti-fatigue products is achieved through at least one of the following indicators: 1) Reduce serum lactate levels after fatigue; 2) Improve quadriceps muscle weight to body weight ratio; 3) Reshape the area distribution of quadriceps muscle fibers and promote muscle fiber expansion; 4) Promote ATP production in myotube cells; 5) Downregulate 5-HT and 5-HIAA, upregulate DA, and overall downregulate the 5-HT / DA ratio.

6. An anti-fatigue product comprising the Gynostemma pentaphyllum extract according to claim 1.

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

8. The product according to claim 6 or 7, characterized in that The products described are foods, dietary supplements, pharmaceuticals, 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, capsule, tablet, effervescent tablet, powder injection, water injection or injection; The medicine includes a single dosage form or a compound dosage form.

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