CN113135978B - Active ingredient dammarane type triterpenoid saponin of gynostemma pentaphylla and separation and application thereof - Google Patents
Active ingredient dammarane type triterpenoid saponin of gynostemma pentaphylla and separation and application thereof Download PDFInfo
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Abstract
The invention provides a gynostemma pentaphylla active component dammarane type triterpenoid saponin compound and a separation method thereof. Experiments show that the separated 8 dammarane type triterpene saponin compounds can obviously prolong the time of resisting normal pressure closed hypoxia and sodium nitrite hypoxia of mice, reduce the death rate of acute hypoxia and have better anti-hypoxia effect; the falling time of the mouse on the rotating rod can be obviously prolonged, the weight bearing swimming time of the mouse is prolonged, and the anti-fatigue effect is better; the hypoxia can cause cognitive dysfunction of mice, and the water maze experiment shows that the pretreatment of the gynostemma pentaphylla active site can protect the learning cognitive ability of the mice to a certain extent, so that the gynostemma pentaphylla active site can be used for preparing the medicines for resisting fatigue, resisting hypoxia and improving learning cognitive.
Description
Technical Field
The invention relates to a separation of an active part and main active components of gynostemma pentaphylla, in particular to a dammarane type triterpenoid saponin as an active component of gynostemma pentaphylla with the functions of resisting fatigue and anoxia and improving learning and cognition and a separation method thereof, belonging to the field of separation of plant active components and the field of medical health care.
Background
Gynostemma pentaphylla (Gynostemmapentaphyllum(Thunb.) Makino), a botanical climbing grass of the genus Gynostemma of the family Cucurbitaceae, the whole herb was used as the drug and was included in the 2015 edition of Chinese pharmacopoeia, which is a part of medicinal materials and decoction pieces that are not collected in the formulation preparation. Gynostemma pentaphylla has the effects of delaying aging, resisting cancer, improving mental activities, improving brain functions, enhancing human immunity, regulating endocrine, reducing cholesterol and transaminase, inhibiting ulcer, relieving tension, tranquilizing mind, relieving pain and the like, and is called as 'long-life herb' for folk.
The gypenoside is the main active component of the gynostemma pentaphylla, and the gynostemma pentaphylla contains dammarane type triterpenoid saponin natural products similar to the ginsenoside. Gynostemma pentaphylla is the only plant containing ginsenoside except for the plant of the genus Panax in the family Araliaceae, and the content of ginsenoside in the excellent plant is even higher than that of ginseng, but the research on the chemical components and the biological activity of the Gynostemma pentaphylla has no ginseng system. At present, the research on gynostemma pentaphylla is a gynostemma pentaphylla extract or total saponins of gynostemma pentaphylla, and the preparation and activity evaluation of functional active sites in the extract are not reported.
Disclosure of Invention
The invention aims to provide a method for separating active sites of gynostemma pentaphylla for resisting fatigue and anoxia and improving learning cognitive functions, 8 new compound active ingredients are obtained, and the active sites and the active ingredients are simultaneously subjected to fatigue resistance, anoxia resistance and learning cognitive function improvement.
Preparation of active site and active component
(1) Pulverizing dried herba Gynostemmatis, extracting with 5-20 times of water or 70% ethanol-water under reflux for 3 times, each for 1-2 hr, mixing extractive solutions, concentrating until no alcohol smell exists, and eluting with macroporous adsorbent resin to obtain active fraction of herba Gynostemmatis, which is labeled as JB 2020.
(2) Dissolving the active part JB2020 of Gynostemma pentaphyllum Makino in water, and extracting with petroleum ether (60-90 deg.C), ethyl acetate and n-butanol respectively to obtain ethyl acetate phase and n-butanol phase.
(3) Separating the ethyl acetate phase by using a silica gel column, and carrying out gradient elution by using chloroform-methanol (70: 1-10:1, v/v) to obtain 7 parts Fr. A-Fr. G.
Fr. D (methanol/water = 85/15, v/v is mobile phase, flow rate is 10 mL/min; chromatographic column model is Daisogel C18, chromatographic column length × diameter is 250 mm × 20 mm), to obtain compound 1 (peak time: 37 min), compound 2 (peak time 39 min) and compound 3 (peak time 57 min) in this order.
Fr. G liquid phase separation (methanol/water = 88/12, v/v is mobile phase, flow rate is 10 mL/min; chromatography column model is Megres C18, length of chromatography column x diameter is 250 mm x 20 mm) to obtain compound 8 (time to peak 13 min).
(4) The n-butanol phase is dissolved at 1M H2SO4Is heated and refluxed for 6 hours, and NaHCO is used after the reaction is finished3Neutralizing the saturated aqueous solution, separating the concentrated solution by using a silica gel column, and performing gradient elution by using chloroform-methanol (20: 1-2:1, v/v) to sequentially obtain 3 parts Fr. A-Fr. C; fr. B is separated by C18 reverse phase silica gel column, methanol/water (50: 50-100)0, v/v) gradient elution to obtain 5 parts Fr. 1-Fr. 5 in sequence.
Liquid phase separation of fraction Fr.1 (methanol/water = 79/21, v/v is mobile phase, flow rate is 10 mL/min; chromatography column model is Megres C18, length of chromatography column multiplied by diameter is 250 mm multiplied by 20 mm) to obtain compound 6 (peak time 79 min);
separating the fraction Fr.2 with silica gel column, and gradient eluting with chloroform-methanol (20: 1-2:1, v/v) to obtain fraction Fr.2.1. Fr.2.1 liquid phase separation (methanol/water = 80/20, v/v is the mobile phase, flow rate is 10 mL/min; column model Daisogel C18, column length × diameter 250 mm × 20 mm) gives compound 7 (peak time 28 min).
The fraction Fr.3 is separated by silica gel column, and gradient elution is carried out with chloroform-methanol (40: 1-10:1, v/v) to obtain 5 fractions Fr. 3.1.1-Fr. 3.5.5. Fr. 3.1.1 liquid phase separation (methanol/water = 91/9, v/v is mobile phase, flow rate is 10 mL/min; chromatography column model is Megres C18 column, length x diameter of chromatography column is 250 mm x 20 mm) to get compound 4 (time to peak 12 min).
Fr.4 liquid phase separation (methanol/water =93/7, v/v is the mobile phase, flow rate is 10 mL/min; column model is Daisogel C18 column, column length × diameter is 250 mm × 20 mm) to give compound 5 (mass 3.0 mg, time to peak 29 min).
The compounds 1-8 obtained from JB2020 are dammarane type triterpene saponins, and the structural formula is as follows:
secondly, the activity research and evaluation of the active site JB2020 and 8 active ingredients of gynostemma pentaphylla
1 anti-fatigue Activity
1.1 rod-turning fatigue test
Four groups of 9-10 mice were set, and the groups were positive control group, JB202050mg/kg group, JB2020100mg/kg group, and JB2020200mg/kg group. The positive control is 100mg/kg red Yijiao capsule.
After the mouse adapts to the mouse room for 3 days, the gavage is started, the gavage is continuously performed for 7 days, the mouse is trained for 3-5 days before the rod rotation experiment under the training condition of 10r/min and 10min each time, the rod rotation experiment is performed after the last gavage is finished for 6h, and the time that the mouse falls off from the upper of the rod rotation for the third time is recorded.
The experimental results are shown in fig. 1: JB2020 has good antifatigue effect.
Weight bearing swimming experiment
Four groups of mice are arranged, wherein each group comprises 9-10 mice, and the mice are respectively a positive control group, a JB202050mg/kg group, a JB2020100mg/kg group and a JB2020200mg/kg group. The positive control is 100mg/kg red Yijiao capsule.
After the mice are adapted to the mouse room for 3 days, the gavage is started, the gavage is continuously performed for 7 days, the weight-bearing swimming experiment is performed after the last gavage is finished for 6 hours, a weight with the weight being 5% of the weight of the mice is hung at the tail of the mice under the conditions that the water depth is 30cm and the water temperature is 25 ℃, and the swimming time of the mice is recorded by 10s when the mice cannot float out of the water surface.
The experimental results are shown in fig. 2: the 60% active part (GP) of the gypenoside has the anti-fatigue effect, and the anti-fatigue effect is equivalent to that of the positive control Hongyi capsule at the same concentration (200 mg/kg) at the concentration of 200mg/kg, and the dosage is dependent.
The experiments show that the gynostemma pentaphylla active site prepared by the method can obviously prolong the falling time of a mouse on a rotating rod, prolong the weight swimming time of the mouse and have better anti-fatigue effect.
Anti-hypoxic activity
2.1JB2020 treatment hypoxia protection of PC12 cells
Grouping experiments: the treatment is divided into a normal control group and a hypoxia treatment group, and the treatment is respectively carried out on 12 groups, namely, a control group, a GP-1 mug/mL group, a GP-3 mug/mL group, a GP-10 mug/mL group, a GP-30 mug/mL group and a GP-60 mug/mL group.
Cell culture: PC12 cells were cultured in RPM 1640 complete medium (Gibco, Life Technologies, Grand Island, NY) containing 10% fetal bovine serum (Hyclone, Utah, USA), 100U/mL penicillin and 100. mu.g/mL streptomycin (Gibco, Life Technologies, Grand Island, NY), placed in a 5% carbon dioxide incubator, cultured at 37 ℃ and replaced every two daysPrimary culture medium. The cell hypoxia treatment conditions were as follows: the cells were placed at 37 ℃ with 0.3% O2，5%CO2And 92% N2Cultured in a hypoxic incubator.
Cell viability assay: cell viability was measured using the CCK-8 kit (CCK8, Dojindo, Kumamoto, Japan). PC12 cells were seeded at 80000 cells per well in 48-well plates and after 24h of incubation, cells were pretreated with varying doses of JB2020 or other fractions for 6h and then treated in a hypoxic (0.3% O2) incubator for 24 h. Old medium was aspirated, CCK-8 was added to complete medium at 10% ratio, mixed well and added to 48 well plates at 200 μ L/well and incubated at 37 ℃ for 1 hour. Then, absorbance was measured at a wavelength of 450nm using a microplate reader (Bio-Rad, USA).
The protective effect of JB2020 treatment on PC12 cell hypoxia is shown in fig. 3: the cell viability of the JB2020 high-dose group of the composition before and after hypoxia treatment reaches the same level, and has strong hypoxia protection effect, so the sample is used for further related research.
Effect of treatment on the cell cycle of PC12
Grouping experiments: the treatment is divided into a normal control group and a hypoxia treatment group, and the control group (control group), the GP-30 mu g/mL group and the GP-60 mu g/mL group are respectively arranged for 6 groups.
The cell sample pellet was collected, approximately 1mL of pre-cooled PBS (phosphate buffered saline) was added to resuspend the cells, centrifuged again, the supernatant carefully aspirated, approximately 50 μ LPBS remained to avoid aspiration of the cells, and the bottom of the tube was flicked to disperse the cells properly to avoid clumping. Adding into 1mL of precooled 70% ethanol, gently blowing and uniformly mixing, fixing at 4 ℃ for more than 30min, centrifuging for 3-5min at 1000g, sucking and removing supernatant, adding 1mL of precooled PBS, re-suspending cells, centrifuging again, carefully sucking and removing supernatant, and remaining about 50 mu LPBS to avoid sucking away cells, and gently flicking the bottom of a centrifuge tube to properly disperse cells and avoid cell agglomeration. Preparing a proper amount of propidium iodide staining solution, adding 0.5mL of the propidium iodide staining solution into each tube of cell samples, slowly and fully suspending cell precipitates, and carrying out warm bath at 37 ℃ in the dark for 30 min. Subsequently, the cells can be stored at 4 ℃ or in an ice bath protected from light. The flow cytometer detects red fluorescence at the 488nm wavelength of the excitation wavelength, and simultaneously detects the light scattering condition.
The test results are shown in fig. 4 and 5: the cell cycle is blocked at the G0G1 stage due to the hypoxia treatment, and the number of cells at the S stage is increased after GYP treatment, which shows that JB2020 can also improve the cell block caused by hypoxia, thereby playing the role of resisting hypoxia.
Western Blotting experiment of treatment groups
Grouping experiments: the treatment is divided into a normal control group and a hypoxia treatment group, and the control group (control group), the GP-30 mu g/mL group and the GP-60 mu g/mL group are respectively arranged for 6 groups.
Collecting a cell sample, adding a Laemmli sample buffer lysate, fully lysing cells, boiling and centrifuging to collect supernatant, and measuring the protein concentration by using BCA (bicinchoninic acid); adding beta-mercaptoethanol according to the volume ratio of 5 percent; preparing separation gel and concentrated gel with proper concentration according to the molecular weight of the target protein; pouring electrophoresis liquid, carrying out electrophoresis at 70V at the early stage, and increasing the voltage to 140V after the protein enters the separation gel; after electrophoresis, 80V is transferred to a PVDF (polyvinylidene fluoride) film; TBST buffer (containing three substances of Tris-HCl, NaCl and tween20, which is a buffer solution commonly used for Western Blotting experiment) is washed, and then 5% skimmed milk powder is sealed for 1 h; incubating the primary antibody at 4 ℃ overnight, and incubating the secondary antibody after washing the membrane; and developing in a dark room.
The test results are shown in fig. 6: as can be seen from the expression amount of the protein, JB2020 treatment can increase the expression of p-ERK (Phospho-ERK, which refers to phosphorylation-activated extracellular regulated protein kinase) and p-AMPK (Phospho-AMPK, which refers to phosphorylation adenylate-activated protein kinase) proteins, thereby resulting in an increase in cell viability.
The experiments show that the gynostemma pentaphylla active site prepared by the method can obviously prolong the time of resisting normal-pressure closed hypoxia and sodium nitrite hypoxia of a mouse, reduce the death rate of acute hypoxia and has a better anti-hypoxia effect. The hypoxia protection function is evaluated by JB2020 treatment on PC12 cell hypoxia protection effect, JB2020 treatment on PC12 cell cycle influence and Western Blotting experiment of JB2020 treatment group, and the results show that JB2020 can improve cell cycle block caused by hypoxia, thereby playing the role of hypoxia protection.
Improving learning cognitive function
Grouping experiments: AD transgenic mice were randomly divided into control (control) and JB 2020-treated groups of 12 mice each.
Morris water maze test: the water maze device is a round water pool with the diameter of 100cm, the water depth of 50cm, the water temperature of 25 +/-1 ℃, and the indoor illumination and the object arrangement are kept consistent. The mouse head is placed in water towards the pool wall, the placing position is random, angles of 60 degrees, 120 degrees, 180 degrees and 360 degrees can be taken as initial positions, a 60s guiding experiment is started, if the mouse cannot find the platform within 60s, the mouse is guided to reach the platform, and the mouse stays on the platform for 10 s. Each training is carried out for 4 times every day, the time interval of each training is 15-20 min, and the training lasts for 4 days. Beginning on day 7, the platform was removed and the mice were placed in water from the original 4 positions. The trajectory of the mouse's activity in water and the escape latency for the mouse to reach the target platform were recorded (fig. 7).
The experiments show that JB2020 can obviously shorten the escape latency of AD transgenic mice, which indicates that JB2020 has an improving effect on learning and memory ability.
Activity study and evaluation of three, 8 active ingredients
1. Anti-fatigue Activity
The experimental operation and the experimental steps are the same as those of the anti-fatigue experiment of the active site JB 2020.
The experimental results are shown in fig. 8: all 8 active compounds showed an anti-fatigue effect.
Weight bearing swimming experiment
The experimental operation and the experimental steps are the same as those of the active site JB2020 load swimming experiment.
The results of the experiment are shown in FIG. 9: all 8 active compounds have an anti-fatigue effect.
Hypoxia protection of PC12 cells by treatment with active compound
Experimental procedures and experimental procedures experiments on the hypoxia protection effect of the active site JB2020 treatment on PC12 cells.
The experimental results are shown in fig. 10: compared with a hypoxia control group, after 8 active compounds are added with drugs, the cell activity is obviously enhanced, and the hypoxia protective effect is stronger.
Based on the active site of the gynostemma pentaphylla and the functions of 8 active compounds, the gynostemma pentaphylla extract can be used for preparing medicines for resisting fatigue and anoxia and improving learning and cognitive functions.
Drawings
Fig. 1 shows the results of the JB2020 rod-rotating fatigue test.
FIG. 2 shows the JB2020 weight swimming test results.
FIG. 3 shows the hypoxic protective effect of JB2020 treatment on PC12 cells.
Figure 4 shows the effect of JB2020 treatment on the PC12 cell cycle.
Figure 5 shows the effect of JB2020 treatment on the PC12 cell cycle (per% of).
FIG. 6 shows the Western Blotting experiment results of JB2020 treatment group.
Fig. 7 shows the results of experiments by JB2020 on improvement of learning cognitive function in mice.
FIG. 8 shows the results of bar-rotating fatigue experiments with 8 active compounds.
FIG. 9 shows the results of a weight swimming test with 8 active compounds.
FIG. 10 shows the cell viability of 8 active compounds.
Detailed Description
The method for extracting the active site of Gynostemma pentaphyllum and the activity thereof according to the present invention are further described by the following specific examples.
Pulverizing the aerial parts of dried herba Gynostemmatis, extracting with 70% ethanol-water under reflux for 3 times, each for 1-2 hr, mixing the extractive solutions, concentrating until no alcohol smell is produced, and purifying with D101 macroporous adsorbent resin to obtain active component JB 2020. Dissolving JB2020 in water, and extracting with petroleum ether (60-90 deg.C), ethyl acetate, and n-butanol respectively to obtain petroleum ether phase, ethyl acetate phase, n-butanol phase and water phase.
The ethyl acetate phase (200G) was separated by silica gel column and gradient eluted with chloroform-methanol (70: 1 to 10:1, v/v) to give 7 fractions (Fr. A to Fr. G). Fr. D was separated using a preparative liquid phase (methanol/water = 85/15, v/v is mobile phase, flow rate is 10 mL/min; column model is Daisogel C18, column length × diameter is 250 mm × 20 mm) to give compound 1 (mass: 2.9 mg, time to peak: 37 min), compound 2 (mass: 3.3 mg, time to peak: 39 min) and compound 3 (mass: 2.4 mg, time to peak: 57 min). Fr. G was separated by preparative liquid chromatography (methanol/water = 88/12, v/v as mobile phase, flow rate 10 mL/min; column model Megres C18, column length × diameter 250 mm × 20 mm) to give compound 8 (mass: 3.0 mg, time to peak 13 min).
The n-butanol phase (20 g) was dissolved in 200 mL of 1M H2SO4Is heated and refluxed for 6 hours, and NaHCO is used after the reaction is finished3Neutralizing with saturated water solution, separating the concentrated solution with silica gel column, and gradient eluting with chloroform-methanol (20: 1-2:1, v/v) to obtain 3 parts (Fr. A-Fr. C). Fr. B is separated by C18 reverse phase silica gel column, and gradient elution is carried out by methanol/water (50: 50-100: 0, v/v) to obtain 5 parts Fr. 1-Fr. 5. Fraction Fr.1 was separated by preparative liquid chromatography (methanol/water = 79/21, v/v as mobile phase, flow rate 10 mL/min; column model Megres C18, column length × diameter 250 mm × 20 mm) to give compound 6 (mass 2.4 mg, time to peak 79 min). Fraction Fr.2 was separated by silica gel column and gradient eluted with chloroform-methanol (20: 1-2:1, v/v) to give fraction Fr. 2.1.1. Fr. 2.1.1 separation with preparative liquid phase (methanol/water = 80/20, v/v as mobile phase, flow rate 10 mL/min; type of column Daisogel C18, length x diameter of column 250 mm x 20 mm) gave compound 7 (mass 2.4 mg, time to peak 28 min). The fraction Fr.3 was separated by silica gel column chromatography, and gradient eluted with chloroform-methanol (40: 1-10:1, v/v) to obtain 5 fractions (Fr. 3.1.1-Fr. 3.5.5). Fr. 3.1.1 separation by preparative liquid phase (methanol/water = 91/9, v/v as mobile phase, flow rate 10 mL/min; column model Megres C18, column length × diameter 250 mm × 20 mm) gave compound 4 (mass 19.3 mg, time to peak 12 min). Fr.4 by preparative liquid phase separation (methanol/water =93/7, v/v as mobile phase, flow rate 10 mL/min; type of column, Daisogel C18 column, length of column, x straightDiameter 250 mm. times.20 mm) to give Compound 5 (mass 3.0 mg, peak appearance time 29 min).
The experimental data for compound 1 are as follows: white amorphous powder; value of optical rotationα]
+ 68 (concentration)c0.2, methanol as solvent); infrared Spectrum IR (film)ν max 3419, 2933, 1748, 1645, 1540, 1455, 1080 cm-1(ii) a Hydrogen spectrum1H and carbon spectra13C NMR is shown in Table 1; high resolution mass spectrum HRESIMSm/z 523.3393 [M + Na]+ (calcd. for C31H48O5Na, 523.3394)。
The experimental data for compound 2 are as follows: white amorphous powder; value of optical rotationα]
+ 290 (concentration)c0.1, methanol as solvent); infrared Spectrum IR (film)ν max 3425, 2939, 1754, 1653, 1537, 1452, 1083 cm-1(ii) a Hydrogen spectrum1H and carbon spectra13C NMR is shown in Table 1; high resolution mass spectrum HRESIMSm/z 523.3379 [M + Na]+ (calcd. for C31H48O5Na, 523.3394)。
The experimental data for compound 3 are as follows: white amorphous powder; value of optical rotationα]
+ 72 (concentration)c0.1, methanol as solvent); infrared Spectrum IR (film)ν max 3394, 2958, 2848, 2091, 1638, 1465, 1055 cm-1(ii) a Hydrogen spectrum1H and carbon spectra13C NMR is shown in Table 1; high resolution mass spectrum HRESIMSm/z 505.3270 [M + Na]+ (calcd. for C31H46O4Na, 505.3288)。
The experimental data for compound 4 are as follows: white amorphous powder; value of optical rotationα]
+ 18 (concentration)c0.1, methanol as solvent); infrared Spectrum IR (film)ν max 3399, 2935, 1719, 1643, 1076 cm-1(ii) a Hydrogen spectrum1H and carbon spectra13C NMR is shown in Table 2; high resolution mass spectrum HRESIMSm/z 491.3146 [M + Na]+ (calcd. for C30H44O4Na, 491.3132)。
The experimental data for compound 5 are as follows: white amorphous powder; value of optical rotationα]
+ 25 (concentration)c0.1, methanol as solvent); infrared Spectrum IR (film)ν max 3402, 2923, 2870, 1735, 1647, 1070 cm-1(ii) a Hydrogen spectrum1H and carbon spectra13C NMR is shown in Table 2; high resolution mass spectrum HRESIMSm/z 491.3124 [M + Na]+ (calcd. for C30H44O4Na, 491.3132)。
The experimental data for compound 6 are as follows: white amorphous powder; value of optical rotationα]
+ 12 (concentration)c0.1, methanol as solvent); infrared Spectrum IR (film)ν max 3380, 2932, 2870, 1729, 1641, 1453, 1371, 1044 cm-1(ii) a Hydrogen spectrum1H and carbon spectra13C NMR is shown in Table 2; high resolution mass spectrum HRESIMSm/z 525.3541 [M + Na]+(calcd. for C31H50O5Na, 525.3550)。
The experimental data for compound 7 are as follows: white amorphous powder; value of optical rotationα]
+ 58 (concentration)c0.2, methanol as solvent); infrared Spectrum IR (film)ν max 3435, 2932, 2865, 1634, 1387, 1073 cm-1(ii) a Hydrogen spectrum1H and carbon spectra13C NMR is shown in the table3; high resolution mass spectrum HRESIMS m/z 641.4045 [M + Na]+ (calcd. for C36H58O8Na, 641.4024)。
The experimental data for compound 8 are as follows: white amorphous powder, optical rotation valueα]
+ 28 (concentration)c0.067, methanol as solvent); infrared Spectrum IR (film)ν max 3407, 2936, 2870, 1636, 1447, 1075, 1034 cm-1(ii) a Hydrogen spectrum1H and carbon spectra13C NMR is shown in Table 3; high resolution mass spectrum HRESIMSm/z 659.4161 [M + Na]+ (calcd. for C36H60O9Na, 659.4130)。
Claims (5)
1. The active component of the gynostemma pentaphylla is dammarane type triterpenoid saponin compound, and the structural formula of the compound is any one of the compounds shown as the following formula:
2. the method for separating the active ingredient of the gynostemma pentaphylla, namely the dammarane type triterpenoid saponin compound, as claimed in claim 1, comprises the following steps:
(1) pulverizing dried herba Gynostemmatis, extracting with 5-20 times of water or 70% ethanol-water under reflux for 3 times, each for 1-2 hr, mixing extractive solutions, concentrating until no alcohol smell exists, and eluting with macroporous adsorbent resin to obtain active fraction of herba Gynostemmatis;
(2) dissolving the active part of Gynostemma pentaphyllum Makino in water, and extracting with ethyl acetate and n-butanol respectively to obtain ethyl acetate phase and n-butanol phase;
(3) separating the ethyl acetate phase by using a silica gel column, and carrying out gradient elution by using chloroform-methanol mixed solution to obtain 7 parts Fr. A-Fr. G; wherein Fr. D is subjected to liquid phase separation to sequentially obtain a compound 1, a compound 2 and a compound 3; fr. G, liquid phase separation to obtain compound 8; in the chloroform-methanol mixed solution, the volume ratio of chloroform to methanol is 70: 1-10: 1; fr. D liquid phase separation conditions: methanol/water = 85/15 as a mobile phase, with a flow rate of 10 mL/min; the type of the chromatographic column is Daisogel C18 column, and the length multiplied by the diameter of the chromatographic column is 250 mm multiplied by 20 mm; fr. G liquid phase separation conditions: methanol/water = 88/12 as a mobile phase, with a flow rate of 10 mL/min; the type of the chromatographic column is a Megres C18 column, and the length multiplied by the diameter of the chromatographic column is 250 mm multiplied by 20 mm;
(4) the n-butanol phase is dissolved at 1M H2SO4Is heated and refluxed for 6 hours, and NaHCO is used after the reaction is finished3Neutralizing the saturated aqueous solution, separating the concentrated solution by using a silica gel column, and performing gradient elution by using chloroform-methanol to obtain 3 parts Fr. A1-Fr. C1; the volume ratio of chloroform to methanol is 20:1-2: 1; wherein Fr. B1 is separated by C18 reverse phase silica gel column, and gradient elution is carried out by methanol/water mixed solution to obtain 5 parts Fr. 1-Fr. 5; in the methanol/water mixed solution, the volume ratio of methanol to water is 50: 50-100: 0;
liquid phase separation of fraction Fr.1 to give compound 6; conditions for liquid phase separation at site Fr.1 to give compound 6: methanol/water = 79/21 as a mobile phase, with a flow rate of 10 mL/min; the type of the chromatographic column is a Megres C18 column, and the length multiplied by the diameter of the chromatographic column is 250 mm multiplied by 20 mm; the structural formula of compound 6 is:
separating the part Fr.2 with silica gel column, and gradient eluting with chloroform-methanol to obtain part Fr.2.1; the volume ratio of chloroform to methanol is 20:1-2: 1; fr.2.1 liquid phase separation to give compound 7; fr.2.1 conditions for liquid phase separation to give compound 7: methanol/water = 80/20 as a mobile phase, with a flow rate of 10 mL/min; the type of the chromatographic column is Daisogel C18 column, and the length multiplied by the diameter of the chromatographic column is 250 mm multiplied by 20 mm;
separating the part Fr.3 by using a silica gel column, and performing gradient elution by using chloroform-methanol to obtain 5 parts Fr. 3.1.1-Fr. 3.5.5; the volume ratio of chloroform to methanol is 40:1-10: 1; fr. 3.1.1 liquid phase separation to obtain compound 4; wherein Fr. 3.1.1 conditions for liquid phase separation to give Compound 4: methanol/water = 91/9 as a mobile phase, with a flow rate of 10 mL/min; the type of the chromatographic column is a Megres C18 column, and the length multiplied by the diameter of the chromatographic column is 250 mm multiplied by 20 mm;
liquid phase separation of fraction Fr.4 to obtain compound 5; site fr.4 conditions for liquid phase separation to give compound 5: methanol/water =93/7 as a mobile phase, with a flow rate of 10 mL/min; the column model was a Daisogel C18 column, the column length X diameter 250 mm X20 mm.
3. The use of the dammarane-type triterpene saponin compound as the active ingredient of Gynostemma pentaphyllum Makino as claimed in claim 1 in the preparation of anti-anoxia drugs.
4. The use of the dammarane-type triterpene saponin compound as the active ingredient of Gynostemma pentaphyllum Makino as claimed in claim 1 in the preparation of antifatigue medicine.
5. The use of the dammarane-type triterpene saponin compound as the active ingredient of Gynostemma pentaphyllum Makino as claimed in claim 1 in preparing a medicine for improving learning and cognition ability.
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