Preparation method and application of silverweed cinquefoil root anti-hypoxia active component

Abstract

The invention provides a preparation method and application of a silverweed cinquefoil root anti-hypoxia active component. The preparation method comprises the following steps: preparing active parts of potentilla anserine through a liquid-liquid-liquid three-phase extraction technology, constructing an HPLC fingerprint spectrum of the active parts, combining an in-vitro hypoxia H9c2 myocardial cell model, investigating anti-hypoxia drug effects of different parts, constructing a spectrum-effect relationship based on the anti-hypoxia activity of the potentilla anserine active parts, screening anti-hypoxia positive correlation components of the potentilla anserine by adopting Pearson correlation analysis and OPLS-DA, and screening the anti-hypoxia positive correlation components of the potentilla anserine. The anti-hypoxia positive correlation active component is prepared by adopting semi-preparative liquid chromatography, the anti-hypoxia activity of the anti-hypoxia positive correlation active component is verified, and the best anti-hypoxia drug effect ratio of the prepared compound is obtained by adopting uniform design and cell survival rate experiments.

Description

Technical Field

[0001] The present invention belongs to the fields of natural product chemistry and traditional Chinese medicine chemistry, and particularly relates to a preparation method and application of anti-hypoxia active components of Potentilla anserina L.. Background Art

[0002] Potentilla anserina L. is the root of Potentilla anserina L. of the genus Potentilla in the Rosaceae family, also known as Potentilla anserina L., ginseng fruit, longevity grass, etc. The root of Potentilla anserina L. extends underground, and spindle-shaped or oval tubers grow at the lower part of the root. The stem is creeping and takes root at the nodes. The basal leaves are interrupted pinnate compound leaves, with leaflets opposite or alternate. The upper surface is green, and the lower surface is densely covered with silver-white villi closely attached. Single flowers are axillary, with yellow petals. The achene is ovoid, with pits and grooves on the back. The flowering and fruiting periods are from July to September. Potentilla anserina L. likes a humid and well-drained growth environment and is mainly distributed in Qinghai, Gansu and other places, growing on river banks, roadsides, mountain slopes and meadows at an altitude of 500-4100 meters.

[0003] Since ancient times, Potentilla anserina L. has been closely related to the lives of local ethnic groups. It was recorded in the ancient Tibetan medicine books "Miao Yin Ben Cao" and "Yu Tuo Ben Cao" written in the middle of the 8th century AD that "the tuberous roots of Potentilla anserina L. are all edible, and their own properties and effects are cool. Mixed and ground into a hot dough, eating it can stop heat diarrhea". It was also recorded in "Xining Prefecture New Annals" in the Qing Dynasty that "Potentilla anserina L. is produced in the wild, shaped like hemp roots and purple in color. Eating it is beneficial to people, and it is also called the longevity fruit". The description in the Tibetan medicine classic "Jing Zhu Ben Cao" is more specific: "Potentilla anserina L. tastes sweet and is cool in nature. Both humans and animals can eat it. In autumn, its nature becomes warm, so autumn Potentilla anserina L. has good quality. Spring Potentilla anserina L. is cool in nature, and its efficacy is to relieve diarrhea". Modern research also shows that Potentilla anserina L. contains rich and diverse chemical substances and nutritional components. Tibetan patent medicines containing Potentilla anserina L. include Siwei Xuelianhua Granules, Liuwei Zhuanggu Granules, etc., which have the effects of promoting blood circulation and warming the meridian, removing turbidity and lipid, nourishing the liver and kidney, strengthening tendons and bones, etc. However, as a characteristic biological resource on the Qinghai-Tibet Plateau, Potentilla anserina L. has not been deeply studied and fully explored at present. Its scientific research value is great, and its development prospect in the application of medicine and food is very broad.

[0004] The present invention prepares different active parts of Potentilla anserina L. by liquid-liquid-liquid three-phase extraction method, constructs fingerprint spectra of each active part of Potentilla anserina L. by HPLC technology, and combines with the pharmacodynamic experiment of H9c2 cell hypoxia model to construct the "spectrum-effect relationship" of Potentilla anserina L. against hypoxia, screen and isolate and purify the anti-hypoxia active components of Potentilla anserina L., laying a research foundation for improving the quality standard of Potentilla anserina L. and the research and development of new anti-hypoxia drugs, in order to provide safer, more effective and less side-effect new anti-hypoxia drugs for different types of hypoxia patients clinically, and provide a scientific basis and support for improving the scientific and technological added value of Potentilla anserina L. and the utilization rate of resources and promoting the local economy. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a preparation method and application of anti-hypoxic active components of Potentilla anserina L.. To achieve the above object, the present invention adopts the following technical solutions:

[0006] 1. Preparation method of anti-hypoxic active fraction of Potentilla anserina L., using the following method:

[0007] (1) Preparation of Potentilla anserina L. extract: Wash the underground part samples of Potentilla anserina L., dry, crush, pass through a 100-mesh sieve, accurately weigh 10 g of Potentilla anserina L. powder into a round-bottom flask, add ethanol according to the solid-liquid ratio, reflux extract, filter, concentrate under reduced pressure, and place in a vacuum drying oven to dry for 24 h until dry to obtain Potentilla anserina L. extract.

[0008] (2) Establishment of liquid-liquid-liquid three-phase system: Select organic solvents with different polarities to establish a liquid-liquid-liquid three-phase extraction system to extract the chemical components in the crude extract of Potentilla anserina L. and prepare different active fractions of Potentilla anserina L.. Referring to the preliminary experiment exploration, a total of 3 different liquid-liquid-liquid three-phase extraction systems are determined, namely: n-hexane∶chloroform∶acetonitrile∶water, n-hexane∶ethyl acetate∶acetonitrile∶water, petroleum ether∶acetonitrile∶dichloromethane∶water.

[0009] (3) Extraction of anti-hypoxic active fraction of Potentilla anserina L.: Accurately weigh the Potentilla anserina L. extract, mix according to the above solvent ratio with a sample loading amount of 0.08 g / mL, let stand at room temperature for a period of time, and then concentrate each phase extraction liquid to dryness under reduced pressure to obtain the active fraction of Potentilla anserina L.. Denote the upper phase of n-hexane∶chloroform∶acetonitrile∶water as M1, the middle phase as M2, and the lower phase as M3; the upper phase of n-hexane∶ethyl acetate∶acetonitrile∶water as M4, the middle phase as M5, and the lower phase as M6; the upper phase of petroleum ether∶acetonitrile∶dichloromethane∶water as M7, the middle phase as M8, and the lower phase as M9; the alcohol extract as M10.

[0010] 2. Application of the above-mentioned Potentilla anserina L. M3 fraction in improving the survival rate of hypoxic H9c2 cells, using 5 μg / mL as the dosing concentration for subsequent experiments, and the cell survival rate reaches 73.12±2.37%.

[0011] 3. Application of the above-mentioned Potentilla anserina L. M3 fraction in inhibiting the secretion of HIF-1α in hypoxic H9c2 cells, using 5 μg / mL as the dosing concentration for subsequent experiments, and the content of HIF-1α in the cells is the lowest, which is 49.16±3.38 ng / mL.

[0012] 4. Application of the above-mentioned Potentilla anserina L. M9 fraction in inhibiting the secretion of IL-6 in hypoxic H9c2 cells, using 5 μg / mL as the dosing concentration for subsequent experiments, and the content of IL-6 in the cells is relatively low, which is 62.44±1.35 pg / mL.

[0013] 5. Application of the M9 part of Potentilla anserina L. in inhibiting the secretion of TNF-α in hypoxic H9c2 cells. The subsequent experimental administration concentration is 5 μg / mL. The content of TNF-α in the cells is relatively low, which is 42.62 ± 3.06 pg / mL.

[0014] 6. Application of the M3 part of Potentilla anserina L. in increasing the content of SOD in hypoxic H9c2 cells. The subsequent experimental administration concentration is 5 μg / mL. The SOD activity in the cells is the strongest, which is 16.05 ± 1.05 U / mg.

[0015] 7. Application of monomeric compounds of Potentilla anserina L. in promoting the proliferation of H9c2 cells. The monomeric compounds are quercitrin, (-)-epigallocatechin, quercetin 3-O-glucuronide, and isoquercitrin. The usage concentration is 0.5 - 5 μg / mL.

[0016] 8. Application of monomeric compounds of Potentilla anserina L. in inhibiting the content of HIF-1α in H9c2 cells. The monomeric compounds are quercitrin, (-)-epigallocatechin, quercetin 3-O-glucuronide, and isoquercitrin. Among them, quercitrin has the most significant inhibitory effect. The content of HIF-1α in the cells is 98.74 ± 1.32 ng / mL.

[0017] 9. Application of the compound of monomeric compounds of Potentilla anserina L. in increasing the survival rate of hypoxic H9c2 cells. When the concentration is 50 μg / mL, the ratio of quercitrin: (-)-epigallocatechin: quercetin 3-O-glucuronide: isoquercitrin is 3:2:3:1, which has a significant effect on the cell survival rate.

[0018] The present invention has the following beneficial effects:

[0019] 1. The results of exploring the change in the survival rate of the in vitro H9c2 cell hypoxia model before and after drug intervention show that: in the range of the administration concentration of 0 - 5 μg / mL, the effects of each active part of Potentilla anserina L. on the survival rate of hypoxic H9c2 cells show a dose-effect relationship. At the same administration concentration, there are differences in the effects of each active part on the survival rate of H9c2 cells, indicating that liquid-liquid-liquid three-phase extraction can effectively enrich the anti-hypoxic activity in Potentilla anserina L. Among them, when the administration concentration is 5 μg / mL, the promoting effect of the M3 part on the proliferation of H9c2 cells is the most obvious.

[0020] 2. The results of the changes in the contents of HIF-1α, IL-6, TNF-α, and SOD in the in vitro H9c2 hypoxic cell model before and after drug intervention showed that at a drug concentration of 5 μg / mL, each active fraction could inhibit the expression of HIF-1α, IL-6, and TNF-α and increase the content of SOD, and there were differences in the effects of each fraction on the relevant factor indexes; compared with the model group, M3 could significantly inhibit the expression level of intracellular HIF-1α and increase SOD activity (P<0.01), and the M9 fraction could significantly inhibit the expression levels of IL-6 and TNF-α in the cells (P<0.01).

[0021] 3. The fingerprint maps of the active fractions of Potentilla anserina were constructed by HPLC. Combining the results of the in vitro pharmacodynamics experiments on H9c2 cells, Pearson and OPLS-DA were used to study the spectrum-effect relationship to screen the anti-hypoxic pharmacodynamic material basis of Potentilla anserina. The results of Pearson correlation analysis showed that peaks 2, 3, 4, 9, 11, 12, 13, and 14 were significantly positively correlated with the anti-hypoxic activity of Potentilla anserina; the results of OPLS-DA multiple regression analysis showed that peaks 1, 2, 3, 4, 6, 7, 10, 11, 12, 14, 15, and 16 were the positive correlation characteristic peaks of the anti-hypoxic activity of Potentilla anserina. Mapping the analysis results of the two to each other, the positive correlation characteristic peaks 2, 3, 4, 11, 12, and 14 of the anti-hypoxic activity of Potentilla anserina were obtained. Among them, the relative peak areas of peaks 11 and 14 were larger, and the anti-hypoxic pharmacodynamic correlation was high, which could be used as the Q-marker for the quality evaluation of the anti-hypoxic effect of Potentilla anserina.

[0022] 4. Semi-preparative liquid chromatography was used for separation and purification, and 4 compounds were identified as quercitrin, gallocatechin, quercetin 3-O-glucuronide, and isoquercitrin by mass spectrometry and nuclear magnetic resonance spectroscopy. The results of the in vitro pharmacodynamics experiments on H9c2 cells showed that in the range of drug concentration from 0.5 to 5 μg / mL, the anti-hypoxic activities of the 4 compounds were different. Among them, quercitrin and isoquercitrin had higher anti-hypoxic activities. At a drug concentration of 0.5 μg / mL, they could significantly increase the survival rate of hypoxic H9c2 cells; gallocatechin and quercetin 3-O-glucuronide had a significant promoting effect on the proliferation of H9c2 cells at a drug concentration of 5 μg / mL. Under the condition of the same drug concentration, quercitrin had the most significant inhibitory effect on the expression of HIF-1α protein in H9c2 cells, with a content of 98.74±1.32 ng / mL (P<0.01). The uniform design experiment showed that the ratio of quercitrin:gallocatechin:quercetin 3-O-glucuronide:isoquercitrin of 3:2:3:1 had a significant effect on the cell survival rate. Description of the Drawings

[0023] Figure 1 It is a diagram of the changes in the morphology of H9c2 cells under different hypoxic times.

[0024] Figure 2 Effect of different hypoxia times on the survival rate of H9c2 cells.

[0025] Figure 3 Effect of different concentrations of each active fraction on the survival rate of H9c2 cells.

[0026] Figure 4 Change in the content of HIF-1α in hypoxic H9c2 cells.

[0027] Figure 5 Change in the content of IL-6 in hypoxic H9c2 cells.

[0028] Figure 6 Change in the content of TNF-α in hypoxic H9c2 cells.

[0029] Figure 7 Change in the content of SOD in hypoxic H9c2 cells.

[0030] Figure 8 HPLC chromatogram of the chemical components of the active fraction M3 of Potentilla anserina L.

[0031] Figure 9 HPLC chromatogram of pre-separation of active ingredients.

[0032] Figure 10 HPLC chromatogram of the active ingredients of Potentilla anserina L.

[0033] Figure 11 Effect of monomeric compounds on the survival rate of hypoxic H9c2 cells.

[0034] Figure 12 Effect of monomeric compounds on the content of HIF-1α in hypoxic H9c2 cells.

[0035] Figure 13 Survival rate of hypoxic H9c2 cells under drug intervention.

[0036] Figure 14 Chemical structure of quercitrin.

[0037] Figure 15 Chemical structure of (-)-epigallocatechin.

[0038] Figure 16 Chemical structure of quercetin 3-O-glucuronide.

[0039] Figure 17 Chemical structure of isoquercitrin. Specific implementation mode

[0040] To make the objectives, technical solutions and advantages of the invention clearer, the following specifically describes the embodiments of the present invention in conjunction with the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the present invention shown in the drawings and described according to the drawings are merely exemplary, and the present invention is not limited to these embodiments. Here, it should also be noted that in order to avoid obscuring the technical solution of the present invention due to unnecessary details, only the structures and / or processing steps closely related to the solution of the present invention are shown in the drawings, while other details of little relevance are omitted.

[0041] Example 1

[0042] This example provides a preparation method for the anti-hypoxia active components of Potentilla anserina L. and a functional analysis method for the active components, specifically including:

[0043] 1. Preparation of Potentilla anserina L. extract

[0044] Wash the underground part samples of Potentilla anserina L. (purchased from Qinghai Jiukang Traditional Chinese Medicine Market), dry, pulverize, pass through a 100-mesh sieve, accurately weigh 10 g of Potentilla anserina L. powder and place it in a round-bottom flask, add ethanol according to the solid-liquid ratio, reflux extract, filter, concentrate under reduced pressure, and place it in a vacuum drying oven to dry for 24 h until dry to obtain Potentilla anserina L. extract.

[0045] 2. Preparation of the active part of Potentilla anserina L. by liquid-liquid-liquid three-phase extraction

[0046] Select organic solvents with different polarities to establish a liquid-liquid-liquid three-phase extraction system, extract the chemical components in the crude extract of Potentilla anserina L., and prepare different active parts of Potentilla anserina L. Referring to the preliminary experiment exploration, a total of 3 different liquid-liquid-liquid three-phase extraction systems are determined, namely: System 1 (n-hexane∶chloroform∶acetonitrile∶water), System 2 (n-hexane∶ethyl acetate∶acetonitrile∶water), System 3 (petroleum ether∶acetonitrile∶dichloromethane∶water). Accurately weigh the Potentilla anserina L. extract, mix it according to the solvent ratios of System 1, System 2, and System 3 at a sample loading amount of 0.08 g / mL, let it stand at room temperature for a period of time, and then concentrate each phase extraction liquid to dryness under reduced pressure to obtain the active part of Potentilla anserina L. Denote the upper phase of System 1 as M1, the middle phase as M2, and the lower phase as M3; denote the upper phase of System 2 as M4, the middle phase as M5, and the lower phase as M6; denote the upper phase of System 3 as M7, the middle phase as M8, and the lower phase as M9; denote the alcohol extract as M10 for subsequent experiments.

[0047] 3. Cell pretreatment

[0048] 3.1 Cell resuscitation and culture

[0049] The H9c2 cells are a cell line subcloned from a cloned cell line of BD1X rat embryonic heart tissue and were purchased from Wuhan Zishan Biotechnology Co., Ltd. (STCC30008). The cryopreserved H9c2 cells were quickly thawed at 37°C until there were no ice crystals, 5 - 8 mL of complete medium (DMEM high-glucose medium containing 10% FBS and 1% double antibiotics) was added, centrifuged at 1200 r / min for 3 - 5 min, the supernatant was removed, 5 mL of complete medium was added, and the cells were repeatedly pipetted with a Pasteur pipette until a single-cell suspension was obtained, and then placed in a cell culture incubator at 37°C and 5% CO2 for culture.

[0050] 3.2 Cell passage

[0051] Take H9c2 cells with a confluence density of 85% - 95%. After removing the original culture medium, add 2 mL of PBS, gently shake the culture flask to wash the cells, and aspirate the PBS; add 1 - 2 mL of 0.25% trypsin digestion solution, gently shake the culture flask to soak all the cells; digest the cells at room temperature until the cell state retracts and becomes round under the microscope, the gap increases and some cells float when gently shaken, then add 3 mL of DMEM complete medium to terminate the digestion and pipette the cells to make them detach from the wall, making the cells as much as possible into a single-cell suspension; collect the suspension, centrifuge at 1200 r / min for 3 - 5 min, aspirate the supernatant, add 1 - 2 mL of complete medium to resuspend the cells, add complete medium according to the required proportion for passage, and after resuspension, inoculate into a new culture flask to complete the passage.

[0052] 3.3 Cell cryopreservation

[0053] Digest the cells, add an appropriate amount of cell cryopreservation solution to resuspend the cells, adjust the cell suspension concentration to 1×106 / mL, transfer the suspension to a cryopreservation tube and seal it, and perform cryopreservation using programmed cooling, place it at 4°C for 30 min, at -20°C for 1 h, in an -80°C refrigerator overnight, and finally transfer it to liquid nitrogen for long-term storage

[89] .

[0054] 4. Determination of the growth characteristics of H9c2 cells

[0055] Adjust the cell density to 3×103 / mL, inoculate 100 μL / well into a 96-well plate, set 6 replicates for each group, and use PBS to seal the edges to prevent edge effects; after the cells adhere, add 10 μL of CCK-8 solution, incubate in a 37°C, 5% CO2 cell culture incubator for 2 h, and measure the absorbance (OD) of H9c2 cells at different growth times (0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d) at 450 nm with an enzyme-labeled instrument. Calculate the number of cells in each group according to the formula: number of cells (104 / mL) = (OD value of the experimental group / OD value of the control group) × 0.3.

[0056] 5. Construction and screening of the hypoxia model of H9c2 cells

[0057] Digest the H9c2 cells in logarithmic growth phase, add complete medium to prepare a cell suspension with a density of about 1×104 / mL, inoculate 100 μL per well into a 96-well plate, and incubate in a 37°C, 5% CO 2 Incubate in a constant temperature incubator for 24 h until the cells adhere. Aspirate and discard the original medium from the cells in the normoxia group and hypoxia group and replace it with serum-free DMEM high-glucose medium. The cells in the hypoxia group are induced under hypoxic conditions (37°C, 5% CO2, 2% O2, 93% N2) for 12 h, 24 h, and 48 h. Add 10 μL of CCK-8 solution to each well and incubate for 2 h. Detect the absorbance (OD) at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Set 6 replicates for each group and repeat the experiment 3 times. Calculate the survival rate of H9c2 cells under different hypoxic induction times to determine the optimal modeling time.

[0058] 6. Effects of different active parts of Potentilla anserina L. on hypoxic H9c2 cells

[0059] 6.1 Detection of the effects of different active parts on the survival rate of H9c2 cells by CCK-8 method

[0060] Digest the H9c2 cells in logarithmic growth phase, add complete medium to prepare a cell suspension with a density of about 1×104 / mL, inoculate 100 μL per well into a 96-well plate, and incubate in a 37°C, 5% CO2 constant temperature incubator for 24 h until the cells adhere. Use serum-free DMEM high-glucose medium to prepare test drugs (containing 0.1% DMSO) with final concentrations of 0, 0.5, 1, 2.5, and 5 μg / mL for different active parts (M1-M9) of Potentilla anserina L., and sterilize through a 0.22 μm filter membrane. Aspirate and discard the original medium from the normoxia group and hypoxia group, and replace it with test drugs at different concentrations. The hypoxia group is placed in a three-gas incubator at 37°C, 5% CO2, 2% O2, and 93% N2 and cultured for another 24 h. Add 10 μL of CCK-8 solution to each well and incubate for 2 h under the original conditions. Detect the absorbance (OD) at 450 nm with an ELISA reader. Set 6 replicates for each group and repeat the experiment 3 times to calculate the survival rate of H9c2 cells.

[0061] 6.2 Effects of different active parts on the contents of HIF-α, IL-6, TNF-α, and SOD in H9c2 cells

[0062] Digest the H9c2 cells in logarithmic growth phase, add complete medium to prepare a cell suspension with a density of about 1×104 / mL, inoculate 2 mL per well into a 6-well plate, and incubate at 37°C, 5% CO 2Incubate in a constant temperature incubator for 24 h until the cells adhere. Use serum-free high-glucose DMEM medium to prepare the test drugs (containing 0.1% DMSO) with the final concentration of 5 μg / mL for each active part (M1-M9) of Potentilla anserina L. Discard the original medium from both the normoxia group and the hypoxia group, replace it with the test drug with the final concentration of 5 μg / mL, and place the hypoxia group in a three-gas incubator at 37 °C, 5% CO2, 2% O2, and 93% N2 for continued culture for 24 h. Centrifuge at 1000 r / min for 10 min to remove cell precipitates and polymers, collect the cell supernatant for subsequent detection, set 6 replicates for each measurement group, and perform operations and measurements according to the instructions of the kit, and calculate the content of each cytokine.

[0063] 7. Pre-separation of anti-hypoxia active components of Potentilla anserina L.

[0064] Take 10 g of the active part M3 of Potentilla anserina L., add 100 mL of distilled water to prepare a turbid solution, extract with n-butanol, concentrate the supernatant of the extract under reduced pressure to dryness, dissolve it in an appropriate amount of methanol, and perform HPLC analysis under the above chromatographic conditions.

[0065] 8. Separation and purification of anti-hypoxia active components of Potentilla anserina L. by semi-preparative liquid chromatography

[0066] Use ultrapure water to prepare a solution with a concentration of 200 mg / mL of the above extract, separate and differentiate it using a Hanbang semi-preparative high-performance liquid chromatograph, select a Hedera-ODS-2C18 column (250 mm × 10 mm, 10 μm), elute with 0.1% trifluoroacetic acid (A)-acetonitrile (B), detect at 254 nm, collect the eluate of the target component, freeze and concentrate to dryness, and perform structure identification after purity verification. The elution procedure is as follows:

[0067] (1) Compound 1: Hedera-ODS-2C18 column (250 mm × 10 mm, 10 μm); column temperature 35 °C; mobile phase is 0.1% trifluoroacetic acid aqueous solution (A)-acetonitrile (B), isocratic elution: 0 - 20 min, 8% B; injection volume is 1 mL; flow rate is 4 mL / min; ultraviolet detection wavelength is 254 nm.

[0068] (2) Compounds 2, 3, 4: Hedera-ODS-2C18 column (250 mm × 10 mm, 10 μm); column temperature 35 °C; mobile phase is 0.1% trifluoroacetic acid aqueous solution (A)-acetonitrile (B), isocratic elution: 0 - 40 min, 20% B; injection volume is 1 mL; flow rate is 4 mL / min; ultraviolet detection wavelength is 254 nm.

[0069] 9. Exploration of anti-hypoxia activity of monomeric compounds

[0070] The prepared compound was formulated into test drugs with final concentrations of 5, 2.5, 1, and 0.5 μg / mL (containing 0.1% DMSO) using serum-free high-glucose DMEM medium, and sterilized by passing through a 0.22 μm filter membrane. The survival rate of hypoxic H9c2 cells and the content of HIF-1α were measured according to the above method, and the prepared compound was subjected to a three-level four-factor compound uniform design using the DPS data processing system. Level 1 (N1): 1:3:2:3; Level 2 (N2): 2:1:1:2; Level 3 (N3): 3:2:3:1. The compound drugs were formulated into test drugs with final concentrations of 5, 12.5, 25, and 50 μg / mL (containing 0.1% DMSO), and sterilized by passing through a 0.22 μm filter membrane. The cell survival rate was measured according to the above method.

[0071] 10. Nuclear magnetic resonance spectroscopy

[0072] The sample was dissolved in deuterated methanol and placed in a 3 mm diameter probe tube, and measured using a Bruker nuclear magnetic resonance spectrometer under the condition of 600 MHz.

[0073] Example 2

[0074] This example provides the preparation results of the anti-hypoxic active ingredients of Potentilla anserina L. described in Example 1 and the functional analysis results of the active ingredients, specifically including:

[0075] 1. Selection of liquid-liquid-liquid three-phase extraction system

[0076] By changing the ratio between the solvents in the liquid-liquid-liquid three-phase extraction system, the effects on the number of phases formed in the system, the phase ratio, and the system formation time were explored. Taking the formation of three liquid phases, an average phase ratio, and a moderate system formation time as the screening criteria, a total of 3 different liquid-liquid-liquid three-phase extraction systems were determined: System 1 (n-hexane∶chloroform∶acetonitrile∶water = 7:3:5:5), System 2 (n-hexane∶ethyl acetate∶acetonitrile∶water = 6:4:5:5), System 3 (petroleum ether∶acetonitrile∶dichloromethane∶water = 5:1:5:5). With 0.08 g / mL as the upper loading limit, the chemical components in the crude extract of Potentilla anserina L. were enriched.

[0077] 2. Effect of different hypoxia induction times on the survival rate of H9c2 cells

[0078] The hypoxic H9c2 cardiomyocytes were placed in a three-gas incubator at 37 °C, 5% CO2, 2% O2, and 93% N2, and hypoxic induction was carried out for 12, 24, and 48 h, and the changes in the survival rate of each group of cells were detected by the CCK-8 method. The cell morphological changes were as Figure 1As shown. By observing the cell morphology, under normoxic conditions, H9c2 cells just inoculated into the well plate are mostly translucent regular circles. Within 6-8 hours after inoculation, a small number of cells begin to adhere to the wall, the cell volume begins to increase, and the cell body gradually becomes full. After culturing for 24 hours, H9c2 cells show clustered growth, showing slender branch-like growth, and most cells show a cardiomyocyte-like morphology (fusiform). At this time, the cells are basically attached to the wall, and a small number of them float freely in the culture medium. With the extension of the hypoxia induction time, the number of detached cells in the model group gradually increases. When the induction time reaches 12 hours, vacuoles appear in the attached cardiomyocytes and the cell body is shortened; when the induction time reaches 24 hours, the number of detached cells increases further, the cell body shrinks, and large gaps appear between cells; when the induction time reaches 48 hours, most of the cells float in the culture medium, and broken cells appear, and the cell morphology is severely damaged.

[0079] The changes in cell viability were shown in Figure 2 The results showed that compared with the control group, the survival rate of H9c2 cells in the hypoxia-induced group showed a decreasing trend to varying degrees, which was statistically significant (P < 0.05). When the hypoxia time was 12h, the cell survival rate was 84.79 ± 4.87%, and most cells still survived at this time; when the hypoxia time was 24h, the cell survival rate reached 48.91 ± 5.16%, indicating that the cells gradually became fragile due to the hypoxic environment; when the hypoxia time was 48h, the cell survival rate was extremely low, reaching 7.11 ± 1.38%, indicating that most cells had died under this condition and were not suitable for model construction. Therefore, the hypoxia induction time was finally selected as 24h for subsequent experimental studies.

[0080] 3. Effects of active fractions of Potentilla anserina on the survival rate of hypoxic H9c2 cells

[0081] The cell survival rate can directly reflect the protective effect of each active part of Potentilla anserina on hypoxic H9c2 cells. Figure 3 As shown: the cell survival rate in the model group decreased significantly, indicating that the model was successfully established; the protective effects of each active part of Potentilla anserina on hypoxic H9c2 cells were different. With the increase of the concentration of the test drug, the cell survival rate increased. There was a dose-effect relationship between each active part and the cell survival rate, and the cell survival rate reached the highest at a concentration of 5μg / mL, with the best anti-hypoxia effect; the M3 part had the most significant effect on hypoxic cells before and after administration, and the cell survival rate reached 73.12±2.37%. Therefore, in order to ensure that each active part exerts the best protective effect, 5μg / mL was used as the subsequent experimental administration concentration.

[0082] 4. Effects of each active site on the content of HIF-α, IL-6, TNF-α, and SOD in cells

[0083] 4.1 Effects of Different Active Parts of Potentilla anserina L. on the Content of HIF-1α in Hypoxic H9c2 Cells

[0084] The linear regression equation of the HIF-1α standard curve was Y = 0.0029X + 0.0397, with R2 = 0.996. The good regression fitting degree indicated that the kit had good performance and could be used for experimental detection. As Figure 4 shown, after 24 h of hypoxia induction, compared with the control group, the content of HIF-1α in the model group increased significantly, indicating successful modeling (P < 0.01); compared with the model group, the contents of HIF-1α in the M1, M2, M4, M7, and M8 parts decreased, showing significant differences (P < 0.05); the contents of HIF-1α in the M3, M5, M6, and M9 parts decreased significantly compared with the model group, showing extremely significant differences (P < 0.01). Compared with other experimental groups, the M3 part had the best inhibitory effect on HIF-1α secretion, and the content of HIF-1α in the cells was the lowest, which was 49.16 ± 3.38 ng / mL.

[0085] 4.2 Effects of Different Active Parts of Potentilla anserina L. on the Content of IL-6 in Hypoxic H9c2 Cells

[0086] A standard curve was drawn with the IL-6 content as the X-axis and the absorbance as the Y-axis, and the IL-6 contents of each group were calculated according to the standard curve. The linear regression equation of the standard curve was Y = 0.017X + 0.0358, with R2 = 0.9983. The good regression fitting degree indicated that the kit had good performance and could be used for experimental detection. The effects of different active parts on the content of IL-6 in hypoxic H9c2 cells were as Figure 5 shown. After 24 h of hypoxia induction, compared with the control group, the IL-6 content in the model group increased from 55.03 ± 3.88 pg / mL to 105.56 ± 5.75 pg / mL, indicating successful modeling (P < 0.01); compared with the model group, the IL-6 contents in the M1, M2, M4, M6, M7, and M8 parts decreased, showing significant differences (P < 0.05); the IL-6 contents in the M3, M5, and M9 parts decreased significantly compared with the model group, showing extremely significant differences (P < 0.01). Compared with other experimental groups, the M9 part had the best inhibitory effect on IL-6 secretion, and the content of IL-6 in the cells was relatively low, which was 62.44 ± 1.35 pg / mL.

[0087] 4.3 Effects of Different Active Parts of Potentilla anserina L. on the Content of TNF-α in Hypoxic H9c2 Cells

[0088] A standard curve was drawn with the TNF-α content as the X-axis and the absorbance as the Y-axis, and the TNF-α contents of each group were calculated according to the standard curve. The linear regression equation of the standard curve was Y = 0.00037X - 0.0449, with R2 = 0.9954. The good regression fitting degree indicated that the kit had good performance and could be used for experimental detection. AsFigure 6 As shown in the figure, after 24 hours of hypoxia induction, compared with the control group, the content of TNF-α in the model group increased from 39.06±2.98 ng / mL to 70.33±2.93 ng / mL, indicating successful modeling (P<0.01); compared with the model group, the content of TNF-α in the M1, M4, M7, and M8 parts decreased, showing significant differences (P<0.05); the content of TNF-α in the M2, M3, M5, M6, and M9 parts decreased significantly compared with the model group, showing extremely significant differences (P<0.01). Compared with other experimental groups, the best inhibitory effect on TNF-α secretion was in the M9 part, and the content of TNF-α in the cells was 42.62±3.06 pg / mL.

[0089] 4.4 Effects of different active parts of Potentilla anserina L. on SOD content in hypoxic H9c2 cells

[0090] The linear regression equation of the standard curve was Y = 0.028X + 0.136, and R2 = 0.9933, indicating good regression fitting, which showed that the kit had good performance and could be used for experimental detection. As Figure 7 shown in the figure, compared with the control group, the SOD activity of the cells in the model group decreased to 5.38±0.15 U / mg; compared with the model group, different active parts of Potentilla anserina L. showed different promoting effects on SOD activity, and significant differences were found in M1, M2, M4, M7, and M8 (P<0.05); extremely significant differences were found in the M3, M5, M6, and M9 parts (P<0.01). Compared with other experimental groups, the M3 part had a stronger promoting effect on SOD activity, with an SOD activity of 16.05±1.05 U / mg; the M8 part had a weaker promoting effect on SOD activity, with an SOD activity of 7.77±0.48 U / mg.

[0091] 5 Pre-separation results of active parts of Potentilla anserina L.

[0092] Since the chemical components contained in Potentilla anserina L. are complex, it is difficult to separate and prepare active ingredients from the crude extract of Potentilla anserina L. Therefore, the positively correlated characteristic peaks of anti-hypoxia activity of Potentilla anserina L. were compared with the HPLC fingerprints of each active part, and the parts with better enrichment of active ingredients were selected for separation and purification. As Figure 8 shown in the figure, the enrichment of positively correlated characteristic peak compounds in the active part M3 was better. Compounds 1, 2, 3, and 4 corresponded to positively correlated characteristic peaks 4, 11, 12, and 14 respectively. Since the four compounds had relatively large polarities, n-butanol was selected for extraction to pre-separate the chemical components of the positively correlated characteristic peaks of anti-hypoxia activity of Potentilla anserina L. As Figure 9 shown in the figure, the chemical components in the n-butanol extract were mainly concentrated before 75 min, and the active ingredients 1, 2, 3, and 4 all had good absorption values, which could be used for separation and purification work.

[0093] 6. Isolation of the anti - hypoxia active components of Potentilla anserina L

[0094] After drying the obtained sample in an oven at 40 °C, 2 g of the sample was weighed and dissolved in 5 mL of methanol, and then filtered through a 0.22 - μm microporous filter membrane. According to the conditions in "5.2.2", a semi - preparative liquid chromatograph was used to separate and purify the 4 active components. As Figure 10 shown, the obtained compounds were analyzed by HPLC under the above conditions, and the peak area ratios of the 4 compounds were all greater than 99%, indicating that the compounds had high purity and could be used for subsequent experiments.

[0095] 7. Exploration of the anti - hypoxia activity of monomeric compounds based on the H9c2 cell hypoxia model

[0096] 7.1 Effect of monomeric compounds on cell viability

[0097] The CCK - 8 method was used to determine the viability of hypoxic H9c2 cells under drug intervention, to explore the protective effect of monomeric compounds on hypoxic H9c2 cells, and at the same time to avoid the influence of cytotoxicity on the experiment. The experimental results are as Figure 11 shown. Compared with the control group, the cell viability in the model group was significantly reduced after 24 h of hypoxia induction, indicating that the model was successfully established; within the range of 0.5 - 5 μg / mL, compound 1 and compound 4 could significantly promote the proliferation of H9c2 cells and significantly increase the cell viability (P < 0.05 or < 0.01); compound 2 and compound 3 had no significant promoting effect on cell proliferation at low concentrations (0.5 - 2.5 μg / mL), and when the concentration was 5 μg / mL, they could significantly increase the cell viability (P < 0.05). The 4 compounds showed a dose - effect relationship within the range of 0.5 - 5 μg / mL, and at a concentration of 5 μg / mL, they had no cytotoxicity and had a significant promoting effect on cell proliferation.

[0098] 7.2 Effect of monomeric compounds on the content of HIF - 1α

[0099] As Figure 12 shown, compared with the control group, the content of HIF - α in cells in the model group was significantly increased after 24 h of hypoxia induction, indicating that the model was successfully established; when the administration concentration was 5 μg / mL, the 4 monomeric compounds could significantly inhibit the expression of HIF - 1α protein and reduce the content of HIF - 1α; the inhibitory effect of compound 1 was the most significant, and the content of HIF - 1α in cells was 98.74 ± 1.32 ng / mL.

[0100] 7.3 Effect of compound compounds on cell viability

[0101] The CCK-8 method was used to determine the survival rate of hypoxic H9c2 cells under drug intervention, explore the protective effect of the compound monomer on hypoxic H9c2 cells, and avoid the influence of cytotoxicity on the experiment. The experimental results are as Figure 13 shown. Compared with the control group, the cell survival rate in the model group decreased significantly after 24 h of hypoxia induction, indicating successful modeling. In the range of 50-5 μg / mL at three levels, it could significantly promote the proliferation of H9c2 cells and significantly increase the cell survival rate (P < 0.01). The compound at three levels showed a dose-effect relationship in the range of 50-5 μg / mL, and there was no cytotoxicity at a concentration of 50 μg / mL and it had a significant promoting effect on cell proliferation, with the most significant effect at the N3 level.

[0102] 7.4 Structural identification of the anti-hypoxic active components of Potentilla anserina

[0103] Based on the nuclear magnetic resonance spectroscopy data and mass spectrometry information of the compounds, the structural identification of the active components isolated from Potentilla anserina was carried out. As Figure 14 shown, compound 1 was: quercitrin. As Figure 15 shown, compound 2 was: gallocatechin. As Figure 16 shown, compound 3 was: quercetin 3-O-glucuronide. As Figure 17 shown, compound 4 was: isoquercitrin.

[0104] The above are only the specific implementation manners of this application. It should be noted that for those of ordinary skill in the art, without departing from the principle of this application, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of this application.

Claims

1. A method for preparing the anti-hypoxia active fraction of Potentilla anserina, characterized in that The preparation method is as follows: (1) Preparation of Potentilla anserina extract: Wash the underground part of Potentilla anserina sample, dry it, crush it, pass it through a 100-mesh sieve, accurately weigh 10 g of Potentilla anserina powder and put it into a round-bottom flask, add ethanol according to the solid-liquid ratio, reflux extract, filter, concentrate under reduced pressure, and dry it in a vacuum drying oven for 24 hours to obtain Potentilla anserina extract; (2) Liquid-liquid-liquid three-phase establishment: Organic solvents with different polarities were selected to establish a liquid-liquid-liquid three-phase extraction system, and the chemical components in the crude extract of Potentilla anserina were extracted to prepare different active parts of Potentilla anserina. Based on the preliminary experimental research, three different liquid-liquid-liquid three-phase extraction systems were determined, namely: n-hexane: chloroform: acetonitrile: water, n-hexane: ethyl acetate: acetonitrile: water, and petroleum ether: acetonitrile: dichloromethane: water; (3) Extraction of the anti-hypoxic active part of Potentilla anserina: accurately weigh Potentilla anserina extract, mix it with a loading amount of 0.08 g / mL according to the above solvent ratio, let it stand at room temperature for a period of time, then reduce the pressure of each phase of extract to dryness to obtain the active part of Potentilla anserina; the upper phase of n-hexane: chloroform: acetonitrile: water is recorded as M1, the middle phase is recorded as M2, and the lower phase is recorded as M3; the upper phase of n-hexane: ethyl acetate: acetonitrile: water is recorded as M4, the middle phase is recorded as M5, and the lower phase is recorded as M6; the upper phase of petroleum ether: acetonitrile: dichloromethane: water is recorded as M7, the middle phase is recorded as M8, and the lower phase is recorded as M9; the alcohol extract is recorded as M10.

2. The use of the Potentilla anserina M3 part of claim 1 to improve the survival rate of hypoxic H9c2 cells, characterized in that When 5 μg / mL of Potentilla anserina M3 fraction was used as the dosing concentration, the cell survival rate reached 73.12±2.37%.

3. The use of the Potentilla anserina M3 part of claim 1 in inhibiting the secretion of HIF-1α in hypoxic H9c2 cells, characterized in that When the dosage concentration of 5 μg / mL was used for the M3 part of Potentilla anserina, the HIF-1α content in the cells was the lowest, which was 49.16±3.38 ng / mL.

4. The use of the Potentilla anserina M9 part of claim 1 to inhibit IL-6 secretion from hypoxic H9c2 cells, characterized in that When the dosage concentration of 5 μg / mL was used for the M9 part of Potentilla anserina, the IL-6 content in the cells was relatively low, at 62.44±1.35 pg / mL.

5. The use of the Potentilla anserina M9 part of claim 1 in inhibiting TNF-α secretion in hypoxic H9c2 cells, characterized in that When the dosage concentration of 5 μg / mL of the Potentilla anserina M9 part was used, the TNF-α content in the cells was relatively low, at 42.62±3.06 pg / mL.

6. The use of the Potentilla anserina M3 part as claimed in claim 1 to increase the SOD content of hypoxic H9c2 cells, characterized in that When the dosage concentration of 5 μg / mL was used for the M3 part of Potentilla anserina, the SOD activity in the cells was the strongest, which was 16.05±1.05U / mg.

7. The use of a monomer compound of Potentilla anserina to promote the proliferation of H9c2 cells, characterized in that The monomer compound is any one of quercetin, gallocatechin, quercetin 3-O-glucoside and isoquercetin, and the dosage concentration is 0.5-5 μg / mL.

8. The use of a monomer compound of Potentilla anserina to inhibit the HIF-1α content of H9c2 cells, characterized in that The monomer compound is any one of quercetin, epigallocatechin gallate, quercetin 3-O-glucoside, and isoquercetin, among which quercetin has the most significant inhibitory effect, and the cell HIF-1α content is 98.74±1.32 ng / mL.

9. Application of a compound of Potentilla anserinae to improve the survival rate of hypoxic H9c2 cells, characterized in that The cell survival rate was most significantly improved when the ratio of 50 μg / mL quercetin:gallocatechin gallate:quercetin 3-O-glucoside:isoquercetin was 3:2:3:1.

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