Flos puerariae biological activity quality control and evaluation method based on reactive oxygen species (ROS) level detection

Abstract

The invention relates to a pueraria flower quality control and evaluation method based on reactive oxygen species (ROS) detection, and can effectively solve the problem that an existing pueraria flower quality detection method is difficult to reflect the overall efficacy of pueraria flowers. The method comprises the following steps: preparing a pueraria flower alcohol extract, constructing an alcohol damage cell model, preparing a cell culture medium containing gradient pueraria flower extract, culturing cells, and carrying out ROS level detection and statistics. The invention relates to a control group, a model group and a kudzuvine flower extract group, the internal quality of the control group, the model group and the kudzuvine flower extract group are jointly controlled from multiple angles, the current quality control and evaluation method is improved, a new technical approach can be provided for quality control and quality evaluation research of other alcohol effect dispelling and liver protecting traditional Chinese medicine decoction pieces, and the method is easy to operate, high in practicability, wide in application range and easy to popularize. The method is an innovation in traditional Chinese medicine quality control and evaluation.

Description

Technical Field

[0001] The invention belongs to the technical field of quality control of traditional Chinese medicines, and particularly relates to a quality control evaluation method for Pueraria lobata medicinal materials. Background Art

[0002] As my country's urbanization process gradually deepens, natural resources are becoming increasingly scarce, and the proportion of artificial cultivation of Chinese medicinal materials has increased significantly. The domestic supply of Chinese medicinal materials now exceeds 70% from artificial cultivation. However, the use of Chinese medicinal materials is based on thousands of years of practical experience, and their traditional sources mainly rely on wild collection. In the process of modern artificial cultivation, the application of methods such as mulching, greenhouse cultivation, fertilizer application, irrigation technology, and soilless cultivation has accelerated the growth rate of Chinese medicinal materials, but it has also raised questions about whether their efficacy has decreased. At present, the testing standards for Chinese herbal medicines are mostly focused on the detection of a single component or a few components, which makes it difficult to reflect the overall efficacy of Chinese medicinal materials with complex ingredients. Therefore, the bioactivity detection method that can reflect the efficacy of drugs, which is commonly used in the testing of biological products, provides a new idea for improving the quality control of Chinese medicinal materials. The "Technical Guidelines for Quality Research of New Chinese Medicines (Trial)" clearly states: "It is encouraged to explore and carry out bioactivity determination research and establish bioactivity determination methods as a substitute or supplement for conventional physical and chemical methods."

[0003] Pueraria flowers, the dried flower buds of Pueraria lobata (Willd.) Ohwi or Pueraria thomsonii Benth., are commonly used in traditional Chinese medicine for hangover relief. The current national inspection standard, the "Ministry of Health Drug Standards: Chinese Medicinal Materials, Volume 1" (1992 edition), only covers properties and flavor. Currently, 24 provincial inspection standards have been issued, adding new inspection items such as impurities, moisture, ash, extracts, and identification. However, these indicators cannot reflect the overall pharmacological properties of kudzu flowers. Establishing a bioactivity test method that can reflect pharmacological efficacy will not only facilitate scientific evaluation of kudzu flowers but also guide farmers in cultivating highly effective cultivated Chinese medicinal materials.

[0004] Both Ben Jing Feng Yuan and Ming Yi Bie Lu record that kudzu flower has the medicinal effect of detoxifying alcohol. Modern pathological research shows that alcohol is primarily metabolized in the liver, making the liver the primary target organ for alcohol poisoning. In cases of heavy or chronic alcohol consumption, liver cells produce large amounts of highly reactive substances such as acetaldehyde and oxygen free radicals due to alcohol metabolism, triggering cellular oxidative stress, which in turn leads to pathological phenomena such as inflammation and apoptosis. Research on the mechanism of kudzu flower's alcohol detoxification effect, both domestic and international, has demonstrated that its components possess antioxidant properties, effectively mitigating alcohol-induced cellular damage. Therefore, examining the effects of kudzu flower on reactive oxygen species (ROS) levels in an alcohol-induced liver cell injury model can scientifically demonstrate its efficacy in detoxifying alcohol. SUMMARY

[0005] In view of the above, in order to overcome the defects of the prior art, the purpose of the present application is to provide a quality detection method for Flos Puerariae based on reactive oxygen species (ROS) detection, which effectively realizes the evaluation of the medicinal effect of Flos Puerariae.

[0006] In the first aspect, the present application provides a quality detection method for Flos Puerariae based on reactive oxygen species (ROS) detection, which comprises the following steps:

[0007] (1) preparing an alcohol extract of Flos Puerariae;

[0008] (2) establishing an alcohol-damaged liver cell model;

[0009] (3) establishing control group, model group and Flos Puerariae extract group cells;

[0010] (4) detecting reactive oxygen species;

[0011] (5) result statistics.

[0012] In some embodiments, step (1) comprises the following steps:

[0013] The Flos Puerariae is ground into powder, a certain amount is weighed, soaked in 5-15 times the volume of alcohol solvent for 20-60 min, and extracted by reflux for 2 times, 1-2 h for the first time and 0.5-1.5 h for the second time using 6-10 times the volume of the same solvent. After the extraction is completed, the filtrate is filtered and combined, the solvent is recovered and vacuum dried, the obtained dry matter is ultrasonically dissolved with an appropriate amount of low-toxicity solvent, and filtered through a 0.22 μm filter membrane to remove bacteria for standby use. The volume multiple of the alcohol solvent refers to 1 g of Flos Puerariae corresponding to 1 mL of solvent.

[0014] Preferably, the alcohol solvent is selected from methanol, ethanol, propanol, isopropanol and n-butanol, more preferably ethanol.

[0015] Preferably, the concentration of the ethanol is 50%-90%, preferably 60%-80%, more preferably 70%.

[0016] Preferably, the volume of the alcohol solvent used for soaking is 8-12 times, more preferably 10 times.

[0017] Preferably, the first extraction time is 1.5 h.

[0018] Preferably, the volume of the solvent used for the second extraction is 7-9 times, more preferably 8 times.

[0019] Preferably, the low-toxicity solvent is selected from DMSO (dimethyl sulfoxide) and NMP (N-methyl pyrrolidone), more preferably DMSO (dimethyl sulfoxide).

[0020] In some embodiments, step (2) comprises the following steps:

[0021] Hepatocytes were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C, 5% CO2, and saturated humidity. Hepatocytes in the logarithmic growth phase were cultured at 1×10 4 Cells were seeded at a density of 100 cells / well in a 96-well plate. After culturing for 4 hours, alcohol was added to the culture medium and cultured for a long time as alcohol-damaged cell model.

[0022] Preferably, the liver-like cells are liver-related cells such as hepatoma cells, hepatocytes, transfected cell lines, and preferably HepG2 cells.

[0023] Preferably, the final concentration of alcohol in the culture medium is 100-1000 mM, more preferably 400-800 mM, most preferably 600 mM.

[0024] In some embodiments, step (3) comprises the following steps:

[0025] After the liver cells were cultured normally for 4 hours, at least 4 wells of cells were selected and cultured for 24 hours without adding alcohol or kudzu flower alcohol extract as the control group; at least 4 wells of cells were selected and cultured for 24 hours with only alcohol added as the model group; in addition, while adding alcohol, kudzu flower alcohol extract was added to the culture medium at final concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mg of crude drug / mL, respectively. At least 4 wells were selected for each concentration and cultured for 24 hours as the kudzu flower extract group.

[0026] In some embodiments, step (4) comprises the following steps:

[0027] After the incubation period, the culture medium was discarded, and the cells were washed twice with PBS. Serum-free culture medium containing 10 μM DCFH-DA was then added and incubated for an additional 30 minutes. After the culture medium was discarded, the cells were washed twice with PBS, pipetted into PBS, and collected by centrifugation at 1000 rpm for 5 minutes. ROS levels were measured in the control, model, and kudzu flower extract groups.

[0028] Preferably, the method for detecting ROS levels includes fluorescence staining, chemiluminescence, electron paramagnetic resonance, electrochemical biosensor, chromatography, and spectrophotometry. More preferably, the method for detecting ROS levels is fluorescence staining.

[0029] Preferably, the specific operation of the fluorescent staining method is: using a microplate reader to measure the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0030] In some embodiments, step (5) comprises the following steps:

[0031] The ROS levels of the control group, the model group, and each concentration of the Pueraria lobata extract group are compared between groups using one-way ANOVA. The experimental results should meet the following requirements: the ROS level of the model group should be significantly higher than that of the control group (P<0.05); when the concentration of the Pueraria lobata extract is increased to a certain level, the ROS level of the extract group should be significantly lower than that of the model group (P<0.05). Moreover, the lower the extract concentration at which a significant difference in ROS level is produced compared to the model group, the stronger the efficacy of Pueraria lobata and the better the quality.

[0032] In a second aspect, the present application provides an application of the detection method of the first aspect in the quality detection of Pueraria lobata medicinal materials.

[0033] In some embodiments, the detection method is also applicable to the quality detection of other alcohol-eliminating and liver-protecting medicinal materials other than Pueraria lobata.

[0034] The medicinal materials include, but are not limited to, Pueraria lobata, Hovenia dulcis Thunb, Schisandra chinensis, and Alpinia officinarum.

[0035] Compared with the prior art, the present application has the following advantages:

[0036] The present application first establishes a Pueraria lobata biological activity detection method for the effect of the extract on the ROS level in the model cell, which basically meets the requirements of the "experimental conditions", "experimental design", "results and statistics", and "result determination" in the "Guiding Principles for Biological Activity Determination of Traditional Chinese Medicines in the Chinese Pharmacopoeia 2025 (Volume 4)", and completes the methodological validation of "influence factor investigation", "method applicability investigation", and "precision investigation", etc. It can be used as a supplement and improvement to the existing chemical determination method, and can provide a new technical approach for the quality control and quality evaluation of other alcohol-eliminating and liver-protecting traditional Chinese medicines. It is easy to operate, has strong practicality, has a wide application range, and is an innovation in the quality control and evaluation of traditional Chinese medicines. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a process roadmap of the Pueraria lobata biological activity detection method based on ROS detection of the present application.

[0038] Figure 2 It is a result schematic diagram of the present application. The control group is alcohol "-" and Pueraria lobata extract "0"; the model group is alcohol "+" and Pueraria lobata extract "0"; #P<0.05 compared with the control group; *P<0.05 compared with the model group.

[0039] Figure 3These are the test results of the effects of 10 batches of kudzu flower samples of the present invention on the ROS levels of model cells. The control group consisted of alcohol "-" and kudzu flower extract "0"; the model group consisted of alcohol "+" and kudzu flower extract "0"; compared with the control group: #P<0.05; compared with the model group: *P<0.05.

[0040] Figure 4 These are the results of six tests on the effect of the same batch of kudzu flower samples of the present invention on the ROS level of model cells at the same extract concentration. The control group was alcohol "-" and kudzu flower extract "0"; the model group was alcohol "+" and kudzu flower extract "0"; compared with the control group: #P<0.05; compared with the model group: *P<0.05. DETAILED DESCRIPTION

[0041] The following examples are intended to enable those skilled in the art to more fully understand the technical solutions and implementation effects of the present invention, but the scope of protection of the present invention is not limited thereto. This section further describes the present invention in detail in conjunction with specific implementation cases, and its technical features and advantages will be clearly reflected in the description. It should be pointed out that the embodiments are only exemplary descriptions and are not intended to limit the scope of the claims of the present invention. Any detailed adjustments, equivalent replacements, or adaptive improvements based on the core principles of the present invention fall within the substantive protection scope of the present invention.

[0042] Example 1 Quality testing of Pueraria lobata medicinal material

[0043] (1) Preparation of Pueraria lobata flower ethanol extract:

[0044] For a batch of kudzu flower powder, 200g was weighed and soaked in 10 volumes (i.e., 1g of kudzu flower corresponds to 10mL) of 70% ethanol for 40 minutes. The powder was then refluxed and extracted twice, the first time for 1.5 hours, and the second time for 1 hour using 8 volumes (i.e., 1g of kudzu flower corresponds to 8mL) of 70% ethanol. After the extractions were complete, the filtrates were filtered, the ethanol recovered, and the powder was vacuum-dried. The resulting dried product was solubilized with an appropriate amount of DMSO and sterilized by filtration through a 0.22μm filter.

[0045] (2) Culture of HepG2 cancer cells and establishment of model cells:

[0046] HepG2 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C, 5% CO2, and saturated humidity. HepG2 cells in the logarithmic growth phase were cultured at 1×10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate. After culturing for 4 hours, alcohol with a final concentration of 600 mM was added to the culture medium and cultured for further culturing as alcohol-damaged cell model cells.

[0047] (3) Establishment of cells in the control group, model group, and kudzu flower extract group:

[0048] After HepG2 cells were cultured normally for 4 hours, at least 4 wells of cells were selected, and no alcohol or Pueraria lobata flower alcohol extract was added, and the cells were cultured for another 24 hours as the control group; at least 4 wells of cells were added with only alcohol and cultured for another 24 hours as the model group; in addition, while adding alcohol, Pueraria lobata flower alcohol extract was added to the culture medium at final concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mg of crude drug / mL, respectively, and at least 4 wells were selected for each concentration, and the cells were cultured for another 24 hours as the Pueraria lobata flower extract group.

[0049] (4) ROS detection:

[0050] After the incubation period, the medium was discarded, and the cells were washed twice with PBS. Serum-free medium containing 10 μM DCFH-DA was then added and incubated for an additional 30 minutes. The medium was discarded, and the cells were washed twice with PBS. The cells were pipetted into PBS and collected by centrifugation at 1000 rpm for 5 minutes. Fluorescence intensity was measured using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0051] (5) Results statistics

[0052] One-way ANOVA was used to compare ROS levels in the control group, model group, and groups with various concentrations of Pueraria lobata extract. The experimental results should meet the following requirements: ROS levels in the model group should be significantly increased compared to the control group (P < 0.05); after the Pueraria lobata extract concentration reaches a certain level, ROS levels in the extract group should be significantly decreased compared to the model group (P < 0.05). Furthermore, the lower the extract concentration at which ROS levels are significantly different from those in the model group, the stronger the efficacy and better the quality of the Pueraria lobata extract.

[0053] according to Figure 2 It can be seen that the ROS level in the model group was significantly higher than that in the control group (P<0.05), indicating that the model was successful. When the concentration of kudzu flower extract reached 2.0 mg crude drug / mL, the ROS level was significantly lower than that in the model group (P<0.05), which reflects the quality of this batch of kudzu flowers in terms of the overall level of efficacy.

[0054] Example 2 Quality testing of different batches of Pueraria lobata medicinal materials

[0055] Take 10 different batches of Pueraria lobata medicinal materials and use the same method as in Example 1 to conduct quality inspection. The results are as follows: Figure 3 A- Figure 3 As shown in J.

[0056] according to Figure 3 A- Figure 3 It can be known that, in the detection process of each batch of Pueraria lobata medicine, the ROS level of the model group is significantly higher than that of the control group (P<0.05), and the ROS level of the model group of each batch is relatively close, having good repeatability. However, the extract concentration at which the ROS level of each batch of Pueraria lobata medicine shows significant difference compared with the model group is not the same, which is 2.0 mg of crude drug / mL, 1.5 mg of crude drug / mL, 2.0 mg of crude drug / mL, 2.0 mg of crude drug / mL, 1.5 mg of crude drug / mL, 2.0 mg of crude drug / mL, 1.0 mg of crude drug / mL, 1.5 mg of crude drug / mL, 1.5 mg of crude drug / mL, and 1.5 mg of crude drug / mL, respectively.

[0057] It can be seen that the present method can accurately detect the quality level of different Pueraria lobata medicines.

[0058] Example 3 Multiple repeated detection of the same batch of Pueraria lobata medicine

[0059] The same batch of Pueraria lobata medicine was subjected to 6 experiments by using the same method as in Example 1, and the concentration of Pueraria lobata extract was 2.0 mg of crude drug / mL. As shown in Table 2, in each experiment, the ROS level of the model group was relatively close, and the ROS level of the Pueraria lobata extract group was also relatively close, which was significantly lower than that of the model group (P<0.05). Figure 4

[0060] It can be seen that the present method has good reproducibility.

[0061] Finally, it should be noted that the above examples are only used to illustrate and not to limit the technical solutions of the present application. Although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the present application can still be modified or replaced equivalently without departing from the spirit and scope of the present application, and any modification or partial replacement should be covered in the scope of the claims of the present application.​

Claims

1. A method for detecting the quality of kudzu flower based on reactive oxygen species (ROS) detection, characterized in that: The following steps are involved: (1) preparing kudzu flower ethanol extract; (2) Establish a liver cell model of alcohol damage; (3) Establishment of cells in the control group, model group, and Pueraria lobata flower extract group; (4) Reactive oxygen species detection; (5) Result statistics.

2. The detection method according to claim 1, wherein The step (1) is as follows: The kudzu flower was powdered, a certain mass was weighed, and the mixture was soaked in 5-15 times the volume of an alcohol solvent for 20-60 minutes, and refluxed for extraction twice, with the first extraction taking 1-2 hours and the second extraction taking 6-10 times the volume of the same solvent for 0.5-1.5 hours; after the extraction was completed, the filtrate was filtered and combined, the solvent was recovered and vacuum dried, the obtained dry matter was solubilized with an appropriate amount of a low-toxic solvent, and sterilized by filtration through a 0.22 μm filter membrane for later use; the volume multiple of the alcohol solvent refers to 1 g of kudzu flower corresponding to 1 mL of solvent.

3. The detection method according to claim 2, characterized in that In the step (1), The alcohol solvent is selected from methanol, ethanol, propanol, isopropanol, n-butanol, preferably ethanol; and / or, The volume of the alcohol solvent used in the soaking is 8-12 times, preferably 10 times; and / or, The first extraction time is 1.5 hours; and / or, The volume of the solvent used in the second extraction is 7-9 times, more preferably 8 times; and / or, The low-toxicity solvent is selected from DMSO (dimethyl sulfoxide) and NMP (N-methylpyrrolidone), preferably DMSO (dimethyl sulfoxide).

4. The detection method according to claim 3, characterized in that The concentration of the ethanol is 50%-90%, preferably 60%-80%, more preferably 70%.

5. The detection method according to any one of claims 1 to 4, characterized in that The step (2) is: Hepatocytes were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C, 5% CO2 and saturated humidity. Hepatocytes in the logarithmic growth phase were cultured at 1×10 4 Cells were seeded at a density of 100 cells / well in a 96-well plate. After culturing for 4 hours, alcohol was added to the culture medium and cultured for a long time as alcohol-damaged cell model. Preferably, the liver-like cells are liver-related cells such as hepatoma cells, hepatocytes, transfected cell lines, and preferably HepG2 cells; and / or, Preferably, the final concentration of alcohol in the culture medium is 100-1000 mM, more preferably 400-800 mM, most preferably 600 mM.

6. The detection method according to any one of claims 1 to 5, characterized in that The step (3) is: After the liver cells were cultured normally for 4 hours, at least 4 wells of cells were selected and cultured for 24 hours without adding alcohol or kudzu flower alcohol extract as the control group; at least 4 wells of cells were selected and cultured for 24 hours with only alcohol added as the model group; in addition, while adding alcohol, kudzu flower alcohol extract was added to the culture medium at final concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mg of crude drug / mL, respectively. At least 4 wells were selected for each concentration and cultured for 24 hours as the kudzu flower extract group.

7. The detection method according to any one of claims 1 to 6, characterized in that The step (4) is: After the culture, the culture medium was discarded, the cells were washed twice with PBS, and then serum-free culture medium containing 10 μM DCFH-DA was added and incubated for another 30 min. After the culture medium was discarded, the cells were washed twice with PBS, pipetted into PBS, and collected by centrifugation at 1000 rpm for 5 min. The ROS levels in the control group, model group, and kudzu flower extract group were detected.

8. The detection method according to claim 7, characterized in that The method for detecting the ROS level includes fluorescence staining, chemiluminescence, electron paramagnetic resonance, electrochemical biosensor, chromatography, and spectrophotometry. Preferably, the method for detecting the ROS level is fluorescence staining; Preferably, the specific operation of the fluorescence staining method is: using a microplate reader to measure the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

9. The detection method according to claims 1-8, characterized in that The step (5) is: One-way ANOVA was used to compare the ROS levels of the control group, model group, and groups with different concentrations of Pueraria lobata extract. The experimental results should meet the following requirements: the ROS level in the model group should be significantly increased compared with the control group (P < 0.05); after the concentration of Pueraria lobata extract increased to a certain level, the ROS level in the extract group should be significantly decreased compared with the model group (P < 0.05); moreover, the lower the extract concentration at which the ROS level was significantly different from that of the model group, the stronger the efficacy of Pueraria lobata and the better the quality.

10. Use of the detection method according to any one of claims 1 to 9 in quality detection of Pueraria lobata medicinal material.

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