Windproof quality evaluation system based on multi-index component content and its construction method

Abstract

The present invention discloses a quality evaluation system of Saposhnikovia divaricata based on the content of multiple index components and a construction method thereof, and belongs to the technical field of drug analysis. The construction method of the quality evaluation system of Saposhnikovia divaricata based on the content of multiple index components of the present invention comprises the following steps: collecting Saposhnikovia divaricata medicinal materials from different origins and different growth years; determining the content of index components in Saposhnikovia divaricata medicinal materials by liquid chromatography; establishing an inflammation model, studying the relationship between index components and anti-inflammatory effects, and finding out the components that contribute the most to the anti-inflammatory effects. The quality evaluation system of Saposhnikovia divaricata disclosed by the present invention evaluates the quality of Saposhnikovia divaricata medicinal materials by detecting the content of 10 index components of Saposhnikovia divaricata. The present invention establishes a method for determining the multi-index content of Saposhnikovia divaricata medicinal materials, which reflects the characteristics of comprehensiveness and stability of traditional Chinese medicine, and screens out the active components that play the pharmacological role of Saposhnikovia divaricata through anti-inflammatory experiments, and establishes a quality evaluation system of Saposhnikovia divaricata medicinal materials based on the content of multiple index components, which is conducive to improving the quality of Saposhnikovia divaricata medicinal materials and the stability of clinical efficacy.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and in particular relates to a siler quality evaluation system based on multi-index component content and a construction method thereof. Background Art

[0002] Saposhnikovia divaricata (Turcz.) Schisck. is a perennial herbaceous plant of the Umbelliferae family. Its unbolted dry root is used as medicine. It tastes spicy and sweet, is warm in nature, and enters the bladder, liver, and spleen meridians. It has the effects of dispelling wind and relieving exterior symptoms, removing dampness and relieving pain, and stopping spasms. It is often used to treat fever, headache, allergic rhinitis, rheumatoid arthritis, and immune system diseases. So far, more than 100 compounds have been isolated from Saposhnikovia, including chromones and coumarin compounds. Due to the influence of factors such as the growth environment, harvest time, and germplasm resources, the content of the active ingredients of Saposhnikovia from different origins and their clinical efficacy are quite different, so the quality control of Saposhnikovia is particularly important.

[0003] As a complex multi-component system, Chinese medicine has the characteristics of multi-component and multi-target effects. It is difficult to fully reflect the intrinsic quality of medicinal materials by detecting a single component. Therefore, multi-index and multi-component quality determination has become a development trend. In the 2020 edition of the Chinese Pharmacopoeia, under the "Saposhnikovia divaricata" medicinal material, cimicifuga glycosides and 5-O-methylvisaminol glycosides are used as Chinese medicine indicator components for evaluating the quality of Saposhnikovia divaricata medicinal materials, and the sum of the two is stipulated to be no less than 0.24%. However, compounds such as cimicifuga, chamotol glycosides, psoralen, chrysophanol toxins, bergamot lactones, and imperatorin, etc., are characteristic components of the quality of Saposhnikovia divaricata medicinal materials of different origins, different years, and different commodity specifications, and they all have certain antibacterial, antipyretic and anti-inflammatory effects. Therefore, using only cimicifuga glycosides and 5-O-methylvisaminol glycosides as quality markers (Q-Markers) of Saposhnikovia divaricata medicinal materials cannot fully evaluate the intrinsic quality of Saposhnikovia divaricata. For this reason, it is urgent to establish a complete Saposhnikovia divaricata quality evaluation system.

[0004] In recent years, the incidence of diseases related to inflammation has increased year by year. At present, the main anti-inflammatory drugs used in clinical practice include non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, etc. and adrenocortical hormones, but both have many adverse reactions. For this reason, finding a safe and effective anti-inflammatory drug has become a research hotspot. Clinical and experimental confirmation of many traditional Chinese medicines have good anti-inflammatory effects, and small toxic side effects. Modern research shows that Saposhnikovia divaricata has biological activities such as anti-tumor, anti-inflammatory, antioxidant, and lipid metabolism promotion, which is inseparable from the various biologically active ingredients such as chromones and coumarins contained in Saposhnikovia divaricata. For this reason, revealing the relationship between traditional Chinese medicine ingredients and their efficacy from an overall perspective can more comprehensively reveal the material basis and mechanism of the efficacy of traditional Chinese medicine. Therefore, constructing a quality evaluation system for multi-index component content to ensure the quality of medicinal materials has become a problem to be solved by technicians in this field. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing a quality evaluation system of Saposhnikovia divaricata based on the content of multiple index components, and to screen out a group of medicinal components related to anti-inflammatory effects by correlating the chemical component content with the efficacy index, thereby effectively ensuring the quality of the medicinal material and providing a basis for the subsequent further development and utilization of Saposhnikovia divaricata.

[0006] Another object of the present invention is to provide a windproof quality evaluation system constructed using the construction method.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The present invention discloses a method for constructing a windproof quality evaluation system based on the content of multiple index components, comprising the following steps:

[0009] Step 1. Collect the Fangfeng herbs from different origins and different growing years;

[0010] Step 2. Determine the content of index components in the Radix Saposhnikoviae Radix by liquid chromatography;

[0011] Step 3. Establish an inflammation model, study the relationship between the index components and the anti-inflammatory effect, and find out the components that contribute most to the anti-inflammatory effect.

[0012] In some embodiments of the present invention, liquid chromatography is used to determine the following index components of the Fangfeng medicinal material: cimicifuga glycosides, 5-O-methylvisaminol glycosides, cimicifuga, chelidonol glycosides, psoralen, zanthoxylum toxin, bergamot lactone, imperatorin, 5-O-methylvisaminol, and chelidonol.

[0013] In some embodiments of the present invention, the same chromatographic conditions are used to determine the index components of Saposhnikovia divaricata.

[0014] Preferably, the chromatographic conditions include:18 As the stationary phase, water was used as mobile phase A and acetonitrile was used as mobile phase B for gradient elution. The gradient elution program was:

[0015] 0min, 75~85%A, 15-25%B,

[0016] 7.5~8.5min, 55~65%A, 35~45%B,

[0017] 19.5~20.5min, 40~50%A, 50~60%B,

[0018] 24.5~25.5min, 40~50%A, 50~60%B,

[0019] 34.5~35.5min, 30~40%A, 60~70%B,

[0020] 39.5~40.5min, 15~25%A, 75~85%B,

[0021] Preferably, the gradient elution program is:

[0022] 0min, 75~85%A, 15-25%B,

[0023] 8min, 55~65%A, 35~45%B,

[0024] 20min, 40~50%A, 50~60%B,

[0025] 25min, 40~50%A, 50~60%B,

[0026] 35min, 30~40%A, 60~70%B,

[0027] 40min, 15~25%A, 75~85%B;

[0028] More preferably, the gradient elution program is:

[0029] 0min, 80% A, 20% B,

[0030] 8min, 59.5% A, 40.5% B,

[0031] 20min, 45% A, 55% B,

[0032] 25min, 45% A, 55% B,

[0033] 35min, 35% A, 65% B,

[0034] 40min, 20%A, 80%B.

[0035] In some embodiments of the present invention, during liquid chromatography determination, the mobile phase flow rate is 0.8 to 1.2 mL / min, the column temperature is 22 to 27° C., and the wavelength is 254 nm;

[0036] Preferably, the liquid chromatography determination comprises the following steps:

[0037] Preparation of the test solution: Take 0.1-0.3g of Radix Saposhnikoviae in a conical flask, add 3-12mL of methanol solution, extract by ultrasonic for 10-120min, cool, make up the weight loss with methanol, shake well, filter through a 0.22μm filter membrane, and take the filtrate to obtain the solution;

[0038] Preparation of reference substance solution: accurately weigh the mixed reference substance and prepare 1 mL of methanol solution containing 0.11 μg of cimicifuga glycosides, 0.17 μg of 5-O-methylvisaminol glycosides, 0.018 μg of cimicifuga, 0.014 μg of hyaluronic acid glycosides, 0.002 μg of 5-O-methylvisaminol, 0.002 μg of psoralen, 0.003 μg of xanthoxylum toxin, 0.002 μg of hyaluronic acid, 0.001 μg of bergamot lactone, and 0.004 μg of imperatorin.

[0039] Take 5-20 μL of the test solution and reference solution respectively, inject and measure.

[0040] In the present invention, a DAD detector is used to perform full wavelength scanning on the test sample, and 254 nm is selected as the detection wavelength by comprehensively considering the peak shape, baseline, separation effect, etc. of each component.

[0041] In some embodiments of the present invention, in step 2, based on the content measurement results, cluster analysis is performed on the Fangfeng medicinal materials of different origins and different growth years to analyze the relationship between the origin of the medicinal materials and the quality of the medicinal materials.

[0042] In some embodiments of the present invention, in step 2, the contents of index components of the fangfeng medicinal materials from different origins and different growth years are standardized and then subjected to principal component analysis to find out the comprehensive quality characteristics that can fully reflect the fangfeng medicinal materials.

[0043] In some embodiments of the present invention, the inflammation model is a lipopolysaccharide-induced cellular inflammation model, preferably, the inflammation model is a lipopolysaccharide-induced RAW264.7 cell inflammation model.

[0044] In some embodiments of the present invention, in step 3, the ethanol extract of Saposhnikovia divaricata is co-cultured with lipopolysaccharide-induced RAW264.7 cells for 12 to 48 hours, preferably 24 hours, and then the contents of NO, IL-6 and IL-1β in the cell supernatant after culture are determined;

[0045] Preferably, a normal control group and a model control group are set up simultaneously during the culture.

[0046] Preferably, the method for preparing the siler vine alcohol extract comprises: taking siler vine powder, adding methanol, ultrasonically extracting, filtering, concentrating the filtrate, and freeze-drying the concentrate to obtain the siler vine alcohol extract;

[0047] More preferably, 2 to 10 g of fangfeng powder is taken, methanol is added at a solid-liquid ratio of 1:15 to 60, ultrasonic extraction is performed for 20 to 120 minutes, and then the mixture is filtered, the filtrate is concentrated, and the concentrate is freeze-dried to obtain a fangfeng alcohol extract.

[0048] In one embodiment of the present invention, fangfeng powder is taken, methanol is added at a solid-liquid ratio of 1:30, and ultrasonic extraction is performed for 60 minutes.

[0049] In the present invention, the preparation method of the alcohol extract of Saposhnikovia divaricata is consistent with the preparation method of the test solution for liquid chromatography determination. The applicant investigated two commonly used extraction methods, namely, ultrasound and heating reflux, with the extraction solvents being 50% methanol, 70% methanol, 90% methanol, 100% methanol, and 100% ethanol, and the solid-liquid ratio being 1:20 g·mL -1 , 1:30g·mL -1 , 1:40g·mL -1 The effects of ultrasonic time of 40min, 50min and 60min on the extraction rate of 10 chemical components in Saposhnikovia divaricata were analyzed. The results showed that the extraction solvent was 100% methanol and the liquid-to-solid ratio was 1:30g·mL -1 , the extraction rate of chemical components in Saposhnikovia divaricata was the highest when ultrasonic extraction was performed for 60 minutes.

[0050] In some embodiments of the present invention, grey relational analysis is performed based on the results of the measured NO, IL-6 and IL-1β contents;

[0051] or / and bivariate correlation analysis was performed between the chemical components of Saposhnikovia divaricata and the contents of NO, IL-6 and IL-1β to determine which chemical components can significantly reduce the inflammatory response and are positively correlated with the anti-inflammatory activity;

[0052] Preferably, the contents of NO, IL-6 and IL-1β are used as reference sequences, and the contents of chemical components are used as comparison sequences, and the grey correlation coefficient between the reference sequence and the comparison sequence is calculated to find out the relationship between each chemical component and the efficacy;

[0053] Preferably, principal component and grey relational analysis are combined to find out the main index components that exert anti-inflammatory activity.

[0054] The windproof quality evaluation system constructed by the above construction method disclosed in the present invention;

[0055] Preferably, the quality evaluation system evaluates the quality of the Fangfeng medicinal material by detecting the content of 10 indicator components in the Fangfeng medicinal material, and the 10 indicator components are cimicifuga glycosides, 5-O-methylvisaminol glycosides, cimicifuga, chelidonol glycosides, psoralen, zanthoxylum toxin, bergamot lactone, imperatorin, 5-O-methylvisaminol, and chelidonol.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] The present invention establishes a multi-index content determination method for fangfeng medicinal materials, and the content determination involves two types of components, chromones and coumarins, which reflects the characteristics of Chinese medicine such as comprehensiveness and stability. The active ingredients that play the pharmacological effects of fangfeng are screened out through anti-inflammatory experiments, and a fangfeng medicinal material quality evaluation system based on the content of multiple index components is established, which is beneficial to improving the quality of fangfeng medicinal materials and the stability of clinical efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Attached Figure 1 The HPLC chart of the mixed reference substance (A), sample solution (B) and blank solvent (C) of Example 1; the names corresponding to the figure marks in the figure are: 1: cimicifuga glycosides; 2: 5-O-methylvisaminol glycosides; 3: cimicifuga; 4: chamoyl glycosides; 5: 5-O-methylvisaminol; 6: psoralen; 7: Zanthoxylum toxin; 8: chamoyl glycosides; 9: bergamot lactones; 10: imperatorin.

[0059] Figure 2 This is a stacked column chart of the content of various indicators of Fangfeng medicinal materials; the Chinese names corresponding to the English in the figure are:

[0060] prim-O-glucosylcimifugin: prim-O-glucosylcimifugin;

[0061] 5-O-methylvisammioside: 5-O-methylvisammioside;

[0062] Cimifugin: cimifugin;

[0063] sec-O-glucosylhamaudol: hamaudol glycoside;

[0064] 5-O-methylvisamminol: 5-O-methylvisamminol;

[0065] psoralen: psoralen;

[0066] xanthotoxin: Zanthoxylum bungeanum toxin;

[0067] hamaudol: hamaudol;

[0068] bergapten: bergamot lactone;

[0069] imperatorin: imperatorin.

[0070] Figure 3 This is the cluster analysis result of 17 batches of Fangfeng medicinal materials. The Chinese names corresponding to the English names in the figure are:

[0071] XJ: Xinjiang; NM: Inner Mongolia; JL: Jilin; HB: Hebei. DETAILED DESCRIPTION

[0072] The present invention is further described in detail below in conjunction with specific examples and accompanying drawings, which are provided to explain the present invention rather than to limit it. Where specific conditions are not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0073] The material information used in the examples of the present invention is as follows:

[0074] RAW264.7 special culture medium (batch number WHAA23A124, Wuhan Punosai Life Science Technology Co., Ltd.), IL-6 ELISA test kit (batch number 202309025, Shanghai Enzyme Absorption Biotechnology Co., Ltd.), IL-1β ELISA test kit (batch number E20231009-20174B, Shanghai Enzyme Biological Technology Co., Ltd.), NO test kit (batch number 0000561647, Promega, USA), cimicifuga glycosides, 5-O-methylvisaminol glycosides, cimicifuga, chelidonol glycosides, psoralen, Zanthoxylum toxin, bergamot endonucleic acid Ester, imperatorin reference substance, batch numbers are 111522-202214, 111523-202212, 111710-200602, 111714-200501, 110739-202319, 112077-202101, 112078-202101, 110826-202219, all purchased from China Food and Drug Inspection Institutes; 5-O-methylvisaminol and chelidonol reference substances, batch numbers are B23192 and B23109, respectively, purchased from Yuanye Biotechnology Co., Ltd.; acetonitrile (Thermo Fisher Scientific, USA).

[0075] The specific sources of 17 batches of medicinal materials from different origins and different growth years used in the embodiments of the present invention are shown in the following table:

[0076] Table 1 Information on 17 batches of Radix Saposhnikoviae samples

[0077]

[0078]

[0079] Example 1

[0080] This embodiment discloses an investigation on the content determination method of 10 index components of the Fangfeng medicinal material, and the 10 index components are: cimicifuga glycosides, 5-O-methylvisaminol glycosides, cimicifuga, chelidonol glycosides, psoralen, zanthoxylum toxin, bergamot lactones, imperatorin, 5-O-methylvisaminol, and chelidonol.

[0081] 1. Chromatographic conditions

[0082] Chromatographic column SupfexAQ-C 18 , 4.6mm×250mm, 5μm;

[0083] Mobile phase: water (A)-acetonitrile (B), gradient elution (0-8 min, 20%-40.5% B, 8-20 min, 40.5%-55% B, 20-25 min, 55% B, 25-35 min, 55%-65% B, 35-40 min, 65%-80% B);

[0084] Flow rate: 1 mL min -1 ; Column temperature 25℃; Wavelength 254nm; Injection volume: 15μL.

[0085] 2. Preparation of mixed reference solution

[0086] Accurately weigh the mixed reference substance and prepare it into 1mL of methanol solution containing 0.11μg of cimicifuga glycosides, 0.17μg of 5-O-methylvisaminol glycosides, 0.018μg of cimicifuga glycosides, 0.014μg of 5-O-methylvisaminol glycosides, 0.002μg of psoralen, 0.003μg of zanthoxylum toxin, 0.002μg of visaminol, 0.001μg of bergamot lactone, and 0.004μg of imperatorin.

[0087] 3. Preparation of test solution

[0088] Take about 0.2 g of the powder of the Radix Saposhnikoviae Fibrosum, weigh it accurately, put it in a conical flask, add 6 mL of methanol, extract it by ultrasonic for 1 hour, let it cool, make up the weight loss with methanol, shake it well, filter it through a 0.22 μm filter membrane, and take the filtrate to obtain the product.

[0089] 4 Methodological investigation

[0090] 4.1 Preparation of standard curve

[0091] 1 μL, 5 μL, 10 μL, 15 μL and 20 μL of the mixed reference solution were accurately pipetted respectively and injected into the liquid chromatograph. The measurement was performed according to the chromatographic conditions shown under “1. Chromatographic conditions”. Linear regression was performed with the input amount (X, μg) as the abscissa and the peak area (Y) as the ordinate. The linear regression equation and correlation coefficient were calculated. The results are shown in Table 2.

[0092] Table 2 Regression equations, linear ranges and correlation coefficients of the 10 components in Saposhnikovia divaricata

[0093]

[0094]

[0095] 4.2 Specificity Experiment

[0096] Take the mixed reference solution, sample solution, and blank solvent (100% methanol by volume) and inject 15 μL of each according to the chromatographic conditions shown under "1. Chromatographic conditions". The results are as follows: Figure 1 As shown, the blank solvent has no interference with the detection of samples.

[0097] 4.3 Precision experiment

[0098] Accurately pipette 15 μL of the same reference solution and inject it 6 times continuously according to the chromatographic conditions shown under "1. Chromatographic conditions". The peak area RSD values ​​of 10 substances repeated 6 times are shown in the following table:

[0099] Table 3 Precision test results

[0100]

[0101]

[0102] As can be seen from the above table, the method of the present invention has good precision.

[0103] 4.4 Stability test

[0104] Accurately pipette 15 μL of the same test solution and inject it at 0, 2, 4, 8, 12, and 24 hours respectively. Determine according to the chromatographic conditions shown under "1. Chromatographic conditions". Repeat 6 times for 10 kinds of substances. The RSD values ​​of the peak areas are shown in the following table:

[0105] Table 4 Stability test results

[0106] Element RSD Cimicifuga glycosides 1.15％ 5-O-Methylvisaminol 0.8％ Cimicifuga 0.75％ Hemoglobin 0.98％ 5-O-Methylvisaminol 1.32％ Psoralen 0.77％ Zanthoxylum bungeanum toxin 0.84％ Hemoglobin 4.16％ Bergamot lactone 1.68％ Imperatorin 1.29％

[0107] As can be seen from the above table, the method of the present invention has good stability.

[0108] 4.5 Repeatability Experiment

[0109] Weigh 6 portions of the same batch of test samples accurately, prepare the test samples according to the method under "3. Preparation of test sample solution", and measure according to the chromatographic conditions shown under "1. Chromatographic conditions". The RSD of the peak areas of 10 substances repeated 6 times is shown in the following table:

[0110] Table 5 Results of repeatability test

[0111]

[0112]

[0113] It can be seen from the above table that the method of the present invention has good repeatability.

[0114] Example 2

[0115] In this example, 17 batches of Saposhnikovia divaricata samples from different origins and different growth years were used to prepare the test solution according to the method under "3. Preparation of test solution" in Example 1, and the contents of 10 chemical components were determined according to the chromatographic conditions shown under "1. Chromatographic conditions" in Example 1. The stacked bar chart of the contents of various indicators of Saposhnikovia divaricata medicinal materials is shown in the attached figure. Figure 2 shown.

[0116] The total content of cimicifuga and 5-O-methylvisaminol glycoside in 17 batches of Saposhnikovia divaricata samples ranged from 2.93 to 12.09 mg·g -1 , all meet the requirement of the 2020 edition of the Chinese Pharmacopoeia that the sum of the two components, cimicifuga glycosides and 5-O-methylvisaminol glycosides, should not be less than 0.24%. Among them, S5, a two-year-old siler produced in Chifeng, Inner Mongolia, has the highest content of 10 chemical components, which is 13.06 mg·g -1 ; S13 The content of 10 chemical components of the one-year-old Fangfeng medicinal material in Anshan, Hebei Province was the lowest, at 3.41 mg·g -1 , the difference between the two is 3.83 times. The contents of 10 components in Saposhnikovia divaricata vary greatly, and the contents from high to low are 5-O-methylvisaminol glycosides> cimicifuga glycosides> cimicifuga glycosides> chamotol glycosides> 5-O-methylvisaminol> Zanthoxylum toxin> imperatorin> bergamot lactone> psoralen> chamotol.

[0117] Example 3

[0118] In this example, the contents of 10 index components in 17 batches of Saposhnikovia divaricata medicinal materials measured in Example 2 were clustered using Metaboanalyst 5.0. The results are as follows: Figure 3 shown.

[0119] The 2-year-old Saposhnikovia sample S5 from Inner Mongolia and the 2-year-old Saposhnikovia samples S8-S11 from Baicheng, Jilin Province were clustered into the first category, which were rich in cimicifuga glycosides, 5-O-methylvisaminol glycosides, cimicifuga, chelidonol glycosides and 5-O-methylvisaminol.

[0120] The one-year-old Saposhnikovia samples S1~S4 produced in Inner Mongolia were clustered into the second largest category, which were rich in psoralen, xanthoxylum toxin, chelidonol, and bergamot lactone.

[0121] The windproof samples S12 to S14 from Hebei Province were clustered into the third category, with relatively high contents of psoralen, xanthoxylum bungeanum and bergamot lactone.

[0122] The one-year-old Saposhnikovia samples S6-S7 from Jilin and the Saposhnikovia samples S15-S17 from Xinjiang were clustered into the fourth category, with relatively low contents of 10 chemical components.

[0123] The results of cluster analysis showed that the quality of Saposhnikovia divaricata had certain regional characteristics, but there might be differences between different batches of herbs from the same origin, which might be because factors such as growing years, soil, and precipitation affected the accumulation of secondary metabolites in Saposhnikovia divaricata.

[0124] Example 4

[0125] In this example, the contents of the 10 index components in the 17 batches of Saposhnikovia divaricata medicinal materials measured in Example 2 were standardized (Z-score method) by SPSS 22.0 statistical software and then principal component analysis was performed. The results are shown in Tables 6 and 7.

[0126] Table 6 Eigenvalues ​​and cumulative variance contribution rates

[0127] Element Eigenvalue variance% Cumulative contribution rate% 1 4.403 44.033 44.033 2 2.865 28.648 72.681 3 1.061 10.611 83.292

[0128] Table 7 Comprehensive scores of 17 batches of Fangfeng medicinal materials samples

[0129] sample F-number sample F-number S1 -2.00 S10 2.41 S2 -0.51 S11 4.39 S3 -1.91 S12 -2.53 S4 0.16 S13 -1.92 S5 3.77 S14 -1.72 S6 -1.91 S15 -0.71 S7 -0.74 S16 -0.03 S8 1.56 S17 -0.16 S9 1.88

[0130] According to the eigenvalue and contribution rate analysis, the first three eigenvalues ​​were all greater than 1, and represented 44.033%, 28.648%, and 10.611% of the total variance of the variables, respectively, with a cumulative contribution rate of 83.292%, indicating that these three principal components are the main factors affecting the quality evaluation of Saposhnikovia divaricata, and can comprehensively reflect the comprehensive quality characteristics of Saposhnikovia divaricata medicinal materials. The comprehensive score value was calculated based on the linear combination expression obtained by principal component analysis. The comprehensive scores of the two-year-old Saposhnikovia divaricata samples S5, S8, S9, S10, and S11 from Inner Mongolia and Jilin were above 1, indicating good quality; the comprehensive scores of the one-year-old Saposhnikovia divaricata samples S1 and S12 from Inner Mongolia and Jilin were low, indicating relatively poor quality.

[0131] Example 4

[0132] This example discloses an experiment on the effects of different Saposhnikovia extracts of the present invention on the RAW264.7 cell inflammation model caused by lipopolysaccharide.

[0133] 1. Preparation of drug extract

[0134] Take 5g of Fangfeng powder, add methanol solution at a liquid-to-solid ratio of 1:30, extract by ultrasonic for 1h, concentrate by rotary evaporation to 10mL, and freeze-dry to obtain freeze-dried powder of different batches of extracts. Take 10mg of freeze-dried powder and dissolve it in 4mL RAW264.7 medium to make a concentration of 2.5mg·mL -1 The mother solution was stored at 4°C away from light.

[0135] 2. Experiments on the effects of cell inflammation models

[0136] 2.1 Effects on cell activity

[0137] RAW264.7 cells in the logarithmic growth phase were taken, 2.5×10 4 Cells were inoculated in 96-well plates. A normal group, a blank control group containing only complete medium, and a drug-added group were set up. In the drug-added group, each drug was set up with 4 concentration groups, which were diluted with complete medium to 200, 400, 600, and 800 μg mL -1 Concentration of siler alcohol extract group.

[0138] After 24 hours of drug intervention, the CCK-8 method was used to detect the effects of different concentrations of ethanol extracts of Saposhnikovia divaricata on the activity of RAW264.7 cells. The calculation formula is as follows:

[0139]

[0140] The results are shown in the following table:

[0141] Table 8 Relative survival rate of RAW 264.7 cells by extracts of Saposhnikovia divaricata from different origins

[0142]

[0143]

[0144] The results showed that when the concentration of the alcohol extract of Saposhnikovia divaricata was 200 μg·mL-1, the cell survival rate was greater than 80%, indicating that when the concentration was 200 μg / mL and below, the dosage concentration was within the safe range and could be used for subsequent experiments.

[0145] 2.2 Anti-inflammatory activity assay

[0146] The cells were cultured at 2.5 × 10 4The cells were inoculated into 96-well plates and cultured for 24 h. The supernatant was discarded and the normal control group and model control group (LPS concentration was 5 μg mL -1 ) and the ethanol extract of Saposhnikovia divaricata group (ethanol extract of Saposhnikovia divaricata and 5 μg·mL -1 LPS) co-intervention cells. After drug intervention for 24 hours, the cell supernatant was taken to detect the NO, IL-6 and IL-1β contents according to the kit instructions. The results are shown in the following table:

[0147] Table 9 Anti-inflammatory activity results of 17 batches of Saposhnikovia divaricata samples

[0148]

[0149]

[0150] Note: Compared with the normal control group, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01

[0151] Compared with the normal control group, the levels of NO, IL-6 and IL-1β in the model control group were significantly increased (P<0.01), indicating that the RAW264.7 inflammatory model was successfully established; compared with the model control group, the tested drug groups could reduce the levels of NO, IL-6 and IL-1β to varying degrees, indicating that the alcohol extract of Saposhnikovia divaricata at a concentration of 200 μg·mL -1 It has a certain anti-inflammatory effect. Among them, the samples S8-S11 and S17 of Saposhnikovia divaricata have a strong inhibitory effect on the expression of inflammatory factor NO in cells, S7, S9-S11 and S17 have a strong inhibitory effect on the expression of inflammatory factor IL-6 in cells, and S5, S8-S11 have a strong inhibitory effect on the expression of inflammatory factor IL-1β. It may be because the samples S5, S8-S11 of Saposhnikovia divaricata are rich in cimicifuga glycosides, 5-O-methylvisaminol glycosides, cimicifuga, chelidonol glycosides and 5-O-methylvisaminol and have a high overall quality, and the samples S7 and S17 have other low-component and high-activity substances, so the anti-inflammatory activity is strong.

[0152] Example 5 Bivariate Correlation Analysis

[0153] The correlation analysis between the 10 chemical components and the expression levels of NO, IL-6 and IL-1β was performed, and the results are shown in Table 10.

[0154] Table 10 Correlation coefficients between chemical composition variable group and pharmacodynamic variable group

[0155]

[0156] Note: “*” indicates significant P<0.05; “**” indicates extremely significant P<0.01.

[0157] As shown in Table 10, cimicifuga glycosides, 5-O-methylvisaminol glycosides, cimicifuga, hyaluronic acid glycosides, and 5-O-methylvisaminol were negatively correlated with NO expression;

[0158] 5-O-methylvisaminol glycoside, 5-O-methylvisaminol, and imperatorin were negatively correlated with IL-6 expression;

[0159] Cimicifuga glycosides, 5-O-methylvisaminol glycosides, and chelidonol glycosides were negatively correlated with the expression of IL-1β.

[0160] The results showed that the compounds cimicifuga glycosides, 5-O-methylvisaminol glycosides, cimicifuga, chamaedryl glycosides, 5-O-methylvisaminol, and imperatorin were positively correlated with the anti-inflammatory activity and were the main active ingredients of Saposhnikovia divaricata to exert anti-inflammatory effects.

[0161] The data in Table 9 were dimensionlessly processed by the initialization method. Then, the levels of NO, IL-6 and IL-1β were used as reference sequences, and the chemical component contents were used as comparison sequences. The grey correlation coefficients between the reference sequences and the comparison sequences were calculated. The results are shown in Table 11.

[0162] Table 11 Grey correlation between chemical component contents and anti-inflammatory indexes of Fangfeng medicinal materials

[0163]

[0164]

[0165] As shown in Table 10, the order of contribution of components with greater correlation with NO expression is as follows: chamoyl glycosides > 5-O-methylvisaminol > 5-O-methylvisaminol glycosides > cimicifuga glycosides > cimicifuga > Zanthoxylum toxin > bergamot lactones > imperatorin > psoralen > chamoyl glycosides; the order of contribution of components with greater correlation with IL-6 expression is as follows: chamoyl glycosides > 5-O-methylvisaminol > 5-O-methylvisaminol glycosides > cimicifuga glycosides > cimicifuga > Zanthoxylum toxin > imperatorin > bergamot lactones > psoralen > chamoyl glycosides; the order of contribution of components with greater correlation with IL-1β expression is as follows: chamoyl glycosides > 5-O-methylvisaminol > cimicifuga > 5-O-methylvisaminol glycosides > cimicifuga glycosides > Zanthoxylum toxin > imperatorin > bergamot lactones > psoralen > chamoyl glycosides. The correlation between each index and each efficacy index was greater than 0.6, indicating that the in vitro anti-inflammatory effect of Saposhnikovia divaricata was the result of the synergistic effect of multiple components. Among them, the correlation coefficients of cimicifuga glycosides, 5-O-methylvisaminol glycosides, cimicifuga, chelidonol glycosides, and 5-O-methylvisaminol with NO, IL-6, and IL-1β ranked high and were all greater than 0.8, indicating that these five chemical components were closely related to the three inflammatory indicators and were the main chemical components reflecting the efficacy of Saposhnikovia divaricata.

[0166] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a windproof quality evaluation system based on the content of multiple index components. It is characterized in that The steps include: Step 1. Collect the Fangfeng herbs from different origins and different growing years; Step 2. Use liquid chromatography to determine the content of the index components in the Fangfeng medicinal material; according to the content measurement results, cluster analysis is performed on the Fangfeng medicinal materials from different origins and different growth years to analyze the relationship between the origin of the medicinal material and the quality of the medicinal material; Step 3. Establish a lipopolysaccharide-induced cellular inflammation model, study the relationship between the index components and the anti-inflammatory effect, and find out the components that contribute most to the anti-inflammatory effect; In the step 2, liquid chromatography is used to determine the following index components of the Fangfeng medicinal material: cimicifuga glycosides, 5-O-methylvisaminol glycosides, cimicifuga, chelidonol glycosides, psoralen, zanthoxylum toxin, bergamot lactone, imperatorin, 5-O-methylvisaminol, chelidonol; The same chromatographic conditions were used to determine the various index components of Saposhnikovia divaricata. The chromatographic conditions included: ODS C 18 As the stationary phase, water was used as the mobile phase A and acetonitrile was used as the mobile phase B for gradient elution. The gradient elution program was: 0 min, 75-85% A, 15-25% B, 8 min, 55~65%A, 35~45%B, 20min, 40~50%A, 50~60%B, 25min, 40~50%A, 50~60%B, 35min, 30~40%A, 60~70%B, 40min, 15-25%A, 75-85%B; In the step 3, the ethanol extract of Saposhnikovia divaricata is co-cultured with RAW264.7 cells induced by lipopolysaccharide for 12 to 48 hours, and then the contents of NO, IL-6 and IL-1β in the cultured cell supernatant are determined; grey correlation analysis is performed based on the results of the determined NO, IL-6 and IL-1β contents; and bivariate correlation analysis is performed between the chemical components of Saposhnikovia divaricata and the contents of NO, IL-6 and IL-1β; In step 3, the method for preparing the siler powder comprises: taking siler powder, adding methanol, ultrasonically extracting, filtering, concentrating the filtrate, and freeze-drying the concentrate to obtain the siler alcohol extract.

2. A method for constructing a windproof quality evaluation system based on multi-index component content according to claim 1, It is characterized in that The gradient elution program was: 0 min, 80%A, 20%B, 8 min, 59.5%A, 40.5%B, 20min, 45%A, 55%B, 25min, 45%A, 55%B, 35min, 35%A, 65%B, 40min, 20%A, 80%B.

3. A method for constructing a windproof quality evaluation system based on multi-index component content according to claim 2, It is characterized in that During liquid chromatography determination, the mobile phase flow rate was 0.8~1.2 mL / min, the column temperature was 22~27℃, and the wavelength was 254 nm.

4. A method for constructing a windproof quality evaluation system based on multi-index component content according to claim 3, It is characterized in that The liquid chromatography determination includes the following steps: Preparation of the test solution: Take 0.1-0.3 g of Radix Saposhnikoviae in a conical flask, add 3-12 mL of methanol solution, extract by ultrasonic for 10-120 min, cool, make up the weight loss with methanol, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the solution; Preparation of reference substance solution: accurately weigh the mixed reference substance and prepare 1 mL of methanol solution containing 0.11 μg of cimicifuga glycosides, 0.17 μg of 5-O-methylvisaminol glycosides, 0.018 μg of cimicifuga, 0.014 μg of hyaluronic acid glycosides, 0.002 μg of 5-O-methylvisaminol, 0.002 μg of psoralen, 0.003 μg of xanthoxylum toxin, 0.002 μg of hyaluronic acid, 0.001 μg of bergamot lactone, and 0.004 μg of imperatorin; Take 5~20 μL of the test solution and reference solution respectively, inject and measure.

5. The method for constructing a windproof quality evaluation system based on multi-index component content according to claim 1, It is characterized in that In the step 2, the content of index components of the fangfeng medicinal materials from different origins and different growing years is standardized and then subjected to principal component analysis to find out the comprehensive quality characteristics that can fully reflect the fangfeng medicinal materials.

6. A method for constructing a windproof quality evaluation system based on multi-index component content according to claim 1, It is characterized in that The inflammation model is a RAW264.7 cell inflammation model induced by lipopolysaccharide.

7. A method for constructing a windproof quality evaluation system based on multi-index component content according to claim 5, It is characterized in that In the step 3, the ethanol extract of Saposhnikovia divaricata is co-cultured with RAW264.7 cells induced by lipopolysaccharide for 24 hours, and then the contents of NO, IL-6 and IL-1β in the cell supernatant after culture are determined.

8. A method for constructing a windproof quality evaluation system based on multi-index component content according to claim 5, It is characterized in that In step 3, a normal control group and a model control group are set up simultaneously during the culture.

9. The method for constructing a windproof quality evaluation system based on multi-index component content according to claim 5, It is characterized in that In the step 3, 2-10 g of radix fangfeng powder is taken, methanol is added at a solid-liquid ratio of 1:15-60, ultrasonic extraction is performed for 20-120 min, the mixture is filtered, the filtrate is concentrated, and the concentrated solution is freeze-dried to obtain a radix fangfeng alcohol extract.

10. A method for constructing a windproof quality evaluation system based on multi-index component content according to claim 7, It is characterized in that Taking the contents of NO, IL-6 and IL-1β as reference sequences and the contents of chemical components as comparison sequences, the grey correlation coefficient between the reference sequence and the comparison sequence was calculated to find out the relationship between each chemical component and the efficacy.

11. A method for constructing a windproof quality evaluation system based on multi-index component content according to claim 7, It is characterized in that Combining principal component and grey relational analysis, the main index components that exert anti-inflammatory activity were found.

12. A windproof quality evaluation system constructed according to the construction method according to any one of claims 1-11.

13. The windproof quality evaluation system according to claim 12, It is characterized in that The quality evaluation system evaluates the quality of the Fangfeng medicinal material by detecting the contents of 10 indicator components in the Fangfeng medicinal material, and the 10 indicator components are cimicifuga glycosides, 5-O-methylvisaminol glycosides, cimicifuga, chelidonol glycosides, psoralen, zanthoxylum toxin, bergamot lactone, imperatorin, 5-O-methylvisaminol, and chelidonol.

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