Fomes officinalis quality control method

Abstract

The invention belongs to the technical field of quality control, and provides a fomes officinalis quality control method which comprises the following steps: 1) analyzing high performance liquid characteristic chromatograms of fomes officinalis samples with different colors; 2) determining the Ali red degree value; 3) analyzing cell viability of fomes officinalis ames samples with different colors; 4) analyzing the correlation between the chromatic value and the content of the effective component; 5) determining LD50 of the fomes officinalis crude product and processed product mice through a single-time administration acute toxicity experiment of the mice; according to the fomes officinalis quality control method provided by the invention, the problems of insufficient fomes officinalis quality control means and low detection precision in the prior art are effectively solved; according to the method, various quality indexes of fomes officinalis ames are systematically and strictly controlled, and the stability and reliability of fomes officinalis ames products are ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of quality control, and particularly relates to a method for quality control of Fomes officinalis. Background Art

[0002] Fomes officinalis (Vill.ex Fr.) Ames is the dried mycelium of the medicinal pore fungus in the family Polyporaceae, and is a genuine medicinal material with a long application history in traditional Chinese medicine and Uyghur medicine.

[0003] However, there are relatively few research reports on the quality standards of Fomes officinalis at present, resulting in the lack of unified and scientific standards for its quality control. Traditionally, in ancient Uyghur medical books, the quality grades of Fomes officinalis were mainly described based on color. It was considered that white Fomes officinalis had the best quality, followed by yellow and red, while black had the worst quality and might be toxic and should be removed. Although this color-based identification method has certain reference value to a certain extent, its subjectivity and inaccuracy limit its application in modern medicinal material quality control.

[0004] At present, the quality evaluation methods of traditional Chinese medicinal materials are constantly developing, but a complete, scientific and widely accepted standard system has not yet been formed. Especially for medicinal materials such as Fomes officinalis with significant color differences, the traditional color identification method is difficult to meet the needs of modern medicinal material quality control. Therefore, it is particularly important to develop a method based on scientific principles that can objectively and accurately evaluate the quality of Fomes officinalis.

[0005] In recent years, with the development of chromatographic technology, spectroscopic technology and biotechnology, the quality control means of traditional Chinese medicinal materials have been greatly enriched and improved. The application of modern analytical means such as high performance liquid chromatography (HPLC) fingerprint technology, color difference meter measurement technology and cytotoxicity experiment provides new ideas and methods for the quality control of traditional Chinese medicinal materials. Especially based on the research idea of "association between appearance and essence", that is, by analyzing the correlation between the apparent color of medicinal materials and their internal chemical components, biological activities and toxicity, it provides a new perspective for the quality control of traditional Chinese medicinal materials.

[0006] Therefore, those skilled in the art have proposed a method for quality control of Fomes officinalis to achieve a scientific and objective evaluation of the quality of Fomes officinalis medicinal materials, and provide new scientific bases and technical means for the quality control of Fomes officinalis medicinal materials to solve the problems raised in the background art Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a method for quality control of Fomes officinalis, by establishing a quality control standard of Fomes officinalis based on color quantization values, including:

[0008] S1. Preparation of different - colored samples of Fomitopsis officinalis: Take the raw medicinal materials of Fomitopsis officinalis, divide them into different - colored samples according to the color from light to dark. Break the dry products into pieces, sieve and grind them into powders, and pass them through a 100 - mesh sieve to obtain Fomitopsis officinalis powder samples, which are numbered from A1 to An in order of color depth, where n is the total number of different colors of Fomitopsis officinalis samples.

[0009] S2. Preparation of test solution: Weigh precisely a certain amount of each of the different - colored Fomitopsis officinalis powder samples obtained in step S1, place them in a stoppered Erlenmeyer flask, add an appropriate amount of methanol or other suitable solvents, carry out ultrasonic extraction, make up the weight loss after cooling, and filter to obtain the test solution.

[0010] S3. Analysis of the high - performance liquid chromatography (HPLC) characteristic chromatogram of different - colored Fomitopsis officinalis samples: Using high - performance liquid chromatography, select appropriate chromatographic columns and mobile - phase conditions, carry out gradient elution on the test solution obtained in step S2, and record the chromatogram data.

[0011] S4. Determination of the chromaticity value of Fomitopsis officinalis: Use a color difference meter to measure different - colored Fomitopsis officinalis powder samples, record their L*, a*, b* values, and calculate the chroma E*ab value, which is used as a quantitative index for the chromaticity value of Fomitopsis officinalis.

[0012] S5. Analysis of the cell activity of different - colored Fomitopsis officinalis samples: Use an appropriate cell line, such as PC12 cells, to establish an oxidative damage model. Through the MTT method or other appropriate cell - activity detection methods, detect the effect of different - colored Fomitopsis officinalis samples on the survival rate of oxidatively damaged cells and evaluate their cell activity.

[0013] S6. Analysis of the correlation between chromaticity values and the content of active ingredients: Use statistical software to perform Pearson correlation analysis on the chromaticity parameters L*, a*, b*, E*ab values and the content of active ingredients of different - colored Fomitopsis officinalis samples, as well as principal component analysis (PCA) and orthogonal partial least - squares discriminant analysis (OPLS - DA) to screen out differential markers.

[0014] S7. Conduct acute toxicity experiments: Through single - dose acute toxicity experiments, determine the LD 50 value of the raw and processed products of Fomitopsis officinalis on experimental animals, such as mice. According to the LD 50 value, grade the acute toxicity of the test substances, and combine the poisoning symptoms and pathological changes to comprehensively evaluate the acute toxicity characteristics and possible toxic target organs of the test substances.

[0015] S8. Establish a quality control standard for Fomitopsis officinalis based on color quantization values: According to the results of steps S4 to S7, combined with the correlation between the chromaticity value, the content of active ingredients, cell activity, and acute toxicity of Fomitopsis officinalis samples, formulate a quality control standard for Fomitopsis officinalis based on color quantization values.

[0016] Preferably, in S2, 0.5 g of the powder samples of different colors of Fomitopsis officinalis are accurately weighed, placed in a 100-ml stoppered Erlenmeyer flask, 25 mL of methanol is added, ultrasonic extraction is carried out for 15 min, after cooling to room temperature, the weight loss is made up, and the test solution is obtained by filtration.

[0017] Preferably, in S3, the high performance liquid chromatography method adopted has a chromatographic column of C18 (250 mm × 4.6 mm, 5 μm), the mobile phase is 0.2% formic acid solution (A) and acetonitrile (B), and the gradient elution program is 0 - 2 min, 5% B; 2 - 10 min, 5% - 52% B; 10 - 30 min, 52% - 65% B; 30 - 45 min, 65% B; 45 - 50 min, 65% - 90% B; 50 - 52 min, 90% - 100% B; 52 - 57 min, 100% B; 57 - 62 min, 100% - 5% B, the detection wavelength is 254 nm, the column temperature is 30 °C, the flow rate is 1 mL / min, and the injection volume is 10 μL.

[0018] Preferably, the cell line adopted in step S5 is PC12 cells, an oxidative damage model is established by H2O2 induction, and the MTT method is used to detect the survival rate of PC12 cells with oxidative damage by different color samples of Fomitopsis officinalis.

[0019] Preferably, the statistical software used in step S6 is SPSS 23.0 or higher version, and SIMCA 14.1 or higher version. By performing Pearson correlation analysis, PCA and OPLS-DA analysis on the chromaticity parameters and active ingredient contents of different color samples of Fomitopsis officinalis, differential markers are screened out.

[0020] Preferably, the experimental animals used in step S7 are KM mice. The LD 50 value of the raw product and processed product of Fomitopsis officinalis is determined by a single-dose acute toxicity experiment, and combined with the poisoning symptoms and pathological changes, the acute toxicity characteristics and possible toxic target organs of the test substance are comprehensively evaluated.

[0021] Preferably, the quality control standard of Fomitopsis officinalis established in step S8 based on the color quantization value includes the chromaticity value range, active ingredient content range, cell activity evaluation standard and acute toxicity grading standard of Fomitopsis officinalis samples.

[0022] High performance liquid chromatography (HPLC): used in this article to analyze the high performance liquid characteristic chromatogram of different color samples of Fomitopsis officinalis to determine its internal chemical components.

[0023] Color difference meter: used in this article to measure the chromaticity value of Fomitopsis officinalis to quantify its color characteristics.

[0024] PC12 cells: used in this article to establish an oxidative damage model to evaluate the effects of different-colored samples of Fomitopsis officinalis on the survival rate of oxidatively damaged cells.

[0025] MTT method: used in this article to detect the survival rate of PC12 cells with oxidative damage by different-colored samples of Fomitopsis officinalis.

[0026] Pearson correlation analysis: used in this article to analyze the correlation between the chromaticity parameters and the contents of active ingredients of different-colored samples of Fomitopsis officinalis.

[0027] Principal component analysis (PCA): used in this article to distinguish different-colored samples of Fomitopsis officinalis and reveal the variation rules of their internal chemical components.

[0028] Orthogonal partial least squares discriminant analysis (OPLS-DA): used in this article to further analyze the differences between different-colored samples of Fomitopsis officinalis and screen out differential markers.

[0029] LD 50 : used in this article to determine the acute toxicity of raw and processed Fomitopsis officinalis on mice to evaluate its safety.

[0030] H2O2: used in this article as an inducer to establish a PC12 cell oxidative damage model to evaluate the antioxidant activity of different-colored samples of Fomitopsis officinalis.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. By providing a method for quality control of Fomitopsis officinalis, the present invention effectively solves the problems of insufficient quality control means and low detection accuracy in the prior art; this method systematically and strictly controls various quality indicators of Fomitopsis officinalis, ensuring the stability and reliability of Fomitopsis officinalis products.

[0033] 2. By adopting advanced detection technologies and algorithms, the present invention improves the accuracy and efficiency of Fomitopsis officinalis quality detection; compared with traditional manual detection methods, the method of the present invention has a higher degree of automation and intelligence level, greatly reducing human errors and missed detection rates.

[0034] 3. The method of the present invention is not only applicable to the quality control of Fomitopsis officinalis, but also can provide useful references for the quality control of other Chinese medicinal materials; the popularization and application of this method will help improve the overall quality control level of the Chinese medicinal materials industry and promote the healthy development of the Chinese medicinal materials industry. Description of the Drawings

[0035] Figure 1 It is a diagram of different-colored samples of Fomitopsis officinalis of the present invention; wherein A is the raw medicine;

[0036] Figure 2 The spectrum diagram of Inonotus obliquus of the present invention; wherein, S1-S5 are A1-A4 and crude drugs respectively.

[0037] Figure 3 The morphological diagram of the influence of test substance A1 of the present invention on the activity of PC12 cells with oxidative damage; wherein, A: normal control group, B: model control group, C: 70 μg / mL group, D: 60 μg / mL group, E: 50 μg / mL group, F: 40 μg / mL group, G: 30 μg / mL group, H: 20 μg / mL group, I: 10 μg / mL group.

[0038] Figure 4 The morphological diagram of the influence of test substance A2 of the present invention on the activity of PC12 cells with oxidative damage; wherein, A: normal control group, B: model control group, C: 120 μg / mL group, D: 110 μg / mL group, E: 100 μg / mL group, F: 90 μg / mL group, G: 80 μg / mL group, H: 70 μg / mL group, I: 60 μg / mL group.

[0039] Figure 5 The PCA score schematic diagram of different color samples of Inonotus obliquus of the present invention.

[0040] Figure 6 The OPLS-DA score scatter schematic diagram of different color samples of Inonotus obliquus of the present invention.

[0041] Figure 7 The OPLS-DA VIP value schematic diagram of different color samples of Inonotus obliquus of the present invention.

[0042] Figure 8 The schematic diagram of the model cross-validation result of the present invention. Detailed implementation manners

[0043] The following further describes the implementation manners of the present invention in detail with reference to the drawings and embodiments. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0044] Embodiment 1: The present invention provides a method for quality control of Inonotus obliquus, by establishing a quality control standard for Inonotus obliquus based on color quantization values, including the following steps:

[0045] 1) Analyze the high-performance liquid chromatography characteristic chromatograms of different color samples of Inonotus obliquus;

[0046] 2) Measure the chromaticity values of Inonotus obliquus;

[0047] 3) Analyze the cell activities of different color samples of Inonotus obliquus;

[0048] 4) Analyze the correlation between chromaticity values and the contents of active ingredients;

[0049] 5) Through the acute toxicity experiment of single-dose administration to mice, the LD of Fomitopsis officinalis in mice was determined 50 .

[0050] Specifically, in the present invention, a color difference meter will be used to measure Fomitopsis officinalis powders of different colors, and the L*, a*, and b* values of Fomitopsis officinalis will be read and recorded. Among them, the L* value represents the brightness of the color, and the larger the value, the higher the brightness; the a* value represents the red-green value, a positive number indicates a red bias, and a negative number indicates a green bias; the b* value represents the yellow-blue value, a positive number indicates a yellow bias, and a negative number indicates a blue bias; the chroma E*ab value = (a*2 + b*2)1 / 2. Repeat 3 times and take the average value.

[0051] Specifically, in the present invention, H2O2 will be used as an inducer to establish a PC12 cell oxidative damage model, and the MTT method will be used to detect the survival rate of the oxidative damaged PC12 cells by the active test substance, and the protective effect of the test substance on the oxidative damaged model cells will be observed.

[0052] Specifically, in the present invention, the SPSS 23.0 analysis software will be used to conduct a Pearson correlation analysis on the spatial chromaticity parameters of different color samples of Fomitopsis officinalis, namely the L*, a*, b*, and E*ab values and the contents of its 11 common components.

[0053] Specifically, in the present invention, the contents of 11 components of different color samples of Fomitopsis officinalis will be imported into the SIMCA 14.1 software and the SPSS 21.0 software for principal component analysis.

[0054] Specifically, on the basis of the classification of PCA, the present invention will further conduct supervised orthogonal partial least squares discriminant analysis (OPLS-DA). The SIMCA 14.1 software will be used to establish a data model of OPLS-DA, obtain an OPLS-DA score plot, establish a variable importance in projection (VIP) value plot, and use this to screen the differential markers of Fomitopsis officinalis color samples.

[0055] Specifically, in the present invention, through the acute toxicity experiment of single-dose administration to mice, the LD of Fomitopsis officinalis in mice was determined 50 .

[0056] Example 1

[0057] I. Materials and Instruments

[0058] 1. Source of medicinal materials: Fomitopsis officinalis medicinal materials were purchased from Xinjiang Ensar Uyghur Medicine Decoction Pieces Co., Ltd. and were identified as Fomitopsis officinalis by Researcher Zhu Jun of the Xinjiang Uygur Autonomous Region Institute of Traditional Chinese Medicine and Ethnic Medicine, meeting the Xinjiang Uygur Autonomous Region Decoction Piece Standard 2020 Edition.

[0059] 2. Test materials: PC12 cells (rat adrenal pheochromocytoma cells), sourced from the Chinese Academy of Sciences Cell Bank.

[0060] II. Experimental Methods

[0061] 1. HPLC Characteristic Chromatograms of Different Color Samples of Fomitopsis officinalis

[0062] 1.1 Preparation of Different Color Samples of Fomitopsis officinalis

[0063] Take the crude medicinal materials of Fomitopsis officinalis, separate different color samples of the same batch of Fomitopsis officinalis, break the dry products, sieve and grind them on a sieve, and pass part of the powder through a 100-mesh sieve to obtain Fomitopsis officinalis powder. The different color samples of Fomitopsis officinalis are numbered as A1, A2, A3, and A4 in order from light to dark, as Figure 1 shown;

[0064] 1.1.1 Preparation of Test Solution

[0065] Precisely weigh 0.5 g of the powders of different color samples of Fomitopsis officinalis, place them in a 100-ml stoppered triangular flask, precisely add 25 mL of methanol, weigh, ultrasonically extract for 15 min, wait until it cools to room temperature, add methanol to make up for the weight loss, filter the liquid with filter paper, and pass the filtrate through a 0.45-μm microporous filter membrane. Take the subsequent filtrate as the test solution.

[0066] 1.1.2 Chromatographic Conditions

[0067] Use a C18 (250 mm×4.6 mm, 5 μm) chromatographic column, the mobile phase is 0.2% formic acid solution (A)-acetonitrile (B), gradient elution (0 - 2 min, 5% B; 2 - 10 min, 5% - 52% B; 10 - 30 min, 52% - 65% B; 30 - 45 min, 65% B; 45 - 50 min, 65% - 90% B; 50 - 52 min, 90% - 100% B; 52 - 57 min, 100% B; 57 - 62 min, 100% - 5% B); the detection wavelength is 254 nm; the column temperature is 30°C; the flow rate is 1 mL / min; the injection volume is 10 μL. Use Empower software to set the injection method and record the chromatogram data.

[0068] 1.1.3 Fingerprint Analysis

[0069] Analyze the chromatograms of different color samples of Fomitopsis officinalis by HPLC, and a total of 11 common peaks are obtained. The chromatogram is as Figure 2 shown; by comparing the fingerprint with the chromatographic peak area, there are 11 common peaks, so 11 common components are determined.

[0070] 1.2 Determination of Color Value of Fomitopsis officinalis

[0071] Measure the powder of Fomitopsis officinalis with different colors using a color difference meter, read out and record the L*, a*, and b* values of Fomitopsis officinalis. Among them, the L* value represents the brightness of the color, and the larger the value, the higher the brightness; the a* value represents the red-green value, with positive numbers indicating a red bias and negative numbers indicating a green bias; the b* value represents the yellow-blue value, with positive numbers indicating a yellow bias and negative numbers indicating a blue bias

[10] ; the chroma E*ab value = (a*2 + b*2)1 / 2. Repeat 3 times and take the average. The results are shown in Table 1;

[0072] Table 1 Determination results of chromaticity values (n = 3)

[0073]

[0074] By measuring the chromaticity values of different color samples of Fomitopsis officinalis, the L* and E*ab of samples A1 - A4 show a downward trend, while a* and b* show a trend of rising first and then falling. The closer the color is to black, the smaller the brightness L*, and the smaller the overall chromaticity value E*ab.

[0075] 1.3 Analysis of cell viability of different color samples of Fomitopsis officinalis

[0076] Establish a PC12 cell oxidative damage model using H2O2 as an inducer, and detect the survival rate of the oxidative damaged PC12 cells by the MTT method to observe the protective effect of the test substance on the oxidative damaged model cells.

[0077] 1.3.1 Cell culture

[0078] PC12 cells are cultured in a 25 cm 2 cell culture flask. The cell culture medium is DMEM high-glucose medium with a 10% FBS + 1% penicillin-streptomycin mixed solution. It is placed in a 37°C, 5% CO2 cell culture incubator for routine culture. Passage culture is carried out at a ratio of 1:3 every 3 - 4 days, and the culture medium is changed on the day after passage.

[0079] 1.3.2 Effects of different color samples of Fomitopsis officinalis on the proliferation of PC12 cells

[0080] Collect PC12 cells in the logarithmic growth phase, digest them routinely, centrifuge, count, and adjust the cell concentration to 1×10 5Cells were inoculated at a density of

[0081] Table 2 Effects of samples of different colors on the survival rate of PC12 cells (n = 3)

[0082]

[0083] After the four test substances acted on PC12 cells, when the concentrations of test substances A1 and A2 were as low as 50 and 100 μg / mL respectively, test substances A1 and A2 showed a certain promoting effect on the activity of PC12 cells. As the administration concentrations of test substances A1 and A2 decreased, there was no obvious promoting effect on the activity of PC12 cells. Therefore, test substances A1 and A2 with concentrations of about 50 and 100 μg / mL were selected for subsequent experiments. Each concentration group of test substances A3 and A4 had obvious cytotoxic effects on PC12 cells. Therefore, the protective effects of test substances A3 and A4 on H2O2-induced oxidative damage of PC12 cells were not studied in the subsequent experiments.

[0084] 1.3.3 Establishment of PC12 cell oxidative damage model

[0085] PC12 cells in the logarithmic growth phase were collected, digested conventionally, centrifuged, counted, and the cell concentration was adjusted to 1×10 5Inoculate at a density of [[ID=]],

[0086] After H2O2 acted on PC12 cells for 24 h, H2O2 at each concentration had a certain inhibitory effect on the activity of PC12 cells. Among them, the inhibitory effect on the activity of PC12 cells was the most significant at a concentration of 800 μmol / L. When the concentration reached 400 μmol / L, the average cell survival rate was 66.64%. When the concentration was lower than 400 μmol / L, the cell activity was above 80%. Therefore, for subsequent experiments, a concentration of 400 μmol / L H2O2 was selected as the concentration for establishing the oxidative damage model of PC12 cells.

[0087] 1.3.4 Effects of different colored samples of Fomes officinalis on the proliferation of PC12 cells with oxidative damage

[0088] The oxidative damage model of PC12 cells was established as described in item 2.3.3. The test substances A1 and A2 were dissolved with the help of dimethyl sulfoxide and then serially diluted to the required concentrations with high-glucose DMEM culture medium containing 10% FBS. After the cell model was established, 100 μL of cell culture medium was added to the blank group and the model control group. The corresponding concentrations of the test substances were added to each experimental group in a 96-well plate. The final concentrations of test substance A1 were 10, 20, 30, 40, 50, 60, and 70 μg / mL, and the final concentrations of test substance A2 were 60, 70, 80, 90, 100, 110, and 120 μg / mL. There were 6 replicates for each concentration group, and the cells were continuously cultured in a cell incubator. After observing the cell growth under a microscope after 24 h, the MTT method was used to detect the cell survival rate, and the experiment was repeated 3 times. The results are as Figure 3 、 Figure 4 shown;

[0089] When the test substances A1 and A2 are within a certain concentration range, they have a certain promoting effect on the activity of PC12 cells with oxidative damage. The results show that the optimal concentration of the white test substance for promoting the activity of PC12 cells with oxidative damage is 50 μg / mL, and the optimal concentration of the yellow test substance for promoting the activity of PC12 cells with oxidative damage is 120 μg / mL. The white test substance has a better effect on promoting cell activity compared with the yellow test substance.

[0090] 1.4 Correlation analysis between chromaticity value and active ingredient content

[0091] Literature research shows that there is a certain correlation between the change of appearance color and the content of its pharmacodynamic components. Pearson correlation analysis was carried out on the spatial chromaticity parameters L*, a*, b*, E*ab values of different color samples of Fomes officinalis and the content of its 11 common components by using SPSS 23.0 analysis software. The results are shown in Table 3;

[0092] Table 3 Results of correlation analysis between content of color samples and chromaticity values

[0093]

[0094] Note: 1) P < 0.1, 2) P < 0.05

[0095] The results show that C1 shows a high correlation with L* and E*ab in this experiment (P < 0.01), and C2, C8, C9, C10 show a significant correlation with L* and b* (P < 0.05). C1 shows a very significant negative correlation with lightness L* and chroma E*ab. As the color of Fomes officinalis deepens, the lightness L* and chroma E*ab gradually decrease, and the content of C1 also increases. C8, C9, C10 show a positive correlation with lightness L* and chroma E*ab. As the color of Fomes officinalis deepens, the lightness L* and chroma E*ab gradually decrease, and C8, C9, C10 become lower and lower.

[0096] 1.5 Principal component analysis (PCA)

[0097] Principal component analysis is an unsupervised data processing method. In order to distinguish different color samples, the content of 11 components of different color samples of Fomes officinalis was imported into SIMCA 14.1 software and SPSS 21.0 software for principal component analysis.

[0098] The results of the eigenvalues and variance contribution rates of PCA analysis show that the variance contribution rate of the first principal component is 57.056%, the second principal component is 29.818%, the third principal component is 13.126%, and the cumulative variance contribution rate of the first three principal components is 100%; the main information sources of the first 3 principal components include C1-C11, indicating that the first 3 principal components can reflect the information of the samples, and it is reasonable to use the first 3 principal components to analyze the overall data.

[0099] The data was imported into SIMCA14.1 software to establish a PCA model, and a PCA score plot was obtained. The R2X (cumulative explanatory ability parameter) and Q2 (prediction ability parameter) in the established PCA model were 0.869 and 0.96 respectively, both greater than 0.5, indicating that the established model has good accuracy and prediction ability. It can be seen from the PCA score plot that the samples were divided into 4 categories, and A1, A2, A3, and A4 were each divided into a separate category. It is proved that the PCA method can be used to distinguish different color samples of Fomitopsis officinalis. The results are as Figure 5 shown.

[0100] 1.6 Orthogonal partial least squares discriminant analysis (OPLS-DA)

[0101] In order to better present the inter-group differences between different samples, based on the classification of PCA, a supervised orthogonal partial least squares discriminant analysis (OPLS-DA) was further carried out. The SIMCA14.1 software was used to establish a data model for OPLS-DA, and an OPLS-DA score plot was obtained. A variable importance in projection (VIP) value plot was established to screen the differential markers of Fomitopsis officinalis color samples. The results are as Figure 6 、 7 shown,

[0102] The independent variable fitting index (R2x) of the established OPLS-DA model is 0.98, the dependent variable fitting index (R2y) is 0.874, and the model prediction index (Q2) is 0.607. R2 and Q2 exceeding 0.5 indicate that the model fitting result is acceptable and can be used for the analysis of Fomitopsis officinalis samples. It can be seen from the OPLS-DA score plot that the samples were divided into 4 categories, and its classification result is consistent with that of PCA, proving the reliability of the results. In the VIP plot, within the 95% confidence interval, generally VIP value > 1 is selected as the index for screening differential components as Figure 7 shown, and the components with VIP value > 1 are C6, C3, C5, C1, C9, C8, C10, C7. After 200 permutation tests (Permutation test), as Figure 8 , the intersection point of the Q2 regression line and the vertical axis is less than zero, indicating that the model does not have overfitting and the model verification is effective.

[0103] Example 2

[0104] I. Materials and Instruments

[0105] 1. Source of medicinal materials: The medicinal material of Fomes officinalis was purchased from Xinjiang Ensar Uyghur Medicine Decoction Pieces Pharmaceutical Co., Ltd. (batch number ALH-YP-210124), and was identified as Fomes officinalis by Researcher Zhu Jun of the Xinjiang Uyghur Autonomous Region Institute of Traditional Chinese Medicine and Ethnic Medicine, meeting the Xinjiang Uyghur Autonomous Region Decoction Piece Standard (2020 Edition).

[0106] 2. Solvent: The drinking water for experimental animals, pure water. The quality control refers to the requirements of "drinking water" under "Animal Feeding and Management Requirements". Prepare the medicinal liquid required for the drug administration group, and the control group is given the same volume of this solvent.

[0107] 3. Experimental animals

[0108] Selection and breeding of experimental animals: KM mice, SPF grade, weighing 18 - 20 g, with an equal number of males and females. They were purchased from the Xinjiang Experimental Animal Research Center, grade: SPF. License number: SCXK(Xin)2023 - 0001. The animals were raised in plastic cages for mice, with no more than 5 animals of the same sex in each cage, drinking water and feeding freely, at a room temperature of 20°C - 26°C, relative humidity of 40% - 70%, 12 h of light and 12 h of darkness; before the experiment, they were adaptively raised for 3 days, and animals meeting the requirements were selected as experimental animals through general behavior observation.

[0109] During grouping, animals that did not meet the inclusion criteria were excluded; for the extra animals that met the inclusion criteria, animals with body weights not meeting the experimental requirements were excluded. The excluded animals were sacrificed by cervical dislocation and frozen in the animal body freezing freezer, and the corpses were handed over to the Urumqi Municipal Solid Waste Disposal Center for disposal.

[0110] II. Test Methods

[0111] 1. Dose setting and its basis

[0112] Clinical dose: 1 g / 60 kg = 0.02 g / kg. Note: It causes hoarseness of the throat.

[0113] Before this test, the maximum preparation concentration (Cmax) of this test article was approximately 20.5% (0.205 g of the test article / ml).

[0114] Preliminary test results: The purpose of the preliminary test was to find out the doses at which 0% and 100% of the animals died or 10% and 90% of the animals died. From the preliminary test results, the 0% death dose was 2.05 g / kg, and the 90% death dose was 8.2 g / kg.

[0115] Preliminary test observation: After the animals were administered the drug, they became rigid, tremored, were dull, and lay prone. Animals died within three days after drug administration. Gross examination of the dead animals showed obvious flatulence in the gastrointestinal tract and mucus filling in the abdominal cavity, as shown in Table 4;

[0116] Table 4 Results of Acute Toxicity Preliminary Experiment of Raw Fomitopsis officinalis

[0117]

[0118] Formal experiment: LD of raw Fomitopsis officinalis 50 Determination, setting 5 doses, see Table 5 below;

[0119] Table 5 Acute Toxicity Dose Setting of Raw Fomitopsis officinalis

[0120]

[0121] Basis for dose setting:

[0122] This test sample is a toxic medicinal material. In the formal acute toxicity test, "LD 50 test" will be carried out, that is, 8.20, 5.74, 4.02, 2.81, 1.97 g of raw medicinal herbs / kg, dose interval: 0.7, maximum administration volume 40 ml / Kg, once a day, orally administered to mice, observe the toxic reaction, and calculate LD 50 .

[0123] 1.2 Animal grouping

[0124] Group setting: Set 5 groups, that is, 8.20, 5.74, 4.02, 2.81, 1.97 g of raw medicinal herbs / kg, dose interval: 0.7. Grouping method: After the adaptation period, the mice are fasted for 12 - 16 hours, their fasting weights are measured, and the stratified random method is used to stratify the 50 animals included in the experiment according to this weight and group them by gender respectively.

[0125] 1.3 Administration route and method

[0126] Administration route and basis: The same as the clinical administration route (oral), this experiment uses oral administration to mice. Administration concentration: The Fomitopsis officinalis administration group is given a liquid medicine with a concentration equivalent to 0.205 g of the test sample / ml. The control group is given an equal volume of drinking water for experimental animals. 40 ml / Kg / time. The control group and Fomitopsis officinalis are administered once a day. Administration time: Immediately after weighing the fasting weight and grouping.

[0127] 1.4 Selection basis for test stage and duration

[0128] Referring to the "Technical Guidelines for the Study of Single - Dose Toxicity of Drugs" (issued by CFDA on May 13, 2014), observe continuously for 2 weeks after single - dose administration. Taking the administration day as D0 and the next day as D1, observe until D14, and conduct autopsy on the surviving animals on D15.

[0129] 1.5 Observation, examination and detection

[0130] 1.5.1 Observation of toxic symptoms and general status

[0131] (1) Observation content: Observe and record the general condition, hair color, activities, gait, expression, feces, urine, etc. of the animals. Observe whether there are any poisoning symptoms, the time of occurrence, the duration, and the recovery situation, and record the dead or dying animals.

[0132] (2) On the day of drug administration: Observe closely for 4 hours after drug administration.

[0133] (3) During the observation period after drug administration: Observe twice a day.

[0134] 1.5.2 Treatment at the end of the observation period

[0135] On D14, after weighing the body weight and the remaining feed, fast the animals in the afternoon without depriving them of water. On the next day of fasting (fasting time: 12 - 16 hours), weigh the fasting body weight, transfer the animals to the dissection room for euthanasia. Euthanize by cervical dislocation. Conduct gross pathological observation, observe the changes of various organs (including but not limited to the heart, liver, spleen, lungs, kidneys, adrenal glands, brain, stomach, intestines, testes, prostate, ovaries, uterus), take the brain, lungs, liver, spleen, kidneys, gastrointestinal tract, fix them with 10% neutral formalin, embed them in paraffin, section them, stain them with HE (Haematoxylin & Eosin), and conduct histopathological examination under a microscope.

[0136] 1.5.3 Examination of dead and dying animals during the experiment

[0137] For the animals that died during the experiment, conduct necropsy in a timely manner; during the necropsy, conduct gross pathological observation, and the examination content is the same.

[0138] 1.6 Data statistics and analysis

[0139] Calculate LD using SPSS statistical software 50 .

[0140] Combined with the above toxicity symptoms, general condition observation, and necropsy results, comprehensively judge the acute toxicity of the crude product of Fomes officinalis by oral administration to mice.

[0141] 3. Summary and conclusion

[0142] 3.1 Experiment overview

[0143] Determination of LD of the crude product of Fomes officinalis, that is, administer to mice orally at 8.20, 5.74, 4.02, 2.81, 1.97 g crude drug / kg, dose interval: 0.7, maximum administration volume 40 ml / Kg, once a day, observe the toxicity reaction, and calculate LD 50 . See Table 6; 50 .

[0144] Table 6 LD of the crude product of Fomes officinalis 50 Results

[0145]

[0146] The data of 1 - 4 groups were statistically analyzed by SPSS to obtain LD 50 which was 5.2 g / kg. The LD 50 95% confidence limit was 4.3 - 6.4 g / kg.

[0147] 3.2 Test results

[0148] After the animals were administered the drug, they showed catalepsy, tremors, dullness, and lying prone for about 6 hours, and began to move slowly after 6 hours. Deaths of animals began to occur 2 hours after drug administration, and animals died within four days after drug administration. Gross examination of the dead animals showed obvious flatulence in the gastrointestinal tract and the abdominal cavity was filled with mucus.

[0149] On D14 after drug administration, all surviving animals were fasted overnight (12 hours), and the mice were dissected the next day. The main visceral tissues of the mice were observed macroscopically, and all the mice had mild flatulence in the gastrointestinal tract.

[0150] The crude product of Fomes officinalis has a certain effect on the gastrointestinal tract of mice.

[0151] Conclusion: The LD 50 of the crude product of Fomes officinalis was 5.2 g / kg (5200 mg / kg). The LD 50 > 5000 mg / kg, belonging to the practically non - toxic level.

[0152] Example 3

[0153] I. Materials and Instruments

[0154] The materials and instruments were the same as those in Example 2.

[0155] II. Test Methods

[0156] 1. Dose Setting and Its Basis

[0157] The processed product of Fomes officinalis (freeze - pulverized); Usage and dosage: 0.5 - 1 g; Clinical dose: 1 g / 60 kg = 0.02 g / kg

[0158] Before this test, the maximum preparation concentration (Cmax) of this test article was explored to be approximately 25.6% (0.256 g of test article / ml), as shown in Table 7;

[0159] Table 7 Acute Toxicity Preliminary Experiment of the Processed Product of Fomes officinalis

[0160]

[0161] One dose was set for the formal test, as shown in Table 8 below:

[0162] Table 8 Dose Setting of Acute Toxicity Experiment of the Processed Product of Fomes officinalis

[0163] Dose Dose Clinical dose Dosing volume Frequency of administration Concentration of dispensed drug g (crude drug) / Kg g (test article) / Kg Multiple ml / Kg / time times / day g (test article) / ml 2.05 2.05 102.5 40 1 0.256

[0164] Dosage setting basis:

[0165] According to the preliminary test results, the formal acute toxicity test will conduct the "maximum tolerated dose test", that is, mice are orally administered at the maximum formulated concentration of 25.6%, the maximum dosing volume of 40 ml / Kg, once a day, and the toxic reactions are observed.

[0166] 1.2 Animal grouping

[0167] Grouping method: After the adaptation period, the mice are fasted for 12 - 16 hours, their fasting weights are weighed, and the stratified random method is used to group the 40 animals included in the test by gender according to this weight stratification.

[0168] 1.3 Administration route and method

[0169] Administration route and basis: The same as the clinical administration route (oral), this test uses oral administration to mice. Administration concentration: The group administered with the processed product of Fomes officinalis Ames is given a medicinal solution equivalent to a concentration of 0.256 g of the test substance / ml. The control group is given an equal volume of drinking water for experimental animals. 40 ml / Kg / time. Both the control group and Fomes officinalis Ames are administered once a day. Administration time: Immediately after weighing the fasting weight and grouping.

[0170] 1.4 Selection basis for test stage and duration

[0171] Referring to the "Technical Guidelines for the Study of Single - Dose Toxicity of Drugs" (issued by CFDA on May 13, 2014), continuous observation is carried out for 2 weeks after single - dose administration. Taking the administration day as D0 and the next day as D1, observation is carried out until D14, and the surviving animals are dissected on D15.

[0172] 1.5 Observation, examination and detection

[0173] The observation of toxic symptoms and general status is the same as that in Example 2.

[0174] Data statistics is the same as that in Example 2.

[0175] Combined with the above results of body weight and food intake data, as well as the results of observation of toxic symptoms, general status and autopsy, comprehensively judge the acute toxicity of the processed product of Fomes officinalis Ames orally administered to mice.

[0176] 3. Summary and conclusion

[0177] 3.1 Test overview

[0178] Test overview: The processed product of Fomes officinalis Ames is orally administered to KM mice at the maximum formulated concentration of 25.6%, the maximum dosing volume of 40 ml / Kg, once a day, for acute toxicity observation.

[0179] 3.2 Test Results

[0180] The results showed that in the acute toxicity test of orally administering the processed product of Fomitopsis officinalis to mice, it had varying degrees of effects on the body weight and food intake of male mice. The food intake decreased significantly on days D0 - 1 after administration, which affected the decrease in body weight. The significant decrease in food intake was caused by flatulence in the gastrointestinal tract. With the gradual recovery after administration, on days D10 - 14, the food intake increased and the body weight also increased accordingly. On D14 after administration, all surviving animals were fasted overnight (12 hours), and the mice were dissected the next day. Macroscopic observation of the main visceral tissues of the mice showed that almost all the mice had mild gastrointestinal flatulence.

[0181] Conclusion: The processed product of Fomitopsis officinalis was orally administered to KM mice at the maximum formulated concentration of 25.6% and the maximum administration volume of 40 ml / Kg, once a day.

[0182] On the day of administration, it was visible that the food intake of the animals decreased significantly, which could cause gastrointestinal flatulence in the animals, and no other obvious toxic reactions occurred. Its maximum non-toxic dose for mice was 2.05 g / Kg (102.5 times the clinical dose). It is safe to take more than 100 times the tolerated human dosage orally.

[0183] In summary: The present invention adopted the research method of "associating the exterior and interior", deeply explored the Fomitopsis officinalis samples, revealed the correlation between the chromaticity change and the change in the internal quality of Fomitopsis officinalis, and provided a new method for optimizing the quality uniformity of Fomitopsis officinalis.

[0184] Based on the ancient records and traditional identification methods which both rely on color, the present invention measured the chromaticity of Fomitopsis officinalis samples with different colors. The results of the present invention showed that among the samples, A1 had the lightest color, the highest lightness L* and chroma E*ab values; while the A4 sample had the darkest color and also the highest lightness L* and chroma E*ab values. This indicates that the Fomitopsis officinalis samples after color separation treatment are easy to identify with the naked eye, which helps to control the quality of Fomitopsis officinalis. The chromaticity values corresponding to the A1 sample are L* = 90.48, a* = 0.61, b* = 7.2, E*ab = 90.76806983, which can be used as the chromaticity reference values for the effective parts of Fomitopsis officinalis samples. By analyzing the chromaticity and component content of Fomitopsis officinalis, the present invention initially suggests that L* > 81.48 and E*ab > 83.0618.

[0185] The present invention uses Pearson correlation analysis to explore the correlation between the color changes and component contents of different color samples of Fomitopsis officinalis. The results show that as the lightness L* and chroma E*ab gradually decrease, the content of C1 gradually increases, while the contents of C8, C9, and C10 are positively correlated with the lightness L* and chroma E*ab. This proves that the component changes of different color samples of Fomitopsis officinalis can be indicated by the changes in chromaticity values. Through PCA and OPLS-DA analysis, the four batches of samples are divided into four categories, among which the VIP values of C6, C3, C5, C1, C9, C8, C10, and C7 are relatively high and can be used as markers for distinguishing different color samples of Fomitopsis officinalis.

[0186] Through fingerprint analysis in the present invention, the content of component C1 in sample A4 is relatively high, while the contents of C8, C9, and C10 in sample A1 are relatively high. It is speculated by ultraviolet absorption spectrum that component C1 may be a fatty acid component. Literature reports that fatty acid components represented by larch in Fomitopsis officinalis have adverse reactions of gastrointestinal irritation and are worthy of further in-depth study. It is speculated that C8, C9, and C10 may be triterpenic acid components, which are also the effective active components of Fomitopsis officinalis. However, due to the lack of standard products, it is impossible to confirm them, and the relationship between the chemical components of Fomitopsis officinalis of different colors can be further explored in the future.

[0187] The research results of the present invention show that A1 has a promoting effect on the activity of PC12 cells with oxidative damage in the concentration ranges of 10 - 70 μg / mL and 60 - 120 μg / mL respectively, and the optimal concentrations are 50 μg / mL and 120 μg / mL respectively. H2O2 can initiate the free radical chain reaction in cells, peroxidize biological macromolecules such as cell membrane liposomes, proteins, and DNA, resulting in a significant decrease in the levels of antioxidant enzymes SOD and antioxidant GSHx in cells, while the contents of peroxide by-products MDA and LDH increase, ultimately leading to cell apoptosis. A3 and A4 both have obvious cytotoxic effects on PC12 cells, which is consistent with the ancient records that "the white ones are of the best quality, the yellow and red ones are of secondary quality, and the black ones are of the worst quality and are toxic and should be removed".

[0188] The LD 50 of the crude product of Fomitopsis officinalis in the present invention is 5.2 g / kg (5200 mg / kg), and LD50 > 5000 mg / kg, belonging to the actually non-toxic level.

[0189] In the present invention, the processed product of Fomitopsis officinalis is orally administered to KM mice at a maximum formulation concentration of 25.6% and a maximum administration volume of 40 ml / Kg, once a day. On the day of administration, it can be seen that the food intake of the animals decreases significantly, which can cause flatulence in the animals, and no other obvious toxic reactions occur. The maximum non-toxic dose for mice is 2.05 g / Kg (102.5 times the clinical dose), and it is safe to take more than 100 times the tolerated human dosage orally.

[0190] Based on the idea of "internal and external association", the present invention studied the dynamic change law of the internal components and apparent color of Fomes officinalis by measuring the content and chromaticity value of different color samples of Fomes officinalis, classified different color samples of Fomes officinalis by chemometric methods, and found the index components with differences in the process of different color samples of Fomes officinalis, which can relatively objectively reflect the digital expression of the color characteristics of Fomes officinalis. Combining with the determination of the content of the internal active ingredients, the uniformity of the quality of Fomes officinalis was optimized.

[0191] The embodiments of the present invention are given for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for quality control of Alihong, characterized in that: include: 1) Analyze the HPLC characteristic chromatograms of different color samples of Alihong; 2) Determine the redness value of Ali; 3) Analyze the cell activity of samples with different colors of Alihong; 4) Analyze the correlation between chromaticity value and active ingredient content; 5) The LD50 of raw and processed Alihong products in mice was determined by single-dose acute toxicity test in mice 50 , according to the calculated LD 50 The acute toxicity of the test substance was graded based on the value; the acute toxicity characteristics and target organs of the test substance were comprehensively evaluated based on the poisoning symptoms and pathological changes of mice.

2. A method for controlling the quality of Alihong as claimed in claim 1, characterized in that: Preparation of different color samples of Alihong: Take Alihong raw medicinal materials, separate the different color samples of Alihong, crush the dry product, sieve and grind it on a basket, pass the powder part through a 100-mesh sieve to obtain Alihong powder; the different color samples of Alihong are numbered A1, A2, A3, and A4 in order from light to dark.

3. A method for controlling the quality of Alihong as claimed in claim 1, characterized in that: Preparation of test solution: Accurately weigh 0.5 g of each powder of different colors of Alihong, place in a 100 ml stoppered conical flask, accurately add 25 ml of methanol, weigh, ultrasonically extract for 15 min, wait for cooling to room temperature, add methanol to make up for the weight loss, filter the liquid with filter paper, pass the filtrate through a 0.45 μm microporous filter membrane, and take the filtrate as the test solution.

4. A method for controlling the quality of Alihong as claimed in claim 1, characterized in that: Chromatographic conditions: C18 chromatographic column, mobile phase of 0.2% formic acid solution-acetonitrile, gradient elution; detection wavelength of 254nm; column temperature of 30℃; flow rate of 1mL / min; injection volume of 10μL; Empower software was used to set the injection method and record the chromatographic data.

5. A method for controlling the quality of Alihong as claimed in claim 1, characterized in that: Use a colorimeter to measure Ali red powder of different colors, read out the L*, a*, and b* values ​​of Ali red and record them; the L* value represents the brightness of the color, the larger the value, the higher the brightness; the a* value represents the red-green value, a positive number represents redder, and a negative number represents greener; the b* value represents the yellow-blue value, a positive number represents yellower, and a negative number represents bluish; the chroma E*ab value = (a*2+b*2)1 / 2, repeat 3 times and take the average value.

6. A method for controlling the quality of Alihong as claimed in claim 1, characterized in that: The PC12 cell oxidative damage model was established using H2O2 as an inducer. The survival rate of the active test substances on the oxidatively damaged PC12 cells was detected by MTT method to observe the protective effect of the test substances on the oxidatively damaged model cells.

7. A method for controlling the quality of Alihong as claimed in claim 1, characterized in that: SPSS.23.0 analysis software was used to conduct Pearson correlation analysis on the spatial chromaticity parameters, L*, a*, b*, E*ab values ​​of different color samples of Alihong and the contents of its 11 common components.

8. A method for controlling the quality of Alihong as claimed in claim 1, characterized in that: The contents of 11 component categories of different color samples of Alihong were imported into SIMCA14.1 software and SPSS21.0 software for principal component analysis.

9. A method for controlling the quality of Alihong as claimed in claim 1, characterized in that: On the basis of PCA classification, supervised orthogonal partial least squares discriminant analysis was carried out, and the OPLS-DA data model was established using SIMCA14.1 software to obtain the OPLS-DA score graph and the variable importance projection value graph to screen the differential markers of Alihong color samples.

10. A method for controlling the quality of Alihong as claimed in claim 1, characterized in that: The raw and processed products of Alihong were orally administered to mice for acute toxicity test; The toxic reactions produced at the maximum concentration and maximum dosage, as well as their severity, incubation period and duration, as well as mortality were observed, and the toxic effects of Alihong processed products in the acute toxicity test of oral administration to mice were preliminarily clarified.

Images