CN113640432A

CN113640432A - Quality evaluation method of loins strengthening and body building pills

Info

Publication number: CN113640432A
Application number: CN202111189534.XA
Authority: CN
Inventors: 任琦; 许妍; 陈希; 付辉政; 肖小武; 洪挺; 周志强; 赵雯; 丁银平; 刘敏; 郑盈莹; 沈敏
Original assignee: Jiangxi Institute For Drug Control
Current assignee: Jiangxi Institute For Drug Control
Priority date: 2021-10-13
Filing date: 2021-10-13
Publication date: 2021-11-12

Abstract

The invention belongs to the technical field of traditional Chinese medicine quality control, and particularly relates to a quality evaluation method of loins strengthening and body building pills. The quality evaluation method comprises the following steps: preparing a mixed reference substance solution, preparing a test substance solution, establishing a fingerprint spectrum, evaluating similarity, screening a quality marker and measuring the content of the quality marker. The invention successfully establishes the UPLC fingerprint of the loins-strengthening and body-building pill, determines 4 quality markers by combining 3 chemical pattern recognition analysis methods of cluster analysis, principal component analysis and orthogonal partial least square-discriminant analysis, and simultaneously determines the content of 2 quality markers of the characteristic chemical component specnuezhenide of glossy privet fruit (steamed with wine) and the characteristic chemical component stilbene glucoside of prepared fleece-flower root in the formula.

Description

Quality evaluation method of loins strengthening and body building pills

Technical Field

The invention belongs to the technical field of traditional Chinese medicine quality control, and particularly relates to a quality evaluation method of loins strengthening and body building pills.

Background

The traditional Chinese medicine fingerprint can reflect the spectrum of the integral characteristics of the active ingredients of the traditional Chinese medicine, and has the characteristics of high analysis speed, strong specificity, good reproducibility and the like. The quality markers (Q-markers) can be used for comprehensive evaluation of quality of Chinese medicinal materials based on overall, systematic and quantitative standards, and are closely related to properties, processing technology, pharmacological action, biological metabolism, etc. The chemical pattern recognition can process and comprehensively analyze and evaluate the multidimensional information of the fingerprint, comprehensively and objectively reflect the overall information of the traditional Chinese medicine, and plays an important role in quality control and quality evaluation research of the traditional Chinese medicine.

The waist strengthening and body building pill consists of glossy privet fruit (steamed with wine), cherokee rose fruit, philippine flemingia root, rhizoma polygonati, rhizoma cibotii, prepared rehmannia root and prepared polygonum multiflorum, has the effects of strengthening waist and body building, and is widely used for treating soreness and weakness of waist and legs, palpitation and dim eyesight, tinnitus and dizziness, impotence and spermatorrhea and the like clinically. The existing standard of loins-strengthening and body-building pills is recorded in the first part of 2020 edition of Chinese pharmacopoeia. At present, quality control research of the method stays in measuring the content of a single component of specnuezhenide by an HPLC method, detection indexes are few, and the whole quality of the loins-strengthening and body-building pill is difficult to effectively control. Due to the complex and diverse components of the loins-strengthening and body-building pill, the treatment effect can be achieved only by the mutual synergy of the components in clinical medication, but the product quality is difficult to control by the existing standard, and the stability and the uniformity of the product process cannot be ensured. At present, only the quality standard research of the loins-strengthening and body-building pills and the report of determining heavy metals and harmful elements in the loins-strengthening and body-building pills by microwave digestion-inductively coupled plasma mass spectrometry are reported in the quality analysis literature, and the research report of establishing the fingerprint spectrum of the loins-strengthening and body-building pills and the screening method of the quality marker is not found in the domestic and foreign literatures.

Disclosure of Invention

The invention aims to provide a quality evaluation method of the loins-strengthening and body-building pill, wherein the method successfully establishes the UPLC fingerprint of the loins-strengthening and body-building pill, screens out 4 quality markers, simultaneously measures the content of 2 quality markers in a preparation, and can be used as a quality control and evaluation method of the loins-strengthening and body-building pill.

The invention provides a quality evaluation method of loins-strengthening and body-building pills, which comprises the following steps:

(1) preparation of mixed control solution: precisely weighing appropriate amount of control substances of salidroside, stilbene glycoside, specnuezhenide, genistein, emodin and physcion, putting into a volumetric flask, adding methanol to prepare a mixed solution containing salidroside 15 μ g/mL, stilbene glycoside 12 μ g/mL, specnuezhenide 60 μ g/mL, genistein 1 μ g/mL, emodin 7 μ g/mL and physcion 5 μ g/mL, namely a mixed control solution;

(2) preparation of a test solution: precisely weighing 2g of the sheared and uniformly mixed loins-strengthening and body-building pills, placing the pills in a conical flask with a plug, adding 50mL of methanol solution, weighing, ultrasonically treating, cooling, weighing again, complementing the loss weight with the methanol solution, and filtering with a 0.22 mu m microporous membrane to obtain a subsequent filtrate, namely a test solution;

(3) establishing a fingerprint and evaluating similarity: respectively measuring the reference substance solution in the step (1) and the test substance solution in the step (2) by using UPLC, wherein the mobile phase is a methanol (A) -0.1% formic acid solution (B) elution system to obtain the finger print of the loins strengthening and fitness pill, and introducing the obtained finger print into a traditional Chinese medicine chromatography finger print similarity evaluation system for similarity evaluation;

(4) and (3) content determination: precisely measuring the test solution in the step (2), determining by adopting the chromatographic condition of the UPLC in the step (3), calculating the content of the component to be measured according to an external standard method, and performing quality evaluation and judgment.

In the technical scheme, the UPLC fingerprint of the loins-strengthening and body-building pill is successfully established by optimizing UPLC chromatographic conditions, the fingerprint has 15 common peaks, and is subjected to identification and attribution analysis to identify six characteristic peaks, the separation effect of each characteristic peak is good, the quality of a product can be comprehensively reflected, and reference basis is provided for quality control and standard revision of the loins-strengthening and body-building pill.

Further, in the above technical scheme, after the establishment of the fingerprint in step (3) is completed, the method further comprises a screening step of a quality marker, specifically: and (3) performing principal component analysis on the test solution obtained in the step (2) by adopting a principal component analysis method, performing orthogonal partial least squares discriminant analysis model analysis on the basis of the principal component analysis to obtain a VIP image, and determining the quality marker of the loins-strengthening and fitness-building pill according to the VIP value.

According to the technical scheme, four quality markers are screened out by combining three chemical mode identification analysis methods of cluster analysis, principal component analysis and orthogonal partial least square-discriminant analysis, and meanwhile, a corresponding quality control system can be established by combining the content determination method, so that the uniformity and the stability of the product are improved, and a scientific evaluation method is further provided for quality monitoring of the loins-strengthening and body-building pills.

Further, in the step (2) of the technical scheme, the concentration of the methanol solution is 50-100%, and the ultrasonic treatment time is 15-60 min. Specifically, in the technical scheme, the concentration of the methanol solution can be 50%, 70%, 80%, 90% and 100%, and diluted ethanol and 70% ethanol can also be used for substitution; the ultrasonic treatment can be 15min, 30min, 45min, 60min, or can be replaced by heating and refluxing for 1 h.

Preferably, in the technical scheme, the concentration of the methanol solution is 80%, the ultrasonic treatment time is 30min, the ultrasonic power is 500W, and the frequency is 40 kHz.

Further, in the step (3) of the above technical solution, the UPLC chromatography conditions are:

in the step (3), the UPLC chromatographic conditions are as follows: the chromatographic column is Acquity UPLC Hss T3 (the specification is 100 multiplied by 2.1mm, 1.8 mu m); the detection wavelength is 224-320nm, and the wavelength conversion is carried out; the column temperature is 25-45 ℃; the sample injection amount is 0.5-5.0 muL; the flow rate is 0.1-0.8 mL/min; the mobile phase takes methanol as a mobile phase A and takes 0.1 percent formic acid as a mobile phase B to carry out gradient elution; the gradient elution procedure was:

when the time is 0-20min, the volume ratio of the mobile phase A is increased from 5% to 90%, and the volume ratio of the mobile phase B is decreased from 95% to 10%;

when the time is 20-25min, the volume ratio of the mobile phase A is 90%, and the volume ratio of the mobile phase B is 10%.

Preferably, in the above technical solution, the UPLC chromatography conditions are:

the chromatographic column is Acquity UPLC Hss T3 (the specification is 100 multiplied by 2.1mm, 1.8 mu m); the detection wavelength is 224-320nm, and the wavelength conversion is carried out; the column temperature was 40 ℃; the sample injection amount is 2.0 mu L; the flow rate is 0.2 mL/min; the mobile phase takes methanol as a mobile phase A and takes 0.1 percent formic acid as a mobile phase B to carry out gradient elution; the gradient elution procedure was:

Further, in the above technical solution, the wavelength conversion method is as follows:

the wavelength is 320nm when 0-4.1 min;

at wavelength of 254nm for 4.1-4.8 min;

at wavelength of 224nm when the time is 4.8-7.0 min;

the wavelength is 320nm when 7.0-11.0 min;

at 11.0-16.0min, the wavelength is 254 nm;

the wavelength is 320nm when the time is 16.0-19.0 min;

the wavelength is 288nm when 19.0-25.0 min.

In the technical scheme, as the ultraviolet absorption wavelengths of all effective components in the loins-strengthening and body-building pill are different, in order to enable the response value of each peak to reach a larger value, the invention adopts a detection wavelength conversion mode for detection.

Further, in the step (3) of the above technical scheme, 15 common peaks exist in the fingerprint of the loin-strengthening and body-building pill, and 6 characteristic peaks are identified by reference substance comparison and ultraviolet spectrograms, wherein the peak No. 4 is salidroside, the peak No. 6 is stilbene glycoside, the peak No. 8 is specnuezhenide, the peak No. 12 is genistein, the peak No. 13 is emodin, and the peak No. 15 is physcion.

Further, in the step (4) of the technical scheme, when the VIP value is more than 1, the component is determined as a quality marker of the loins strengthening and fitness pill. In the technical scheme, the VIP map can visually represent the difference components influencing the difference between the sample groups, and when VIP is more than 1, the VIP is the quality difference marker component and can be determined as the quality marker.

Further, the quality markers of the loins strengthening and body building pill in the technical scheme comprise stilbene glucoside and specnuezhenide.

Compared with the prior art, the method has the beneficial effects that:

1. according to the invention, the UPLC fingerprint of the loins-strengthening and body-building pill is successfully established by optimizing UPLC chromatographic conditions, the fingerprint has 15 common peaks, and is subjected to identification and attribution analysis to identify six characteristic peaks, the separation effect of each characteristic peak is good, the quality of the product can be comprehensively reflected, and the monitoring of the quality of the loins-strengthening and body-building pill is facilitated;

2. according to the invention, four quality markers are screened out by combining three chemical mode identification analysis methods of cluster analysis, principal component analysis and orthogonal partial least square-discriminant analysis, and the content of the quality marker components of specnuezhenide and stilbene glucoside in the loin-strengthening body-building pill is measured, so that a scientific evaluation method is further provided for quality monitoring of the loin-strengthening body-building pill;

3. the quality evaluation method is rapid, accurate, simple, convenient and efficient, has good repeatability, can be used as a quality control and evaluation method of the loins-strengthening and body-building pill, and provides a reference basis for the quality control and standard revision of the loins-strengthening and body-building pill in the later period.

Drawings

FIG. 1 is a diagram of the absorption spectrum of salidroside of the present invention;

FIG. 2 is a graph of stilbene glycoside absorption spectrum of the present invention;

FIG. 3 is a diagram showing an absorption spectrum of specnuezhenide according to the present invention;

FIG. 4 is a graph of the absorption spectrum of genistein according to the present invention;

FIG. 5 is an absorption spectrum of emodin according to the present invention;

FIG. 6 is an absorption spectrum of physcion according to the present invention;

FIG. 7 is a UPLC chromatogram of waist strengthening and body building pills determined by Acquity UPLC Hss T3 chromatographic columns (1.8 μm, 100X 2.1 mm);

FIG. 8 is UPLC chromatogram of waist strengthening and body building pills determined by Phenomenex Kinetex ® chromatographic columns (1.7 μm, 100X 2.1 mm) of the invention;

FIG. 9 is UPLC chromatogram of Zhuangyao Jiangyan pill measured by Agilent Eclipse Plus C18 RRHD chromatographic column (1.8 μm, 150X 3.0 mm);

FIG. 10 is a UPLC chromatogram of a negative control of the present invention;

FIG. 11 is a comparison fingerprint of the loins strengthening and body building pill of the present invention;

FIG. 12 is a chromatogram peak assignment and attribution chart of the present invention;

FIG. 13 is a CA tree diagram of 11 batches of loins strengthening and body building pills according to the present invention;

FIG. 14 is a PCA scatter plot of 11 batches of loins strengthening and fitness pill samples of the present invention;

FIG. 15 is a scatter plot of 11 batches of waist strengthening and body building pills OPLS-DA according to the present invention;

FIG. 16 is a diagram of the OPLS-DA replacement test of 11 batches of loins strengthening and body building pills according to the present invention;

FIG. 17 is a diagram of 11 batches of waist strengthening and body building pills OPLS-DA VIP of the present invention;

fig. 18 shows UPLC chromatograms of the mixed control (a), sample (B), and negative sample (C) of the present invention, wherein 1 is stilbene glycoside and 2 is specnuezhenide.

Detailed Description

The technical features of the present invention described above and those described in detail below (as an embodiment) can be combined with each other to form a new or preferred technical solution, but the present invention is not limited to these embodiments, and the embodiments also do not limit the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The formulations according to the following examples are all commercially available products and are commercially available, unless otherwise specified.

The invention is described in further detail below with reference to the figures and examples:

experimental instruments and materials:

1. instrument for measuring the position of a moving object

Ultra high performance liquid chromatograph model Agilent1290 infinity (dad) (Agilent technologies ltd, usa); Milli-Q Direct 16 ultrapure water instrument (Millipore, Germany, China Co., Ltd.); one hundred thousand balances, model Sartorius BT 25S; one in ten thousandth of a balance of the type Sartorius BSA 124S-CW (Sartorius, germany).

2. Material

Salidroside (Zhongzhonghuan, batch No. 110818-200404); stilbene glucoside (Zhongzhou institute, batch No. 110844-201109); specnuezhenide (middle school, lot number: 111926-; genistein (Zhongzhong department, lot number: 111704-201703); emodin (middle institute, batch number: 110756-201512); physcion (Zhongzhou province, batch No.: 110758-201616). Seven medicinal materials of glossy privet fruit (steamed with wine), rhizoma polygonati, prepared rehmannia root, cherokee rose fruit, east Asian tree fern rhizome, prepared fleece-flower root and philippine flemingia root are provided by Jiangxi Jiuhua pharmaceutical industry Co., Ltd and are identified as certified products by the Xigxi province drug inspection and detection research institute's Fanghui administration and minister Zeng. The methanol and the acetonitrile are chromatographically pure, the water is ultrapure water, and the others are analytically pure. 11 samples of Zhuangyao Jiangyang Wan were obtained from Jiangxi Jiuhua pharmaceutical Co., Ltd (batch Nos. 190101, 190102, 190301, 160102, 160302, 160603, 180701, 180702, 180901 and 180902) and Jiangxi Tiantian pharmaceutical Co., Ltd (batch No. 20160701).

Example 1: establishment of fingerprint and similarity evaluation

1. Preparation of the solution

1.1 preparation of Mixed control solutions

Taking a proper amount of salidroside reference substance, stilbene glycoside reference substance, specnuezhenide reference substance, genistein reference substance, emodin reference substance and physcion reference substance, precisely weighing, respectively adding methanol to prepare mixed solutions each containing 15 μ g of salidroside, 12 μ g of stilbene glycoside, 60 μ g of specnuezhenide, 1 μ g of genistein, 7 μ g of emodin and 5 μ g of physcion per 1mL, and shaking up to obtain the final product.

1.2 preparation of test solutions

Taking samples of the loins strengthening and body building pills, cutting into pieces, mixing uniformly, precisely weighing 2g, placing in a conical flask with a plug, precisely adding 50mL of 80% methanol, weighing, carrying out ultrasonic treatment for 30min, cooling, weighing again, supplementing the loss weight with 80% methanol, filtering with a 0.22 mu m microporous membrane, and taking the subsequent filtrate to obtain the traditional Chinese medicine composition.

1.3 preparation of Single herb solution

Collecting fructus Ligustri Lucidi (steamed with wine), rhizoma Polygonati, radix rehmanniae Preparata, fructus Rosae Laevigatae, rhizoma Cibotii, radix Polygoni Multiflori Preparata, and radix Flemingiae Philippinensis, and preparing according to the method of 1.2.

2. Chromatographic conditions

The chromatographic column is octadecyl silane bonded silica gel as a filler, and the type is Acquity UPLC Hss T3 (the column length is 100mm, the inner diameter is 2.1mm, and the grain diameter is 1.8 μm); the mobile phase is methanol (A) -0.1% formic acid solution (B), and gradient elution is carried out (0-20 min, 5% -90% A; 20-25min, 90% A); the detection wavelength is wavelength conversion (0-4.1 min, 320nm, 4.1-4.8min, 254nm, 4.8-7.0min, 224nm, 7.0-11.0min, 320nm, 11.0-16.0min, 254nm, 16.0-19.0min, 320nm, 19.0-25.0min, 288 nm); the column temperature was 40 ℃; the sample injection amount is 2.0 mu L; the flow rate was 0.2 mL/min.

3. Condition optimization

3.1 selection of detection wavelength

Due to the complexity and diversity of the traditional Chinese medicine components, different chemical components have different ultraviolet absorption wavelengths. The maximum absorption wavelength of salidroside is 224 +/-2 nm, the maximum absorption wavelength of stilbene glycoside is 320 +/-2 nm, the maximum absorption wavelength of specnuezhenide is 224 +/-2 nm, the maximum absorption wavelength of genistein is 260 +/-2 nm, the maximum absorption wavelength of emodin is 288 +/-2 nm, the maximum absorption wavelength of physcion is 288 +/-2 nm, the absorption spectra are respectively shown in figures 1-6, and in order to make the response value of each peak larger, the test adopts a wavelength conversion mode for detection, as shown in table 1.

Table 1 detection wavelength conversion table

3.2 selection of the Mobile phase

The experiments of the invention found that the best gradient elution effect was performed according to the specification of table 2, using methanol as mobile phase a and 0.1% formic acid solution as mobile phase B.

TABLE 2 mobile phase gradient elution Table

3.3 examination of extraction solvent

Taking 6 parts of loins-strengthening and body-building pills (Jiangxi Bantiantianyao pharmaceutical industry Co., Ltd., batch number: 180901), precisely adding 50% methanol, 70% methanol, 80% methanol, diluted ethanol and 70% ethanol solution into each 2g part of loins-strengthening and body-building pills, respectively 50ml, carrying out ultrasonic treatment for 30 minutes, cooling, weighing, respectively supplementing the lost weight with the corresponding solution, shaking uniformly, filtering by using a microporous filter membrane (0.22 mu m), taking the subsequent filtrate, and observing an extraction solvent through UPLC analysis, wherein the 6 solvent extraction samples can achieve good separation, wherein each component of 80% methanol has good response and better separation degree, therefore, 80% methanol is preferably used as the extraction solvent.

3.4 examination of extraction methods and time

5 parts of loins-strengthening and body-building pills (Jiangxi Bantiantianyao pharmaceutical Co., Ltd., batch No. 180901) are precisely weighed, and are respectively treated by ultrasonic treatment (power: 500W, frequency: 40 kHz) for 15min, 30min, 45min and 60min and heating reflux for 1h, and the extraction method and time are examined, and the results are shown in Table 3.

According to the investigation result, the samples can be well separated by two extraction modes, the operation is convenient and the time is saved, therefore, the preferred extraction method of the invention is ultrasonic treatment (power: 500W, frequency: 40 kHz) for 30 min.

TABLE 3 examination results of different extraction methods

3.5 different brands of column durability Studies

And taking the same test solution, comparing chromatographic columns of different brands, and inspecting the durability of the method. An Agilent1290 series high performance liquid chromatograph is adopted to analyze and record chromatograms by using Acquity UPLC Hss T3 chromatographic columns (1.8 mu m, 100 multiplied by 2.1 mm), Phenomenex Kinetex chromatographic columns (1.7 mu m, 100 multiplied by 2.1 mm) and Agilent Eclipse Plus C18 RRHD chromatographic columns (1.8 mu m, 150 multiplied by 3.0 mm), respectively, and the chromatograms are shown in FIGS. 7-9.

The results of comparing chromatograms obtained from three chromatographic columns of different brands show that the Waters Acquity UPLC Tss T3 chromatographic columns show that the number of chromatographic peaks is large, the peak shape is good, and the separation effect is optimal. Therefore, the Waters Acquity UPLC Tss T3 chromatographic column is preferably used as the chromatographic column for the finger print research of the loins-strengthening body-building pills.

3.6 blank solvent test

80% methanol was taken, measured according to the above "2. chromatographic conditions", 2. mu.L of sample was injected, and a chromatogram was recorded, as shown in FIG. 10. The result shows that the blank solvent does not have a chromatographic peak corresponding to the test solution in the chromatogram.

3.7 precision test

A sample (Jiangxi Bantiantian pharmaceutical Co., Ltd., lot: 180901) was taken, prepared by the method of "preparation of sample solution 1.2" described above, and subjected to continuous sampling 6 times under the "chromatographic conditions 2" described above, and the retention time and the integrated peak area of each chromatographic peak were measured and recorded. The relative retention time RSD of each chromatographic peak is calculated to be less than 1.00 percent and the relative peak area RSD is calculated to be less than 5.00 percent by taking the No. 8 chromatographic peak (specnuezhenide) as a reference peak (S), and the mutual similarity of the spectra is more than 0.90, so that the result shows that the precision of the instrument is good.

3.8 repeatability test

6 parts of a sample (Jiangxi Bantianyao pharmaceutical Co., Ltd., batch No. 180901) was taken, prepared by the method of "preparation of test sample solution 1.2" described above, and measured under the "chromatographic conditions 2" described above, and the retention time and peak area of each chromatographic peak were recorded. The method is characterized in that a No. 8 chromatographic peak (specnuezhenide) is taken as a reference peak (S), the relative retention time RSD of each chromatographic peak is calculated to be less than 1.00 percent, the relative peak area RSD is calculated to be less than 5.00 percent, and the mutual similarity of the spectra is larger than 0.90, so that the method is good in reproducibility.

3.9 stability test

6 portions of a sample (Jiangxi Bantiantianyao Co., Ltd., batch No. 180901) were taken, prepared by the method of "preparation of test solution 1.2" described above, and finger prints were measured at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h under the "chromatographic conditions 2" described above, and the retention time and peak area of each chromatographic peak were recorded. And (3) calculating the relative retention time RSD of each chromatographic peak by taking the No. 8 chromatographic peak (specnuezhenide) as a reference peak (S), wherein the relative peak area RSD is less than 1.00 percent, the relative peak area RSD is less than 5.00 percent, and the mutual similarity of the spectra is more than 0.90, and the result shows that the method is stable within at least 24 h.

4. Establishment of fingerprint

Taking 11 batches of loins-strengthening and body-building pill sample solutions, measuring according to the 2. chromatographic conditions, and recording chromatograms. The obtained data is imported into a 2012 version of the traditional Chinese medicine chromatography fingerprint similarity evaluation system, the time window width is selected to be 0.10min, the generation method of the comparison map is the median, the comparison map is obtained after automatic matching, and 15 common peaks are determined in total as shown in fig. 11.

5. Evaluation of similarity

Introducing chromatogram data of 11 batches of loins strengthening and body building pill samples into a 2012 version of 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system', calculating the similarity between the samples and a comparison fingerprint, and respectively obtaining results of 1.000, 0.999, 0.994, 0.811, 0.837, 0.803, 0.855, 0.851, 0.936, 0.906 and 0.720. The results show that the similarity of 11 batches of samples is lower than 0.85 for 4 batches of samples.

The difference of the production processes of different production enterprises is larger, and the difference of the samples produced by the same production enterprise is also larger.

Example 2: identification and attribution of common peaks and selection of reference peaks

3 solutions were prepared according to the method of example 1 and the results are shown in FIG. 12, measured according to the chromatographic conditions of example 1.

6 characteristic peaks are identified by adopting the retention time of a reference substance and an ultraviolet spectrogram, and are respectively salidroside No. 4, stilbene glucoside No. 6, specnuezhenide No. 8, genistein No. 12, emodin No. 13 and emodin methyl ether No. 15.

Comparing the test sample with chromatogram of medicinal materials, and assigning 15 common peaks, wherein fructus Ligustri Lucidi (steamed with wine) provides

peaks

4, 5, 7, 8, and 10; cherokee rose fruit provides peak No. 9; philippine flemingia root provides

peaks

12 and 14; rhizoma Polygonati provides peak No. 1; rehmanniae radix Preparata provides

peaks

1, 2, 3, 11; the prepared fleece flower root provides peaks No. 6, 13 and 15.

Wherein, the No. 8 peak is the characteristic component of glossy privet fruit (steamed with wine), and the peak has moderate retention time, strong absorption and good separation degree, therefore, the No. 8 peak is taken as a reference peak in the invention.

Example 3: screening for quality markers

1. Cluster Analysis (CA)

A test sample solution is prepared and measured according to the method of example 1, the peak areas of 15 common peaks of 11 batches of loins-strengthening and body-building pill samples are quantified relative to the sample weighing and then standardized to form an 11 x 15-order original matrix, IBM SPSS staticisics 23.0 software is used, the group connection is used as a clustering method, the measuring degree adopts the squared Euclidean distance, and the system clustering analysis is carried out, wherein a CA tree diagram is shown in FIG. 13.

The results show that when the euclidean cluster is 10, 11 samples can be classified into 3 classes: class I: S1-S3, the similarity is more than 0.990; and II: S4-S10, wherein the similarity is more than 0.800; class III: and S11, wherein the similarity is 0.720. This result is consistent with the similarity evaluation result in example 1.

2. Principal Component Analysis (PCA)

After standardized processing is carried out on the quantized common peak-peak areas by IBM SPSS statics 23.0 software, main component analysis is carried out on 11 batches of loins-strengthening and body-building pill samples, and the characteristic value of a correlation coefficient matrix and the variance contribution rate of the characteristic value are calculated. 3 main components (characteristic value is more than 1) are extracted, the cumulative variance contribution rate is 89.915%, and the main components can represent most information of the fingerprint spectrum common peak. The main component dispersion point score chart was drawn by SIMCA 14.1 software, as shown in fig. 14.

The results show that 11 batches of loins strengthening and body building pill samples can be divided into 3 types, and the results are consistent with the clustering analysis results.

3. Quadrature partial least squares discriminant analysis (OPLS-DA)

In order to further screen out components with larger contribution, the OPLS-DA model is established by adopting SIMCA 14.1 software on the basis of main component analysis, and as a result, the X matrix setting percentage parameter R2X is 0.843, the model stability parameter R2Y is 1, the model prediction capability parameter Q2 is 0.869 and is more than 0.5, and the difference between R2 and Q2 is less than 0.2, which indicates that the established model has stronger interpretation rate, stability and predictability. The scatter-point score plots of 11 test samples were obtained by fitting analysis with the OPLS-DA model, as shown in FIG. 15.

In order to verify whether the established OPLS-DA model has overfitting, the variable parameters of the classification matrix are randomly arranged 200 times for replacement test, as shown in FIG. 16. The results show that the intercepts of the R2 and Q2 fit lines on the Y coordinate axis are respectively 0.129 (which should be less than 0.3) and-0.761 (which should be less than 0.05), which indicates that the model has no overfitting phenomenon and is reliable.

The VIP map can visually represent the difference components influencing the difference between the sample groups, when VIP is more than 1, the VIP map is the marker component of the quality difference, and the VIP map in the embodiment is shown in FIG. 17. The results showed that the VIP values of peak 6 (stilbene glycoside), peak 3, peak 2, and peak 8 (specnuezhenide) were all greater than 1, and that the mass difference marker components were 4 mass markers of the loin-strengthening and fitness pill, while peak 6 was stilbene glycoside and peak 8 was specnuezhenide, and peak 2 and peak 3 were not determined due to the absence of the control.

Therefore, the uniformity and stability of the product can be improved by establishing a corresponding quality control system.

Example 4: determination of content

1. Chromatographic conditions and preparation of test solution

The chromatographic conditions and the test solution preparation method are the same as those in example 1.

2. Determination of sample content

Taking 11 batches of loins-strengthening and body-building pills to be tested solution, carrying out UPLC (ultra performance liquid chromatography) measurement, recording peak areas, and calculating the content of each component to be tested according to an external standard method, wherein the results are shown in Table 4.

TABLE 4 determination of the two Mass marker contents (mg. g)^-1，n=2）

The results show that the content of stilbene glucoside and specnuezhenide in the sample is high, and the determination is easy. The quality of the loins strengthening and body building pill can be evaluated by the content of two quality marker components of stilbene glucoside and specnuezhenide.

Example 5: methodology investigation

Preparation of a reference solution: precisely weighing appropriate amount of stilbene glycoside reference substance and specnuezhenide reference substance, placing into volumetric flask, adding methanol to obtain solution containing stilbene glycoside 12 μ g per 1mL and specnuezhenide 60 μ g per 1mL, and shaking.

The test solution and the negative control solution (lacking fructus Ligustri Lucidi and radix Polygoni Multiflori Preparata) were prepared according to the method of example 1.

1. System applicability and specificity testing

mu.L of each control solution, 2. mu.L of each test solution and negative control solution were sampled and analyzed, and the chromatogram was recorded (as shown in FIG. 18) under the chromatographic conditions of example 1. As a result, the degree of separation between two mass markers and adjacent chromatographic peaks was greater than 1.5; the theoretical plate number is more than 10000 according to stilbene glucoside and specnuezhenide peaks; negative sample solution has no interference, which shows that the specificity of the method is good.

2. Investigation of linear relationships

Each control solution was diluted 0, 2, 4, 8, 20 and 40 times, and the dilutions were measured under the chromatographic conditions in example 1 by using the peak area Y as the ordinate and the concentration X (mg. mL-1) as the abscissa, to prepare a standard curve, and the results are shown in Table 5.

TABLE 5 regression equation and Linear Range for two Mass markers

The results show that 2 characteristic components are in good linear relationship with peak area between 0 and 40 times dilution.

3. Precision test of instrument

Precisely measuring 5mL of each reference substance solution, respectively placing in a 10 mL volumetric flask, adding methanol to a constant volume to a scale, taking the solution, carrying out continuous sample injection measurement for 6 times according to the chromatographic conditions in example 1, recording a chromatogram, and calculating RSD of peak areas of 2 mass markers.

As a result, the RSD (n = 6) of the peak areas of stilbene glycoside and specnuezhenide was 0.10% and 0.28%, respectively, indicating that the precision of the instrument was good.

4. Stability test

2g of loins-strengthening and body-building pills (Jiangxi Bantiantianyao pharmaceutical Co., Ltd., batch number: 180901) are precisely weighed, prepared according to the method of the test solution in example 1, measured for 0 h, 2 h, 4 h, 8 h, 12 h and 24 h according to the chromatographic conditions in example 1, and the chromatogram is recorded and the RSD of the peak area of 2 mass markers is calculated.

Results RSD (n = 6) of the peak areas of stilbene glycoside and specnuezhenide were 1.4% and 1.5%, respectively, indicating that the test solution had good stability at room temperature for 24 h.

5. Repeatability test

2g of loins-strengthening and body-building pills (Jiangxi Bantiantianyao pharmaceutical Co., Ltd., batch number: 180901) are taken, 6 parts in total are precisely weighed, the pills are prepared according to the method of the test solution in the example 1, the chromatographic conditions are measured according to the example 1, the chromatogram and the peak area are recorded, and the content of 2 quality marker components and the RSD thereof are calculated according to an external standard method.

As a result, the average contents of stilbene glycoside and specnuezhenide were 0.2804mg g-1, 1.7804mg g-1, and RSD (n = 6) were 1.4% and 0.72%, respectively, indicating that the reproducibility of the method was good.

6. Sample application recovery test

1 g of loins-strengthening and body-building pills (Jiangxi Bantiantianyao pharmaceutical Co., Ltd., batch No. 180901) are taken and weighed precisely, 50mL of standard solution (prepared by the above preparation method of the mixed reference solution, containing stilbene glucoside 11.4322 μ g/mL and specnuezhenide glucoside 70.0328 μ g/mL) is precisely added respectively by sample-adding recovery test, the test sample is prepared according to the method of example 1, the chromatographic conditions are determined according to the example 1, the recovery rate is calculated, and the results are shown in Table 6. The accuracy of the measurement method of the present invention was good.

TABLE 6 results of recovery test: (n=6）

In conclusion, the invention successfully establishes the UPLC fingerprint of the loins-strengthening and body-building pill, determines 4 quality markers by combining 3 chemical pattern recognition analysis methods of cluster analysis, principal component analysis and orthogonal partial least square-discriminant analysis, and simultaneously determines the content of 2 quality markers of the characteristic chemical component specnuezhenide of glossy privet fruit (steamed with wine) and the characteristic chemical component stilbene glucoside of prepared fleece-flower root.

Finally, it should be emphasized that the above-described preferred embodiments of the present invention are merely examples of implementations, rather than limitations, and that many variations and modifications of the invention are possible to those skilled in the art, without departing from the spirit and scope of the invention.

Claims

1. A quality evaluation method of the loins strengthening and body building pill is characterized by comprising the following steps:

(3) establishing a fingerprint and evaluating similarity: respectively measuring the reference substance solution in the step (1) and the test substance solution in the step (2) by using UPLC (ultra performance liquid chromatography), wherein a mobile phase is a methanol-0.1% formic acid solution elution system to obtain the fingerprint of the loins-strengthening and body-building pill, and introducing the obtained fingerprint into a traditional Chinese medicine chromatography fingerprint similarity evaluation system for similarity evaluation;

2. The quality evaluation method of the loins strengthening and body building pill according to claim 1, characterized by further comprising a step of screening quality markers after the establishment of the fingerprint spectrum in the step (3), specifically: and (3) performing principal component analysis on the test solution obtained in the step (2) by adopting a principal component analysis method, performing orthogonal partial least squares discriminant analysis model analysis on the basis of the principal component analysis to obtain a VIP image, and determining the quality marker of the loins-strengthening and fitness-building pill according to the VIP value.

3. The quality evaluation method of the loins strengthening and body building pill according to claim 1, wherein in the step (2), the concentration of the methanol solution is 50-100%, and the ultrasonic treatment time is 15-60 min.

4. The quality evaluation method of the loins strengthening and body building pill according to claim 3, wherein the concentration of the methanol solution is 80%, the ultrasonic treatment time is 30min, the ultrasonic power is 500W, and the frequency is 40 kHz.

5. The quality evaluation method of the loins strengthening and body building pill according to claim 1, wherein in the step (3), the UPLC chromatographic conditions are as follows: the chromatographic column is Acquity UPLC Hss T3 with the specification of 100 multiplied by 2.1mm and 1.8 mu m; the detection wavelength is 224-320nm, and the wavelength conversion is carried out; the column temperature is 25-45 ℃; the sample injection amount is 0.5-5.0 muL; the flow rate is 0.1-0.8 mL/min; the mobile phase takes methanol as a mobile phase A and takes 0.1 percent formic acid as a mobile phase B to carry out gradient elution; the gradient elution procedure was:

6. The quality evaluation method of the loins strengthening and body building pill according to claim 5, wherein the UPLC chromatographic conditions are as follows: the chromatographic column is Acquity UPLC Hss T3 with the specification of 100 multiplied by 2.1mm and 1.8 mu m; the detection wavelength is 224-320nm, and the wavelength conversion is carried out; the column temperature was 40 ℃; the sample injection amount is 2.0 mu L; the flow rate is 0.2 mL/min; the mobile phase takes methanol as a mobile phase A and takes 0.1 percent formic acid as a mobile phase B to carry out gradient elution; the gradient elution procedure was:

7. The quality evaluation method of the loins strengthening and body building pill according to any one of claims 5 or 6, wherein the wavelength conversion method is as follows:

the wavelength is 320nm when 0-4.1 min;

at wavelength of 254nm for 4.1-4.8 min;

at wavelength of 224nm when the time is 4.8-7.0 min;

the wavelength is 320nm when 7.0-11.0 min;

at 11.0-16.0min, the wavelength is 254 nm;

the wavelength is 320nm when the time is 16.0-19.0 min;

the wavelength is 288nm when 19.0-25.0 min.

8. The method for evaluating the quality of Zhuangyao Jiangyan Wan as claimed in claim 1, wherein in step (3), 15 common peaks exist in the fingerprint of the Zhuangyao Jiangyan Wan, 6 characteristic peaks are identified by comparison with a reference substance and an ultraviolet spectrum, wherein the peak 4 is salidroside, the peak 6 is stilbene glycoside, the peak 8 is specnuezhenide, the peak 12 is genistein, the peak 13 is emodin, and the peak 15 is physcion.

9. The method for evaluating the quality of the loins strengthening and fitness pill as claimed in claim 2, wherein in the step (4), when the VIP value is more than 1, the ingredient is determined as the quality marker of the loins strengthening and fitness pill.

10. The method for evaluating the quality of the loins strengthening and fitness pill according to claim 9, wherein the loins strengthening and fitness pill quality markers comprise stilbene glycoside and specnuezhenide.