CN110596284B - Chinese magnoliavine fruit quality detection method based on spectrum-effect relationship - Google Patents
Chinese magnoliavine fruit quality detection method based on spectrum-effect relationship Download PDFInfo
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Abstract
The invention discloses a schisandra quality detection method based on a spectrum effect relationship, which comprises the following steps: firstly, establishing a UPLC-MS fingerprint of an MRSA (methicillin-resistant Staphylococcus aureus) in-vitro antibacterial active component of a schisandra fruit test product to be tested, screening out a characteristic peak, then substituting the peak area of the screened characteristic peak into a spectrum effect mathematical model for calculation, and judging the quality of the schisandra fruit test product according to the result obtained by calculation. The method adopts a mode of combining the fingerprint spectrum and the mathematical model to judge the quality of the schisandra chinensis product, can avoid the interference of third-party factors, does not need to carry out complicated pharmacological experiments, avoids the individual deviation of animals, and can basically quickly and clearly determine the antibacterial effect of the MRSA (methicillin resistant Staphylococcus aureus) active part of the schisandra chinensis.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine quality standard detection, and particularly relates to a schisandra quality detection method based on a spectrum effect relationship.
Background
Fructus Schisandrae chinensis is dried mature fruit of Schisandra chinensis (Turcz.) Baill of Magnoliaceae, and is known as "North fructus Schisandrae chinensis", and is distributed in Heilongjiang, Jilin, Liaoning, inner Mongolia, Hebei, Shanxi, Ningxia, Gansu, Shandong, etc. It is sour and sweet in taste, warm in nature, and has effects of astringing, arresting discharge, invigorating qi, promoting fluid production, invigorating kidney, and calming heart. Clinically, it is usually used for chronic cough, dyspnea of deficiency type, nocturnal emission, enuresis, frequent micturition, chronic diarrhea, spontaneous perspiration, night sweat, thirst due to body fluid consumption, internal heat, diabetes, palpitation, insomnia, etc. The existing research shows that schisandra chinensis extracted by 70% ethanol is sequentially subjected to petroleum ether (60-90 ℃), chloroform and ethyl acetate sectional extraction, and an ethyl acetate extraction Component is subjected to AB-8 macroporous adsorption resin separation and water elution to obtain a Component (EEW Component) which is an MRSA (methicillin resistant Staphylococcus aureus) in-vitro antibacterial activity site, and has strong antibacterial activity on antibiotic sensitive Staphylococcus aureus and clinically-separated drug-resistant MRSA.
At present, the quality standard of the schisandra chinensis is mostly qualitative or quantitative analysis aiming at a few active ingredients, and the research on the relation between the schisandra chinensis and the pharmacodynamic activity is not provided, in particular to a method for detecting the superiority and inferiority of the antibacterial activity of the schisandra chinensis.
Disclosure of Invention
The invention adopts a multiple regression analysis method to perform correlation analysis on UPLC-MS characteristic fingerprint spectrum of anti-MRSA in-vitro antibacterial active components (EEW Component) of schisandra chinensis and the drug effect of in-vitro antibacterial activity, constructs the spectrum-effect relationship of the antibacterial active parts of schisandra chinensis, and can be used for judging the quality of schisandra chinensis.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the method for detecting the quality of the schisandra chinensis based on the spectrum effect relationship comprises the following steps:
step 1, establishing a UPLC-MS fingerprint of an MRSA (methicillin resistant Staphylococcus aureus) in-vitro antibacterial active component of a schisandra chinensis test sample to be tested;
and 3, substituting the characteristic peak area screened out in the step 2 into a spectral efficiency mathematical model for calculation, and judging the quality of the schisandra fruit test product according to the result obtained by calculation.
Further, in the step 2, a traditional Chinese medicine chromatogram fingerprint similarity evaluation system is adopted to complete the screening of the characteristic peaks.
Further, the preparation process of the MRSA in-vitro bacteriostatic active component comprises the following steps: taking dried and crushed schisandra chinensis medicinal materials, adding 70% ethanol for reflux extraction, concentrating an extracting solution to obtain a schisandra chinensis ethanol extract, adding purified water for suspension, respectively and sequentially extracting with petroleum ether, chloroform and ethyl acetate with equal volume, combining ethyl acetate extracting solutions, concentrating to dryness to obtain an ethyl acetate extract, suspending the ethyl acetate extract in the purified water, adsorbing and eluting with pretreated AB-8 macroporous adsorption resin, collecting an eluting component, concentrating to dryness, and vacuum drying to obtain the schisandra chinensis ethanol extract.
Further, the chromatographic conditions for UPLC-MS in step 1 are as follows:
the preparation method of the test solution comprises the following steps: dissolving 0.0050g sample in 2mL methanol, filtering with 0.22 μm microporous membrane before sample introduction, and collecting filtrate as sample solution;
UPLC chromatographic separation conditions: the chromatographic column is Acquisty UPLC BEH C18, the column temperature is 45 ℃, the volume flow rate is 0.3mL min < -1 >, the sample injection amount is 2 mu L, the mobile phase is acetonitrile A-0.1% formic acid B, and the phase A proportion changes along with the time: 0-3 min, 0-0%, 3-10 min, 0-15%, 10-12 min, 15-80%, 12-12.1 min, 80-0%;
MS conditions: the electrospray ionization ion source has stronger negative ion mode scanning mass spectrum response, the capillary voltage is 3.0kV, the taper hole voltage is 30V, the ion source temperature is 100 ℃, the desolvation gas temperature is 400 ℃, and the taper hole gas flow is 50 L.h-1Flow rate of desolvation agent 700 L.h-1The low energy scanning transmission collision energy is 6eV, the high energy scanning transmission collision energy is 20eV, and the scanning mass range is 20-1000 m/z.
Further, the chromatographic retention time of the characteristic peaks in the fingerprint is respectively as follows: 1.13min, 1.98min, 2.29min, 3.26min, 3.71min, 6.09min, 6.99min, 7.91min, 8.22min, 8.41min, 8.60min, 9.03 min.
Further, the spectral efficiency model is as follows:
Y＝0.792-6.715*10-5X1+4.977*10-6X2+3.243*10-6X5-1.846*10-5X6-2.133*10-6X8+3.173*10-5X9，R2＝1,P<0.05。
wherein, X1Represents the peak area of chromatographic peak with retention time of 1.13min, X2Represents the peak area of chromatographic peak with retention time of 1.98min, X5Represents the peak area of chromatographic peak with retention time of 3.71min, X6Represents the peak area of chromatographic peak with retention time of 6.09min, X8Represents the peak area of chromatographic peak with retention time of 7.91min, X9Represents the peak area of chromatographic peak with retention time of 8.22min, and Y is the bacteriostasis coefficient of the anti-MRSA active part of the schisandra fruit.
Calculating the corresponding bacteriostatic coefficient of the MRSA-resistant active site of the test sample according to the established mathematical model of the spectrum effect relationship; and when Y is more than 0.6, judging that the quality of the test sample of the schisandra chinensis is good.
Has the beneficial effects that: according to the invention, by establishing a mathematical model, fingerprint data and the bacteriostatic coefficient of the anti-MRSA active part of the schisandra chinensis are correlated, and 6 components in the schisandra chinensis are analyzed to obtain large contribution to the anti-MRSA bacteriostatic activity, so that the invention further discloses that the antibacterial active pharmacodynamic substance of the schisandra chinensis is based on a plurality of active components, and 2 compounds with large contribution are preliminarily identified, and are all large-polarity organic acids. The method adopts the combination of the fingerprint and the mathematical model to judge the quality of the schisandra product, can avoid the interference of third party factors, does not need to carry out complicated pharmacological experiments, avoids the individual deviation of animals, and can basically and clearly determine the antibacterial effect of the MRSA-resistant active part of the schisandra.
Drawings
FIG. 1 is UPLC-MS characteristic fingerprint of EEW Component site of Schisandra chinensis in different producing areas in example 1.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples.
Example 1
Establishing a detection model for schisandra quality based on spectral efficiency relationship
1. Drugs and reagents
Fructus Schisandrae (9 batches of medicinal materials are produced and harvested for the time shown in Table 1) is identified as dry mature fruit of Schisandra chinensis S.chinensis of Magnoliaceae by Wuxi traditional Chinese medicine Hospital Hemin Shi Yi, and specimen (No. YXB 20161101-YXB 20161109) is stored in traditional Chinese medicine research room; vancomycin (Lilly, Spain, batch No. C752996); citric acid (China institute for food and drug assay, batch No. 111679-201602).
TABLE 1 Schisandra chinensis medicinal material source
Acetonitrile (Fisher Scientific, USA, chromatographic purity); formic acid (Sigma-Aldrich, USA, chromatographic purity); water (Millipore corporation, usa, ultrapure water); AB-8 macroporous adsorbent resin (Guangfu Fine chemical research institute, Tianjin); other reagents were analytically pure. MH agar medium (Kyoto Loop Microbiol technologies, Inc., lot number 3105584); MH broth medium (guangdong cyclokay microbiological science ltd., lot No. 3105299); methicillin-resistant staphylococcus aureus ATCC 43300 (guangdong province collection of microorganisms, lot number 20160510).
2. Preparation of anti-MRSA active site (EEW Component) of fructus Schisandrae
The method comprises the following steps: weighing 10g of schisandra chinensis medicinal material which is dried and crushed at 65 ℃, wherein the material-liquid ratio is 1: 10 adding 70% ethanol, extracting under reflux for 3h, extracting at 80 deg.C for 2 times, mixing the extractive solutions for 2 times, and concentrating to dry to obtain fructus Schisandrae ethanol extract. Adding 100mL of purified water into the suspension, respectively and sequentially extracting the suspension for 4 times by using petroleum ether (60-90 ℃), chloroform and ethyl acetate which are equal in volume, combining ethyl acetate extraction liquid, and concentrating the ethyl acetate extraction liquid until the ethyl acetate extraction liquid is dried to obtain an ethyl acetate extract. The ethyl acetate extract was suspended in purified water to prepare 10 g.L -1The pretreated AB-8 macroporous adsorption resin is added into the sample loading liquid, the dosage of the macroporous resin is 60g, and the material-liquid ratio is 3: 1, the sample loading rate is 2 mL/min-1Standing for adsorption for 3 hr, eluting with 3 times of column volume of purified water at flow rate of 4 mL/min-1. Collecting purified water eluate Component, concentrating to dry, and vacuum drying to obtain AB-8 macroporous adsorbent resin separated fructus Schisandrae ethyl acetate extract purified water eluate Component (EEW Component).
The prepared ethyl acetate extracts of 9 batches of schisandra chinensis medicinal materials are 0.2912g, 0.3215g, 0.3345g, 0.3235g, 0.3457g, 0.3267g, 0.3412g, 0.3089g and 0.3243g respectively, and the yields are 2.912%, 3.215%, 3.345%, 3.235%, 3.457%, 3.267%, 3.412%, 3.089% and 3.243% respectively. The EEW Component of 9 batches of schisandra chinensis medicinal materials is 0.1973g, 0.2007g, 0.2234g, 0.2283g, 0.2324g, 0.2289g, 0.2323g, 0.2087 and 0.2134 respectively, and the yield is 1.973%, 2.007%, 2.234%, 2.283%, 2.324%, 2.289%, 2.323%, 2.087% and 2.134% respectively.
3. Determination of antibacterial efficacy of anti-MRSA active site (EEW Component) of fructus Schisandrae chinensis medicinal materials in different producing areas
(1) Preparation of the bacterial suspension
Selecting single typical colony of methicillin-resistant staphylococcus aureus ATCC 43300, culturing in MH broth culture medium at 37 deg.C for 24 hr, and correcting the bacterial suspension to 0.1 McLeod turbidity unit with turbidimeter, wherein the bacterial content is 3 × 10 7CFU·mL-1And (4) standby.
(2) Pharmacodynamic determination of anti-MRSA (methicillin resistant Staphylococcus aureus) in vitro antibacterial activity of schisandra EEW Component part
The in vitro antibacterial activity is measured according to a pipe-disc method recommended by 'Chinese pharmacopoeia' of 2015 edition, a culture dish with the diameter of 90mm is taken, 20mL of melted MH agar culture medium is added, the mixture is uniformly spread and placed, cooled and solidified to be used as a bottom layer. Heating MH agar culture medium to melt, cooling to 50 deg.C, adding prepared experimental bacterial suspension to make bacterial content be 1.0%, shaking, adding 5mL into culture dish, and uniformly spreading on bottom layer to obtain bacterial layer. After the culture medium is placed, cooled and solidified, 4 stainless steel small tubes (with the inner diameter of 6.0mm +/-0.1 mm, the height of 10.0mm +/-0.1 mm and the outer diameter of 7.8mm +/-0.1 mm) are uniformly placed on the bacterial layer at equal intervals. Collecting 9 batches of fructus Schisandrae, preparing 9 batches of fructus Schisandrae EEW Component parts for test according to the method of 2, precisely weighing, adding sterile water to obtain 100 g.L-1A test solution. 0.1mL of test solution was added to each stainless steel vial. The test of each test solution is independently repeated for 3 times, the culture dish is placed in a biochemical incubator for 24 hours at 37 ℃, the diameter of the inhibition zone is measured by a vernier caliper, and the average value of the diameter of the inhibition zone of the test solution at the EEW Component part of 9 batches of schisandra chinensis is measured. Adding vancomycin positive control group into each dish, adding 10 g.L into stainless steel tubule -10.1mL of vancomycin. A negative control group was added to a stainless steel vial with 0.1mL of sterile aqueous solution. And judging the sensitivity of each component to bacteria according to the bacteriostatic coefficient, wherein the higher the bacteriostatic coefficient is, the stronger the bacteriostatic activity is. The bacteriostasis coefficient is the diameter of the bacteriostasis zone of the test solution/the diameter of the bacteriostasis zone of vancomycin in the same culture dish.
Mean value plus or minus standard deviation for bacteriostatic activity experimental results
Data between groups was shown to be analyzed by one-way ANOVA using origin7.5 statistical software, P<0.05 indicates that the significant difference is statistically significant, P<0.01 indicates that the most significant difference is statistically significant.
TABLE 29 anti-MRSA bacteriostatic activity of Schisandra chinensis EEW Component parts ((S))
n＝3)
Note: p <0.01 compared to negative control group.
4. Establishment of UPLC-MS characteristic fingerprint spectrum of schisandra EEW Component part
(1) Preparation of test solutions
Taking 9 batches of schisandra EEW Component part samples prepared in the experiment for determining the bacteriostatic efficacy, precisely weighing 0.0050g and 2mL of methanol for dissolving, and filtering by a 0.22 mu m microporous filter membrane before sample injection.
(2) Chromatographic separation conditions
Chromatographic column Acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm, Waters Corp.); the column temperature is 45 ℃; the volume flow rate is 0.3 mL/min-1(ii) a The sample injection amount is 2 mu L; the mobile phase was acetonitrile (a) -0.1% formic acid (B). Phase a ratio varies with time: 0-3 min, 0% -0%; 0-15% for 3-10 min; 10-12 min, 15% -80%; 12-12.1 min, 80% -0%.
(3) Conditions of Mass Spectrometry
Electrospray ionization ion Source (ESI), negative ion mode scanning mass spectrometry response is stronger, Capillary voltage (Capillary)3.0kV, Cone-hole voltage (Sampling Cone)30V, ion Source Temperature (Source Temperature)100 ℃, Desolvation Temperature (Desolvation Temperature)400 ℃, Cone-hole Gas Flow (Cone Gas Flow) 50 L.h-1, Desolvation Gas Flow (Desolvation Gas Flow)700 L.h-1. The low energy scan (function 1) transmits collision energy of 6eV, the high energy scan (function 2) transmits collision energy of 20eV, and the scan mass range is m/z 20-1000.
And (3) analyzing and processing the 9 batches of schisandra EEW Component active site samples by using MassLynx V4.1 to a negative ion mode total ion flow diagram, wherein a UPLC-MS characteristic fingerprint spectrum is shown in figure 1. The integrated chromatography software (Waters corporation) was used to automatically Integrate, and each peak area in the characteristic fingerprint was derived, and 11 common peaks were matched according to the retention time of each peak in the characteristic fingerprint of each sample, as shown in table 3.
TABLE 3 common peak area of EEW Component part of Schisandra chinensis in different producing areas
(4) Spectral efficiency relationship analysis
Establishment of multiple Regression (PLSR) equation
Taking the peak area of each peak representing chemical components in the characteristic fingerprint as an independent variable X, taking the bacteriostasis coefficient of the MRSA resistant active part of the schisandra as a dependent variable Y, and carrying out multiple linear regression analysis on the independent variable and the dependent variable Y by adopting SPSS19.0 software to obtain a regression equation Y which is 0.792-6.715X 10 -5X1+4.977*10-6X2+3.243*10-6X5-1.846*10-5X6-2.133*10-6X8+3.173*10- 5X9，R2＝1,P<0.05。
The regression coefficient represents the contribution of each variable to the drug effect, the larger the regression coefficient is, the larger the contribution to the drug effect is, the positive independent variable of the coefficient is positively correlated with the drug effect, and the negative coefficient is negatively correlated with the drug effect. Model diagnostic parameter R2＝1，P<0.05, which shows that the regression model has stronger fitting interpretation capability and model prediction capability.
In regression analysis, the contribution of each characteristic common peak to the bacteriostatic activity is evaluated by using regression coefficients, and X is indicated according to the regression coefficients of each characteristic peak2、X5、X9Is in positive correlation with the bacteriostatic activity, X1、X6、X8The compounds represented by peaks 2, 5 and 9 on the UPLC-MS characteristic map are considered to be anti-MRSA antibacterial active substances (group) of schisandra chinensis. Molecular formula matching software Elemental compositionTM(Waters corporation) calculation processing, combining the relative molecular mass and relative retention time of each compound, attributing each main molecular ion peak and high-energy collision fragment signal through literature search and relevant database,and carrying out UPLC-MS/MS compound qualitative identification on the characteristic peak 2 and the characteristic peak 3. The primary identification result shows that the peak 2 is citric acid and the peak 3 is malic acid isomer, and the compound identification result is shown in Table 4.
Table 4 identification of the composition of the compounds
Note: ingredients confirmed by control
Second, method for detecting quality of schisandra chinensis by using mathematical model based on spectral efficiency relationship
1. Instrument and reagent
Acquisty UPLC ultra high performance liquid chromatograph (Waters Corporation, USA), MALDI SYNAPT QTOF-MS quadrupole time of flight mass spectrometry (Waters Corporation, UK), vancomycin (Lilly, Spanish, batch No. C752996); citric acid (China institute for food and drug assay, batch No. 111679-201602).
Fructus Schisandrae (3 batches, purchased from Suzhou Tianling Chinese medicinal decoction pieces Co., Ltd.) was identified as dry mature fruit of Schisandra chinensis S.chinensis of Magnoliaceae by TCM physicians in Wuxi city, and 10g of dried and pulverized Schisandra chinensis at 65 deg.C was weighed.
Methicillin-resistant staphylococcus aureus ATCC 43300 (guangdong province collection of microorganisms, lot number 20160510). Selecting single typical colony of methicillin-resistant staphylococcus aureus ATCC 43300, culturing in MH broth culture medium at 37 deg.C for 24 hr, and correcting the bacterial suspension to 0.1 McLeod turbidity unit with turbidimeter, wherein the bacterial content is 3 × 107CFU·mL-1And (5) standby.
2. Preparation of test solutions
Preparation of anti-MRSA active site (EEW Component) from fructus Schisandrae 3. Weighing 10g of schisandra chinensis medicinal material which is dried and crushed at 65 ℃, wherein the material-liquid ratio is 1: 10 adding 70% ethanol, extracting under reflux for 3h, extracting at 80 deg.C for 2 times, mixing the extractive solutions for 2 times, and concentrating to dry to obtain fructus Schisandrae ethanol extract. Adding 100mL of purified water into the suspension, respectively extracting the suspension for 4 times by respectively using petroleum ether (60-90 ℃), chloroform and ethyl acetate with equal volumes, and combining the ethyl acetate The extract was concentrated to dryness to obtain an ethyl acetate extract. The ethyl acetate extract was suspended in purified water to prepare 10 g.L-1The pretreated AB-8 macroporous adsorption resin is added into the sample loading liquid, the dosage of the macroporous resin is 60g, and the material-liquid ratio is 3: 1, the sample loading rate is 2 mL/min-1Standing for adsorption for 3 hr, eluting with 3 times of column volume of purified water at flow rate of 4 mL/min-1. Collecting purified water eluate Component, concentrating to dry, and vacuum drying to obtain AB-8 macroporous adsorbent resin separated fructus Schisandrae ethyl acetate extract purified water eluate Component (EEW Component). 0.0050g and 2mL of methanol were precisely weighed and dissolved, and the solution was filtered through a 0.22 μm microporous membrane before injection.
Chromatographic conditions are as follows: chromatographic column Acquity UPLC BEH C18(100 mm. times.2.1 mm, 1.7 μm, Waters Co.); the column temperature is 45 ℃; the volume flow rate is 0.3 mL/min-1(ii) a The sample injection amount is 2 mu L; the mobile phase was acetonitrile (a) -0.1% formic acid (B). Phase a ratio varies with time: 0-3 min, 0% -0%; 0-15% for 3-10 min; 10-12 min, 15% -80%; 12-12.1 min, 80% -0%.
Mass spectrum conditions: electrospray ionization ion Source (ESI), stronger negative ion mode scanning mass spectrum response, 3.0kV Capillary voltage (Capillary), 30V Cone-hole voltage (Sampling Cone), 100 ℃ ion Source Temperature (Source Temperature), 400 ℃ Desolvation Temperature (Desolvation Temperature), and 50 L.h.cone-hole gas flow (Cone gas flow) -1The Desolvation Gas Flow rate (Desolvation Gas Flow) was 700 L.h-1. The low energy scan (function 1) transmits collision energy of 6eV, the high energy scan (function 2) transmits collision energy of 20eV, and the scan mass range is m/z 20-1000.
After sample injection, 12 common peaks are matched and are respectively X1、X2、X3、X4、X5、X6、X7、X8、X9、X10、X11、X12。
The retention time and peak area of each common peak are shown in table 5 below:
TABLE 5 Table of retention time of common peak and area of peak
Substituting the peak area into the model equation
Y＝0.792-6.715*10-5X1+4.977*10-6X2+3.243*10-6X5-1.846*10-5X6-2.133*10-6X8+3.173*10-5X9，R2＝1,P<0.05, calculating the bacteriostatic coefficient of the sample, YSample 1＝0.7427，YSample 2＝0.7069，YSample 30.7008. And judging whether the bacteriostatic effect is good or bad according to the Y value of the calculation result, wherein the bacteriostatic coefficients of the 3 batches of schisandra chinensis in the embodiment are all larger than 0.6, which indicates that the quality of the schisandra chinensis decoction pieces is better.
In order to verify the practicability of the model, the in-vitro antibacterial activity is determined by adopting a tube-disc method, the in-vitro antibacterial activity is determined according to the tube-disc method recommended by 'Chinese pharmacopoeia' of 2015 edition, a culture dish with the diameter of 90mm is taken, 20mL of molten MH agar culture medium is added, the culture dish is uniformly spread, and the culture dish is placed, cooled and solidified to serve as a bottom layer. Heating MH agar culture medium to melt, cooling to 50 deg.C, adding prepared experimental bacterial suspension to make bacterial content be 1.0%, shaking, adding 5mL into culture dish, and uniformly spreading on bottom layer to obtain bacterial layer. After the culture medium is placed, cooled and solidified, 4 stainless steel small tubes (with the inner diameter of 6.0mm +/-0.1 mm, the height of 10.0mm +/-0.1 mm and the outer diameter of 7.8mm +/-0.1 mm) are uniformly placed on the bacterial layer at equal intervals. Taking 3 batches of fructus Schisandrae chinensis medicinal materials, precisely weighing the prepared EEW Component parts of the 3 batches of fructus Schisandrae chinensis, adding sterile water, and preparing into 100 g.L -1A test solution. To each stainless steel vial was added 0.1mL of test solution. The test of each test solution is independently repeated for 3 times, the culture dish is placed in a biochemical incubator for 24 hours at 37 ℃, the diameter of the inhibition zone is measured by a vernier caliper, and the average value of the diameter of the inhibition zone of the test solutions at the EEW Component parts of 3 batches of schisandra chinensis is measured. Adding vancomycin positive control group into each dish, adding 10 g.L into stainless steel tubule-10.1mL of vancomycin. A negative control group was added to a stainless steel vial with 0.1mL of sterile aqueous solution. Judging each component pair according to the bacteriostasis coefficientThe sensitivity of bacteria is higher, and the higher the bacteriostasis coefficient is, the stronger the bacteriostasis activity is. And judging the sensitivity of each batch of components to MRSA according to the bacteriostasis coefficient (the bacteriostasis coefficient is the diameter of the bacteriostasis zone of the test solution/the diameter of the bacteriostasis zone of the vancomycin in the same culture dish). A comparison was made between the mathematical model and the experimental data results, as shown in table 6 below.
TABLE 6 verification of mathematical models
The bacteriostasis coefficient of the verification sample obtained by substituting the peak area in the established mathematical model is basically consistent with the measured value, and the overall judgment result is more accurate, so the model has a better drug effect evaluation result.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (1)
1. The schisandra quality detection method based on the spectrum effect relationship is characterized by comprising the following steps: the method comprises the following steps:
step 1, establishing a UPLC-MS fingerprint of an MRSA (methicillin resistant Staphylococcus aureus) in-vitro antibacterial active component of a schisandra chinensis test sample to be tested;
the preparation process of the MRSA in-vitro bacteriostatic active component comprises the following steps: taking dried and crushed schisandra chinensis medicinal materials, adding 70% ethanol for reflux extraction, concentrating an extracting solution to obtain a schisandra chinensis ethanol extract, adding purified water for suspension, respectively and sequentially extracting with petroleum ether, chloroform and ethyl acetate with equal volume, combining ethyl acetate extracting solutions, concentrating to dryness to obtain an ethyl acetate extract, suspending the ethyl acetate extract in the purified water, adsorbing and eluting with pretreated AB-8 macroporous adsorption resin, collecting an eluting component, concentrating to dryness, and vacuum drying to obtain the schisandra chinensis ethanol extract;
the chromatographic conditions for UPLC-MS were as follows:
the preparation method of the test solution comprises the following steps: dissolving 0.0050g sample in 2mL methanol, filtering with 0.22 μm microporous membrane before sample introduction, and collecting filtrate as sample solution;
UPLC chromatographic separation conditions: the chromatographic column is Acquisty UPLC BEH C18, the column temperature is 45 ℃, the volume flow rate is 0.3mL min < -1 >, the sample injection amount is 2 mu L, the mobile phase is acetonitrile A-0.1% formic acid B, and the phase A proportion changes along with the time: 0-3 min, 0-0%, 3-10 min, 0-15%, 10-12 min, 15-80%, 12-12.1 min, 80-0%;
MS conditions: the electrospray ionization ion source has stronger negative ion mode scanning mass spectrum response, the capillary voltage is 3.0kV, the taper hole voltage is 30V, the ion source temperature is 100 ℃, the desolvation gas temperature is 400 ℃, and the taper hole gas flow is 50 L.h-1Flow rate of desolvation agent 700 L.h-1The low energy scanning transmission collision energy is 6eV, the high energy scanning transmission collision energy is 20eV, and the scanning mass range is 20-1000 m/z;
step 2, screening out the characteristic peaks of the fingerprint obtained in the step 1;
screening the characteristic peaks by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, wherein the chromatogram retention time of the characteristic peaks in the fingerprint is respectively as follows: 1.13min, 1.98min, 2.29min, 3.26min, 3.71min, 6.09min, 6.99min, 7.91min, 8.22min, 8.41min, 8.60min, 9.03 min;
step 3, substituting the characteristic peak area screened out in the step 2 into a spectral efficiency mathematical model for calculation, and judging the quality of the schisandra fruit test product according to the result obtained by calculation;
the spectral efficiency mathematical model is as follows:
Y＝0.792-6.715*10-5X1+4.977*10-6X2+3.243*10-6X5-1.846*10-5X6-2.133*10-6X8+3.173*10-5X9，R2＝1,P<0.05；
wherein, X1Represents the peak area of chromatographic peak with retention time of 1.13min, X2The peak area of chromatographic peak representing retention time of 1.98min, X5Represents the peak area of chromatographic peak with retention time of 3.71min, X 6Represents the peak area of chromatographic peak with retention time of 6.09min, X8Represents the peak area of chromatographic peak with retention time of 7.91min, X9Representing the peak area of a chromatographic peak with retention time of 8.22min, and Y is the bacteriostasis coefficient of the anti-MRSA active part of the schisandra chinensis;
the method for judging the quality of the schisandra chinensis comprises the following steps: and when the Y of the test sample is more than 0.6, judging that the quality of the test sample of the schisandra chinensis is good.
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