CN112525978A - Method for screening active ingredients of pilose antler for promoting testicular interstitial cells to secrete testosterone - Google Patents
Method for screening active ingredients of pilose antler for promoting testicular interstitial cells to secrete testosterone Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
Abstract
A method for screening active ingredients of pilose antler for promoting testicular interstitial cells to secrete testosterone belongs to the technical field of traditional Chinese medicine material identification. The invention uses electrospray tandem mass spectrometry (ESI-MS/MS) as a technical means to detect chemical components in 6 batches of each fresh pilose antler product and each traditional processed product, measures the testosterone secretion of rat testicular interstitial cells promoted by each sample through an ELISA method, performs data analysis by using a chemometric method, and correlates with a Principal Component Analysis (PCA), a partial least squares discriminant analysis (PLS-DA) and an orthogonal partial least squares discriminant analysis (OPLS-DA) method to screen out main components of the pilose antler promoting the testosterone secretion of the testicular interstitial cells, thereby ensuring the accuracy of experimental results. The method not only can provide a basis for the research of the active ingredients of the pilose antler for promoting the testicular interstitial cells to secrete the testosterone, but also provides a certain thought for the research of other traditional Chinese medicine ingredients.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a method for screening active ingredients of pilose antler for promoting testicular interstitial cells to secrete testosterone.
Background
Cornu Cervi Pantotrichum (Cervi Cornu Pantotrichum) is one of the traditional famous drugs in China, and is the young horn of ungossified dense-married couple hair of male deer (Cervus nippon Temminck) or red deer (Cervus elaphus Linnaeus) belonging to Cervidae. In China, the application history is thousands of years. Sweet, salty and warm in nature. It enters kidney and liver meridians. It is listed as a Chinese herb in Shen nong Ben Cao Jing, and has the actions of invigorating kidney yang, tonifying essence and blood, strengthening tendons and bones, etc. The testicular interstitial cell is an endocrine cell existing in the testicular interstitium, and has the main function of secreting testosterone, and the content of the secreted testosterone accounts for 95 percent of the total testosterone. The improvement of the secretion of androgen and the regulation of physiological function are expressed by the capability of testosterone secretion and proliferation of testicular interstitial cells, so that the effects of strengthening yang and producing sperm of pilose antler are closely related to the testosterone level of the body.
Electrospray ionization tandem mass spectrometry, as a soft ionization technique, has become an important means for studying the structure of natural products. The direct injection electrospray tandem mass spectrometry only needs a small amount of samples, and does not need to separate and process analytes, so that the analysis steps are saved, and the detection efficiency is improved. If the method can be combined with a chemometric method for application, the composition and the structure of chemical components in complex systems of analyzed traditional Chinese medicines and the like can be associated with the corresponding activity of the chemical components, so that the aim of quickly screening the active components is fulfilled.
The basis of the drug effect substance of the pilose antler is mainly polypeptide components, only amino acid is considered, and the active component for promoting testosterone secretion in the pilose antler can not be screened out comprehensively and effectively, so the invention provides a method for screening the active component for promoting testosterone secretion in the pilose antler by using an electrospray tandem mass spectrometry (ESI-MS/MS).
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a method for screening active ingredients of pilose antler for promoting testosterone secretion by testicular interstitial cells, which is used for solving the technical problem that the active ingredients of pilose antler for promoting testosterone secretion cannot be screened out comprehensively and effectively only by investigating amino acid.
A method for screening active ingredients of cornu Cervi Pantotrichum for promoting testosterone secretion by leydig cell comprises the following steps,
step one, preparing a fresh pilose antler product and a traditional pilose antler processed product sample;
step two, preparing pilose antler fresh product freeze-dried powder and pilose antler traditional processed product sample freeze-dried powder;
step three, preparing fresh pilose antler filtrate and traditional pilose antler processed product filtrate for mass spectrometry;
fourthly, respectively carrying out electrospray tandem mass spectrometry on the fresh pilose antler filtrate and the traditional pilose antler processed product filtrate to obtain a primary mass spectrometry fingerprint spectrogram of the fresh pilose antler filtrate and a primary mass spectrometry fingerprint spectrogram of the traditional pilose antler processed product filtrate, and exporting the spectrums to obtain primary mass spectrometry data of the fresh pilose antler processed product filtrate, primary mass spectrometry data of the traditional pilose antler processed product filtrate, secondary mass spectrometry data of an ion peak of the fresh pilose antler processed product filtrate and secondary mass spectrometry data of an ion peak of the traditional pilose antler processed product filtrate;
step five, separating and culturing the rat testicular interstitial cells for activity determination;
step six, respectively measuring the testosterone secretion of rat testicular interstitial cells by using a mouse testosterone (T) kit and a light absorption value after color development;
and seventhly, arranging the primary mass spectrum data of the fresh pilose antler filtrate obtained in the fourth step, the primary mass spectrum data of the traditional pilose antler processed product filtrate, the secondary mass spectrum data of the ion peak of the fresh pilose antler processed product filtrate and the secondary mass spectrum data of the ion peak of the traditional pilose antler processed product filtrate by using an Excell table, introducing the data into Simca-p14 software, and obtaining the potential active ingredient of the pilose antler for promoting the testicular interstitial cells to secrete testosterone by using a multivariate statistical analysis method, and obtaining the active ingredient structure of the pilose antler for promoting the testicular interstitial cells to secrete the testosterone by combining the secondary mass spectrum data of the ion peak of the fresh pilose antler processed product filtrate and the secondary mass spectrum data of the ion peak of the traditional pilose antler.
The method for preparing the fresh pilose antler product and the traditional pilose antler processed product sample in the first step comprises the following steps:
singing fresh cornu Cervi Pantotrichum with alcohol burner, removing fuzz, cutting into 2 parts, wrapping 1 part with preservative film to obtain conventional processed product sample, taking 1 part as fresh cornu Cervi Pantotrichum sample,
and (3) putting the wrapped pilose antler into boiling water, boiling and frying for 4-8 times, each time for 1-3 min, discharging blood from the tip of the pilose antler by using a No. 16 needle connected with nitrogen after each boiling and frying, putting the pilose antler into an oven at 50-90 ℃ after the last boiling and frying for discharging blood, baking for 6-10 h, taking out the pilose antler, cutting the pilose antler into small pieces, washing for 4-6 times, and storing the small pieces in a constant-temperature refrigerator at 4 ℃ as a traditional processed product for later use.
The method for preparing the pilose antler fresh product freeze-dried powder and the pilose antler traditional processed product sample freeze-dried powder in the step two comprises the following steps:
respectively taking 10 g-30 g of each of the fresh pilose antler product obtained in the step one and the traditional pilose antler processed product sample, respectively carrying out tissue crushing by using water with the weight of 10 times-20 times of that of the pilose antler sample, carrying out extraction at 4 ℃ for 24 h-36 h, centrifuging at the rotating speed of 3600r/min for 10 min-30 min, then carrying out microfiltration on supernatant, and freeze-drying the microfiltrate to respectively obtain the fresh pilose antler product freeze-dried powder and the traditional pilose antler processed product freeze-dried powder.
The method for preparing the fresh pilose antler filtrate and the traditional pilose antler processed product filtrate for mass spectrometry in the third step comprises the following steps:
and taking 1mg of the fresh pilose antler freeze-dried powder and the traditional pilose antler processed product freeze-dried powder in the second step respectively, dissolving the fresh pilose antler freeze-dried powder and the traditional pilose antler processed product freeze-dried powder respectively by using methanol with the mass concentration of 60-80%, fixing the volume to 1ml, centrifuging the mixture for 10min at the rotating speed of 12000r/min, taking supernatant, and filtering the supernatant by using an alcohol film of 0.45um to respectively obtain fresh pilose antler processed product filtrate and traditional pilose antler processed product filtrate for mass spectrometry.
The mass range of the device used in the electrospray tandem mass spectrometry in the third step is determined to be m/z 200-500 through correction before experiments, a positive ion mode is adopted, sample injection is carried out by a flow injection pump, the flow rate is 5 mu L/min, the temperature of dry air is 350 ℃, the flow rate of dry air is 9ml/min, the pressure of atomized air is 0.24Mpa (35.0psi), and the capillary voltage is 3.5 kV.
The method for separating and culturing the rat testicular interstitial cells for activity determination in the fifth step comprises the following steps:
taking testis of SD male adult rats with the weight of 250-300 g, randomly grouping, respectively putting the testis into TypeI collagenase, carrying out oscillatory digestion for 20-40 min in a constant temperature oscillator at 35-38 ℃, diluting with 2-4 times of DMEM culture medium to stop digestion, filtering and centrifuging to prepare suspensions, sucking out each suspension, respectively inoculating into a culture bottle, and culturing in a 37 ℃ CO2 incubator for 24h to obtain testis interstitial cell suspension for experiments;
the method for measuring the testosterone secretion amount of the fresh pilose antler filtrate and the traditional pilose antler processed product filtrate for promoting the rat testicular interstitial cells in the sixth step comprises the following steps:
the density after the culture in the fifth step is 10 mu L/hole to 120 mu L/hole5The cell suspension was seeded in 96-well plates at 37 ℃ with 5% CO2After culturing for 1-2 days under the condition, dividing the 96-well plate into a test sample group, a positive group and a blank control group,
adding 100 μ L/hole of test solution into test group, respectively, wherein the test solution is fresh filtrate of cornu Cervi Pantotrichum and conventional filtrate of cornu Cervi Pantotrichum, respectively to obtain test group containing fresh filtrate of cornu Cervi Pantotrichum and test group containing conventional filtrate of cornu Cervi Pantotrichum,
adding DMEM/F12 culture solution containing human chorionic gonadotropin HCG with final concentration of 1U/mL into the positive groups respectively at a rate of 100 μ L/well,
the blank control group was added with 100. mu.L/well of DMEM/F12 culture solution,
the 96-well plate was then incubated at 37 ℃ with 5% CO2Culturing for 24 hours under the condition, respectively absorbing cell supernatant of each group for carrying out a mouse testosterone (T) kit experiment, respectively adding standard products and samples carried in the mouse testosterone (T) kit with different concentrations into an enzyme label plate coated with a rat testosterone monoclonal antibody in advance, respectively adding biotin-labeled secondary antibody and enzyme label reagent, reacting for 50-80 min at 37 ℃ to form immune complex, washing the plate for 4-6 times by using washing liquid to remove unbound enzyme, respectively and sequentially adding a substrate A and a substrate B carried by the mouse testosterone (T) kit, developing for 10min at 37 ℃, then adding a stop solution carried by the mouse testosterone (T) kit, measuring the light absorption value under 450nm within 10min, and obtaining the secretion content of fresh pilose antler and traditional processed pilose antler products to testicular interstitial cells.
The multivariate statistical analysis method in the seventh step comprises a Principal Component Analysis (PCA) method, an orthogonal partial least squares discriminant analysis (OPLS-DA) method, a partial least squares discriminant analysis (PLS-DA) method and a gray correlation analysis (GRA).
The method for obtaining the active component structure of the antler for promoting the testicular interstitial cells to secrete testosterone in the seventh step comprises the following steps:
firstly, exporting primary mass spectrum data of a fresh pilose antler product filtrate, primary mass spectrum data of a traditional pilose antler product filtrate, secondary mass spectrum data of an ion peak of the fresh pilose antler product filtrate and secondary mass spectrum data of the ion peak of the traditional pilose antler product filtrate;
secondly, using SIMCA14.1 software to obtain a score scatter diagram of the fresh pilose antler products and the traditional pilose antler processed products by using mass spectrum peak abundance of each derived primary mass spectrum data in the range of m/z 200-500 as a variable and using a Principal Component Analysis (PCA) method for each derived primary mass spectrum data, wherein each point in the diagram represents 1 sample, and the fresh pilose antler products and the traditional pilose antler processed products have difference;
taking a partial least squares discriminant analysis (PLS-DA) model with a fitting degree meeting the requirement as a supervised orthogonal partial least squares discriminant analysis (OPLS-DA) model, reducing the OPLS-DA to obtain a scatter diagram, wherein the scatter diagram comprises the fresh pilose antler product and the traditional pilose antler processed product, the difference between the two groups is maximized, the variation rule between the groups is highlighted, and simultaneously, a VIP (variable projection importance) value is adopted to screen components with larger difference,
setting the components corresponding to the variable with the VIP value greater than 1 as main different components of the fresh pilose antler product and the traditional pilose antler processed product;
thirdly, obtaining active ingredients of the testosterone secretion promoting activity of the pilose antler sample by a gray correlation analysis (GRA) method, a partial least squares discriminant analysis (PLS-DA) method and an electrospray ionization mass spectrometry fingerprint of the fresh and processed pilose antler products
a. Calculating the grey correlation degree of data derivation in the Excel table, and determining the sequence of contribution of each component to the testosterone secretion action from large to small according to the obtained correlation value, wherein the correlation degree of the chemical components represented by 4 ion peaks and the testosterone secretion activity is more than 0.85, so that the testosterone secretion action of Leydig cells of the pilose antler is the result of the joint action of multiple components;
b. extracting main components for multiple times and iterating through SIMCA14.1 software, fitting a mathematical equation between the abundance data of the common ion peaks and the testosterone secretion activity data in the electrospray mass spectrum primary mass spectrum of the fresh and processed velvet antler products, and obtaining a contribution degree sequence of the chemical components represented by each ion peak to the testosterone secretion activity, wherein the chemical components represented by a partial ion peak are positively correlated with the testosterone secretion activity, and the chemical components represented by a partial ion peak are negatively correlated with the testosterone secretion activity;
c. 2 ion peaks at the first 5 positions of fresh pilose antler products and traditional pilose antler processed products which have the largest contribution to the activity of promoting the secretion of testosterone are coincident, namely m/z416 and 426 which are respectively in positive correlation with the activity;
d. and c, carrying out spectrum recognition on possible components in the step c according to the ion peak secondary mass spectrum data of the fresh pilose antler filtrate and the ion peak secondary mass spectrum data of the pilose antler traditional processed product filtrate, and screening Tyr-Ser-Phe and Phe-Phe-Ile/leu as main active components of the pilose antler for promoting testicular interstitial cells to secrete testosterone.
Through the design scheme, the invention can bring the following beneficial effects:
the method comprises the steps of detecting chemical components in 6 batches of fresh pilose antler products and traditional processed products by taking an electrospray tandem mass spectrometry (ESI-MS/MS) as a technical means, measuring the testosterone secretion amount of rat testicular interstitial cells of each sample by an ELISA method, processing obtained data by using a chemometric method, screening out main components of the pilose antler for promoting the testicular interstitial cells to secrete testosterone, and analyzing obtained results. The method not only can provide a basis for the research of the active ingredients of the pilose antler for promoting the testicular interstitial cells to secrete the testosterone, but also provides a certain thought for the research of other traditional Chinese medicine ingredients.
The method which combines the electrospray tandem mass spectrometry with the multivariate statistical analysis method is utilized for analysis. Compared with the prior art, the method is more comprehensive, detects the components of different processed products of the pilose antler on the whole, and can systematically compare the difference between the fresh pilose antler product and the traditional pilose antler processed product;
the data analysis is carried out by using a chemometric method, and the Principal Component Analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) are correlated with each other, so that the accuracy of an experimental result is ensured; and by combining modern analysis software, the analysis result is accurate and reliable. The invention has universality and provides a method and basis for analyzing and screening other important components in a certain angle.
Drawings
The invention is further described with reference to the following figures and detailed description:
FIG. 1 is an electrospray ionization mass spectrometry fingerprint of a fresh pilose antler product obtained in step four in the embodiment of the invention;
FIG. 2 is an electrospray ionization mass spectrometry fingerprint of a traditional processed product of pilose antler obtained in step four of the embodiment of the invention;
FIG. 3 is a PCA score scatter plot of the fresh velvet antler product obtained in the seventh step of the present invention and the conventional velvet antler processed product;
FIG. 4 is a PLS-DA model verification diagram of fresh cornu Cervi Pantotrichum and processed cornu Cervi Pantotrichum products in accordance with the embodiment of the present invention;
FIG. 5 is a scatter diagram of OPLS-DA scores of the fresh velvet antler product obtained in the seventh step and the conventional velvet antler processed product in the example of the present invention;
FIG. 6 is a first secondary mass spectrum of the deer antler sample obtained in the eighth step of the example of the present invention;
fig. 7 is a second mass spectrum of the antler sample obtained in the eighth step in the example of the invention.
Detailed Description
A method for screening active ingredients of cornu Cervi Pantotrichum for promoting testosterone secretion by leydig cell comprises the following steps,
step one, preparing a fresh pilose antler product and a sample of a traditional pilose antler processed product
Singing fresh cornu Cervi Pantotrichum with alcohol burner, removing fuzz, cutting into 2 parts, wrapping 1 part with preservative film to obtain conventional processed product sample, taking 1 part as fresh cornu Cervi Pantotrichum sample,
putting the wrapped pilose antler into boiling water, boiling and frying for 4-8 times, each time for 1-3 min, discharging blood from the tip of the pilose antler by using a No. 16 needle connected with nitrogen after each boiling and frying, putting the pilose antler into an oven at 50-90 ℃ after the last boiling and frying for discharging blood, baking for 6-10 h, taking out the pilose antler, cutting the pilose antler into small pieces, washing for 4-6 times by using water, and storing the pilose antler in a constant-temperature refrigerator at 4 ℃ as a traditional processed product for later use;
step two, preparation of fresh pilose antler product and pilose antler traditional processed product sample
Respectively taking 10 g-30 g of the fresh pilose antler product obtained in the step one and the traditional pilose antler processed product sample, respectively carrying out tissue crushing by using water with the weight of 10 times-20 times that of the pilose antler sample, carrying out extraction at 4 ℃ for 24 h-36 h, centrifuging at the rotating speed of 3600r/min for 10 min-30 min, then carrying out microfiltration on supernatant, and freeze-drying the micro-filtrate to respectively obtain fresh pilose antler product freeze-dried powder and traditional pilose antler processed product freeze-dried powder;
step three, preparing pilose antler sample for mass spectrometry
Respectively taking 1mg of the fresh pilose antler freeze-dried powder and the traditional pilose antler processed product freeze-dried powder in the second step, respectively dissolving by using methanol with the mass concentration of 60% -80%, fixing the volume to 1ml, centrifuging for 10min at the rotating speed of 12000r/min, taking supernatant, filtering by using an alcohol membrane of 0.45um, and respectively obtaining fresh pilose antler filtered liquid and traditional pilose antler processed product filtered liquid for mass spectrometry;
fourthly, carrying out electrospray tandem mass spectrometry on the fresh pilose antler filtrate and the traditional pilose antler processed product filtrate;
fourthly, respectively carrying out electrospray tandem mass spectrometry on the fresh pilose antler filtrate and the traditional pilose antler processed product filtrate to obtain a primary mass spectrometry fingerprint spectrogram of the fresh pilose antler filtrate and a primary mass spectrometry fingerprint spectrogram of the traditional pilose antler processed product filtrate, and exporting the spectrums to obtain primary mass spectrometry data of the fresh pilose antler processed product filtrate, primary mass spectrometry data of the traditional pilose antler processed product filtrate, secondary mass spectrometry data of an ion peak of the fresh pilose antler processed product filtrate and secondary mass spectrometry data of an ion peak of the traditional pilose antler processed product filtrate;
step five, separating and culturing rat testicular interstitial cells for activity determination
Taking testis of SD male adult rat with weight of 250-300 g, randomly grouping, placing into TypeI collagenase, respectively, digesting in a constant temperature oscillator at 35-38 deg.C for 20-40 min under oscillation, diluting with 2-4 times volume of DMEM medium to stop digestion, filtering, centrifuging to obtain suspension, sucking out each suspension, inoculating into culture bottle, and culturing at 37 deg.C in CO2Culturing in an incubator for 24h to obtain testis interstitial cell suspension for experiments;
step six, determining the influence of the antler on the content of testosterone secreted by the leydig cells
The density after the culture in the fifth step is 10 mu L/hole to 120 mu L/hole5The cell suspension was seeded in 96-well plates at 37 ℃ with 5% CO2After culturing for 1-2 days under the condition, dividing the 96-well plate into a test sample group, a positive group and a blank control group,
adding 100 μ L/hole of test solution into test group, respectively, wherein the test solution is fresh filtrate of cornu Cervi Pantotrichum and conventional filtrate of cornu Cervi Pantotrichum, respectively to obtain test group containing fresh filtrate of cornu Cervi Pantotrichum and test group containing conventional filtrate of cornu Cervi Pantotrichum,
adding DMEM/F12 culture solution containing human chorionic gonadotropin HCG with final concentration of 1U/mL into the positive groups respectively at a rate of 100 μ L/well,
the blank control group was added with 100. mu.L/well of DMEM/F12 culture solution,
the 96-well plate was then incubated at 37 ℃ with 5% CO2Culturing for 24 hours under the condition, respectively absorbing cell supernatant of each group for carrying out a mouse testosterone (T) kit experiment, respectively adding standard products and samples carried in the mouse testosterone (T) kit with different concentrations into an enzyme label plate coated with a rat testosterone monoclonal antibody in advance, respectively adding biotin-labeled secondary antibody and enzyme label reagent, reacting for 50-80 min at 37 ℃ to form an immune complex, washing the plate for 4-6 times by using a washing solution to remove unbound enzyme, respectively and sequentially adding a substrate A and a substrate B carried by the mouse testosterone (T) kit, developing for 10min at 37 ℃, then adding a stop solution carried by the mouse testosterone (T) kit, measuring the light absorption value under 450nm within 10min, and obtaining the secretion content of fresh pilose antler and traditional processed pilose antler products on testicular interstitial cells;
and seventhly, arranging the primary mass spectrum data of the fresh pilose antler filtrate, the primary mass spectrum data of the traditional pilose antler processed product filtrate, the secondary ion peak mass spectrum data of the fresh pilose antler filtrate and the secondary ion peak mass spectrum data of the traditional pilose antler processed product filtrate by using an Excel table, introducing the data into Simca-p14 software, and obtaining the potential active ingredient of the pilose antler for promoting the testicular interstitial cells to secrete testosterone by using a multivariate statistical analysis method, and obtaining the active ingredient structure of the pilose antler for promoting the testicular interstitial cells to secrete testosterone by combining the secondary ion peak mass spectrum data of the fresh pilose antler filtrate and the secondary ion peak mass spectrum data of the traditional pilose antler processed product filtrate.
The multivariate statistical analysis methods include a Principal Component Analysis (PCA) method, a partial least squares discriminant analysis (PLS-DA) method, a gray correlation analysis (GRA) method, and an orthogonal partial least squares discriminant analysis (OPLS-DA) method.
The method for obtaining the active component structure of the antler for promoting the testicular interstitial cells to secrete testosterone in the seventh step comprises the following steps:
firstly, exporting primary mass spectrum data of a fresh pilose antler product filtrate, primary mass spectrum data of a traditional pilose antler product filtrate, secondary mass spectrum data of an ion peak of the fresh pilose antler product filtrate and secondary mass spectrum data of the ion peak of the traditional pilose antler product filtrate;
secondly, using SIMCA14.1 software to obtain a score scatter diagram of the fresh pilose antler products and the traditional pilose antler processed products by using mass spectrum peak abundance of each derived primary mass spectrum data in the range of m/z 200-500 as a variable and using a Principal Component Analysis (PCA) method for each derived primary mass spectrum data, wherein each point in the diagram represents 1 sample, and the fresh pilose antler products and the traditional pilose antler processed products have difference;
taking a partial least squares discriminant analysis (PLS-DA) model with a fitting degree meeting the requirement as a supervised orthogonal partial least squares discriminant analysis (OPLS-DA) model, reducing the OPLS-DA to obtain a scatter diagram, wherein the scatter diagram comprises the fresh pilose antler product and the traditional pilose antler processed product, the difference between the two groups is maximized, the variation rule between the groups is highlighted, and simultaneously, a VIP (variable projection importance) value is adopted to screen components with larger difference,
setting the components corresponding to the variable with the VIP value greater than 1 as main different components of the fresh pilose antler product and the traditional pilose antler processed product;
thirdly, obtaining active ingredients of the testosterone secretion promoting activity of the pilose antler sample by a grey correlation analysis method, a partial least squares discriminant analysis (PLS-DA) method and an electrospray ionization mass spectrum fingerprint of the fresh and processed pilose antler products
a. Deriving data in the Excel table, calculating grey correlation degree, and determining the magnitude sequence of contribution of each component to testosterone secretion action according to the magnitude of the obtained correlation degree value, wherein the correlation degree of chemical components represented by 4 ion peaks and testosterone secretion activity is more than 0.85, so that the Leydig cell testosterone secretion action of the antler is a result of the joint action of multiple components;
b. extracting main components for multiple times and iterating through SIMCA14.1 software, fitting a mathematical equation between the abundance data of the common ion peaks and the testosterone secretion activity data in the electrospray mass spectrum primary mass spectrum of the fresh and processed velvet antler products, and obtaining a contribution degree sequence of the chemical components represented by each ion peak to the testosterone secretion activity, wherein the chemical components represented by a partial ion peak are positively correlated with the testosterone secretion activity, and the chemical components represented by a partial ion peak are negatively correlated with the testosterone secretion activity;
c. 2 ion peaks at the first 5 positions of fresh pilose antler products and traditional pilose antler processed products which have the largest contribution to the activity of promoting the secretion of testosterone are coincident, namely m/z416 and 426 which are respectively in positive correlation with the activity;
d. and c, carrying out spectrum recognition on possible components in the step c according to the ion peak secondary mass spectrum data of the fresh pilose antler filtrate and the ion peak secondary mass spectrum data of the pilose antler traditional processed product filtrate, and screening Tyr-Ser-Phe and Phe-Phe-Ile/leu as main active components of the pilose antler for promoting testicular interstitial cells to secrete testosterone.
The first embodiment,
Step one, burning and unhairing fresh pilose antler by alcohol burner, longitudinally cutting into 2 parts, wherein 1 part is wrapped by a preservative film for traditional processing, and 1 part is used as a fresh product.
Frying the packaged cornu Cervi Pantotrichum in boiling water for 1min each time; cooking and frying for 5 times, discharging blood from the tip of the antler with a No. 16 needle connected with nitrogen after cooking and frying, baking at 70 deg.C for 8h after discharging blood in the last cooking and frying, taking out, cutting into small pieces, washing with water for 5 times, bagging, and storing in a refrigerator at 4 deg.C for use.
And step two, taking 20g of each of the fresh pilose antler products and the traditional processed pilose antler products in the step one, crushing by 10 times of water, and leaching at the constant temperature of 4 ℃ for 24 hours. Centrifuging at 3600r/min for 10min, microfiltering the supernatant, and freeze-drying the microfiltered liquid for later use.
Taking six parts of each cornu Cervi Pantotrichum sample, dissolving each part at 1mg with 70% methanol to constant volume to 1ml, centrifuging (12000r/min, 10min), collecting supernatant, filtering with 0.45um alcohol membrane, and subjecting the filtrate to electrospray mass spectrometry.
Step three, the samples in the step two are respectively tested on a 6320ion-trap electrospray ion trap mass spectrometer in a positive ion mode with a mass range of m/z:200-500, a flow rate: 5 mu L/min; temperature of the drying gas: 350 ℃; flow rate of drying gas: 9 ml/min; the pressure of atomizing gas: 0.24MPa (35.0 psi); capillary voltage: 3.5 kV.
And step four, carrying out a series of operations such as processing and exporting the maps of the 12 samples, and carrying out datamation on the maps to obtain all data required by the next analysis.
And step five, randomly grouping the male adult rats with the body weight of 250 g-300 g. Taking out testis, placing into Type I collagenase, digesting for 30min at 37 deg.C under shaking, and diluting with 3 times volume of DMEM to stop digestion. Filtering, centrifuging to obtain suspension, sucking out, inoculating into culture flask, and placing in 37 deg.C CO2Culturing in an incubator for 24 h. Obtaining the testis interstitial cells for experiments.
Step six, inoculating the seed in a 96-well plate with the density of 105Cells were suspended at 100. mu.L/well for 48 hours, and the culture medium was discarded. Adding a test solution into each group of the test group at 50 mu g/mL/hole, 100 mu g/mL/hole and 200 mu g/mL/hole respectively; adding 100 μ L of DMEM/F12 culture solution containing Human Chorionic Gonadotropin (HCG) to the positive group (the final concentration of HCG is 1U/mL); adding 100 mu L of DMEM/F12 culture solution into the blank control group; each group is respectively provided with 3 multiple holes. At 37 ℃ and 5% CO2After 24 hours of culture under the condition, a standard substance with the concentration of 24ng/mL, 12ng/mL, 6ng/mL, 3ng/mL, 1.5ng/mL and 0ng/mL in a mouse testosterone (T) kit and sample solutions with the concentration of 50 mug/mL/hole, 100 mug/mL/hole and 200 mug/mL/hole respectively are added into an enzyme label plate coated with a rat testosterone monoclonal antibody in advance. Then adding biotin-labeled secondary antibody and enzyme-labeled reagent, and reacting for 60min at 37 ℃ to form immune complex. After washing the plate 5 times with the washing solution, unbound enzyme was removed, and substrate A, B from the mouse testosterone (T) kit was added and developed at 37 ℃ for 10 min. Adding self-contained stop solution in mouse testosterone (T) kit, and measuring the light absorption value at 450nm within 10 min. Obtaining the influence of different antler samples on the high-pass content of the testicular interstitial cell secretion.
And step seven, comprehensively sorting the obtained data by using an Excel table, importing the data into Simca-p14.1 software for multivariate statistical analysis, and obtaining corresponding graphs and data by using Principal Component Analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA).
And step eight, comprehensively analyzing the antler samples by combining the obtained score map and the load map with the composition of different antler samples, taking mass spectrum peak abundance of each sample mass spectrum data m/z within the range of 200-500 as a variable, and using SIMCA14.1 software to perform PCA analysis result on each derived primary mass spectrum data as shown in figure 3. Each point represents 1 sample, and the fresh pilose antler product and the processed pilose antler product have difference to some extent. But PCA analysis does not eliminate random errors that are not relevant for the purpose of the study. Therefore, the adoption of the supervised OPLS-DA can reduce the intra-group difference and highlight the variation rule among groups. Whereas the use of OPLS-DA must be based on the validation of the PLS-DA model.
FIG. 4 shows that the PLS-DA model fits well, with R generated by any one random permutation at the left end2(0.621) and Q2The (-0.115) values are all smaller than the original value at the right end, which indicates that the prediction capability of the original model is larger than that of any randomly arranged y variable, namely the model is effective.
The OPLS-DA analysis can maximize the difference between groups, and as can be seen from figure 5, the fresh and processed products of cornu Cervi Pantotrichum are divided into two categories, which are well distinguished, indicating the difference between processed and processed cornu Cervi Pantotrichum. And simultaneously screening components with larger differences by adopting the size of the VIP value, wherein the variable with the VIP value being more than 1 can play a key role in classifying the samples. The m/ z 258, 409, 416, 408, 426, 375, 315, 392, 483, 393, 354, 453 and 437 obtained by analysis can be used as main different components of the fresh and processed pilose antler products.
And step nine, analyzing the electrospray ionization mass spectrometry fingerprint spectrums of the fresh pilose antler products and the processed products and the active ingredients of the testosterone secretion promoting activity by a grey correlation degree analysis method and a partial least square method.
(1) And (5) carrying out grey correlation calculation after the raw data are subjected to standardized conversion. According to the obtained correlation, the sizes of the contributions of the components to the testosterone secretion promotion effect are determined to be in the order of m/z 408>416>409>426>393>359>274>258>481>315>392>497>437>483>375>453>365>376> 475>368>350>369>363>306>382>358>354> 333. The correlation degree of chemical components represented by 4 ion peaks and the testosterone secretion activity is more than 0.85, so that the testosterone secretion function of the Leydig cells of the pilose antler is realized by the joint action of multiple components.
(2) Extracting main components for multiple times by SIMCA14.1 software and iterating to fit a mathematical equation between the shared ion peak abundance data and testosterone secretion activity data in the electrospray ionization mass spectrum primary mass spectrum of the fresh and processed pilose antler products: y is 0.0600106X258+0.0781771X274-0.086587X306+0.113733X315-0.0797819X333+0.0968694X342-0.0432387X350+0.0388062X354-0.0576641X358+0.00138796X359-0.0175176X363-0.062117X365+0.041837X368+0.052248X369-0.0666202X375+0.0339687X376+0.0597742X382-0.0131358X392+0.00365154X393+0.0131067X408+0.0275998X409+0.0854705X416+0.0868593X426+0.0721696X437+0.0808682X453+0.0650291X475+0.0585195X481+0.0607728X483+0.061128X497The chemical components represented by the ion peaks contribute to the testosterone secretion promoting activity in the order of magnitude: m/z315>342>426>306>416>453>333>274>437>375>475>365>497>483>258>382>481>358>369>350>369>354>376>409>363>392>408>393>359, where m/z 258, 274, 315, 342, 354, 359, 368, 369, 376, 382, 393, 408, 409, 416, 426, 437, 453, 475, 481, 483 and 497 are positively correlated with activity, and m/z 306, 333, 350, 358, 363, 365, 375 and 392 are negatively correlated with activity. A
(3) By combining the results of grey correlation analysis and partial least squares analysis, it can be found that 2 of the first 5 ion peaks which have the greatest contribution to the testosterone secretion activity are coincided, namely m/z416 and 426 respectively, and are positively correlated with the activity
Step ten, performing map recognition on possible components in the step nine according to the secondary mass spectrum result of the pilose antler sample, and conjecturing by combining the prior art and theoretical knowledge
(1) M/z416 in the primary mass spectrogram is an [ M + H ] + ion peak, which indicates that the molecular weight is 415, and the chemical formula is Tyr-Ser-Phe.
(2) M/z426 in the primary mass spectrogram is an [ M + H ] + ion peak, which indicates that the molecular weight is 425 and the chemical formula is Phe-Phe-Ile/leu.
The invention researches the active ingredients of the fresh pilose antler products and the traditional processed products of the pilose antler which promote the testicular interstitial cells to secrete testosterone by combining the electrospray tandem mass spectrometry with a multivariate statistical analysis method. And (3) collecting sample information by adopting an electrospray tandem mass spectrum, and measuring the content of testosterone secreted by the leydig cells by an ELISA method. Analyzing and detecting the obtained components by using Principal Component Analysis (PCA) partial least square method discriminant analysis (PLS-DA) and orthogonal partial least square method discriminant analysis (OPLS-DA) methods, wherein the results show that the chemical compositions of the fresh pilose antler product and the traditional processed pilose antler product are different, and combining partial least square method (PLS) and gray correlation analysis (GRA) to screen Tyr-Ser-Phe and Phe-Phe-Ile/leu as the main active components of the pilose antler for promoting testicular interstitial cells to secrete testosterone.
Claims (9)
1. A method for screening active ingredients of antler for promoting testicular interstitial cells to secrete testosterone is characterized in that: comprises the following steps which are sequentially carried out,
step one, preparing a fresh pilose antler product and a traditional pilose antler processed product sample;
step two, preparing pilose antler fresh product freeze-dried powder and pilose antler traditional processed product sample freeze-dried powder;
step three, preparing fresh pilose antler filtrate and traditional pilose antler processed product filtrate for mass spectrometry;
fourthly, respectively carrying out electrospray tandem mass spectrometry on the fresh pilose antler filtrate and the traditional pilose antler processed product filtrate to obtain a primary mass spectrometry fingerprint spectrogram of the fresh pilose antler filtrate and a primary mass spectrometry fingerprint spectrogram of the traditional pilose antler processed product filtrate, and exporting the spectrums to obtain primary mass spectrometry data of the fresh pilose antler processed product filtrate, primary mass spectrometry data of the traditional pilose antler processed product filtrate, secondary mass spectrometry data of an ion peak of the fresh pilose antler processed product filtrate and secondary mass spectrometry data of an ion peak of the traditional pilose antler processed product filtrate;
step five, separating and culturing the rat testicular interstitial cells for activity determination;
step six, respectively measuring the testosterone secretion of rat testicular interstitial cells by using a mouse testosterone (T) kit and a light absorption value after color development;
and seventhly, arranging the primary mass spectrum data of the fresh pilose antler filtrate obtained in the fourth step, the primary mass spectrum data of the traditional pilose antler processed product filtrate, the secondary mass spectrum data of the ion peak of the fresh pilose antler processed product filtrate and the secondary mass spectrum data of the ion peak of the traditional pilose antler processed product filtrate by using an Excell table, introducing the data into Simca-p14 software, and obtaining the potential active ingredient of the pilose antler for promoting the testicular interstitial cells to secrete testosterone by using a multivariate statistical analysis method, and obtaining the active ingredient structure of the pilose antler for promoting the testicular interstitial cells to secrete the testosterone by combining the secondary mass spectrum data of the ion peak of the fresh pilose antler processed product filtrate and the secondary mass spectrum data of the ion peak of the traditional pilose antler.
2. The method of claim 1, wherein the step of screening the active ingredient for stimulating testosterone secretion from pilose antler mesenchymal cells comprises: the method for preparing the fresh pilose antler product and the traditional pilose antler processed product sample in the first step comprises the following steps:
singing fresh cornu Cervi Pantotrichum with alcohol burner, removing fuzz, cutting into 2 parts, wrapping 1 part with preservative film to obtain conventional processed product sample, taking 1 part as fresh cornu Cervi Pantotrichum sample,
and (3) putting the wrapped pilose antler into boiling water, boiling and frying for 4-8 times, each time for 1-3 min, discharging blood from the tip of the pilose antler by using a No. 16 needle connected with nitrogen after each boiling and frying, putting the pilose antler into an oven at 50-90 ℃ after the last boiling and frying for discharging blood, baking for 6-10 h, taking out the pilose antler, cutting the pilose antler into small pieces, washing for 4-6 times, and storing the small pieces in a constant-temperature refrigerator at 4 ℃ as a traditional processed product for later use.
3. The method of claim 1, wherein the step of screening the active ingredient for stimulating testosterone secretion from pilose antler mesenchymal cells comprises: the method for preparing the pilose antler fresh product freeze-dried powder and the pilose antler traditional processed product sample freeze-dried powder in the step two comprises the following steps:
respectively taking 10 g-30 g of each of the fresh pilose antler product obtained in the step one and the traditional pilose antler processed product sample, respectively carrying out tissue crushing by using water with the weight of 10 times-20 times of that of the pilose antler sample, carrying out extraction at 4 ℃ for 24 h-36 h, centrifuging at the rotating speed of 3600r/min for 10 min-30 min, then carrying out microfiltration on supernatant, and freeze-drying the microfiltrate to respectively obtain the fresh pilose antler product freeze-dried powder and the traditional pilose antler processed product freeze-dried powder.
4. The method of claim 1, wherein the step of screening the active ingredient for stimulating testosterone secretion from pilose antler mesenchymal cells comprises: the method for preparing the fresh pilose antler filtrate and the traditional pilose antler processed product filtrate for mass spectrometry in the third step comprises the following steps:
and taking 1mg of the fresh pilose antler freeze-dried powder and the traditional pilose antler processed product freeze-dried powder in the second step respectively, dissolving the fresh pilose antler freeze-dried powder and the traditional pilose antler processed product freeze-dried powder respectively by using methanol with the mass concentration of 60-80%, fixing the volume to 1ml, centrifuging the mixture for 10min at the rotating speed of 12000r/min, taking supernatant, and filtering the supernatant by using an alcohol film of 0.45um to respectively obtain fresh pilose antler processed product filtrate and traditional pilose antler processed product filtrate for mass spectrometry.
5. The method of claim 1, wherein the step of screening the active ingredient for stimulating testosterone secretion from pilose antler mesenchymal cells comprises: the mass range of the device used in the electrospray tandem mass spectrometry in the third step is determined to be m/z 200-500 through correction before experiments, a positive ion mode is adopted, sample injection is carried out by a flow injection pump, the flow rate is 5 mu L/min, the temperature of dry air is 350 ℃, the flow rate of dry air is 9ml/min, the pressure of atomized air is 0.24Mpa (35.0psi), and the capillary voltage is 3.5 kV.
6. The method of claim 1, wherein the step of screening the active ingredient for stimulating testosterone secretion from pilose antler mesenchymal cells comprises: the method for separating and culturing the rat testicular interstitial cells for activity determination in the fifth step comprises the following steps:
taking testis of SD male adult rats with the weight of 250-300 g, randomly grouping, respectively putting the testis into Type I collagenase, carrying out shake digestion for 20-40 min in a constant temperature oscillator at 35-38 ℃, diluting with 2-4 times of DMEM culture medium to stop digestion, filtering and centrifuging to prepare suspensions, sucking out each suspension, respectively inoculating into a culture bottle, and culturing in a 37 ℃ CO2 incubator for 24h to obtain the testis interstitial cell suspension for experiments.
7. The method of claim 1, wherein the step of screening the active ingredient for stimulating testosterone secretion from pilose antler mesenchymal cells comprises: the method for measuring the testosterone secretion amount of the fresh pilose antler filtrate and the traditional pilose antler processed product filtrate for promoting the rat testicular interstitial cells in the sixth step comprises the following steps:
the density after the culture in the fifth step is 10 mu L/hole to 120 mu L/hole5The cell suspension was seeded in 96-well plates at 37 ℃ with 5% CO2After culturing for 1-2 days under the condition, dividing the 96-well plate into a test sample group, a positive group and a blank control group,
adding 100 μ L/hole of test solution into test group, respectively, wherein the test solution is fresh filtrate of cornu Cervi Pantotrichum and conventional filtrate of cornu Cervi Pantotrichum, respectively to obtain test group containing fresh filtrate of cornu Cervi Pantotrichum and test group containing conventional filtrate of cornu Cervi Pantotrichum,
adding DMEM/F12 culture solution containing human chorionic gonadotropin HCG with final concentration of 1U/mL into the positive groups respectively at a rate of 100 μ L/well,
the blank control group was added with 100. mu.L/well of DMEM/F12 culture solution,
the 96-well plate was then incubated at 37 ℃ with 5% CO2Culturing for 24 hours under the condition, respectively absorbing cell supernatant of each group for carrying out a mouse testosterone (T) kit experiment, respectively adding standard products and samples carried in the mouse testosterone (T) kit with different concentrations into an enzyme label plate coated with a rat testosterone monoclonal antibody in advance, respectively adding biotin-labeled secondary antibody and enzyme label reagent, reacting for 50-80 min at 37 ℃ to form immune complex, washing the plate for 4-6 times by using washing liquid to remove unbound enzyme, respectively and sequentially adding a substrate A and a substrate B carried by the mouse testosterone (T) kit, developing for 10min at 37 ℃, then adding a stop solution carried by the mouse testosterone (T) kit, measuring the light absorption value under 450nm within 10min, and obtaining the secretion content of fresh pilose antler and traditional processed pilose antler products to testicular interstitial cells.
8. The method of claim 1, wherein the step of screening the active ingredient for stimulating testosterone secretion from pilose antler mesenchymal cells comprises: the multivariate statistical analysis method in the seventh step comprises a Principal Component Analysis (PCA) method, an orthogonal partial least squares discriminant analysis (OPLS-DA) method, a partial least squares discriminant analysis (PLS-DA) method and a gray correlation analysis (GRA).
9. The method of claim 8, wherein the step of screening the active ingredient for stimulating testosterone secretion from the pilose antler mesenchymal cells comprises: the method for obtaining the active component structure of the antler for promoting the testicular interstitial cells to secrete testosterone in the seventh step comprises the following steps:
firstly, exporting primary mass spectrum data of a fresh pilose antler product filtrate, primary mass spectrum data of a traditional pilose antler product filtrate, secondary mass spectrum data of an ion peak of the fresh pilose antler product filtrate and secondary mass spectrum data of the ion peak of the traditional pilose antler product filtrate;
secondly, using SIMCA14.1 software to obtain a score scatter diagram of the fresh pilose antler products and the traditional pilose antler processed products by using mass spectrum peak abundance of each derived primary mass spectrum data in the range of m/z 200-500 as a variable and using a Principal Component Analysis (PCA) method for each derived primary mass spectrum data, wherein each point in the diagram represents 1 sample, and the fresh pilose antler products and the traditional pilose antler processed products have difference;
taking a partial least squares discriminant analysis (PLS-DA) model with a fitting degree meeting the requirement as a supervised orthogonal partial least squares discriminant analysis (OPLS-DA) model, reducing the OPLS-DA to obtain a scatter diagram, wherein the scatter diagram comprises the fresh pilose antler product and the traditional pilose antler processed product, the difference between the two groups is maximized, the variation rule between the groups is highlighted, and simultaneously, a VIP (variable projection importance) value is adopted to screen components with larger difference,
setting the components corresponding to the variable with the VIP value greater than 1 as main different components of the fresh pilose antler product and the traditional pilose antler processed product;
thirdly, obtaining active ingredients of the testosterone secretion promoting activity of the pilose antler sample by a gray correlation analysis (GRA) method, a partial least squares discriminant analysis (PLS-DA) method and an electrospray ionization mass spectrometry fingerprint of the fresh and processed pilose antler products
a. Calculating the grey correlation degree of data derivation in the Excel table, and determining the sequence of contribution of each component to the testosterone secretion action from large to small according to the obtained correlation value, wherein the correlation degree of the chemical components represented by 4 ion peaks and the testosterone secretion activity is more than 0.85, so that the testosterone secretion action of Leydig cells of the pilose antler is the result of the joint action of multiple components;
b. extracting main components for multiple times and iterating through SIMCA14.1 software, fitting a mathematical equation between the abundance data of the common ion peaks and the testosterone secretion activity data in the electrospray mass spectrum primary mass spectrum of the fresh and processed velvet antler products, and obtaining a contribution degree sequence of the chemical components represented by each ion peak to the testosterone secretion activity, wherein the chemical components represented by a partial ion peak are positively correlated with the testosterone secretion activity, and the chemical components represented by a partial ion peak are negatively correlated with the testosterone secretion activity;
c. 2 ion peaks at the first 5 positions of fresh pilose antler products and traditional pilose antler processed products which have the largest contribution to the activity of promoting the secretion of testosterone are coincident, namely m/z416 and 426 which are respectively in positive correlation with the activity;
d. and c, carrying out spectrum recognition on possible components in the step c according to the ion peak secondary mass spectrum data of the fresh pilose antler filtrate and the ion peak secondary mass spectrum data of the pilose antler traditional processed product filtrate, and screening Tyr-Ser-Phe and Phe-Phe-Ile/leu as main active components of the pilose antler for promoting testicular interstitial cells to secrete testosterone.
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