CN115372520A - Blood-entering component and component distribution determination method of litsea cubeba oil - Google Patents
Blood-entering component and component distribution determination method of litsea cubeba oil Download PDFInfo
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Abstract
The invention discloses a blood-entering component and a component distribution measuring method of litsea cubeba oil, belonging to the technical field of traditional Chinese medicines, wherein the blood-entering component of the litsea cubeba oil comprises eucalyptol, p-cymene, 4-terpene alcohol, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol and 2- (4-methylphenyl) propan-2-ol; a method for measuring the blood-entering component distribution of litsea cubeba oil is used for measuring the blood-entering component distribution of the litsea cubeba oil and comprises the following steps: s1, solution preparation, S2, liquid medicine preparation, S3, tissue sample collection, S4, tissue sample treatment, S5, standard curve acquisition, S6 and tissue homogenate sample determination; the composition can show the blood-entering components of the litsea cubeba oil and the distribution conditions of various tissues in a rat body, clarify the dynamic characteristics and the tissue exposure degree of the components in the litsea cubeba oil in the rat body and provide a pharmacodynamic substance basis for the deep development and utilization of the litsea cubeba oil.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, in particular to a method for measuring blood-entering components and component distribution of litsea cubeba oil.
Background
The litsea cubeba is dry mature fruit of Cinnamomum migao of Lauraceae, also known as Mao Dan mother, vomiting wood and Chinese star anise belt, mainly produced in Guizhou, guangxi and Yunnan provinces of China, and is first recorded in Bencao gang mu Shi Yi (compendium of materia Medica): the bigger one is expensive, the smaller one is inferior, and the bigger one is called this product. The litsea cubeba oil is volatile oil obtained from litsea cubeba dry fruits by a steam distillation method, is a common medicine for Guizhou minority nationalities, has the characteristics of light yellow clear liquid, has faint scent, pungent taste and slight tingling, has the effects of warming middle-jiao to dispel cold, regulating qi to alleviate pain and the like, is used for treating stomachache, abdominal pain, chest pain, rheumatic arthritis, vomiting, chest distress and the like, and is externally used for expelling mosquitoes and flies. Modern researches show that the main active ingredient of litsea pungens is volatile oil, which can increase myocardial oxygen supply and reduce myocardial oxygen consumption, has the effects of resisting myocardial ischemia, relaxing visceral smooth muscle and vascular smooth muscle, resisting virus, inflammation and pain, inhibiting bacteria, etc. At present, the large fruit litsea cubeba oil is taken as a relevant preparation on the market, and the large fruit litsea cubeba oil has the functions of promoting qi and activating blood circulation, heart and stomach pain relieving capsules, migao heart and le dropping pills, migao essential oil dropping pills and the like, and is mainly used for treating cardiovascular diseases such as coronary heart disease, angina pectoris and the like and gastrointestinal tract diseases in clinic.
At present, in the aspect of quality control of the traditional Chinese medicine volatile oil, the GC-MS or GC technology is mainly adopted to carry out qualitative and quantitative research on chemical components of the volatile oil. In the traditional Chinese medicine quality evaluation system, the integrity of the traditional Chinese medicine is mostly embodied by adopting a fingerprint spectrum technology. The fingerprint spectrum is combined with chemical pattern recognition to carry out deeper mining and discussion on the fingerprint spectrum data, and the method is widely used for identification and quality evaluation of traditional Chinese medicines. Chemical pattern recognition is an important component of chemometrics and can be divided into unsupervised and supervised pattern recognition methods. Unsupervised pattern recognition methods include Cluster Analysis (CA), principal Component Analysis (PCA), and the like; the supervised pattern recognition method comprises Discriminant Analysis (DA), partial least squares-discriminant analysis (PLS-DA) and the like, and can be used for integrating, reducing dimension and classifying multi-index data, so that the quality information of the traditional Chinese medicine is reflected more comprehensively, scientifically and objectively. The traditional Chinese medicine fingerprint spectrum is combined with a multi-component content measuring method, has the characteristics of integrity, characteristics, stability and the like, can be used as one of effective control means for comprehensively evaluating the quality of the traditional Chinese medicine, becomes a mode for comprehensively evaluating the internal quality of the traditional Chinese medicine, is widely accepted at home and abroad, and can be used as a means for controlling the quality of the volatile oil of the traditional Chinese medicine.
The pharmacokinetics of traditional Chinese medicine is a subject which studies the dynamic change rules of traditional Chinese medicine components in vivo Absorption (A), distribution (D), metabolism (M) and Excretion (E) and the time-time relationship thereof by means of the principle of dynamics, and quantitatively describes the relationship by using mathematical functions. The research on the pharmacokinetics of the traditional Chinese medicine can provide a basis for clarifying the pharmacodynamic material basis, the action mechanism of the traditional Chinese medicine and the scientificity of the compatibility of the traditional Chinese medicine compound and the design and optimization of the traditional Chinese medicine administration scheme. The tissue distribution of the traditional Chinese medicine is an important factor influencing the drug metabolism and the drug treatment effect, and the appropriate accumulation part and exposure amount can more accurately provide the safety and effectiveness information of the traditional Chinese medicine. The litsea cubeba oil is a main drug of a plurality of traditional Chinese medicine preparations for treating cardiovascular and cerebrovascular diseases, gastrointestinal diseases and the like, such as qi-regulating and blood-activating dropping pills, heart-stomach pain-relieving capsules, miyao Xinle dropping pills and the like, so that the research on ADME process characteristics of the litsea cubeba oil in organisms can provide quantifiable basis for the research on the drug effect substance basis of the litsea cubeba oil preparation. At present, domestic and foreign literature reports about the litsea cubeba oil mainly focus on extraction and purification, chemical components and pharmacological activity, and the research on pharmacokinetics and tissue distribution of the litsea cubeba oil is not seen, so that the pharmacological and toxicological research of the litsea cubeba oil is slow in progress, and the litsea cubeba oil cannot be clinically applied.
Disclosure of Invention
1. Technical problem to be solved
Aiming at the problems in the prior art, the invention aims to provide a method for measuring the blood-entering component and the component distribution of the litsea cubeba oil, which can show the blood-entering component of the litsea cubeba oil and the distribution condition of each tissue in a rat body, clarify the dynamic characteristics and the tissue exposure degree of the components in the litsea cubeba oil in the rat body and provide a pharmacodynamic substance basis for the deep development and utilization of the litsea cubeba oil.
2. Technical scheme
In order to solve the above problems, the present invention adopts the following technical solutions.
A blood-entering component of Jatropha curcas oil comprises eucalyptol, p-cymene, 4-terpene alcohol, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol, and 2- (4-methylphenyl) propan-2-ol.
A method for measuring the blood-entering component distribution of litsea cubeba oil is used for measuring the blood-entering component distribution of the litsea cubeba oil and comprises the following steps:
s1, preparing solution
1) Preparing a standard solution: accurately weighing a proper amount of eucalyptol, p-cymene, 4-terpene alcohol, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol and 2- (4-methylphenyl) propan-2-ol reference substances, placing the reference substances in a volumetric flask, adding ethyl acetate to dissolve and dilute the reference substances to a scale, preparing a mixed reference substance solution containing eucalyptol, p-cymene, 4-terpene alcohol, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol and 2- (4-methylphenyl) propan-2-ol, and storing the mixed reference substance solution for later use;
2) Preparing an internal standard solution: precisely weighing a cyclohexanone reference substance, placing the cyclohexanone reference substance in a volumetric flask, adding a small amount of ethyl acetate, ultrasonically dissolving and fixing the volume to obtain an internal standard stock solution of cyclohexanone; taking a proper amount of cyclohexanone internal standard stock solution, gradually diluting to prepare an internal standard solution, and storing for later use;
s2, preparing liquid medicine: weighing appropriate amount of oleum Alpiniae Katsumadai, placing in a beaker, adding 0.4-0.6% Tween-80 normal saline solution, and dissolving with ultrasound for 8-12min to obtain medicinal liquid, and mixing immediately;
s3, collecting tissue samples:
1) Selecting a plurality of SD male rats, grouping the SD male rats on average, fasting for 12h before administration, freely drinking water, respectively performing femoral artery bloodletting after administration after the rats take the liquid medicine in the step S2 orally, and then quickly taking out the heart, the liver, the spleen, the lung, the kidney, the brain, the stomach, the intestine, the skin and the muscle;
2) Washing off blood stain and contents on the surface of the tissue by using ice normal saline, drying the tissue by using filter paper, filling the tissue into a self-sealing bag to form a tissue sample, and storing the tissue sample for later use;
s4, processing of the tissue sample: homogenizing the tissue sample in the step S3 by using physiological saline according to the proportion of 1:2 (g: mL), then centrifuging for 8-15min at 7000-9000 r.min < -1 >, taking 100 mu L of rat ground tissue supernatant, placing the rat ground tissue supernatant into a 1.5mL centrifuge tube, sequentially adding 100 mu L of the internal standard solution in the step S1-2, carrying out vortex mixing for 2-5min, then adding 200 mu L of ethyl acetate, carrying out vortex mixing for 2-5min, carrying out centrifugation for 10-15min at 4-6 ℃ and 10000-13000 r.min < -1 >, obtaining a tissue homogenate sample, and storing for later use;
s5, obtaining a standard curve: taking 100 mu L of rat blank liver tissue homogenate, adding 100 mu L of the mixed reference substance solution containing 6 components in the step S1-1, and preparing the mixture into a medicine concentration equivalent to that of a rat tissue homogenate sample; performing linear regression by using the ratio (A/Ai) of the peak area of the component to be detected and the peak area of the cyclohexanone internal standard as a ordinate Y and the concentration (C) of each component as an abscissa X according to the operation of the method for processing the tissue sample under the item S4, and calculating a linear equation, namely a standard curve;
s6, measuring a tissue homogenate sample: and (5) carrying out sample injection measurement on the tissue homogenate sample in the step S4 by a GC-MS SIM method, recording the index components of all samples and the peak area of the cyclohexanone internal standard, and substituting the peak areas into the standard curve in the accompanying step S5 to calculate the concentration of each component to be measured.
Further, the control in step S1-1 was placed in a 10mL volumetric flask, and the mixed control solution was stored at-15 to 25 ℃ for later use, and the volumetric flask in step S1-2 was also 10mL and the storage temperature was also-15 to 25 ℃.
Further, the concentration of the internal standard solution in the step S1-2 is 200-250 ng.mL-1.
Further, the femoral artery bleeding time points in step S3-1 were four, and were 0.167h, 0.5h, 1h, and 1.5h after the administration.
Further, the tissue sample is preserved at-15 to 25 ℃ in step S3-2.
Further, the dose in step S3-1 is 0.365-0.385g/d.
Further, the homogenate sample of the step S4 is stored in a sample bottle containing an inner cannula at a temperature of-15 to 25 ℃.
Further, the chromatographic condition of the GC-MS SIM method in the step S6 is no split-flow sample injection.
Further, the detection voltage of the mass spectrum condition in the GC-MS SIM method in the step S6 is 0.25-0.3KV.
3. Advantageous effects
Compared with the prior art, the invention has the advantages that:
the mass spectrometry is used for exploring the chemical substance composition of the litsea cubeba oil, qualitative and quantitative quality control research is combined, experimental basis is provided for improving and perfecting the quality standard of the litsea cubeba oil, the pharmacokinetic process and the tissue distribution characteristics of 6 components of eucalyptol, p-cymene, 4-terpene alcohol, 4-isopropylcyclohex-2-ene-1-one, alpha-terpineol and 2- (4-methylphenyl) propan-2-ol in organisms of the litsea cubeba oil are preliminarily determined, and research basis is provided for the deep development and utilization of the litsea cubeba oil.
Drawings
FIG. 1 shows the distribution of eucalyptol according to the present invention in rats;
FIG. 2 is the distribution of p-cymene of the present invention in rats;
FIG. 3 is a graph showing the distribution of 4-terpene alcohols of the present invention in rats;
FIG. 4 shows the distribution of 4-isopropylcyclohex-2-en-1-one of the present invention in rat;
FIG. 5 shows the distribution of alpha-terpineol according to the present invention in rats;
FIG. 6 shows the distribution of 2- (4-methylphenyl) propan-2-ol according to the present invention in rats.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention; it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art without any inventive work are within the scope of the present invention.
Example (b):
a blood-entering component of Jatropha curcas oil comprises eucalyptol, p-cymene, 4-terpene alcohol, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol, and 2- (4-methylphenyl) propan-2-ol.
A method for measuring the distribution of blood components of Jatropha curcas oil is used for measuring the distribution of blood components of eucalyptol, p-cymene, 4-terpene alcohol, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol and 2- (4-methylphenyl) propan-2-ol, and comprises the following steps:
1.2.1 solution preparation
1.2.1.1 preparation of Standard solutions
Accurately weighing a proper amount of eucalyptol, p-cymene, 4-terpene alcohol, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol and 2- (4-methylphenyl) propan-2-ol reference substances, placing the reference substances into a 10mL measuring flask, adding ethyl acetate to dissolve and dilute the reference substances to a scale, and preparing a mixed reference substance solution containing eucalyptol (1.378 mg. ML-1), p-cymene (1.110 mg. ML-1), 4-terpene alcohol (1.660 mg. ML-1), 4-isopropylcyclohex-2-en-1-one (1.538 mg. ML-1), alpha-terpineol (1.690 mg. ML-1) and 2- (4-methylphenyl) propan-2-ol (1.694 mg. ML-1), and storing the mixed reference substance solution at the temperature of minus 20 ℃ for later use.
1.2.1.2 preparation of internal Standard solution
Precisely weighing a cyclohexanone reference substance, placing the reference substance into a 10mL volumetric flask, adding a small amount of ethyl acetate, ultrasonically dissolving, and fixing the volume to obtain an internal standard stock solution of cyclohexanone (1.072 mg. ML < -1 >). Taking a proper amount of the cyclohexanone internal standard stock solution, gradually diluting the cyclohexanone internal standard stock solution to prepare 200 ng.mL < -1 > internal standard solution, and storing the internal standard solution at the temperature of minus 20 ℃ for later use.
1.2.2 doses administered
Reference is made to 2012 edition of national food and drug administration standards (qi regulating and blood circulation promoting drop pills): the dripping pill for regulating qi-flowing and promoting blood circulation is administered in an amount of 30 pills (25 mg pill weight) per day, that is, 0.75g per day, 4500g of fructus Litseae Pungentis medicinal material, 11.30% of fructus Litseae Pungentis oil extraction rate (refer to the item 2.5 under the chapter I), and 508g of volatile oil; calculated as follows: 508/1000 × 0.75=0.381g/d, the daily administration amount of the litsea pungens oil is 0.381g/d, and the dosage is within the range of 0.05-0.45 g/d of the oral administration amount of the litsea pungens oil specified in the present 2019 version quality standard of Chinese medicinal materials and national medicinal materials in Guizhou province; namely, the daily dose of a 60kg patient is 6.35 mg/kg-1, and the rat oral dose is 40.05 mg/kg-1 according to the 6.3 times of the equivalent dose of the rat. In this example, the rat equivalent dose of 40.05 mg/kg-1 was defined as a low dose, 80.10 mg/kg-1 as a medium dose, and 160.20 mg/kg-1 as a high dose.
1.2.3 preparation of medicinal liquid
Preparing a liquid medicine: weighing appropriate amount of fructus Alpiniae Katsumadai oil, placing in a beaker, adding 0.5% Tween-80 normal saline solution, and dissolving with ultrasound for 10 min.
1.2.4 GC-MS conditions
Chromatographic conditions are as follows: no shunt sampling; the temperature programmed is detailed in the following table.
Column oven temperature program
Mass spectrum conditions: detecting voltage: the ion information detected by 0.25KV and 6 index components and internal standard is shown in the table below.
Mass spectrum condition of index component and internal standard
1.2.5 Collection of tissue samples
24 SD male rats with the body weight of 220 +/-20 g are divided into four groups, 6 rats are taken, fasting is carried out for 12 hours before administration, water is freely drunk, and after the rats take the litsea pungens oil (80.10 mg. Kg < -1 >) orally, hearts, livers, spleens, lungs, kidneys, brains, stomachs, intestines, skins and muscles are rapidly taken out after bleeding is respectively taken from femoral arteries at four time points of 0.167 hours, 0.5 hours, 1 hour and 1.5 hours after administration. The blood and contents on the tissue surface were washed off with ice saline, dried with filter paper, and packaged in a self-sealing bag and stored at-20 ℃ for further use.
1.2.6 treatment of tissue samples
Homogenizing tissue with physiological saline at a ratio of 1:2 (g: mL), centrifuging at 8000 r.min-1 for 10min, collecting rat ground tissue supernatant 100 μ L, placing in 1.5mL centrifuge tube, sequentially adding internal standard solution 100 μ L, vortex mixing for 2min, adding ethyl acetate 200 μ L, vortex mixing for 2min, centrifuging at 12000 r.min-1 at 4 deg.C for 10min, and storing at-20 deg.C.
1.2.7 obtaining a Standard Curve
100 μ L of rat blank liver homogenate sample was taken, and 100 μ L of a series of control solutions containing 6 components was added to prepare a drug concentration equivalent to that of the rat tissue homogenate sample. The method was carried out according to the tissue sample treatment method under item "1.2.6". And (3) performing linear regression by taking the ratio (A/Ai) of the peak area of the component to be measured and the peak area of the cyclohexanone internal standard as a vertical coordinate Y and the concentration (C) of each component as a horizontal coordinate X, and calculating a linear equation to obtain the standard curve.
The 6 components showed good linear relationship with peak area in the corresponding concentration range. The equation for the standard curve and the lowest limit of quantitation (LLOQ) for rat tissue homogenate samples are detailed in the table below.
Standard curves for 6 Components in each tissue
1.2.8 tissue homogenate sample assay
The rat homogenate samples were removed from the freezer (-20 ℃), thawed at room temperature, processed according to the tissue sample processing method under item "1.2.6", and assayed by GC-MS SIM. And (4) recording the peak areas of the index components and the cyclohexanone internal standard of all the samples, and substituting the peak areas into a following standard curve to calculate the concentration of each component to be measured.
Tissue distribution assay results:
the distribution of 6 components in rat tissues after intragastric administration of litsea macrocarpus oil (80.10 mg. Kg-1) to rats was investigated by GC-MS SIM method. The concentrations of the 6 components in the rat tissues are shown in the following table, and the distribution trends and levels are shown in the figure of the specification.
Distribution of 6 components in rat body (x + -s, n = 6)
Of these, 6 components were detected immediately in the tissue and reached the highest concentration at 0.17 or 0.5h, then rapidly dropped to low levels at 1h in most tissues. The trend is consistent with the results of the traditional Chinese medicine kinetic parameters t1/2 and MRT in the third part. In addition to the gastrointestinal tract, these 6 components showed high exposure in blood-rich heart, liver, kidney, spleen, brain. It is noteworthy that the physical properties of 5 other components besides 4-isopropylcyclohex-2-en-1-one contribute to penetration into the blood brain barrier, and all 5 compounds show significant levels in the brain.
Among them, the content trends of 6 components in each tissue of rat are as follows: eucalyptol is present in the heart, liver, spleen, lung, brain, stomach, and small intestine; p-cymene is contained in liver and small intestine; 4-terpene alcohol is contained in liver, brain and stomach; 4-isopropylcyclohex-2-en-1-one is contained in liver, stomach and small intestine; the alpha-terpineol is contained in heart, liver, spleen, lung, kidney, brain, stomach, small intestine and muscle; 2- (4-methylphenyl) propan-2-ol is contained in the liver, lung, stomach and small intestine; the content of each component in the tissues reaches the highest value within 0.17h, and shows a descending trend within 0.5-1.5 h, which is consistent with the trend phenomenon of the component appearing within 0.033-1.5 h in the third part of medicine time curve diagram 3-2. Eucalyptol is also present in the skin; p-cymene is contained in heart, spleen, lung, kidney, brain, stomach, muscle and skin; 4-terpene alcohol has content in heart, spleen, lung, kidney, small intestine, muscle and skin; 4-isopropylcyclohex-2-en-1-one is present in the spleen, kidney and skin; alpha-terpineol is also present in the skin; 2- (4-methylphenyl) propan-2-ol content in heart, spleen, kidney, brain, muscle, skin; the content of each component in the above tissue is in ascending trend at 0.17-0.5 h, reaches peak at 0.5h, and then rapidly decreases. The content of eucalyptol in kidney and muscle reaches peak value within 1h, and rapidly decreases within 1.5h.
In conclusion, firstly, the scheme establishes and verifies a simple, sensitive and high-specificity method based on GC-MS SIM, which is used for measuring eucalyptol, alpha-terpineol, p-cymene, 4-isopropylcyclohex-2-en-1-one and 2- (4-methylphenyl) propan-2-ol in tissues of rats; all indexes of the methodology meet the analysis requirements of biological samples.
Secondly, the scheme shows the distribution condition of 6 components in vivo after the rats are subjected to intragastric administration of the litsea cubeba oil for the first time, and the result shows that 6 components are quickly absorbed and transferred to tissues and then quickly eliminated; wherein the exposure in gastrointestinal tract and liver is highest, and other 5 components except 4-isopropylcyclohex-2-en-1-one are detected in kidney, heart, spleen, brain, lung, muscle and skin; whereas 4-isopropylcyclohex-2-en-1-one was detected only in stomach, small intestine, kidney, spleen, liver, skin; in addition to 4-isopropylcyclohex-2-en-1-one, 5 other components have absorption-promoting and blood-brain barrier permeability properties.
Finally, the scheme discusses the in vivo treatment process of the main component of the litsea cubeba oil and is helpful to provide reference for the pharmacological and toxicological research and clinical application of the litsea cubeba oil.
The foregoing is only a preferred embodiment of the present invention; the scope of the invention is not limited thereto. Any person skilled in the art should be able to cover the technical scope of the present invention by equivalent or modified solutions and modifications within the technical scope of the present invention.
Claims (10)
1. The blood-entering component of the litsea cubeba oil is characterized in that the blood-entering component comprises eucalyptol, p-cymene, 4-terpene alcohol, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol and 2- (4-methylphenyl) propan-2-ol.
2. A method for measuring the blood-entering component distribution of litsea cubeba oil, which is characterized in that the method is used for measuring the blood-entering component distribution of the litsea cubeba oil of claim 1 and comprises the following steps:
s1, preparing solution
1) Preparing a standard solution: accurately weighing a proper amount of eucalyptol, p-cymene, 4-terpene alcohol, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol and 2- (4-methylphenyl) propan-2-ol reference substances, placing the reference substances in a volumetric flask, adding ethyl acetate to dissolve and dilute the reference substances to a scale, preparing a mixed reference substance solution containing eucalyptol, p-cymene, 4-terpene alcohol, 4-isopropylcyclohex-2-en-1-one, alpha-terpineol and 2- (4-methylphenyl) propan-2-ol, and storing the mixed reference substance solution for later use;
2) Preparing an internal standard solution: precisely weighing a cyclohexanone reference substance, placing the cyclohexanone reference substance in a volumetric flask, adding a small amount of ethyl acetate, ultrasonically dissolving, and fixing the volume to obtain an internal standard stock solution of cyclohexanone; taking a proper amount of cyclohexanone internal standard stock solution, gradually diluting to prepare an internal standard solution, and storing for later use;
s2, preparing liquid medicine: weighing appropriate amount of oleum Alpiniae Katsumadai, placing in a beaker, adding 0.4-0.6% Tween-80 normal saline solution, and dissolving with ultrasound for 8-12min to obtain medicinal liquid, and mixing immediately;
s3, collecting tissue samples:
1) Selecting a plurality of SD male rats, grouping the SD male rats on average, fasting for 12h before administration, freely drinking water, respectively performing femoral artery bloodletting after administration after the rats take the liquid medicine in the step S2 orally, and then quickly taking out the heart, the liver, the spleen, the lung, the kidney, the brain, the stomach, the intestine, the skin and the muscle;
2) Washing blood and contents on the surface of the tissue with ice normal saline, drying the tissue with filter paper, filling the tissue into a self-sealing bag to form a tissue sample, and storing the tissue sample for later use;
s4, processing of the tissue sample: homogenizing the tissue sample in the step S3 by using physiological saline according to the proportion of 1:2 (g: mL), then centrifuging for 8-15min at 7000-9000 r.min < -1 >, taking 100 mu L of rat ground tissue supernatant, placing the rat ground tissue supernatant into a 1.5mL centrifuge tube, sequentially adding 100 mu L of the internal standard solution in the step S1-2, carrying out vortex mixing for 2-5min, then adding 200 mu L of ethyl acetate, carrying out vortex mixing for 2-5min, carrying out centrifugation for 10-15min at 4-6 ℃ and 10000-13000 r.min < -1 >, obtaining a tissue homogenate sample, and storing for later use;
s5, obtaining a standard curve: taking 100 mu L of rat blank liver tissue homogenate, adding 100 mu L of the mixed reference substance solution containing 6 components in the step S1-1, and preparing the mixture into a medicine concentration equivalent to that of a rat tissue homogenate sample; operating according to the processing method of the tissue sample under the item of the step S4, performing linear regression by taking the peak area ratio (A/Ai) of the component to be detected and the cyclohexanone internal standard as a longitudinal coordinate Y and the concentration (C) of each component as a horizontal coordinate X, and calculating a linear equation, namely a standard curve;
s6, measuring a tissue homogenate sample: and (4) carrying out sample injection measurement on the tissue homogenate sample in the step S4 by a GC-MS SIM method, recording index components of all samples and peak areas of cyclohexanone internal standards, and substituting the peak areas into the standard curve in the following step S5 to calculate the concentration of each component to be measured.
3. The method according to claim 2, wherein the control in step S1-1 is placed in a 10mL volumetric flask, and the mixed control solution is stored at-15-25 ℃ for use, the volumetric flask in step S1-2 is also 10mL, and the storage temperature is also-15-25 ℃.
4. The method for measuring the distribution of the components entering the blood of litsea megacarpum oil according to claim 2, wherein the concentration of the internal standard solution in step S1-2 is 200-250 ng-mL "1.
5. The method for determining the distribution of bleeding components of litsea grandiflorum seed oil according to claim 2, wherein the bleeding time points of the femoral artery in step S3-1 are four, 0.167h, 0.5h, 1h and 1.5h after administration.
6. The method for measuring the distribution of components entering the blood of litsea cubeba oil according to claim 2, characterized in that the preservation temperature of the tissue sample in step S3-2 is-15 to 25 ℃.
7. The method for determining the distribution of components entering the blood of litsea cubeba oil according to claim 2, wherein the dose administered in step S3-1 is 0.365-0.385g/d.
8. The method for determining the distribution of blood components of zingiber macrocarpum hance oil according to claim 2, wherein the tissue homogenate sample of the step S4 is stored in a sample bottle with an inner cannula at a temperature of-15 to 25 ℃.
9. The method for determining the distribution of the bleeding components of the litsea cubeba oil according to claim 2, wherein the chromatographic condition of the GC-MS SIM method in the step S6 is no-split-flow sampling.
10. The method for determining the distribution of components entering the blood of zingiber macrocarpum hance oil according to claim 2, wherein the detection voltage of the mass spectrum condition in the GC-MS SIM method in step S6 is 0.25-0.3KV.
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