CN118169289A - Method for detecting components in Ruyi zhenbao pills - Google Patents
Method for detecting components in Ruyi zhenbao pills Download PDFInfo
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- CN118169289A CN118169289A CN202410349836.6A CN202410349836A CN118169289A CN 118169289 A CN118169289 A CN 118169289A CN 202410349836 A CN202410349836 A CN 202410349836A CN 118169289 A CN118169289 A CN 118169289A
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Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a detection method of components in a Ruyi treasure pill body, which comprises a method A for qualitatively detecting blood entering or brain entering components of the Ruyi treasure pill and a method B for quantitatively detecting the blood entering or brain entering components of the Ruyi treasure pill, wherein the blood entering and brain entering prototype components and metabolites of the Ruyi treasure pill body can be comprehensively identified and identified by the qualitative detection method A, so that the metabolism and conversion rules of main active components and active components in the Ruyi treasure pill body are primarily defined; by the quantitative detection method B, the high exposure components of the Ruyi zhenbao pill in the blood plasma and the cerebrospinal fluid at different time points can be accurately quantified, and the pharmacokinetic characteristics of the components in the blood plasma and the cerebrospinal fluid can be conveniently characterized. The detection provides a reliable experimental basis for identifying the pharmacodynamic substance basis of the Ruyi treasure pill, and also lays a chemical substance basis for explaining the action mechanism of the Ruyi treasure pill.
Description
Technical Field
The invention relates to the field of medicine analysis, in particular to a detection method of components in Ruyi zhenbao pills.
Background
As a first choice for treating white vein diseases (which are the general nerve system injury diseases) by Tibetan medicine, the Ruyi zhenbao pill has wide clinical application and plays an important role. Although the Ruyi zhenbao pill has obvious clinical curative effect, the research on the chemical components and the material basis is weak, and the scientific value of the variety is seriously influenced.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides a method for detecting the components in the Ruyi zhenbao pills, and the prototype components and the metabolites in the bodies can be definitely detected by qualitative detection; by quantitative detection, high exposure components can be clarified, and the pharmacokinetic process can be monitored conveniently.
The technical scheme adopted by the invention for solving the technical problems is as follows:
The application provides a detection method of components in Ruyi zhenbao pills, which comprises the following steps: a qualitative detection method A for the components of Ruyi Zhenbao pills entering blood or brain comprises the following detection steps:
a1: collecting a biological sample of an administration organism, and processing to obtain a solution to be tested, wherein the biological sample is a plasma sample or a cerebrospinal fluid sample;
A2: detecting and analyzing the biological sample by adopting an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbit trap high-resolution mass spectrometry combination technology, wherein:
Chromatographic conditions: the C18 column (2.1 mm. Times.150 mm,1.7 μm) was used, mobile phase A was 0.1% aqueous formic acid, mobile phase B was acetonitrile, and gradient elution conditions were: 0-15min,3% -20% B;15-30min,20% -40% B;30-40min,40% -70% B;40-45min,70% -95% B; the column temperature is 25 ℃, the sample injection amount is set to 4 mu L, and the flow rate is set to 0.30mL/min;
Mass spectrometry conditions: electrospray ionization ion source, positive and negative ion mode collection data; the data acquisition range is 100-1500m/z; the cracking voltage is 100V; collision energy 25eV; the difference in collision voltage is 15eV;
A3: mass spectrum data were collected using Xcalibur software to infer the composition of the Ruyi treasure bolus.
Further, in A1, the method for collecting the plasma sample is as follows: collecting abdominal aortic blood of organism after administration for a period of time, standing for 2 hr, centrifuging at 3000r/min for 10min after layering of serum, and freezing supernatant at-80deg.C;
The cerebrospinal fluid sample collection method is as follows: after a period of administration, the cerebral spinal pool is pierced through a micro infusion needle from the occipital macropore, cerebrospinal fluid is extracted and centrifuged for 10min at 4000r/min at 4 ℃ in a precooled centrifuge tube, and the supernatant is taken and stored at-80 ℃ for later use.
Further, the plasma sample treatment method is as follows: thawing the plasma sample in a refrigerator at 4 ℃, swirling for 3min, standing for 5min, centrifuging for 10min at 13500r/min at 4 ℃, and taking supernatant to obtain a solution to be tested; the treatment method of the cerebrospinal fluid sample comprises the following steps: thawing the cerebrospinal fluid sample in a refrigerator at 4 ℃, swirling for 3min, standing for 5min, centrifuging for 10min at 13500r/min at 4 ℃, and taking the supernatant to obtain a solution to be measured.
The detection method of the application also comprises a method B for quantitatively detecting blood entering or brain entering components of the Ruyi zhenbao pill, wherein the blood entering components are glycyrrhizic acid, glycyrrhizin, apigenin-7-O-glucuronide and gallic acid, and the brain entering components are glycyrrhizic acid, apigenin-7-O-glucuronide and gallic acid; the specific detection steps are as follows:
B1: preparing a mixed reference substance solution and an icariin internal standard solution of each component by using methanol;
B2: preparation of a drug-containing sample solution: taking blank plasma or blank cerebrospinal fluid, adding corresponding mixed reference substance solution, internal standard solution and acetonitrile, mixing by vortex, standing for 5min, centrifuging at 13500r/min at 4deg.C for 10min, collecting supernatant, and analyzing by sample injection; preparing a drug administration sample solution, namely taking a processed plasma sample or cerebrospinal fluid sample, adding an internal standard solution and acetonitrile, and processing sample injection according to the method;
B3: detecting and analyzing the sample solution by adopting a UHPLC-MS/MS technology, wherein:
Chromatographic conditions: c18 column (2.1 mm. Times.150 mm,1.7 μm), mobile phase A was 0.1% aqueous formic acid, mobile phase B was acetonitrile, gradient elution conditions: 0-1min,5% -30% B;1-5min,30% -80% B;5-6min,80% -95% B;6-8min,95%% B;8-9min,95% -5% B;9-12min,5% B; the column temperature is 40 ℃, the sample injection amount is set to 2 mu L, and the flow rate is set to 0.30mL/min;
Mass spectrometry conditions: the electrospray ionization ion source is in a negative ion mode, the capillary voltage is 2000V (ESI -), the taper hole voltage is 30V, the ion source temperature is 150 ℃, the taper hole air flow is 150L/h, the desolventizing air temperature is 450 ℃, the desolventizing air flow is 1000L/h, and the monitoring mode is a dynamic multi-reaction monitoring mode.
Further, in B1, the concentration of each component in the mixed reference substance solution is 1.5875-200ng/mL; the concentration of the internal standard solution was 100ng/mL.
Further, in B2, the preparation method of the drug-containing sample solution specifically comprises the following steps: taking 50 mu L of blank plasma or blank cerebrospinal fluid, adding 12.5 mu L of mixed reference substance solution, 2.5 mu L of internal standard solution and 135 mu L of acetonitrile, mixing uniformly by vortex, standing for 5min, centrifuging for 10min at 4 ℃ at 13500r/min, and taking supernatant for sample injection analysis; the preparation method of the administration sample solution comprises thawing plasma sample or cerebrospinal fluid sample in a refrigerator at 4deg.C, swirling for 3min, precisely sucking 50 μl, adding 2.5 μl of internal standard solution and 147.5 μl of cold acetonitrile, swirling and mixing for 3min, standing for 5min, centrifuging at 13500r/min at 4deg.C for 10min, and collecting supernatant for sample injection analysis.
The invention adopts the structure and has the advantages that: firstly, relatively comprehensive identification and identification are carried out on blood entering and brain entering prototype components and metabolites through qualitative detection, and the main active components and the metabolism and conversion rules of the active components in vivo are primarily defined; and secondly, accurately quantifying the high exposure components of the Ruyi zhenbao pill in the plasma and the cerebrospinal fluid at different time points through quantitative detection, and objectively characterizing the pharmacokinetic characteristics of the components in the plasma and the cerebrospinal fluid. The method provides a reliable experimental basis for identifying the drug effect substance basis of the Ruyi zhenbao pill and lays a chemical substance basis for explaining the action mechanism of the Ruyi zhenbao pill. Specific:
(1) 41 blood-entering components and 20 brain-entering components of the Ruyi zhenbao pill can be identified and obtained through a qualitative detection method, and metabolic pathways and products of 6 representative components are obtained through analysis. Wherein the blood-entering component comprises organic acids, flavonoids, volatile oils, amides, ketones, sesquiterpenes, amino acids, phenylpropanoids and monosaccharides, the organic acids in the medicine myrobalan, the medicine myrobalan and the king medicine clove and cinnamon are the most, and the flavonoid components in the medicine herba lagotis brachycis and liquorice paste are the next more; the brain-entering component comprises amino acids, organic acids, alkaloids, phenylpropanoids, volatile oils, phenols, ketones and sesquiterpenes, wherein the organic acids contained in the principal drugs of licorice paste, king's medicine of clove and princess's medicine of myrobalan and fructus Terminaliae Billericae and fructus Phyllanthi are used as main components. The detection result lays a data foundation for deeply analyzing the prescription rule and the drug effect substance foundation of the Ruyi Zhenbao pills.
(2) The content of glycyrrhizic acid, glycyrrhizin, apigenin-7-O-glucuronide and gallic acid 5 in the plasma sample can be measured by a quantitative detection method; and measuring the contents of 4 components of glycyrrhizic acid, apigenin-7-O-glucuronide and gallic acid in the cerebrospinal fluid sample. The detection method has good specificity, daily precision and accuracy in the daytime and stability, and in addition, the pharmacokinetic process of each component in the body can be conveniently monitored.
Drawings
FIG. 1 is a BPI chromatogram of a biological blank plasma sample and a drug administration plasma sample;
FIG. 2 is an analytical chart of glycyrrhizic acid cleavage pathway;
FIG. 3 is a diagram of plasma metabolite analysis of glycyrrhizic acid;
FIG. 4 is an analytical view of the plasma cleavage pathway of glycyrrhizin;
FIG. 5 is a graph of an analysis of plasma metabolites of liquiritin;
FIG. 6 is an analytical view of the galangin plasma cleavage pathway;
FIG. 7 is a graph of analysis of galangin plasma metabolites;
FIG. 8 is an analytical map of the cholic acid plasma cleavage pathway;
FIG. 9 is a chart showing the analysis of the plasma cleavage pathway of gallic acid;
FIG. 10 is a chart of analysis of plasma gallate metabolites;
FIG. 11 is an analytical view of apigenin plasma cleavage pathway;
FIG. 12 is a graph of apigenin plasma metabolite analysis;
FIG. 13 is an analytical view of apigenin-7-O-glucuronide plasma cleavage pathway;
FIG. 14 is a graph of apigenin-7-O-glucuronide plasma metabolite analysis;
FIG. 15 is a BPI chromatogram of a biological blank cerebrospinal fluid sample and a dosing cerebrospinal fluid sample test;
FIG. 16 is a diagram of a glycyrrhizic acid cerebrospinal fluid metabolite analysis;
FIG. 17 is a diagram showing the analysis of the metabolites of glycyrrhizin cerebrospinal fluid;
FIG. 18 is a graph of analysis of galangin cerebrospinal fluid metabolites;
FIG. 19 is a chart of the analysis of the metabolites of gallic acid cerebrospinal fluid;
FIG. 20 is a diagram of apigenin cerebrospinal fluid metabolite analysis;
FIG. 21 is a graph of apigenin-7-O-glucuronide cerebrospinal fluid metabolite analysis;
FIG. 22 is an extract ion flow chromatogram of 5 compounds and icariine as internal standard in plasma;
FIG. 23 is a graph showing the drug time profile of the components in plasma after intragastric administration in SD rats;
FIG. 24 is an extract ion flow chromatogram of 5 compounds and icariine as an internal standard in cerebrospinal fluid;
FIG. 25 is a graph showing the drug time profile of the components in cerebrospinal fluid after intragastric administration in SD rats.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present invention will be described in detail below with reference to the following detailed description and the accompanying drawings.
First material
(1) The experimental instrument is an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbit trap high resolution mass spectrometer; xex TQ Ablute triple quadrupole mass spectrometer; xcalibur data analysis system (Thermo company, usa); massLynx 4.2 SCN1040 data analysis System (Waters, USA); waters ACQUITY UPLC BEH C18 chromatographic column1.7 Μm,2.1mM X150mm, waters, USA); NA-5L nitrogen-air all-in-one (Beijing Zhongxing Huili technology Co., ltd.); QL-901 vortex mixer (kylin medical instruments, inc. of Jiangsu Haimen); one ten million analytical balance (Sidoriko instruments Co., ltd.).
(2) Experimental medicine, ruyi Zhenbao pill medicine powder (company: jinzhengcang medicine stock Co., ltd., batch number: 01230301, specification: 100g medicine powder, 1g medicine powder each corresponds to 1g of the raw medicine); standard substance: apigenin (lot number: DSTDQ 002602), glycyrrhizic acid (lot number: DSTDGQ 000603), apigenin-7-O-glucuronide (lot number: DSTDQ 009001), gallic acid (lot number: DSTDM 000802), and glycyrrhizin (lot number: DSTDG 000902) are obtained from Lemeitian medicine; icariine (lot number: B21576) was purchased from Shanghai Yuan Yes Biotechnology Co. Acetonitrile (mass spectrum grade, thermo company, usa); methanol (chromatographic purity, thermo company, usa); formic acid (HPLC grade, SIGMA ALDRICH company, usa); distilled water (Chengshi, guangzhou).
(3) The experimental animals adopt 96 SPF-class male SD rats with the weight of 200+/-20 g. Provided by beijing vernalia laboratory animal limited, laboratory animal production license number: SCXK (Beijing) 2021-0011, SCXK (Beijing) 2021-0006. Rats were fed uniformly to laboratory animals at the basic theoretical institute of traditional Chinese medicine, academy of Chinese traditional medicine. The animal room is clean and bright, the ventilation condition is good, the room temperature is about 23+/-5 ℃, and the humidity is about 55+/-5%. The padding is replaced every 4 days. Rats eat and drink water freely and are replaced in time every day. Rats were fed adaptively for 1 week before the experimental formally started.
(II) detection method and results
Example 1: qualitative detection method for blood entering and brain entering prototype components and metabolic components of Ruyi zhenbao pills
1.1 Chromatographic conditions and Mass Spectrometry conditions
(1) Chromatographic conditions: waters ACQUITY UPLC BEH C18A chromatographic column (2.1 mm. Times.150 mm,1.7 μm) with mobile phase A being 0.1% aqueous formic acid and mobile phase B being acetonitrile. Gradient elution conditions were set as follows: 0-15min,3% -20% B;15-30min,20% -40% B;30-40min,40% -70% B;40-45min,70% -95% B; the column temperature was set at 25℃and the sample loading was set at 4. Mu.L, with a flow rate of 0.30mL/min. The ultraviolet detection wavelength is 400nm.
(2) Mass spectrometry conditions: data were collected using an electrospray ionization (ESI) ion source, positive and negative ion modes. The data acquisition range m/z is set to 100-1500. The cleavage voltage (DP) was 100V, the Collision Energy (CE) was 25eV, and the collision voltage difference (CES) was 15eV.
1.2 Collection of biological samples
Mixing Ruyi zhenbao pill powder 15g with ultrapure water, and making into solution with mass concentration of 0.36 g/mL. (adult dose is 5 g/day, rat clinical equivalent dose is 0.45g/kg,4 times clinical dose is 1.8g/kg,1 rat lavage 0.36 g)
(1) Method for collecting plasma samples: SD rats were randomly divided into a blank group and a dosing group, 6 animals each, fasted for 12 hours (free drinking water), and the dosing group was given a Ruyi treasure pill suspension according to 1.8g/kg gavage, and the blank group was given the same volume of ultrapure water. After 1.5h of gastric lavage, 10% chloral hydrate is anesthetized, abdominal aorta is taken for blood, standing is carried out for 2h, after serum layering, centrifugation is carried out for 10min at 3000r/min, supernatant is taken, and serum of rats with different groups is mixed by vortex and frozen at-80 ℃ for standby.
(2) The collecting method of the cerebrospinal fluid sample comprises the following steps: 6 SD rats are taken, fasted for 12 hours (free drinking water), then the stomach is filled with 1.8g/kg of Zhenbao pill powder, after 2 hours of administration, 20% of uratan is used for anesthesia, then head and neck shearing and disinfection are carried out, a longitudinal incision is cut along a longitudinal axis by a surgical knife, a micro infusion needle is penetrated into a small brain medullary pool from a occipital bone macropore, cerebrospinal fluid is extracted into a precooled centrifuge tube, and the rats are killed immediately after the extraction. The cerebrospinal fluid is centrifuged at 4000r/min for 10min at 4℃and the supernatant is taken for storage at-80 ℃.
1.3 Pretreatment of biological sample introduction
The treatment method of the plasma sample and the cerebrospinal fluid sample comprises the following steps: after thawing the rat sample in a refrigerator at 4 ℃, vortex for 3min, stand for 5min, centrifuge for 10min at 13500r/min at 4 ℃, and take supernatant for sample injection analysis.
1.4 Blood-entering prototype component and Metabolic component detection analysis
1.4.1 Identification of blood-entering Components
The plasma sample liquid is collected in positive and negative ion modes to obtain mass spectrum data, the Xcalibur software is used for collecting the mass spectrum data, the PBI chromatogram is shown in figure 1, wherein black represents the positive ion mode, red represents the negative ion mode, A represents a blank group, and B represents a dosing group. By analyzing the mass spectrum cleavage laws of different types of compounds, 41 prototype components (see Table 1) and 24 metabolites (see Table 2) were initially identified from plasma, the 24 metabolites are derived from the prototype components of glycyrrhizic acid, glycyrrhizin, galangin, gallic acid, apigenin and apigenin-7-O-glucuronide, and are derived from the medicinal materials of the monarch drug, the lagerstroemia indica and the licorice, the queen drug, the galangal, and the prince drug, the myrobalan.
Specifically, it is identified from the plasma of Ruyi treasure pill administration: 10 organic acid components are mainly from medicinal materials such as fructus Terminaliae Billericae, fructus Chebulae, fructus Tsaoko, flos Caryophylli, glycyrrhrizae radix, and fructus Phyllanthi; 8 flavonoid components mainly come from medicinal materials such as herba Lagotis Brevibacterium, glycyrrhrizae radix, rhizoma Alpiniae Officinarum, etc.; 3 amides mainly from fructus Piperis Longi; 5 volatile oil components mainly from flos Caryophylli and cortex Cinnamomi; in addition, 3 amino acids, 2 alkaloids, 2 phenylpropanoids, 4 ketones, 3 sesquiterpenes and 1 monosaccharide were identified.
TABLE 1 Ruyi zhenbao Wan into blood prototype composition table
TABLE 2 representative blood-entering Components and metabolites thereof
1.4.2 Analysis of representative blood-entering component cleavage and Metabolic rule
(1) Glycyrrhizic acid, which is an important component of chemical components of Glycyrrhrizae radix, is mainly glycyrrhizic acid derivative, and is shown in mass spectrum as [ M-H ] -M/z 821.3965 excimer ion, and M/z 351.0573 fragment is obtained after glycosidic bond is broken and-C 29H42O5 group is lost; the m/z 195.0356 fragment ions were obtained after successive removal of the-C 2O2、-C6H18O11、-C19H30O11 groups, the cleavage route being shown in FIG. 2. The compound is easy to carry out glucuronidation or sulfation after hydration in vivo, partial metabolite is easy to carry out sulfation or acetylation after glucoside removal and oxidation after excimer ion fragment removal, and m/z455.3520 and m/z 471.3111 fragment ions are obtained, and the metabolic analysis is shown in figure 3.
(2) One of the main sources of flavonoid components in the Ruyi Zhenbao pills is the monarch drug liquorice paste, and the glycyrrhizin is a representative component. The liquiritin shows the excimer ion of [ M-H ] -M/z 417.1196 in mass spectrum, the fragment ion M/z 255.0666 with the glucosyl removed mainly appears in the secondary spectrum, and C 7H4O3 is lost to form M/z 119.0503; the characteristic fragments of m/z 135.0089 obtained by RDA cleavage on the C ring further lose CO 2 to form fragment ions of m/z 119.08, and the cleavage route is shown in FIG. 4. Flavonoid components in licorice are susceptible to reduction, methylation, glucuronidation and sulfation type reactions in serum. The glycyrrhizin structure contains 1 molecule of glucose, glucose is easy to lose in vivo, and exists in the form of aglycone, and further generates ion peaks of m/z 431.3146, m/z 255.1590 and m/z 119.0252, and the metabolic analysis is shown in figure 5.
(3) Galangin is the main component of galangal, the prince drug, the excimer ion peak is M/z 271.0600 ([ M+H ] +) and the characteristic fragment ion M/z 225.0556 ([ M-CO2+H ] +) and 197.0596 ([ M-CO2-CO+H ] +) or 153.0192 (RDA, 1,3 A), and the cleavage route is shown in FIG. 6. Galangin is mainly present in plasma as the following metabolites (fig. 6): the peak of C2 excimer ion is M/z 623.3163 ([ M+H ] +) and the molecular formula is C 27H26O17, and the main fragment ions are M/z 447.0918 ([ M-C6H8O6+H ] +) and 271.0593 ([ M-2C 6H8O6 +H ] +) and are combined by the double glucuronic acid; c3 excimer ions were m/z 447.1304, with major fragments m/z 271.0597([M-C6H8O6+H]+)、215.0697([M-C6H8O6-2CO+H]+)、165.0182(RDA,0,3A) and 153.0181 (RDA, 1,3 A), respectively, indicating that glucuronic acid binding of galangin occurred. The molecular formula of C4 is C 21H18O11, the excimer ions are M/z 463.1303 ([ M+H ] +) respectively, oxidation reaction and glucuronic acid combination reaction occur, and the characteristic fragment ions m/z 329.0508([1,3A+C6H8O6+H]+)、287.0543([M-C6H8O6+H]+)、165.0184(0,3A)、153.0187(1,3A) and 121.0289 (0,3 B). The molecular formula of C5 is C 15H10O8 S, and the excimer ion is M/z 351.0186 ([ M+H ] +). The molecular composition of C6 excimer ion is C 15H10O9 S, and the m/z 367.2648 is respectively used for oxidation reaction and sulfuric acid binding reaction. The C7 excimer ion is M/z 285.2075 ([ M+H ] +), and methylation reaction occurs. The main feature fragments are m/z 270.0513([M-CH3+H]+)、242.0564([M-CH3-CO+H]+)、241.0849([M-CO2+H]+)、211.0744([M-CO2-OCH3+H]+)、179.0325(0,3A)、167.0331(1,3A) and 105.0335 (0 ,3 B). The molecular formula of C8 is C 17H12O6, the excimer ion is M/z 313.2390 ([ M+H ] +) and the main fragments are m/z 285.0769([M-CO+H]+)、257.0798([M-2CO+H]+)、207.0288(0,3A)、195.0278(1,3A)、119.0491(1,3B) and 105.0337 (0,3 B), the A ring is subjected to N-acetyl binding reaction, and the metabolic analysis is shown in FIG. 7.
(4) Cholic acid is the main component of the animal drug-representing medicine bezoar derived from the formula, and shows the excimer ion [ M-H ] -M/z 407.2809 in mass spectrum, and the dehydrated fragment ion M/z 389.2199 mainly appears in the secondary spectrum, so that C 5H14O4 is further lost to form M/z 259.1792, and the cleavage path is shown in figure 8.
(5) Gallic acid the main ingredient of Terminalia chebula. The mass spectrum result shows that the gallic acid mainly has an excimer ion peak of m/z 169.0138, and characteristic ion fragments m/z 124.0370 are obtained after the-C 6H10O5 group is lost, and the cleavage mode is shown in figure 9. The metabolic pathways and products thereof after bleeding are shown in FIG. 10.
(6) Apigenin is derived from herba Lagotis Brevibacterium, belonging to flavonoid. The flavonoids mainly undergo cleavage and loss rearrangement reactions with the C ring as the center. In addition, flavonoid aglycones are subject to serial cleavage which is a sequential loss of neutral fragments such as CO, H 2 O, etc. The mass spectrum results show that the apigenin mainly exists in m/z 271.0962 of excimer ions, fragment ions m/z 243.0649 of-CO groups mainly appear in the secondary spectrogram, or fragment ions m/z153.0182 are obtained after RDA cleavage, and the cleavage mode is shown in figure 11. Apigenin metabolism analysis is shown in FIG. 12, and the excimer ion peak of E2 is M/z349.349.2643 ([ M-H ] -). E3 excimer ion peak m/z 2283.0826, E4 gives rise to the excimer ion peak m/z 269.0822, presumably 5-hydroxy-7-methoxyflavan. The excimer ion peak of E5 is M/z 313.1068 ([ M-H ] -), the fragment ion M/z 271 ([ M-H-3CH 2]-)、269([M-H-CO2]-)、179(1,3 [ A-H ] -) and 117 (1,3 [ B-H-2H-CH ] -) are presumed to be dihydroapigenin-4', 5, 7-trimethyl ether.
(7) Apigenin-7-O-glucuronide is derived from the monarch drug Lagotis brachycyrrhiza, m/z 445.0671 excimer ions are shown in mass spectrum, and a secondary spectrum result shows that fragment ions m/z 243.0649 and fragment ions m/z 175.0249 of the component with glycosidic bond broken and lost-C 6H8O10 groups are mainly existed, and the cleavage route is shown in figure 13. The metabolic pathways and products thereof after bleeding are shown in FIG. 14.
1.5 Detection and analysis of brain-entering prototype Components and Metabolic Components
1.5.1 Identification of brain-entering Components
The sample of the cerebrospinal fluid of the Ruyi zhenbao pill is collected in the positive and negative ion modes, and the obtained mass spectrum data is shown in figure 15. 20 prototype components and 16 metabolites were initially identified from the queen drug and the monarch drug of the Ruyi zhenbao pill by analyzing mass spectrum cleavage rules of different types of compounds. Specifically, the identification from the cerebrospinal fluid of the Ruyi treasure pill administration is as follows: 7 organic acid compounds are mainly from myrobalan, fructus Terminaliae Billericae, flos Caryophylli, fructus Phyllanthi and Glycyrrhrizae radix extract. 3 volatile oil components are mainly used for clove-cinnamon medicine pair. 3 alkaloid compounds mainly comprise Tibetan radix aucklandiae, fructus Amomi rotundus, flos Caryophylli and fructus Piperis Longi. In addition, there are 2 alkaloid components, 2 phenylpropanoid components, 1 sesquiterpene component, 1 phenol component and 1 ketone component.
TABLE 3 brain prototype composition table of Ruyi Zhenbao Wan
TABLE 4 representative brain entry ingredients and metabolites thereof
1.5.2 Analysis of representative brain-entering component cleavage and Metabolic rules
(1) Glycyrrhizic acid has metabolism component excimer ion peaks of m/z 455.1883 and m/z 471.1833, and the metabolic pathway is shown in figure 16.
(2) The main ion peaks of the liquiritin into brain component metabolism are m/z 255.1605 and m/z 119.0490, and the metabolic pathway is shown in figure 17.
(3) The main ion peaks of galangin glycoside into brain component metabolism are m/z 623.3163, m/z 351.0551m/z 367.2647, m/z 285.2075 and m/z 313.2390, and the metabolic pathway analysis is shown in figure 18.
(4) Gallic acid mainly undergoes deoxidization and other processes in cerebrospinal fluid, and metabolic pathway analysis is shown in FIG. 19.
(5) Apigenin, the metabolite in apigenin cerebrospinal fluid mainly generates excimer ion peaks with m/z 349.349.2643, m/z 283.0826 and m/z 269.0822, and the metabolic pathway is shown in figure 20.
(6) Apigenin-7-O-glucuronide has the processes of glycosidic bond fracture, methylation, deoxidation and the like in cerebrospinal fluid, and the metabolic pathway analysis is shown in figure 21.
In conclusion, the application obtains 41 blood-entering prototype components and 20 brain-entering prototype components through blood-entering and brain-entering component detection of Ruyi Zhenbao pills, and analyzes and obtains metabolic pathways and products of 6 representative components. Wherein the blood-entering component comprises organic acids, flavonoids, volatile oils, amides, ketones, sesquiterpenes, amino acids, phenylpropanoids and monosaccharides, the organic acids in the medicine myrobalan, the medicine myrobalan and the king medicine clove and cinnamon are the most, and the flavonoid components in the medicine herba lagotis brachycis and liquorice paste are the next more; the brain-entering component comprises amino acids, organic acids, alkaloids, phenylpropanoids, volatile oils, phenols, ketones and sesquiterpenes, wherein the organic acids contained in the principal drugs of licorice paste, king's medicine of clove and princess's medicine of myrobalan and fructus Terminaliae Billericae and fructus Phyllanthi are used as main components. The result lays a data foundation for deeply analyzing the prescription rule and the drug effect substance foundation of the Ruyi treasure pill.
In particular, by comparing the in vivo components with the in vitro components of the Ruyi zhenbao pills, the medicine effect of the components can be presumed, and the method has important significance for researching the action mechanism of medicines. In the brain component type, the ratio of flavonoid components is reduced relative to the ratio of blood components, and the ratio of organic acid components is increased, so that the organic acid components are presumed to be the pharmacodynamic substance basis for directly exerting an intervention effect on the middle-jiao in the treatment of neuropathic pain.
Example 2: method for quantitatively detecting components of Ruyi zhenbao pill for blood entering and brain entering prototype
As well as the characteristics of blood absorption and brain absorption, the medicinal components of the Ruyi zhenbao pill also need to reach a certain exposure in blood plasma and cerebrospinal fluid, and have better absorption, distribution, metabolism, excretion and other pharmacokinetic characteristics. Therefore, in order to further clarify the pharmacodynamic substances of the Ruyi zhenbao pill, the UPLC-Q-TRAP/MS technology is adopted to quantitatively detect blood entering components and brain entering components so as to identify high exposure components and perform pharmacokinetic characterization.
2.1 Chromatographic conditions and Mass Spectrometry conditions
(1) Chromatographic conditions: waters ACQUITY UPLC BEH C18A chromatographic column (2.1 mm. Times.150 mm,1.7 μm), mobile phase A was 0.1% aqueous formic acid, mobile phase B was acetonitrile, gradient elution conditions: 0-1min,5% -30% B;1-5min,30% -80% B;5-6min,80% -95% B;6-8min,95%% B;8-9min,95% -5% B;9-12min,5% B; the column temperature was 40℃and the sample injection amount was set at 2. Mu.L and the flow rate was set at 0.30mL/min.
(2) Mass spectrometry conditions: the electrospray ionization ion source is in a negative ion mode, the capillary voltage is 2000V (ESI -), the taper hole voltage is 30V, the ion source temperature is 150 ℃, the taper hole air flow is 150L/h, the desolventizing air temperature is 450 ℃, the desolventizing air flow is 1000L/h, and the monitoring mode is a dynamic multi-reaction monitoring mode.
2.2 Method for collecting biological samples
Mixing Ruyi zhenbao pill powder 15g with ultrapure water, and making into solution with mass concentration of 0.36 g/mL. (adult dose is 5 g/day, rat clinical equivalent dose is 0.45g/kg,4 times clinical dose is 1.8g/kg,1 rat lavage 0.36 g)
(1) Method for collecting plasma samples: 12 SD rats were taken and fasted for 12 hours before the experiment without water. The medicinal powder suspension of the Ruyi Zhenbao pill is respectively taken from 0.5mL of blood by 0h, 0.08h, 0.25h, 0.5h, 0.75h, 1h, 2h, 4h, 6h, 8h, 12h and 24h through retrobulbar veins of rats, centrifuged for 10min at 4000r/min in a heparinized test tube, and the supernatant is taken and stored in a refrigerator at the temperature of minus 80 ℃ for standby.
(2) The collecting method of the cerebrospinal fluid sample comprises the following steps: 72 SD rats were randomly divided into 12 groups of 6 rats, and no water was fed for 12 hours prior to the experiment. The preparation method comprises the steps of performing gastric lavage, respectively performing anesthesia on 0h, 0.08h, 0.25h, 0.5h, 0.75h, 1h, 2h, 4h, 6h, 8h, 12h and 24h by using 20% uratein, shearing hair on the head and neck, sterilizing, cutting a longitudinal line incision (about 2 cm) along a longitudinal axis by using a surgical knife, penetrating a small infusion needle into a cerebellum medullary canal from a major occipital bone hole, extracting cerebrospinal fluid in a precooled centrifuge tube, and immediately killing a rat after extraction. The cerebrospinal fluid is centrifuged at 4000r/min for 10min at 4 ℃, and the supernatant is separated and stored at-80 ℃ for later use.
2.3 Preparation method of solution
2.3.1 Preparation of the Mixed control solution
Weighing 2mg of glycyrrhizic acid, apigenin-7-O-glucuronide, gallic acid and glycyrrhizin reference substances respectively, adding methanol for dissolution, and fixing the volume into a 10mL volumetric flask to prepare a mixed reference substance stock solution with the concentration of each reference substance of 0.2 mg/mL. Accurately sucking 50 mu L of the mixed reference substance stock solution, and fixing the volume to a 50mL volumetric flask by using methanol to obtain 200ng/mL of mixed reference substance solution.
2.3.2 Preparation of internal Standard solution
Precisely weighing icariin reference substance 2mg, adding methanol, fixing the volume into a 10mL measuring flask, preparing into 0.2mg/mL internal standard stock solution, precisely sucking 25 mu L internal standard stock solution, and fixing the volume of methanol into a 50mL measuring flask to obtain 100ng/mL internal standard solution.
2.3.3 Preparation of series of Mixed control solutions
The mixed reference substance solution of 2.3.1 is diluted with methanol according to the proportion of 1:1 in a gradient way, and the serial mixed reference substance solutions with reference substance concentrations (ng/mL) of 1.5875, 3.175, 6.25, 12.5, 25, 50, 100 and 200 are respectively obtained.
2.3.4 Preparation of low, medium and high three quality control sample solutions
Taking 50 mu L of a rat blank sample (blank plasma or blank cerebrospinal fluid), adding 12.5 mu L of a mixed reference substance solution, 2.5 mu L of an internal standard solution and 135 mu L of acetonitrile, uniformly mixing for 3min by vortex, standing for 5min, centrifuging for 10min at 13500r/min at 4 ℃, and taking a supernatant to obtain a drug-containing sample test solution.
The difference between the low, medium and high quality control sample solutions is that the concentration of the added mixed reference substance solution is 6.25ng/mL, 25ng/mL and 100ng/mL respectively.
2.3.5 Method for processing biological samples
Thawing rat plasma sample or cerebrospinal fluid in refrigerator at 4deg.C, vortexing for 3min, precisely sucking 50 μl, adding internal standard solution 2.5 μl and cold acetonitrile 147.5 μl, vortexing, mixing for 3min, standing for 5min, centrifuging at 13500r/min at 4deg.C for 10min, and collecting supernatant, and analyzing.
2.4 Methodological investigation of quantitative detection of blood-entering Components
2.4.1 Specificity investigation
(1) Preparation of sample test solutions
Sample 1: 50 mu L of rat blank plasma is added with 150 mu L of acetonitrile, mixed for 3min by vortex, stood for 5min, centrifuged for 10min at 13500r/min at 4 ℃, and the supernatant is taken as a blank plasma sample test solution.
Sample 2: a test solution of the plasma sample containing the drug at the concentration of 100ng/mL of the mixed reference substance solution was prepared according to the preparation method of 2.3.4.
Sample 3: a sample of the plasma sample to be administered was obtained according to the treatment method of 2.3.5.
(2) Detection analysis
And carrying out UHPLC-MS/MS analysis on the sample 1, the sample 2 and the sample 3 according to the chromatographic condition and the mass spectrum condition of 2.1 respectively to obtain analysis results of blank plasma test liquid, drug-containing plasma sample test liquid and drug-containing plasma sample test liquid, and the analysis results of the drug-containing plasma sample test liquid are shown in figure 22. In the figure A, B, C represents the extracted ion flow chromatograms of sample 1, sample 2 and sample 3, respectively.
The 5 compounds to be tested and the internal standard compound are not interfered by endogenous substances through investigation, and the method has good specificity and is suitable for content measurement of 5 components.
2.4.2 Linear investigation
Precisely measuring 50 mu L of rat blank plasma, respectively adding 12.5 mu L of mixed reference substance solution, 2.5 mu L of internal standard solution and 135 mu L of acetonitrile, precipitating protein, vortex centrifuging, preparing a series of matrix standard curve samples by taking the concentration of the mixed reference substance solution as a variable, respectively carrying out UHPLC-MS/MS analysis according to the chromatographic condition and the mass spectrum condition of 2.1, carrying out regression calculation by adopting a weighted least square method by taking the mass concentration (ng/mL) of each component as an abscissa (X) and the ratio of the peak area of the corresponding sample to the peak area of the internal standard as an ordinate (Y), drawing a standard curve by adopting a weight of 1/X2, and calculating a linear regression equation of each component, wherein the result is shown in Table 5.
TABLE 5 regression equation and linear range for 5 compounds in plasma
As can be seen from table 5, each compound has a good linear relationship in its corresponding linear range.
2.4.3 Precision and accuracy inspection
Bottom, middle and high quality control sample solutions of blank plasma were prepared according to the method of 2.3.4, and UHPLC-MS/MS analysis was performed according to the chromatographic conditions and mass spectrometric conditions of 2.1, respectively. Continuously injecting samples for 6 times in the same day, and calculating the accuracy in the day; the measurement was continued for 3 days, and the daytime precision was calculated, and the results are shown in Table 6.
Table 6 results of calculation of precision and accuracy within day and day
As can be seen from Table 6, the RSD of the 5 compounds is less than 13.68% in the daily, daytime precision and accuracy, which indicates that the method is stable and feasible.
2.4.4 Stability investigation
Preparing bottom, middle and high quality control sample solutions of blank plasma according to a method of 2.3.4, standing for 12 hours at room temperature, and inspecting short-term stability of the sample; 3 times of freeze thawing cycle at-80 ℃ to examine the freeze thawing stability of the sample; the samples were subjected to cold storage at-80℃for 4 weeks and examined for long-term stability. The calculation results are expressed in terms of Relative Standard Deviation (RSD), and the results are shown in table 7.
Table 7 stability experiment (n=6)
As can be seen from Table 7, the stability RSD of all 5 compounds is less than 15%, which indicates that the method is stable and feasible.
2.4.5 Investigation of matrix Effect and recovery
Sample 1: plasma and control together precipitate proteins
Taking 3 parts of 50 mu L blank plasma, adding 12.5 mu L mixed reference substance solution, 2.5 mu L internal standard solution and 135 mu L acetonitrile into each part, respectively preparing 6 parts of mixed reference substance solution with the concentration of 6.25ng/mL, 25ng/mL and 100ng/mL in parallel at each concentration, precipitating protein, carrying out UHPLC-MS/MS analysis according to the chromatographic condition and the mass spectrum condition of 2.1, and recording the ratio (A) of the peak area of the component to the peak area of the internal standard, wherein the result is shown in Table 8.
Sample 2: adding reference substance into the plasma precipitated protein
In addition, 3 parts of 50 mu L of rat blank plasma are taken, 135 mu L of acetonitrile precipitated protein is added to each part, vortex centrifugation is carried out, 12.5 mu L of mixed reference substance solution and 2.5 mu L of internal standard solution are added, the concentrations of the mixed reference substance solution in the three parts are 6.25ng/mL, 25ng/mL and 100ng/mL respectively, 6 parts are prepared in parallel under each concentration, UHPLC-MS/MS analysis is carried out according to the chromatographic condition and the mass spectrum condition of 2.1, the ratio (B) of the peak area of the recorded component to the peak area of the internal standard is recorded, and the extraction recovery rate=A/B×100%.
Sample 3: methanol plus control together precipitate protein
Taking 3 parts of methanol solution instead of blank plasma, namely taking 3 parts of 50 mu L of methanol, respectively adding 12.5 mu L of mixed reference substance solutions with the concentration of 6.25ng/mL, 25ng/mL and 100ng/mL, preparing 6 parts of mixed reference substance solutions with each concentration in parallel, adding 2.5 mu L of internal standard solution and 135 mu L of acetonitrile precipitated protein, carrying out vortex centrifugation, carrying out UHPLC-MS/MS analysis according to the chromatographic condition and the mass spectrum condition of 2.1, and recording the ratio (C) of the peak area of the component to the peak area of the internal standard, wherein the matrix effect=A/C multiplied by 100 percent.
Table 8 matrix effect and recovery experiments (n=6)
As shown in Table 8, the matrix effect of 5 compounds is 78.55% -114.11%, the extraction recovery rate is 78.95% -109.17%, and the extraction method is stable and is not interfered by the matrix, thus basically meeting the requirements of biological sample analysis methods.
2.5 Pharmacokinetic experiment results for quantitative detection of blood-entering Components
The average drug-time curve of 5 compounds in rats after gavage administration of Ruyi zhenbao pills is shown in figure 23. Blood concentration data were calculated using DAS2.1.1 software and the pharmacokinetic profile of the drug was characterized by parameters such as drug half-life (T1/2), peak concentration (Cmax), peak time (Tmax), and area under the drug-time curve (Area under the time-concentration curve, AUC), and the results are shown in table 9.
Table 9 5 pharmacokinetic parameters of compounds (mean±s.d., n=6
| Pharmacokinetic parameters | Apigenin | Glycyrrhizic acid | Apigenin-7-O-glucuronide | Gallic acid | Liquiritigenin |
| Cmax/ng·L-1 | 75.04±3.95 | 18.21±0.87 | 2032.70±59.68 | 24.23±0.71 | 10.68±1.18 |
| Tmax/h | 8 | 0.25 | 0.08 | 0.25 | 0.5 |
| AUC0-t/ng·L-1·h-1 | 249.74±11.80 | 31.47±1.80 | 374.49±10.99 | 320.09±5.23 | 17.26±7.24 |
| AUC0-∞/ng·L-1·h-1 | 250.30±11.76 | 32.27±1.59 | 375.33±11.36 | 744.36±8.56 | 19.52±9.27 |
| T1/2/h | 2.20±0.07 | 4.15±0.54 | 4.24±0.44 | 12.77±0.44 | 3.63±0.73 |
| MRT/h | 8.13±0.03 | 6.93±0.04 | 1.87±0.13 | 13.14±0.18 | 4.44±1.49 |
From fig. 23 and table 9, each compound was detected in plasma at each time point in rats, with a complete in vivo process. In addition, the apigenin-7-O-glucuronide Tmax is relatively smaller, the absorption speed is high, and the peak can be reached rapidly after entering blood; apigenin absorption is relatively slow; the MRT of the 5 components is more than 1.87h and can reach 13.14h at the highest.
2.6 Methodological investigation of quantitative detection of brain-entering Components
2.6.1 Specificity investigation
(1) Preparation of sample test solutions
Sample 1:50 mu L of blank cerebrospinal fluid of a rat is added with 150 mu L of acetonitrile, mixed by vortex for 3min, kept stand for 5min, centrifuged for 10min at 13500r/min at 4 ℃, and the supernatant is taken as blank cerebrospinal fluid sample test solution.
Sample 2: the preparation method of 2.3.4 is used for preparing the cerebrospinal fluid sample test liquid containing the drug under the concentration of 100ng/mL mixed reference substance solution.
Sample 3: a sample of the cerebrospinal fluid for administration was obtained according to the treatment method of 2.3.5.
(2) Detection analysis
And carrying out UHPLC-MS/MS analysis on the sample 1, the sample 2 and the sample 3 according to the chromatographic condition and the mass spectrum condition of 2.1 respectively to obtain the analysis results of blank cerebrospinal fluid test liquid, the cerebrospinal fluid sample test liquid containing the drug and the cerebrospinal fluid sample test liquid to be administered, which are shown in figure 24. In the figure A, B, C represents the extracted ion flow chromatograms of sample 1, sample 2 and sample 3, respectively.
The 5 compounds to be tested and the internal standard compound are not interfered by endogenous substances through investigation, and the method has good specificity and is suitable for content measurement of 5 components.
2.6.2 Linear investigation
Precisely measuring 50 mu L of rat blank cerebrospinal fluid, respectively adding 12.5 mu L of mixed reference substance solution, 2.5 mu L of internal standard solution and 135 mu L of acetonitrile, precipitating protein, vortex centrifuging, preparing a series of matrix standard curve samples by taking the concentration of the mixed reference substance solution as a variable, respectively carrying out UHPLC-MS/MS analysis according to the chromatographic condition and the mass spectrum condition of 2.1, carrying out regression operation by adopting a weighted least square method by taking the mass concentration (ng/mL) of each component as an abscissa (X) and the ratio of the peak area of the corresponding sample to the peak area of the internal standard as an ordinate (Y), drawing a standard curve by adopting a weighted least square method, and calculating a linear regression equation of each component, wherein the result is shown in Table 10.
Table 10 regression equation and linear Range for 5 Compounds in cerebrospinal fluid
As can be seen from table 10, each compound has a good linear relationship in its corresponding linear range.
2.6.3 Precision and accuracy inspection
Preparing bottom, middle and high quality control sample solutions of blank cerebrospinal fluid according to the method of 2.3.4, and performing UHPLC-MS/MS analysis according to the chromatographic condition and the mass spectrum condition of 2.1 respectively. Continuously injecting samples for 6 times in the same day, and calculating the accuracy in the day; the measurement was continued for 3 days, and the daytime precision was calculated, and the results are shown in Table 11.
Table 11 within day, precision and accuracy experiments between days
As can be seen from Table 11, the RSD of the 5 compounds is less than 12.85% in the daily, daytime precision and accuracy, which indicates that the method is stable and feasible.
2.6.4 Stability investigation
Preparing bottom, middle and high quality control sample solutions of blank cerebrospinal fluid according to a method of 2.3.4, standing at room temperature for 12 hours, and inspecting short-term stability of the sample; 3 times of freeze thawing cycle at-80 ℃ to examine the freeze thawing stability of the sample; the samples were subjected to cold storage at-80℃for 4 weeks and examined for long-term stability. The calculation results are expressed in terms of Relative Standard Deviation (RSD), and the results are shown in table 12.
Table 12 stability experiment (n=6)
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As can be seen from Table 12, the stability RSD of all 5 compounds is less than 10.52%, which indicates that the method is stable and feasible.
2.6.5 Investigation of matrix Effect and recovery
The blank plasma of 2.4.5 was replaced with cerebrospinal fluid and UHPLC-MS/MS analysis was performed using the same preparation and detection methods, and the ratio (A) of the peak area of the component to the peak area of the internal standard was recorded, and the results are shown in Table 13.
Table 13 matrix effect and recovery experiments (n=6)
As shown in Table 13, the matrix effect of 5 compounds is 74.73% -105.76%, the extraction recovery rate is 69.40% -110.84%, and the extraction method is stable and is not interfered by the matrix, thus basically meeting the requirements of biological sample analysis methods.
2.7 Pharmacokinetic experiment results for quantitative detection of brain-entering Components
The average drug-time curve of 4 compounds in rats after gavage administration of Ruyi zhenbao pills is shown in fig. 25. The pharmacokinetic profile of the drug was characterized by parameters such as drug half-life (T1/2), peak concentration (Cmax), peak time (Tmax), and area under the drug-time curve (Area under the time-concentration curve, AUC) calculated using DAS 2.1.1 software, as shown in Table 14.
Table 74 pharmacokinetic parameters of compounds (mean±s.d., n=6)
| Pharmacokinetic parameters | Apigenin | Glycyrrhizic acid | Apigenin-7-O-glucuronide | Gallic acid |
| Cmax/ng·L-1 | 3.99±0.09 | 6.08±0.18 | 12.49±0.51 | 29.61±0.81 |
| Tmax/h | 2 | 2 | 0.75 | 0.08 |
| AUC0-t/ng·L-1·h-1 | 12.89±0.70 | 62.25±3.32 | 9.58±0.20 | 248.52±12.41 |
| AUC0-∞/ng·L-1·h-1 | 13.45±0.91 | 359.96±79.30 | 9.63±0.21 | 504.64±4.91 |
| T1/2/h | 6.21±0.82 | 17.50±2.61 | 3.30±0.32 | 11.72±1.79 |
| MRT/h | 8.15±0.29 | 11.17±0.19 | 6.41±0.25 | 13.14±0.44 |
As is clear from FIG. 23 and Table 9, only 4 components of apigenin, glycyrrhizic acid, gallic acid and apigenin-7-O-glucuronide were detected in cerebrospinal fluid, and no glycyrrhizin was detected.
Wherein, the gallic acid Tmax is relatively small, the absorption speed is high, and the peak can be reached rapidly after entering the brain; apigenin and glycyrrhizic acid are absorbed relatively slowly; MRT of the 4 components is more than 6.41h and the highest MRT can reach 13.14h; the gallic acid content is highest at 5 min; the glycyrrhizic acid content is relatively low, the absorption speed is maximum in 2 hours, and the glycyrrhizic acid can be detected within 6 hours, so that the residence time of the glycyrrhizic acid in cerebrospinal fluid is long.
However, glycyrrhizin, a flavonoid component, is not detected in cerebrospinal fluid, probably because it binds to glucuronic acid and sulfate bonds in plasma, affecting its penetration through the blood brain barrier; the reduced levels of components in the cerebrospinal fluid compared to plasma are related to the barrier that exists between the blood and the cerebrospinal fluid. The main function of the blood brain barrier is to prevent unnecessary substances from entering the brain through blood, protect brain tissues from toxic substances and maintain the internal environment relatively stable, but the existence of the blood brain barrier is also a major obstacle affecting the therapeutic effect of drugs for central nervous system diseases. Its presence prevents 95% of the drugs from entering brain tissue from the blood, which is a significant cause of failure in many central nervous system drug therapies. The Ruyi zhenbao pill is mainly used for treating nervous system diseases, so that the research on the content of active ingredients in cerebrospinal fluid is important for revealing the pharmacodynamic substance basis of the Ruyi zhenbao pill for treating nervous system diseases.
The above embodiments are not to be taken as limiting the scope of the invention, and any alternatives or modifications to the embodiments of the invention will be apparent to those skilled in the art and are intended to fall within the scope of the invention. The present invention is not described in detail in the following, but is well known to those skilled in the art.
Claims (6)
1. The method for detecting the components in the Ruyi zhenbao pill is characterized by comprising the following steps:
a qualitative detection method A for the components of Ruyi Zhenbao pills entering blood or brain comprises the following detection steps:
a1: collecting a biological sample of an administration organism, and processing to obtain a solution to be tested, wherein the biological sample is a plasma sample or a cerebrospinal fluid sample;
A2: detecting and analyzing the biological sample by adopting an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbit trap high-resolution mass spectrometry combination technology, wherein:
Chromatographic conditions: the C18 column (2.1 mm. Times.150 mm,1.7 μm) was used, mobile phase A was 0.1% aqueous formic acid, mobile phase B was acetonitrile, and gradient elution conditions were: 0-15min,3% -20% B;15-30min,20% -40% B;30-40min,40% -70% B;40-45min,70% -95% B; the column temperature is 25 ℃, the sample injection amount is set to 4 mu L, and the flow rate is set to 0.30mL/min;
Mass spectrometry conditions: electrospray ionization ion source, positive and negative ion mode collection data; the data acquisition range is 100-1500m/z; the cracking voltage is 100V; collision energy 25eV; the difference in collision voltage is 15eV;
A3: mass spectrum data were collected using Xcalibur software to infer the composition of the Ruyi treasure bolus.
2. The method for detecting the components in the Ruyi zhenbao pills according to claim 1, wherein in the A1, the method for collecting the plasma sample is as follows: collecting abdominal aortic blood of organism after administration for a period of time, standing for 2 hr, centrifuging at 3000r/min for 10min after layering of serum, and freezing supernatant at-80deg.C;
The cerebrospinal fluid sample collection method is as follows: after a period of administration, the cerebral spinal pool is pierced through a micro infusion needle from the occipital macropore, cerebrospinal fluid is extracted and centrifuged for 10min at 4000r/min at 4 ℃ in a precooled centrifuge tube, and the supernatant is taken and stored at-80 ℃ for later use.
3. The method for detecting the internal components of the Ruyi zhenbao pill according to claim 2, wherein,
The plasma samples were processed as follows: thawing the plasma sample in a refrigerator at 4 ℃, swirling for 3min, standing for 5min, centrifuging for 10min at 13500r/min at 4 ℃, and taking supernatant to obtain a solution to be tested;
The treatment method of the cerebrospinal fluid sample comprises the following steps: thawing the cerebrospinal fluid sample in a refrigerator at 4 ℃, swirling for 3min, standing for 5min, centrifuging for 10min at 13500r/min at 4 ℃, and taking the supernatant to obtain a solution to be measured.
4. The method for detecting the internal components of the Ruyi zhenbao pills according to any one of claims 1 to 3, which is characterized by further comprising a method B for quantitatively detecting blood entering components or brain entering components of the Ruyi zhenbao pills, wherein the blood entering components are glycyrrhizic acid, glycyrrhizin, apigenin-7-O-glucuronide and gallic acid, and the brain entering components are glycyrrhizic acid, apigenin-7-O-glucuronide and gallic acid;
The specific detection steps are as follows:
B1: preparing a mixed reference substance solution and an icariin internal standard solution of each component by using methanol;
B2: preparation of a drug-containing sample solution: taking blank plasma or blank cerebrospinal fluid, adding corresponding mixed reference substance solution, internal standard solution and acetonitrile, mixing by vortex, standing for 5min, centrifuging at 13500r/min at 4deg.C for 10min, collecting supernatant, and analyzing by sample injection; preparing a drug administration sample solution, namely taking a processed plasma sample or cerebrospinal fluid sample, adding an internal standard solution and acetonitrile, and processing sample injection according to the method;
B3: detecting and analyzing the sample solution by adopting a UHPLC-MS/MS technology, wherein:
Chromatographic conditions: c18 column (2.1 mm. Times.150 mm,1.7 μm), mobile phase A was 0.1% aqueous formic acid, mobile phase B was acetonitrile, gradient elution conditions: 0-1min,5% -30% B;1-5min,30% -80% B;5-6min,80% -95% B;6-8min,95%% B;8-9min,95% -5% B;9-12min,5% B; the column temperature is 40 ℃, the sample injection amount is set to 2 mu L, and the flow rate is set to 0.30mL/min;
Mass spectrometry conditions: the electrospray ionization ion source is in a negative ion mode, the capillary voltage is 2000V (ESI -), the taper hole voltage is 30V, the ion source temperature is 150 ℃, the taper hole air flow is 150L/h, the desolventizing air temperature is 450 ℃, the desolventizing air flow is 1000L/h, and the monitoring mode is a dynamic multi-reaction monitoring mode.
5. The method for detecting an in vivo component of a Ruyi treasure pill according to claim 4, wherein in B1, the concentration of each component in the mixed reference solution is 1.5875-200ng/mL; the concentration of the internal standard solution was 100ng/mL.
6. The method for detecting the components in the Ruyi zhenbao pills according to claim 4, wherein the preparation method of the solution containing the medicine sample in the B2 is specifically as follows: taking 50 mu L of blank plasma or blank cerebrospinal fluid, adding 12.5 mu L of mixed reference substance solution, 2.5 mu L of internal standard solution and 135 mu L of acetonitrile, mixing uniformly by vortex, standing for 5min, centrifuging for 10min at 4 ℃ at 13500r/min, and taking supernatant for sample injection analysis; the preparation method of the administration sample solution comprises thawing plasma sample or cerebrospinal fluid sample in a refrigerator at 4deg.C, swirling for 3min, precisely sucking 50 μl, adding 2.5 μl of internal standard solution and 147.5 μl of cold acetonitrile, swirling and mixing for 3min, standing for 5min, centrifuging at 13500r/min at 4deg.C for 10min, and collecting supernatant for sample injection analysis.
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