A method for detecting the components of Ruyi Zhenbao Pills

Abstract

The present invention relates to a method for detecting the in vivo components of Ruyi Zhenbao Pills, which includes a method A for qualitatively detecting the blood- or brain-entering components of Ruyi Zhenbao Pills, and a method B for quantitatively detecting the blood- or brain-entering components of Ruyi Zhenbao Pills. Through the qualitative detection method A, the prototype components and metabolites entering the blood and brain can be more comprehensively identified and identified, and the main active components and the metabolism and transformation rules of the active components in the body are preliminarily clarified; through the quantitative detection method B, the high-exposure components of Ruyi Zhenbao Pills at different time points in plasma and cerebrospinal fluid can be accurately quantified, and the pharmacokinetic characteristics of the above components in plasma and cerebrospinal fluid can be conveniently characterized. The above detection provides a reliable experimental basis for identifying the pharmacodynamic material basis of Ruyi Zhenbao Pills, and also lays a chemical substance foundation for explaining its mechanism of action.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for detecting in vivo components of Ruyi Zhenbao Pills. Background Art

[0002] Ruyi Zhenbao Pills are the first choice of Tibetan medicine for treating white vein disease (a general term for diseases that damage the nervous system). They are widely used in clinical practice and play an important role. Although Ruyi Zhenbao Pills have significant clinical efficacy, the research on their chemical composition and material basis is weak, which seriously affects the scientific value of this variety. Summary of the invention

[0003] In order to make up for the deficiencies of the prior art, the present invention provides a method for detecting the components of Ruyi Zhenbao Pills in vivo. Through qualitative detection, the prototype components and metabolites in the body can be clearly identified; through quantitative detection, the components with high exposure levels can be identified, which is convenient for monitoring the pharmacokinetic process.

[0004] The technical solution adopted by the present invention to solve the above technical problems is:

[0005] The present application provides a method for detecting the components of Ruyi Zhenbao Pills in vivo, including: a method A for qualitatively detecting the components of Ruyi Zhenbao Pills that enter the blood or brain, and the detection steps are as follows:

[0006] A1: Collecting a biological sample from a medicated organism and processing it to obtain a test solution, wherein the biological sample is a plasma sample or a cerebrospinal fluid sample;

[0007] A2: Ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry is used to detect and analyze biological samples, including:

[0008] Chromatographic conditions: C18 chromatographic column (2.1 mm × 150 mm, 1.7 μm) was used, mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, gradient elution conditions were: 0-15 min, 3%-20% B; 15-30 min, 20%-40% B; 30-40 min, 40%-70% B; 40-45 min, 70%-95% B; column temperature was 25 ° C, injection volume was set to 4 μL, flow rate was set to 0.30 mL / min;

[0009] Mass spectrometry conditions: electrospray ionization source, data acquisition in positive and negative ion modes; data acquisition range 100-1500 m / z; fragmentation voltage 100 V; collision energy 25 eV; collision voltage difference 15 eV;

[0010] A3: Use Xcalibur software to collect mass spectrometry data and infer the ingredients of Ruyi Zhenbao Pills.

[0011] Furthermore, in A1, the plasma sample collection method is as follows: after a period of administration, the abdominal aorta blood of the organism is collected, and the blood is allowed to stand for 2 hours. After the serum is separated, the blood is centrifuged at 3000r / min for 10 minutes, and the supernatant is frozen at -80°C for later use;

[0012] The method for collecting cerebrospinal fluid samples is as follows: after a period of administration, puncture the cerebellomedullary cistern through the foramen magnum through a microinfusion needle, extract cerebrospinal fluid into a precooled centrifuge tube, centrifuge at 4000r / min for 10min at 4°C, and store the supernatant at -80°C for later use.

[0013] Furthermore, the processing method of plasma samples is as follows: after the plasma sample is thawed in a 4°C refrigerator, vortexed for 3 minutes, allowed to stand for 5 minutes, centrifuged at 13500 r / min for 10 minutes at 4°C, and the supernatant is obtained to obtain the test solution; the processing method of cerebrospinal fluid samples is as follows: after the cerebrospinal fluid sample is thawed in a 4°C refrigerator, vortexed for 3 minutes, allowed to stand for 5 minutes, centrifuged at 13500 r / min for 10 minutes at 4°C, and the supernatant is obtained to obtain the test solution.

[0014] The detection method of the present application also includes method B for quantitatively detecting the components of Ruyi Zhenbao Pills that enter the blood or brain. The components that enter the blood are glycyrrhizic acid, liquiritin, apigenin, apigenin-7-O-glucuronide and gallic acid, and the components that enter the brain are glycyrrhizic acid, apigenin, apigenin-7-O-glucuronide and gallic acid. The specific detection steps are as follows:

[0015] B1: Prepare the mixed reference solution of each component and the icariin internal standard solution with methanol;

[0016] B2: Preparation of drug-containing sample solution: Take blank plasma or blank cerebrospinal fluid, add the corresponding mixed reference solution, internal standard solution and acetonitrile, vortex mix, let stand for 5 minutes, centrifuge at 13500r / min for 10 minutes at 4°C, and take the supernatant for sampling and analysis; Preparation of drug-containing sample solution: Take the treated plasma sample or cerebrospinal fluid sample, add the internal standard solution and acetonitrile, and process and inject according to the above method;

[0017] B3: The sample solution is tested and analyzed using UHPLC-MS / MS technology, including:

[0018] Chromatographic conditions: C18 chromatographic column (2.1 mm × 150 mm, 1.7 μm), mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, gradient elution conditions: 0-1 min, 5%-30% B; 1-5 min, 30%-80% B; 5-6 min, 80%-95% B; 6-8 min, 95% B; 8-9 min, 95%-5% B; 9-12 min, 5% B; column temperature 40 ° C, injection volume set to 2 μL, flow rate set to 0.30 mL / min;

[0019] Mass spectrometry conditions: electrospray ionization source in negative ion mode, capillary voltage 2000V (ESI - ), cone voltage was 30 V, ion source voltage was 30 V, ion source temperature was 150 ° C, cone gas flow rate was 150 L / h, desolvation gas temperature was 450 ° C, desolvation gas flow rate was 1000 L / h, and the monitoring mode was dynamic multiple reaction monitoring mode.

[0020] Furthermore, in B1, the concentration of each component in the mixed reference solution is 1.5875-200 ng / mL; the concentration of the internal standard solution is 100 ng / mL.

[0021] Further, in B2, the preparation method of the drug-containing sample solution is as follows: take 50 μL of blank plasma or blank cerebrospinal fluid, add 12.5 μL of the corresponding mixed reference solution, 2.5 μL of internal standard solution and 135 μL of acetonitrile, vortex mix, let stand for 5 minutes, centrifuge at 13500 r / min for 10 minutes at 4°C, and take the supernatant for sampling and analysis; the preparation method of the drug sample solution is as follows: thaw the plasma sample or cerebrospinal fluid sample in a 4°C refrigerator, vortex for 3 minutes, accurately pipette 50 μL, add 2.5 μL of internal standard solution and 147.5 μL of cold acetonitrile, vortex mix for 3 minutes, let stand for 5 minutes, centrifuge at 13500 r / min for 10 minutes at 4°C, and take the supernatant for sampling and analysis.

[0022] The present invention adopts the above structure, which has the following advantages: first, through qualitative detection, the prototype components and metabolites entering the blood and brain are more comprehensively identified and authenticated, and the main active ingredients and the metabolism and transformation rules of the active ingredients in the body are preliminarily clarified; second, through quantitative detection, the high-exposure components of Ruyi Zhenbao Pills at different time points in plasma and cerebrospinal fluid are accurately quantified, and the pharmacokinetic characteristics of the above components in plasma and cerebrospinal fluid are objectively characterized. This provides a reliable experimental basis for identifying the material basis of the efficacy of Ruyi Zhenbao Pills, and also lays a chemical substance foundation for explaining its mechanism of action. Specifically:

[0023] (1) Through qualitative detection methods, 41 blood-entering components and 20 brain-entering components of Ruyi Zhenbao Pills were identified, and the metabolic pathways and products of 6 representative components were analyzed. Among them, the blood-entering components include organic acids, flavonoids, volatile oils, amides, ketones, sesquiterpenes, amino acids, phenylpropanoids and monosaccharides. The organic acid components from the prince medicine Terminalia chebula, Terminalia chebula, and the queen medicine Clove and Cinnamon are the most, followed by the flavonoid components from the monarch medicine Short-spiked Rabbit Ear Grass and Licorice Paste; the brain-entering components include amino acids, organic acids, alkaloids, phenylpropanoids, volatile oils, phenols, ketones and sesquiterpenes, among which the organic acid components contained in the monarch medicine Licorice Paste, Queen Medicine Clove, and Prince Medicine Terminalia chebula, Terminalia chebula, and Phyllanthus emblica are the most. The above test results lay a data foundation for in-depth analysis of the formula rules and pharmacodynamic material basis of Ruyi Zhenbao Pills.

[0024] (2) The quantitative detection method can be used to determine the contents of five components, namely glycyrrhizic acid, glycyrrhizin, apigenin, apigenin-7-O-glucuronide and gallic acid, in plasma samples; and the contents of four components, namely glycyrrhizic acid, apigenin, apigenin-7-O-glucuronide and gallic acid, in cerebrospinal fluid samples. The detection method has good specificity, intra-day and inter-day precision and accuracy, and stability. In addition, it is also convenient for monitoring the pharmacokinetic process of each component in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 BPI chromatograms detected for blank plasma samples and administered plasma samples of organisms;

[0026] Figure 2 This is the analytical diagram of the glycyrrhizic acid cleavage pathway;

[0027] Figure 3 This is the analysis diagram of glycyrrhizic acid plasma metabolites;

[0028] Figure 4 This is the analytical diagram of the plasma cleavage pathway of liquiritin;

[0029] Figure 5 This is the analysis chart of plasma metabolites of liquiritin;

[0030] Figure 6 This is the analytical diagram of the plasma cleavage pathway of galangin;

[0031] Figure 7 This is the analysis diagram of plasma metabolites of galangin;

[0032] Figure 8 This is a diagram of the bile acid plasma cleavage pathway;

[0033] Fig. 9 This is a diagram of the plasma cleavage pathway of gallic acid;

[0034] Fig.10 This is the analysis chart of gallic acid plasma metabolites;

[0035] Fig.11 This is the analytical diagram of the plasma cleavage pathway of apigenin;

[0036] Fig.12 This is the analysis diagram of apigenin plasma metabolites;

[0037] Fig.13 This is the analytical diagram of the plasma cleavage pathway of apigenin-7-O-glucuronide;

[0038] Fig.14 This is the plasma metabolite analysis chart of apigenin-7-O-glucuronide;

[0039] Fig.15 BPI chromatograms detected for blank cerebrospinal fluid samples and drug-administered cerebrospinal fluid samples of the organism;

[0040] Fig.16 This is the analysis diagram of glycyrrhizic acid metabolites in cerebrospinal fluid;

[0041] Fig.17 This is the analysis diagram of liquiritin cerebrospinal fluid metabolites;

[0042] Fig.18 This is the analysis diagram of galangin cerebrospinal fluid metabolites;

[0043] Fig.19 This is the analysis diagram of gallic acid cerebrospinal fluid metabolites;

[0044] Fig. 20 This is the analysis diagram of cerebrospinal fluid metabolites of apigenin;

[0045] Fig.21 This is the cerebrospinal fluid metabolite analysis chart of apigenin-7-O-glucuronide;

[0046] Fig. 22 The extracted ion chromatograms of five compounds and internal standard icariin in plasma;

[0047] Fig.23 This is the drug-time curve of each component in the plasma of SD rats after intragastric administration;

[0048] Fig.24 The extracted ion chromatograms of five compounds and internal standard icariin in cerebrospinal fluid;

[0049] Fig.25 This is the drug-time curve of each component in the cerebrospinal fluid of SD rats after oral administration. DETAILED DESCRIPTION

[0050] In order to clearly illustrate the technical features of the present solution, the present invention is described in detail below through specific implementation methods and in conjunction with the accompanying drawings.

[0051] 1. Materials

[0052] (1) Experimental instruments: ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometer; XevoTQ Absolute triple quadrupole mass spectrometer; Xcalibur data analysis system (Thermo Corporation, USA); MassLynx 4.2SCN1040 data analysis system (Waters Corporation, USA); Waters ACQUITY UPLC BEH C18 column ( 1.7μm, 2.1mmX150mm, Waters Company, USA); NA-5L nitrogen-air all-in-one machine (Beijing Zhongxing Huili Technology Co., Ltd.); QL-901 vortex mixer (Qilin Medical Instrument Co., Ltd., Haimen City, Jiangsu Province); 1 / 100,000 analytical balance (Sartorius Scientific Instrument Co., Ltd.).

[0053] (2) Experimental drugs: Ruyi Zhenbao Pills powder (company: Jinhe Tibetan Medicine Co., Ltd., batch number: 01230301, specification: 100 g powder, 1 g powder is equivalent to 1 g original medicinal material); standard substances: apigenin (batch number: DSTDQ002602), glycyrrhizic acid (batch number: DSTDGQ000603), apigenin-7-O-glucuronide (batch number: DSTDQ009001), gallic acid (batch number: DSTDM000802), licorice glycoside (batch number: DSTDG000902) were purchased from Lemeitian Pharmaceutical; icariin (batch number: B21576) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Acetonitrile (mass spectrometry grade, Thermo Fisher Scientific, USA); methanol (chromatographic grade, Thermo Fisher Scientific, USA); formic acid (HPLC grade, Sigma Aldrich, USA); distilled water (Guangzhou Watsons).

[0054] (3) Experimental animals: 96 SPF male SD rats weighing 200±20g were used. Provided by Beijing Weitonglihua Experimental Animal Co., Ltd., the experimental animal production license number is SCXK (Beijing) 2021-0011, SCXK (Beijing) 2021-0006. Rats were uniformly fed in the experimental animal room of the Institute of Basic Theory of Traditional Chinese Medicine, China Academy of Chinese Medical Sciences. The animal room is clean and bright, with good ventilation, room temperature of about 23±5℃, and humidity of about 55±5%. The bedding is changed every 4 days. Rats are free to eat and drink water, which is changed in time every day. Before the formal start of the experiment, the rats are adaptively fed for 1 week.

[0055] (II) Detection methods and results

[0056] Example 1: Method for qualitative detection of the prototype components and metabolic components of Ruyi Zhenbao Pills that enter the blood and brain

[0057] 1.1 Chromatographic and mass spectrometry conditions

[0058] (1) Chromatographic conditions: Waters ACQUITY UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 μm), mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. Gradient elution conditions were set as follows: 0-15 min, 3%-20% B; 15-30 min, 20%-40% B; 30-40 min, 40%-70% B; 40-45 min, 70%-95% B; column temperature was set at 25 °C, injection volume was set at 4 μL, and flow rate was set at 0.30 mL / min. UV detection wavelength was 400 nm.

[0059] (2) Mass spectrometry conditions: Electrospray ionization (ESI) ion source was used to collect data in positive and negative ion modes. The data collection range m / z was set to 100-1500. Fragmentation voltage (DP): 100 V, collision energy (CE): 25 eV, collision voltage difference (CES): 15 eV.

[0060] 1.2 Collection of biological samples

[0061] Take 15g of Ruyi Zhenbao Pills powder, add appropriate amount of ultrapure water, shake and mix evenly, and make a solution with a mass concentration of 0.36g / mL for standby use. (The adult dose is 5g / day, the clinical equivalent dose of rats is 0.45g / kg, and the 4-fold clinical dose is 1.8g / kg. A 250g rat is gavaged with 0.36g)

[0062] (1) Collection method of plasma samples: 12 SD rats were randomly divided into a blank group and a treatment group, with 6 rats in each group. The rats were fasted for 12 hours (free access to water). The treatment group was gavaged with 1.8 g / kg of Ruyi Zhenbao Pill suspension, and the blank group was gavaged with the same volume of ultrapure water. 1.5 hours after gavage, the rats were anesthetized with 10% chloral hydrate, and blood was collected from the abdominal aorta. The blood was allowed to stand for 2 hours. After the serum was separated, it was centrifuged at 3000 r / min for 10 minutes, and the supernatant was collected. The serum of different rats in the same group was vortexed and mixed and frozen at -80°C for later use.

[0063] (2) Method for collecting cerebrospinal fluid samples: 6 SD rats were fasted for 12 h (free access to water), and then gavaged with 1.8 g / kg of Ruyi Zhenbao Pill powder. Two hours after administration, the rats were anesthetized with 20% urethane, and then the hair on the head and neck was trimmed and disinfected. A longitudinal incision was made along the longitudinal axis with a scalpel, and a microinfusion needle was inserted into the cerebellomedullary cistern through the foramen magnum to extract cerebrospinal fluid into a precooled centrifuge tube. After the extraction, the rats were immediately killed. The cerebrospinal fluid was centrifuged at 4000 r / min at 4°C for 10 min, and the supernatant was stored at -80°C.

[0064] 1.3 Biological sample pre-injection treatment

[0065] Processing method of plasma samples and cerebrospinal fluid samples: After the rat samples were thawed in a 4°C refrigerator, they were vortexed for 3 minutes, allowed to stand for 5 minutes, centrifuged at 13500r / min for 10 minutes at 4°C, and the supernatant was taken for sampling and analysis.

[0066] 1.4 Detection and analysis of prototype components and metabolic components entering the blood

[0067] 1.4.1 Identification of blood components

[0068] The plasma samples were collected in positive and negative ion modes to obtain mass spectrometry data. The mass spectrometry data were collected using Xcalibur software. The PBI chromatogram is shown in Figure 1 , where black represents positive ion mode, red represents negative ion mode, A represents blank group, and B represents drug administration group. By analyzing the mass spectrometry fragmentation rules of different types of compounds, 41 prototype components (see Table 1) and 24 metabolites (see Table 2) were preliminarily identified from plasma. The 24 metabolites were derived from prototype components such as glycyrrhizic acid, liquiritin, galangin, gallic acid, apigenin and apigenin-7-O-glucuronide, which were derived from the main medicines of short-spiked rabbit ear grass and liquorice, the queen medicine of galangal, and the prince medicine of terminalia chebula.

[0069] Specifically, from the plasma of Ruyi Zhenbao Pills administration, 10 organic acid components were identified, mainly from medicinal materials such as terminalia chebula, terminalia chebula, amomum villosum, clove, licorice, and emblica; 8 flavonoid components, mainly from medicinal materials such as short-spiked rabbit ear grass, licorice, and galangal; 3 amide components, mainly from piper longum; 5 volatile oil components, mainly from cloves and cinnamon; in addition, 3 amino acid components, 2 alkaloid components, 2 phenylpropanoid components, 4 ketone components, 3 sesquiterpenoid components and 1 monosaccharide component were also identified.

[0070] Table 1 List of the prototype components of Ruyi Zhenbao Pills that enter the blood

[0071]

[0072]

[0073] Table 2 Representative blood components and their metabolites

[0074]

[0075]

[0076] 1.4.2 Analysis of the cleavage and metabolic patterns of representative blood components

[0077] (1) Glycyrrhizic acid: Triterpenoids are an important component of licorice chemical composition, mainly glycyrrhizic acid derivatives. Taking glycyrrhizic acid as an example, the mass spectrum shows a quasi-molecular ion of [MH]-m / z 821.3965, and the -C 29 H 42 After O5 group, m / z 351.0573 fragment was obtained; -C2O2, -C6H 18 O 11 , -C 19 H 30 O 11 The fragment ion m / z 195.0356 was obtained after the group was added. The fragmentation pathway is shown in Figure 2 This type of compound is prone to glucuronidation or sulfation after hydration in the body. Some metabolites are prone to sulfation or acetylation after hydration and oxidation after the quasi-molecular ion fragments remove the glycosides, resulting in m / z 455.3520 and m / z 471.3111 fragment ions. Metabolic analysis is shown in Figure 3 .

[0078] (2) Liquorice glycoside: One of the main sources of flavonoids in Ruyi Zhenbao Pills is the main drug liquorice paste, and licorice glycoside is a representative component. Liquorice glycoside shows a quasi-molecular ion of [MH]-m / z 417.1196 in the mass spectrum. The secondary spectrum mainly shows the fragment ion m / z 255.0666 after the glucose group is removed, and m / z 119.0503 is formed by losing C7H4O3; the characteristic fragment m / z 135.0089 is obtained by RDA cleavage on the C ring, and further CO2 is lost to form a fragment ion of m / z 119.08. The cleavage pathway is shown in Figure 4 The flavonoid components in licorice are prone to reduction, methylation, glucuronidation and sulfation reactions in serum. The structure of liquiritin contains one molecule of glucose, which is easy to lose glucose in the body and exists in the form of aglycone, further generating ion peaks of m / z 431.3146, m / z 255.1590, and m / z 119.0252. Metabolic analysis is shown in Figure 5 .

[0079] (3) Galangin: Galangin is the main component of the queen medicine galangal, with a quasi-molecular ion peak of m / z 271.0600 ([M+H]+), characteristic fragment ions m / z 225.0556 ([M-CO2+H]+), 197.0596 ([M-CO2-CO+H]+) or 153.0192 (RDA, 1，3 A), cleavage pathways such as Figure 6 The main metabolites of galangin in plasma are as follows ( Figure 6 ): The C2 quasi-molecular ion peak is m / z 623.3163 ([M+H]+), and the molecular formula is C27 H 26 O 17 The main fragment ions are m / z 447.0918 ([M-C6H8O6+H]+) and 271.0593 ([M-2C6H8O6+H]+), and diglucuronic acid binding occurs; the C3 quasi-molecular ions are m / z 447.1304, and the main fragments are m / z 271.0597 ([M-C6H8O6+H]+), 215.0697 ([M-C6H8O6-2CO+H]+), 165.0182 (RDA, 0，3 A) and 153.0181 (RDA, 1，3 A), indicating that galangin undergoes glucuronic acid conjugation. The molecular formula of C4 is C 21 H 18 O 11 The quasi-molecular ions are m / z 463.1303 ([M+H]+), oxidation reaction and glucuronic acid binding reaction, characteristic fragment ions m / z 329.0508 ([ 1，3 A+C6H8O6+H]+), 287.0543([M-C6H8O6+H]+), 165.0184( 0，3 A) 153.0187( 1，3 A) and 121.0289( 0，3 B) The molecular formula of C5 is C 15 H 10 O8S, the quasi-molecular ion is m / z 351.0186 ([M+H]+). C6 quasi-molecular ions are m / z 367.2648, and the oxidation reaction and sulfuric acid combination reaction occur, and the molecular composition is C 15 H 10 O9S. The C7 quasi-molecular ion is m / z 285.2075 ([M+H]+), and a methylation reaction occurs. The main characteristic 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 ( 0，3 A) 167.0331( 1，3 A) and 105.0335( 0 ，3 B) The molecular formula of C8 is C 17 H 12 O6, the quasi-molecular ion is m / z 313.2390 ([M+H]+), the main fragments are m / z 285.0769 ([M-CO+H]+), 257.0798 ([M-2CO+H]+), 207.0288 (0，3 A) 195.0278( 1，3 A) 119.0491( 1，3 B) and 105.0337( 0，3 B), Ring A undergoes N-acetyl binding reaction, metabolic analysis see Figure 7 .

[0080] (4) Bile acid: Bile acid is the main component of the representative animal medicine bezoar in the formula. It shows a quasi-molecular ion of [MH]-m / z 407.2809 in the mass spectrum, and the main dehydrated fragment ion m / z 389.2199 appears in the secondary spectrum, and further loss of C5H 14 O4 formation m / z 259.1792, fragmentation pathway see Figure 8 .

[0081] (5) Gallic acid: Gallic acid is the main component of Terminalia chebula. Mass spectrometry results show that gallic acid mainly exists in the quasi-molecular ion peak of m / z 169.0138, with loss of -C6H 10 The characteristic ion fragment m / z 124.0370 was obtained after the O5 group, and the fragmentation mode was as follows Fig. 9 Its metabolic pathway and products after entering the blood are shown in Fig.10 .

[0082] (6) Apigenin: Apigenin is derived from the main herb, short-spiked rabbit ear grass, and belongs to the flavonoids. Flavonoids mainly undergo a breakage and loss rearrangement reaction centered on the C ring. In addition, flavonoid aglycones often undergo a series of fragmentation reactions that successively lose neutral fragments such as CO and H2O. The mass spectrometry results show that apigenin mainly exists in the quasi-molecular ion of m / z 271.0962, and the fragment ion of the -CO group mainly appears in the secondary spectrum at m / z 243.0649, or after RDA cleavage, a fragment ion of m / z153.0182 is obtained. The cleavage method is as follows: Fig.11 . Apigenin metabolism analysis see Fig.12 The quasi-molecular ion peak of E2 is m / z 349.349.2643 ([MH]-). The quasi-molecular ion peak of E3 is m / z 2283.0826, and E4 produces a quasi-molecular ion peak of m / z 269.0822, which is speculated to be 5-hydroxy-7-methoxyflavan. The quasi-molecular ion peak of E5 is m / z 313.1068 ([MH]-), and the fragment ions are m / z 271 ([MH-3CH2]-), 269 ([MH-CO2]-), 179 ( 1，3 [AH]-) and 117( 1，3 [BH-2H-CH]-), which was speculated to be dihydroapigenin-4',5,7-trimethyl ether.

[0083] (7) Apigenin-7-O-glucuronide: Apigenin-7-O-glucuronide is derived from the main herb, short-spiked rabbit ear grass, and shows a quasi-molecular ion of m / z 445.0671 in the mass spectrum. The secondary spectrum results show that the main component is the glycosidic bond breakage loss -C6H8O 10 The fragment ions of the group are m / z 243.0649 and m / z 175.0249, and the fragmentation pathways are as follows Fig.13 Its metabolic pathway and products after entering the blood are shown in Fig.14 .

[0084] 1.5 Detection and analysis of prototype components and metabolic components that enter the brain

[0085] 1.5.1 Identification of brain-entering components

[0086] The mass spectrometry data of Ruyi Zhenbao Pills cerebrospinal fluid samples were collected in positive and negative ion modes. Fig.15 . By analyzing the mass spectrometry fragmentation rules of different types of compounds, 20 prototype components and 16 metabolites were preliminarily identified from the queen and monarch drugs of Ruyi Zhenbao Pills. Specifically, 7 organic acid compounds were identified from the cerebrospinal fluid after administration of Ruyi Zhenbao Pills, mainly from Terminalia chebula, Terminalia chebula, clove, Phyllanthus emblica, and Licorice paste. 3 volatile oil component compounds, mainly from the clove-cinnamon drug pair. 3 alkaloid compounds, mainly from Tibetan costusroot, white cardamom, clove, and Piper longum. In addition, there are 2 alkaloid components, 2 phenylpropanoid components, 1 sesquiterpenoid component, 1 phenolic component and 1 ketone component.

[0087] Table 3. Prototype ingredients of Ruyi Zhenbao Pills that enter the brain

[0088]

[0089] Table 4 Representative brain-entering components and their metabolites

[0090]

[0091] 1.5.2 Analysis of the cleavage and metabolic patterns of representative brain components

[0092] (1) Glycyrrhizic acid: The quasi-molecular ion peaks of glycyrrhizic acid metabolites entering the brain are m / z 455.1883 and m / z 471.1833. The metabolic pathways are as follows: Fig.16 .

[0093] (2) Liquorice glycoside: The main ion peaks of liquorice glycoside metabolites entering the brain are m / z 255.1605 and m / z 119.0490. The metabolic pathways are as follows: Fig.17 .

[0094] (3) Galangin: The main ion peaks of galangin glycoside metabolites entering the brain are m / z 623.3163, m / z 351.0551, m / z 367.2647, m / z 285.2075, and m / z 313.2390. The metabolic pathway analysis is shown in Fig.18 .

[0095] (4) Gallic acid: Gallic acid mainly undergoes deoxygenation and other processes in cerebrospinal fluid. For metabolic pathway analysis, see Fig.19 .

[0096] (5) Apigenin: The metabolites of apigenin in cerebrospinal fluid mainly produce quasi-molecular ion peaks of m / z 349.349.2643, m / z 283.0826, and m / z 269.0822. The metabolic pathways are as follows: Fig. 20 .

[0097] (6) Apigenin-7-O-glucuronide: Apigenin-7-O-glucuronide undergoes glycosidic bond cleavage, methylation, and deoxygenation in cerebrospinal fluid. The metabolic pathway analysis is shown in Fig.21 .

[0098] In summary, this application has obtained 41 prototype components that enter the blood and 20 prototype components that enter the brain by detecting the blood and brain components of Ruyi Zhenbao Pills, and analyzed the metabolic pathways and products of 6 representative components. Among them, the blood-entering components include organic acids, flavonoids, volatile oils, amides, ketones, sesquiterpenes, amino acids, phenylpropanoids and monosaccharide components, with the largest number of organic acid components from Prince Medicine Terminalia Chebula, Hair Terminalia Chebula, and Queen Medicine Cloves and Cinnamon, followed by flavonoid components from Jun Medicine Short Spiked Rabbit Ear Grass and Licorice Paste; Brain-entering components include amino acids, organic acids, alkaloids, phenylpropanoids, volatile oils, phenols, ketones and sesquiterpenes, of which the main organic acid components are contained in Jun Medicine Licorice Paste, Queen Medicine Cloves, and Prince Medicine Terminalia Chebula, Hair Terminalia Chebula, and Phyllanthus Emblica. The above results lay a data foundation for in-depth analysis of the formula rules and pharmacodynamic material basis of Ruyi Zhenbao Pills.

[0099] In particular, by comparing the in vivo components of Ruyi Zhenbao Pills with the in vitro components, it is possible to infer which components are exerting their efficacy, which is of great significance for studying the mechanism of drug action. Among the types of components that enter the brain, the proportion of flavonoid components is reduced compared to the components that enter the blood, while the proportion of organic acid components is increased. Therefore, it is speculated that organic acid components may be the pharmacological material basis for Ruyi Zhenbao Pills to directly intervene in the central nervous system in the treatment of neuropathic pain.

[0100] Embodiment 2: Method for quantitative detection of the prototype components of Ruyi Zhenbao Pills entering blood and brain

[0101] In addition to being absorbed into the blood and brain, the active ingredients of Ruyi Zhenbao Pills also need to reach a certain exposure level in plasma and cerebrospinal fluid, and should have good pharmacokinetic characteristics such as absorption, distribution, metabolism, and excretion. Therefore, in order to further clarify the active substances of Ruyi Zhenbao Pills, UPLC-Q-TRAP / MS technology will be used to quantitatively detect the blood and brain components to identify high-exposure components and perform pharmacokinetic characterization.

[0102] 2.1 Chromatographic and mass spectrometry conditions

[0103] (1) Chromatographic conditions: Waters ACQUITY UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 μm), mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile, gradient elution conditions: 0-1 min, 5%-30% B; 1-5 min, 30%-80% B; 5-6 min, 80%-95% B; 6-8 min, 95% B; 8-9 min, 95%-5% B; 9-12 min, 5% B; column temperature was 40 °C, injection volume was set to 2 μL, and flow rate was set to 0.30 mL / min.

[0104] (2) Mass spectrometry conditions: electrospray ionization source in negative ion mode, capillary voltage 2000 V (ESI - ), cone voltage was 30 V, ion source voltage was 30 V, ion source temperature was 150 ° C, cone gas flow rate was 150 L / h, desolvation gas temperature was 450 ° C, desolvation gas flow rate was 1000 L / h, and the monitoring mode was dynamic multiple reaction monitoring mode.

[0105] 2.2 Collection methods of biological samples

[0106] Take 15g of Ruyi Zhenbao Pills powder, add appropriate amount of ultrapure water, shake and mix evenly, and make a solution with a mass concentration of 0.36g / mL for standby use. (The adult dose is 5g / day, the clinical equivalent dose of rats is 0.45g / kg, and the 4-fold clinical dose is 1.8g / kg. A 250g rat is gavaged with 0.36g)

[0107] (1) Collection method of plasma samples: 12 SD rats were fasted but not watered for 12 hours before the experiment. The Ruyi Zhenbao Pills powder suspension was gavaged intragastrically. About 0.5 mL of blood was collected from the retroorbital vein of the rats at 0 h, 0.08 h, 0.25 h, 0.5 h, 0.75 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration. The blood was placed in a heparinized test tube and centrifuged at 4000 r / min for 10 min. The supernatant was separated and stored in a -80°C refrigerator for later use.

[0108] (2) Method for collecting cerebrospinal fluid samples: 72 SD rats were randomly divided into 12 groups, with 6 rats in each group. The rats were fasted but not watered for 12 hours before the experiment. The rats were gavaged and anesthetized with 20% urethane at 0h, 0.08h, 0.25h, 0.5h, 0.75h, 1h, 2h, 4h, 6h, 8h, 12h, and 24h after the administration. The hair on the head and neck was trimmed and disinfected. A longitudinal incision (about 2cm) was made along the longitudinal axis with a scalpel. A microinfusion needle was inserted into the cerebellomedullary cistern from the foramen magnum to extract cerebrospinal fluid into a precooled centrifuge tube. The rats were killed immediately after the extraction. The cerebrospinal fluid was centrifuged at 4000r / min at 4℃ for 10min, and the supernatant was separated and stored at -80℃ for later use.

[0109] 2.3 Solution preparation method

[0110] 2.3.1 Preparation of mixed reference solution

[0111] Weigh 2 mg of glycyrrhizic acid, apigenin, apigenin-7-O-glucuronide, gallic acid, and liquiritin reference substances, respectively, dissolve them in methanol, and dilute them to a 10 mL volumetric flask to prepare a mixed reference substance stock solution with a concentration of 0.2 mg / mL for each reference substance. Accurately pipette 50 μL of the mixed reference substance stock solution and dilute it to a 50 mL volumetric flask with methanol to obtain a 200 ng / mL mixed reference substance solution.

[0112] 2.3.2 Preparation of internal standard solution

[0113] Accurately weigh 2 mg of icariin reference substance, add methanol, and make up to the volume in a 10 mL volumetric flask to prepare a 0.2 mg / mL internal standard stock solution. Accurately pipette 25 μL of the internal standard stock solution, and make up to the volume in a 50 mL volumetric flask with methanol to obtain a 100 ng / mL internal standard solution.

[0114] 2.3.3 Preparation of a series of mixed reference solutions

[0115] The mixed reference substance solution in 2.3.1 was diluted with methanol in a gradient ratio of 1:1 to obtain a series of mixed reference substance solutions with reference substance concentrations (ng / mL) of 1.5875, 3.175, 6.25, 12.5, 25, 50, 100, and 200, respectively.

[0116] 2.3.4 Preparation of low, medium and high quality control sample solutions

[0117] Take 50 μL of rat blank sample (blank plasma or blank cerebrospinal fluid), add 12.5 μL of mixed reference solution, 2.5 μL of internal standard solution and 135 μL of acetonitrile, vortex mix for 3 min, let stand for 5 min, centrifuge at 13500 r / min for 10 min at 4 °C, take the supernatant, and obtain the drug-containing sample test solution.

[0118] Among them, the difference between the low, medium and high quality control sample solutions is that the concentrations of the mixed reference solution added are 6.25ng / mL, 25ng / mL and 100ng / mL respectively.

[0119] 2.3.5 Biological sample processing methods

[0120] After thawing, rat plasma samples or cerebrospinal fluid were vortexed for 3 minutes in a 4°C refrigerator, 50 μL was accurately pipetted, 2.5 μL of internal standard solution and 147.5 μL of cold acetonitrile were added, vortexed for 3 minutes, allowed to stand for 5 minutes, centrifuged at 13500 r / min for 10 minutes at 4°C, and the supernatant was taken for sampling and analysis.

[0121] 2.4 Methodological study on quantitative detection of blood components

[0122] 2.4.1 Specificity Investigation

[0123] (1) Preparation of sample test solution

[0124] Sample 1: Add 150 μL of acetonitrile to 50 μL of rat blank plasma, vortex mix for 3 min, let stand for 5 min, centrifuge at 13500 r / min for 10 min at 4°C, and take the supernatant as the blank plasma sample test solution.

[0125] Sample 2: Prepare the drug-containing plasma sample test solution at a concentration of 100 ng / mL mixed reference solution according to the preparation method in 2.3.4.

[0126] Sample 3: Obtain the test solution of the administered plasma sample according to the processing method in 2.3.5.

[0127] (2) Detection and analysis

[0128] The above samples 1, 2 and 3 were subjected to UHPLC-MS / MS analysis according to the chromatographic conditions and mass spectrometry conditions of 2.1, respectively, to obtain the analysis results of blank plasma test solution, drug-containing plasma sample test solution and drug-administered plasma sample test solution, see Fig. 22 In the figure, A, B, and C represent the extracted ion chromatograms of sample 1, sample 2, and sample 3, respectively.

[0129] The results showed that the five test compounds and internal standard compounds were not interfered by endogenous substances, and the method had good specificity and was suitable for the determination of the contents of the five components.

[0130] 2.4.2 Linear investigation

[0131] Accurately measure 50 μL of rat blank plasma, add 12.5 μL of mixed reference solution, 2.5 μL of internal standard solution and 135 μL of acetonitrile respectively, precipitate protein, vortex centrifuge, and prepare a series of matrix standard curve samples with the concentration of mixed reference solution as the variable. UHPLC-MS / MS analysis was performed according to the chromatographic conditions and mass spectrometry conditions of 2.1 respectively. The mass concentration of each component (ng / mL) was used as the abscissa (X), and the ratio of the corresponding sample peak area to the internal standard peak area was used as the ordinate (Y). The weighted least squares method was used for regression operation with a weight of 1 / X2. The standard curve was drawn and the linear regression equation of each component was calculated. The results are shown in Table 5.

[0132] Table 5 Regression equations and linear ranges of five compounds in plasma

[0133]

[0134]

[0135] It can be seen from Table 5 that each compound has a good linear relationship within its corresponding linear range.

[0136] 2.4.3 Precision and accuracy investigation

[0137] The base, medium and high quality control sample solutions of blank plasma were prepared according to the method in 2.3.4, and UHPLC-MS / MS analysis was performed according to the chromatographic conditions and mass spectrometry conditions in 2.1. The sample was injected 6 times continuously on the same day to calculate the intra-day precision; the measurement was continued for 3 days to calculate the inter-day precision. The results are shown in Table 6.

[0138] Table 6 Intra-day and inter-day precision and accuracy calculation results

[0139]

[0140] As shown in Table 6, the RSDs of intra-day and inter-day precision and accuracy of the five compounds were all less than 13.68%, indicating that the method was stable and feasible.

[0141] 2.4.4 Stability study

[0142] According to the method in 2.3.4, the bottom, middle and high quality control sample solutions of blank plasma were prepared and placed at room temperature for 12 hours to investigate the short-term stability of the samples; freeze-thaw cycles were performed at -80°C for 3 times to investigate the freeze-thaw stability of the samples; and refrigerated at -80°C for 4 weeks to investigate the long-term stability of the samples. The calculation results are expressed as relative standard deviation (RSD), and the results are shown in Table 7.

[0143] Table 7 Stability test (n=6)

[0144]

[0145]

[0146] As shown in Table 7, the stability RSDs of the five compounds were all less than 15%, indicating that the method was stable and feasible.

[0147] 2.4.5 Investigation of matrix effect and recovery rate

[0148] Sample 1: Plasma + control substance to precipitate protein

[0149] Take 3 portions of 50 μL blank plasma, add 12.5 μL mixed reference solution, 2.5 μL internal standard solution and 135 μL acetonitrile to each portion, the concentrations of the mixed reference solution in the three portions are 6.25 ng / mL, 25 ng / mL and 100 ng / mL, respectively, prepare 6 portions in parallel at each concentration, precipitate protein, vortex centrifuge, and perform UHPLC-MS / MS analysis according to the chromatographic conditions and mass spectrometry conditions of 2.1, and record the ratio of the component peak area to the internal standard peak area (A). The results are shown in Table 8.

[0150] Sample 2: First add plasma precipitate protein and then add reference substance

[0151] Take three portions of 50 μL of rat blank plasma, add 135 uL of acetonitrile to each portion to precipitate protein, vortex centrifuge, and then add 12.5 μL of mixed reference solution and 2.5 μL of internal standard solution. The concentrations of the mixed reference solution in the three portions are 6.25 ng / mL, 25 ng / mL and 100 ng / mL, respectively. Prepare 6 portions in parallel at each concentration, and perform UHPLC-MS / MS analysis according to the chromatographic conditions and mass spectrometry conditions of 2.1. Record the ratio of the component peak area to the internal standard peak area (B), and the extraction recovery rate = A / B×100%.

[0152] Sample 3: Protein precipitation with methanol + reference substance

[0153] Take another methanol solution to replace blank plasma, that is, take 3 portions of 50 μL of methanol, add 12.5 μL of mixed reference solution with concentrations of 6.25 ng / mL, 25 ng / mL, and 100 ng / mL respectively, prepare 6 portions of each concentration in parallel, add 2.5 μL of internal standard solution and 135 μL of acetonitrile to precipitate protein, vortex centrifuge, and perform UHPLC-MS / MS analysis according to the chromatographic conditions and mass spectrometry conditions of 2.1, record the ratio of the component peak area to the internal standard peak area (C), matrix effect = A / C×100%.

[0154] Table 8 Matrix effect and recovery rate experiment (n=6)

[0155]

[0156] It can be seen from Table 8 that the matrix effects of the five compounds are between 78.55% and 114.11%, and the extraction recoveries are between 78.95% and 109.17%, indicating that the extraction method is stable, not affected by the matrix, and basically meets the requirements of the biological sample analysis method.

[0157] 2.5 Pharmacokinetic experimental results of quantitative detection of blood components

[0158] After the rats were intragastrically administered with Ruyi Zhenbao Pills, the average drug-time curves of the five compounds in the rats were as follows: Fig.23 The blood drug concentration data were calculated using DAS2.1.1 software, and the pharmacokinetic characteristics of the drug were characterized by parameters such as drug half-life (T1 / 2), peak concentration (Cmax), time to peak (Tmax) and area under the time-concentration curve (AUC). The results are shown in Table 9.

[0159] Table 9 Kinetic parameters of 5 compounds (Mean±SD, n=6)

[0160] Pharmacokinetic parameters Apigenin Glycyrrhizic acid Apigenin-7-O-glucuronide gallic acid Licorice glycoside <![CDATA[C max / ng·L -1 ]]> 75.04±3.95 18.21±0.87 2032.70±59.68 24.23±0.71 10.68±1.18 <![CDATA[T max / h]]> 8 0.25 0.08 0.25 0.5 <![CDATA[AUC 0-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 <![CDATA[AUC 0-∞ / 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 <![CDATA[T 1 / 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

[0161] pass Fig.23 As shown in Table 9, each compound can be detected in the plasma of rats at each time point, and there is a complete in vivo process. In addition, it can be seen that the Tmax of apigenin-7-O-glucuronide is relatively small, the absorption speed is fast, and it can quickly reach the peak after entering the blood; apigenin is relatively slowly absorbed; the MRT of the five components is greater than 1.87h, and the highest can reach 13.14h.

[0162] 2.6 Methodological study on quantitative detection of brain components

[0163] 2.6.1 Specificity inspection

[0164] (1) Preparation of sample test solution

[0165] Sample 1: Add 150 μL of acetonitrile to 50 μL of blank rat cerebrospinal fluid, vortex mix for 3 min, let stand for 5 min, centrifuge at 13500 r / min for 10 min at 4°C, and take the supernatant as the blank cerebrospinal fluid sample test solution.

[0166] Sample 2: Prepare the drug-containing cerebrospinal fluid sample test solution at a concentration of 100 ng / mL mixed reference solution according to the preparation method in 2.3.4.

[0167] Sample 3: Obtain the cerebrospinal fluid sample for testing according to the method described in 2.3.5.

[0168] (2) Detection and analysis

[0169] The above samples 1, 2 and 3 were subjected to UHPLC-MS / MS analysis according to the chromatographic conditions and mass spectrometry conditions of 2.1, respectively, to obtain the analysis results of blank cerebrospinal fluid test solution, drug-containing cerebrospinal fluid sample test solution and drug-administered cerebrospinal fluid sample test solution, see Fig.24 In the figure, A, B, and C represent the extracted ion chromatograms of sample 1, sample 2, and sample 3, respectively.

[0170] The results showed that the five test compounds and internal standard compounds were not interfered by endogenous substances, and the method had good specificity and was suitable for the determination of the contents of the five components.

[0171] 2.6.2 Linear investigation

[0172] Accurately measure 50 μL of rat blank cerebrospinal fluid, add 12.5 μL of mixed reference solution, 2.5 μL of internal standard solution and 135 μL of acetonitrile respectively, precipitate protein, vortex centrifuge, and prepare a series of matrix standard curve samples with the concentration of mixed reference solution as the variable. UHPLC-MS / MS analysis was performed according to the chromatographic conditions and mass spectrometry conditions of 2.1 respectively. The mass concentration of each component (ng / mL) was used as the abscissa (X), and the ratio of the corresponding sample peak area to the internal standard peak area was used as the ordinate (Y). The weighted least squares method was used for regression operation with a weight of 1 / X2. The standard curve was drawn and the linear regression equation of each component was calculated. The results are shown in Table 10.

[0173] Table 10 Regression equations and linear ranges of five compounds in cerebrospinal fluid

[0174]

[0175]

[0176] It can be seen from Table 10 that each compound has a good linear relationship within its corresponding linear range.

[0177] 2.6.3 Precision and accuracy examination

[0178] The blank cerebrospinal fluid base, medium and high quality control sample solutions were prepared according to the method in 2.3.4, and UHPLC-MS / MS analysis was performed according to the chromatographic conditions and mass spectrometry conditions in 2.1. The sample was injected 6 times continuously on the same day to calculate the intra-day precision; the measurement was continued for 3 days to calculate the inter-day precision. The results are shown in Table 11.

[0179] Table 11 Intra-day and inter-day precision and accuracy experiments

[0180]

[0181] As shown in Table 11, the RSDs of intra-day and inter-day precision and accuracy of the five compounds were all less than 12.85%, indicating that the method was stable and feasible.

[0182] 2.6.4 Stability study

[0183] According to the method in 2.3.4, the bottom, middle and high quality control sample solutions of blank cerebrospinal fluid were prepared and placed at room temperature for 12 hours to investigate the short-term stability of the samples; freeze-thaw cycles were performed at -80°C for 3 times to investigate the freeze-thaw stability of the samples; and refrigerated at -80°C for 4 weeks to investigate the long-term stability of the samples. The calculation results were expressed as relative standard deviation (RSD), and the results are shown in Table 12.

[0184] Table 12 Stability test (n=6)

[0185]

[0186]

[0187] As shown in Table 12, the stability RSDs of the five compounds were all less than 10.52%, indicating that the method was stable and feasible.

[0188] 2.6.5 Investigation of matrix effect and recovery rate

[0189] The blank plasma in 2.4.5 was replaced with cerebrospinal fluid, and UHPLC-MS / MS analysis was performed using the same preparation and detection methods. The ratio of the component peak area to the internal standard peak area (A) was recorded. The results are shown in Table 13.

[0190] Table 13 Matrix effect and recovery rate experiment (n=6)

[0191]

[0192] It can be seen from Table 13 that the matrix effects of the five compounds are between 74.73% and 105.76%, and the extraction recoveries are between 69.40% and 110.84%, indicating that the extraction method is stable, not affected by the matrix, and basically meets the requirements of the biological sample analysis method.

[0193] 2.7 Pharmacokinetic results of quantitative detection of brain-entering components

[0194] After the rats were intragastrically administered with Ruyi Zhenbao Pills, the average drug-time curves of the four compounds in the rats were as follows: Fig.25 The pharmacokinetic characteristics of the drug were characterized by parameters such as drug half-life (T1 / 2), peak concentration (Cmax), time to peak (Tmax) and area under the time-concentration curve (AUC), as shown in Table 14.

[0195] Table 7 Pharmacokinetic parameters of 4 compounds (Mean±SD, n=6)

[0196] Pharmacokinetic parameters Apigenin Glycyrrhizic acid Apigenin-7-O-glucuronide gallic acid <![CDATA[C max / ng·L -1 ]]> 3.99±0.09 6.08±0.18 12.49±0.51 29.61±0.81 <![CDATA[T max / h]]> 2 2 0.75 0.08 <![CDATA[AUC 0-t / ng·L -1 ·h -1 ]]> 12.89±0.70 62.25±3.32 9.58±0.20 248.52±12.41 <![CDATA[AUC 0-∞ / ng·L -1 ·h -1 ]]> 13.45±0.91 359.96±79.30 9.63±0.21 504.64±4.91 <![CDATA[T 1 / 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

[0197] pass Fig.23 As shown in Table 9, only four components, apigenin, glycyrrhizic acid, gallic acid and apigenin-7-O-glucuronide, were detected in the cerebrospinal fluid, while liquiritin was not detected.

[0198] Among them, the Tmax of gallic acid is relatively small, and its absorption speed is fast, reaching the peak quickly after entering the brain; apigenin and glycyrrhizic acid are absorbed relatively slowly; the MRT of the four components is greater than 6.41h, and the highest can reach 13.14h; the gallic acid content is highest at 5min; the glycyrrhizic acid content is relatively low, and its absorption speed into the brain is slow, with the maximum content at 2h, and it can be detected within 6 hours, indicating that glycyrrhizic acid stays in the cerebrospinal fluid for a long time.

[0199] However, the flavonoid component liquiritin was not detected in the cerebrospinal fluid. The reason may be that it is combined with glucuronic acid and sulfate ester bonds in plasma, which affects its penetration through the blood-brain barrier; the content of components in cerebrospinal fluid is lower than that in plasma, which is related to the barrier between blood and cerebrospinal fluid. The main function of the blood-brain barrier is to prevent unnecessary substances from entering the brain through the blood, protect brain tissue from damage by toxic substances and maintain the relative stability of the internal environment, but the existence of the blood-brain barrier is also a major obstacle to the effect of drug treatment of central nervous system diseases. Its existence prevents 95% of drugs from entering brain tissue from the blood, which is an important reason for the failure of many central nervous system drug treatments. Ruyi Zhenbao Pills are mainly used to treat nervous system diseases, so studying the content of active ingredients in cerebrospinal fluid is crucial to revealing the material basis of the efficacy of Ruyi Zhenbao Pills in treating nervous system diseases.

[0200] The above specific implementations cannot be used as limitations on the scope of protection of the present invention. For those skilled in the art, any replacement, improvement or change made to the implementations of the present invention falls within the scope of protection of the present invention. The parts not described in detail in the present invention are all known technologies to those skilled in the art.

Claims

1. A method for detecting the components of Ruyi Zhenbao Pills, characterized in that: include: Method A for qualitative detection of the blood or brain components of Ruyi Zhenbao Pills, the detection steps are as follows: A1: Collecting a biological sample from a medicated organism and processing it to obtain a test solution, wherein the biological sample is a plasma sample or a cerebrospinal fluid sample; A2: Ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry is used to detect and analyze biological samples, including: Chromatographic conditions: A C18 column was used with specifications of 2.1 mm × 150 mm, 1.7 µm; mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile, and gradient elution conditions were: 0-15 min, 3%-20% B; 15-30 min, 20%-40% B; 30-40 min, 40%-70% B; 40-45 min, 70%-95% B; column temperature was 25 °C, injection volume was set to 4 µL, and flow rate was set to 0.30 mL / min; Mass spectrometry conditions: electrospray ionization source, data acquisition in positive and negative ion modes; data acquisition range 100-1500 m / z ; fragmentation voltage 100 V; collision energy 25 eV; collision voltage difference 15 eV; A3: Use Xcalibur software to collect mass spectrometry data and infer the ingredients of Ruyi Zhenbao Pills; Method B for quantitatively detecting the components of Ruyi Zhenbao Pills that enter the blood or brain, wherein the components that enter the blood are glycyrrhizic acid, liquiritin, apigenin, apigenin-7-O-glucuronide and gallic acid, and the components that enter the brain are glycyrrhizic acid, apigenin, apigenin-7-O-glucuronide and gallic acid, and the specific detection steps are as follows: B1: Prepare the mixed reference solution of each component and the icariin internal standard solution with methanol; B2: Preparation of drug-containing sample solution: Take blank plasma or blank cerebrospinal fluid, add the corresponding mixed reference solution, internal standard solution and acetonitrile, vortex mix, let stand for 5 minutes, centrifuge at 13500 r / min for 10 minutes at 4 ℃, and take the supernatant for sampling and analysis; Preparation of drug sample solution: Take the treated plasma sample or cerebrospinal fluid sample, add the internal standard solution and acetonitrile, and process and inject according to the above method; B3: The sample solution is tested and analyzed using UHPLC-MS / MS technology, including: Chromatographic conditions: C18 chromatographic column, with specifications of 2.1mm×150mm, 1.7µm; mobile phase A was 0.1% formic acid aqueous solution, 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; column temperature 40℃, injection volume set to 2µL, flow rate set to 0.30mL / min; Mass spectrometry conditions: electrospray ionization ion source in negative ion mode, capillary voltage of 2000 V, cone voltage of 30 V, ion source voltage of 30 V, ion source temperature of 150 °C, cone gas flow rate of 150 L / h, desolvation gas temperature of 450 °C, desolvation gas flow rate of 1000 L / h, and monitoring mode of dynamic multiple reaction monitoring mode.

2. The method for detecting the in vivo components of Ruyi Zhenbao Pills according to claim 1, characterized in that: In A1, the plasma sample collection method is as follows: after a period of administration, the abdominal aorta blood of the organism is collected, and the blood is allowed to stand for 2 hours. After the serum is separated, the blood is centrifuged at 3000r / min for 10 minutes, and the supernatant is frozen at -80℃ for later use; The method for collecting cerebrospinal fluid samples is as follows: after a period of administration, puncture the cerebellomedullary cistern through the foramen magnum through a microinfusion needle, extract cerebrospinal fluid into a precooled centrifuge tube, centrifuge at 4000r / min for 10min at 4°C, and store the supernatant at -80°C for later use.

3. The method for detecting the in vivo components of Ruyi Zhenbao Pills according to claim 2, characterized in that: The plasma sample was processed as follows: the plasma sample was thawed in a 4°C refrigerator, vortexed for 3 min, allowed to stand for 5 min, centrifuged at 13500 r / min for 10 min at 4°C, and the supernatant was taken to obtain the test solution; Processing method of cerebrospinal fluid samples: After thawing in a 4 ℃ refrigerator, the cerebrospinal fluid sample was vortexed for 3 min, allowed to stand for 5 min, centrifuged at 13500r / min for 10 min at 4 ℃, and the supernatant was taken to obtain the test solution.

4. The method for detecting the in vivo components of Ruyi Zhenbao Pills according to claim 1, characterized in that: In B1, the concentration of each component in the mixed reference solution is 1.5875-200 ng / mL; the concentration of the internal standard solution is 100 ng / mL.

5. The method for detecting the in vivo components of Ruyi Zhenbao Pills according to claim 1, characterized in that: In B2, the preparation method of the drug-containing sample solution is as follows: take 50 μL of blank plasma or blank cerebrospinal fluid, add the corresponding 12.5 μL of mixed reference solution, 2.5 μL of internal standard solution and 135 μL of acetonitrile, vortex mix, let stand for 5 minutes, centrifuge at 13500 r / min for 10 minutes at 4°C, and take the supernatant for sampling and analysis; the preparation method of the drug-administered sample solution is as follows: thaw the plasma sample or cerebrospinal fluid sample in a 4°C refrigerator, vortex for 3 minutes, accurately pipette 50 μL, add 2.5 μL of internal standard solution and 147.5 μL of cold acetonitrile, vortex mix for 3 minutes, let stand for 5 minutes, centrifuge at 13500 r / min for 10 minutes at 4°C, and take the supernatant for sampling and analysis.

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