A pharmacokinetic analysis method of tongwei hemorrhoid liquid medicine

Abstract

The application relates to the technical field of pharmaceutical analysis, and discloses a pharmacokinetic analysis method of Tongwei Xiaozheng medicinal liquid, which adopts Q-Orbitrap high-resolution liquid chromatography-mass spectrometry to analyze the concentration of Jasmonic acid in blood components of the Tongwei Xiaozheng medicinal liquid in rat plasma, establishes a plasma concentration determination method for the blood original component Jasmonic acid, carries out methodological verification such as specificity, precision and accuracy, and then calculates pharmacokinetic parameters. In the Tongwei Xiaozheng medicinal liquid, 92 blood components are identified, 17 of which are original components and 75 of which are metabolites, the application firstly determines that Jasmonic acid is one of the blood original components of the Tongwei Xiaozheng medicinal liquid, and reveals the pharmacokinetic characteristics of the Jasmonic acid in normal and model rats. The method has strong specificity, high sensitivity and good reproducibility, and can provide a scientific basis for the research on the pharmacodynamic material basis of the Tongwei Xiaozheng medicinal liquid, the establishment of quality control standards and the clinical rational use of drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a pharmacokinetic analysis method for a micro-hemorrhoid-relieving liquid. Background Technology

[0002] Tongwei Hemorrhoid-Eliminating Solution is developed based on a traditional folk remedy from Guangxi, closely combined with modern TCM theory. It has been registered with medical institutions in Guangxi Zhuang Autonomous Region for the application of traditional techniques in preparing TCM and ethnic minority medicine preparations and is currently in clinical use. This preparation consists of four TCM herbs—*Gynostemma pentaphyllum*, *Hedyotis diffusa*, *Euphorbia humifusa*, and *Sanguisorba officinalis*—combined in a prescribed ratio. *Gynostemma pentaphyllum* is the principal herb, *Hedyotis diffusa* and *Euphorbia humifusa* are the assistant herbs, and *Sanguisorba officinalis* serves as an adjuvant herb. All components work synergistically to exert their therapeutic effects.

[0003] Ground peach blossom, sweet and pungent in taste, cool in nature, has the functions of dispelling wind and dampness, promoting blood circulation and reducing swelling, clearing heat and detoxifying. Ground peach blossom is particularly good at treating sores and ulcers caused by wind, dampness, and heat, and can dispel wind, clear heat, break up accumulations, disperse toxins, and moisten the intestines. Whether taken internally or applied externally, it has a good therapeutic effect on carbuncles and boils. This formula uses it for its functions of clearing heat, detoxifying, and dispelling wind. It can clear damp heat in the stomach and intestines, reduce swelling and dissipate nodules, moisten the intestines and promote bowel movements, and at the same time has the effect of dispelling wind. It can alleviate various symptoms and is the chief herb in the formula.

[0004] *Houttuynia cordata*, bitter and sour in taste, and slightly cold in nature, has the functions of promoting blood circulation, removing blood stasis, promoting urination, clearing heat, and reducing swelling. It is effective for injuries from falls and blows, joint swelling and pain, fever due to external pathogens, boils, and carbuncles. This formula primarily utilizes its effects of clearing heat, reducing swelling, and promoting blood circulation. Because *Houttuynia cordata* also strengthens muscles and bones, it can alleviate symptoms such as anal swelling and pain, and protruding hemorrhoids caused by insufficient vital energy and weak qi. When used with *Gynostemma pentaphyllum*, it enhances the effects of clearing heat and reducing swelling, improving symptoms such as anal swelling and pain, making it a key herb in the formula.

[0005] Euphorbia humifusa, with a pungent taste and neutral properties, has the functions of clearing heat and detoxifying, cooling blood and stopping bleeding, and promoting diuresis and relieving jaundice. It is particularly effective for treating blood heat and bleeding. Hemorrhoid patients often experience bleeding during bowel movements due to damaged blood vessels, and Euphorbia humifusa is especially effective in treating bleeding caused by the accumulation of heat. Euphorbia humifusa can cool the blood and stop bleeding, improving bleeding symptoms, while also having the effects of clearing heat and removing dampness. Together with Prunus persica and Euphorbia pekinensis, it works to clear heat, reduce swelling, and stop bleeding, making it a key herb in the formula.

[0006] Sanguisorba officinalis, with a bitter, sour, and astringent taste, and slightly cold in nature, has the functions of cooling the blood and stopping bleeding, detoxifying, and astringing sores. It is used for hematochezia, hemorrhoidal bleeding, bloody dysentery, metrorrhagia, burns, carbuncles, and boils. Sanguisorba officinalis can assist Euphorbia humifusa in its hemostatic function, enhancing its blood-cooling and hemostatic effects. It has a good therapeutic effect on hematochezia or bleeding during defecation caused by hemorrhoids, serving as an adjuvant in the formula. Simultaneously, Sanguisorba officinalis enters the large intestine meridian, guiding other herbs directly to the lesion, working together to expel pathogens, thus also serving as a guiding herb in the formula.

[0007] This formula is designed for hemorrhoids caused by damp-heat accumulation and qi stagnation and blood stasis. It works by clearing heat and detoxifying, promoting blood circulation and reducing swelling, and moistening the intestines to relieve constipation. Clinically, it is used for symptoms such as anal swelling, pain, itching, numbness, rectal bleeding, and prolapsed hemorrhoids. The applicant has completed pharmacodynamic studies of this preparation. Through in vitro examination of its antibacterial, anti-inflammatory, analgesic, and hemostatic effects, it has been confirmed that Tongwei Xiaozhi Liquid possesses antibacterial, anti-inflammatory, analgesic, hemostatic, and small intestinal motility-improving functions, demonstrating a good therapeutic effect on hemorrhoids. Simultaneously, toxicity studies of single-dose and repeated-dose administration of Tongwei Xiaozhi Liquid were conducted. The results showed that no toxic reactions occurred in Kunming mice after a single dose of Tongwei Xiaozhi Liquid. In SD rats, repeated administration followed by a 24-hour and 14-day withdrawal period showed no significant effects on blood routine tests, blood biochemical indicators, or organ morphology. However, the blood components of Tongwei Xiaozhi Liquid are currently unclear, the material basis of its pharmacodynamic effects remains ambiguous, and the in vivo pharmacokinetic characteristics of the drug need further investigation. Summary of the Invention

[0008] To address the above shortcomings, this invention establishes an analytical method to clearly identify the blood components of Tongwei Xiaozhi liquid. The concentration of jasmonic acid in rat plasma was determined, and this method was applied to pharmacokinetic studies in rats. This provides a scientific basis for clarifying its potential pharmacodynamic material basis, establishing quality control standards, and promoting its rational clinical application. The specific technical solution is as follows: A pharmacokinetic analysis method for a hemorrhoid-relieving liquid was developed, employing Q-Orbitrap high-resolution liquid chromatography-mass spectrometry (LC-MS / MS) to determine the concentration of jasmonic acid in plasma. The mass spectrometry conditions were as follows: ion source: electrospray ionization (ESI); scanning mode: positive and negative ion switching scan; detection mode: selected reaction monitoring (PRM); resolution: 17500; automatic gain control (AGC) target: 2e5; electrospray voltage: 3.5 kV Neg; capillary temperature: 300 ℃; collision gas: high-purity argon, purity ≥99.999%; collision energy (NCE): 32; sheath gas: nitrogen, purity ≥99.999%, 40 Arb; auxiliary gas: nitrogen, purity ≥99.999%, 350℃. The chromatographic conditions were as follows: column: Atlantis T3 100×2.1 mm, 3 µm, Waters; flow rate: 0.6 mL / min; A phase: 0.1 % formic acid aqueous solution; Phase B: acetonitrile; Column oven temperature: 35℃; Autosampler temperature: 10.0℃; Autosampler injection volume: 5.00 µL; Gradient elution program: 0~2.0 min, 95% Phase A; 2.0~3.8 min, 5% Phase A; 3.8~4.0 min, 5% Phase A; 4.0~5.0 min, 95% Phase A.

[0009] Preferably, the method further includes analyzing the components of Tongwei Xiaozhi medicine entering the bloodstream before determining the concentration of jasmonic acid in the plasma. The mass spectrometry conditions for analyzing the components of Tongwei Xiaozhi medicine entering the bloodstream are as follows: ion source: electrospray ionization source ESI; scanning mode: positive and negative ion switching scan; detection mode: full mass / dd-MS2, wherein the full mass resolution is 70000 and the dd-MS2 resolution is 17500; scan range: 100.0~1500.0 m / z; electrospray voltage: 3.2 kV Positive, Negative; capillary temperature: 300 ℃; collision gas: high-purity argon, purity ≥99.999%; collision energy NCE: 30, 40, 60; sheath gas: nitrogen, purity ≥99.999%, 40 Arb; auxiliary gas: nitrogen, purity ≥99.999%, 15 Arb, 350 ℃; Chromatographic conditions: AQ-C18, 150×2.1mm, 1.8 µm, Welch; Flow rate: 0.30mL / min; Phase A: 0.1% formic acid aqueous solution; Phase B: methanol; Column oven temperature: 35℃; Autosampler temperature: 10.0℃; Autosampler injection volume: 5.00 µL; Gradient elution program: 1~5 min, 98% Phase A; 5~10 min, 80% Phase A; 10~15 min, 50% Phase A; 15~20 min, 20% Phase A; 20~27 min, 5% Phase A; 27~28 min, 5% Phase A; 28~30 min, 98% Phase A.

[0010] Preferably, the method further includes investigating a plasma treatment method for detecting the jasmonic acid content of the blood-entering component after analyzing the blood-entering components of the Tongwei Xiaozhi medicine solution. The plasma treatment method is as follows: Take 50 µL of plasma samples collected at 1 h and 2 h, mix them thoroughly, and prepare three 100 µL mixed plasma aliquots. Add 150 µL of anhydrous methanol, 75% methanol, and 50% methanol respectively, vortex to mix for 10 min, centrifuge at 13000 rpm for 10 min, collect the supernatant, dry it under nitrogen, and redissolve the residue in 100 µL of 100% methanol. Incubate at 4 ℃ at 12000 r·min -1 Centrifuge for 10 minutes and collect the supernatant.

[0011] Preferably, the method for determining the concentration of jasmonic acid in plasma using Q-Orbitrap high-resolution liquid chromatography-mass spectrometry (LC-MS / MS) involves the following plasma preparation: 100 µL of plasma is added to 150 µL of anhydrous methanol, vortexed for 10 min, centrifuged at 10000-15000 rpm for 5-15 min, the supernatant is collected, dried, and the residue is reconstituted with 100 µL of 100% methanol and centrifuged at 12000 r·min at 4 °C. -1 Centrifuge for 10 minutes and collect the supernatant.

[0012] Preferably, the method for determining the concentration of jasmonic acid in plasma using Q-Orbitrap high-resolution liquid chromatography-mass spectrometry includes the following preparation method for the reference solution: accurately weigh an appropriate amount of jasmonic acid reference standard, dissolve the jasmonic acid reference standard in 100% methanol to obtain a 2 mg / mL reference standard stock solution, and then take an appropriate amount of the 2 mg / mL reference standard stock solution and dilute it with 100% methanol to prepare a series of standard solutions with concentrations of 50, 10, 2, 0.5, and 0.1 ng / mL.

[0013] Preferably, the concentration of jasmonic acid in plasma is determined using Q-Orbitrap high-resolution liquid chromatography-mass spectrometry, wherein the internal standard solution is prepared by taking an appropriate amount of ibuprofen reference standard and adding 100% methanol to prepare an ibuprofen internal standard solution of 2.015 µg / L.

[0014] Preferably, the concentration of jasmonic acid in plasma is determined using Q-Orbitrap high-resolution liquid chromatography-mass spectrometry. The plasma collection method is as follows: 1.0 mL of blood is collected from the venous plexus of the rat fundus and placed in a centrifuge tube containing sodium heparin. The collected sample is allowed to stand at 4°C for 60 min, and then centrifuged at 3000 r / min at 4°C for 10 min. The supernatant is then collected and stored at -80°C.

[0015] Preferably, the rats are SPF-grade SD rats. Twenty-four SPF-grade SD rats were randomly divided into a blank control group, a single-dose Tongwei Xiaozhi liquid administration group, a multiple-dose Tongwei Xiaozhi liquid administration group, and an anal swelling model + multiple-dose Tongwei Xiaozhi liquid administration group, with six rats in each group (half male and half female). The rats were acclimatized for 7 days, fasted for 12 hours before the experiment, but allowed free access to water. Each group of rats was administered 20 mL / kg of distilled water / drug via gavage according to their body weight: rats in the single-dose Tongwei Xiaozhi liquid administration group received Tongwei Xiaozhi liquid once daily for 1 day; rats in the multiple-dose Tongwei Xiaozhi liquid administration group and the anal swelling model + multiple-dose Tongwei Xiaozhi liquid administration group received Tongwei Xiaozhi liquid once daily for 7 days; rats in the blank control group were administered the same volume of distilled water via gavage once daily for 7 days. Fasting was maintained for 12 hours before the last administration, but water access was permitted.

[0016] Preferably, in the anal swelling model + Tongwei Xiaozhi drug solution multiple administration group, 1 hour before the last administration, the rats are lightly anesthetized with ether, and a cotton ball soaked in 16µL of croton oil mixture is placed in the rat anus for 30 seconds, while being slowly rotated 5 times to establish the rat anal croton oil swelling model.

[0017] Preferably, the method further includes calculating pharmacokinetic parameters after determining the concentration of jasmonic acid in plasma, the pharmacokinetic parameters including time to peak concentration, terminal elimination half-life, peak concentration, area under the curve, and mean residence time.

[0018] Preferably, a one-compartment model and a non-intravenous injection method are used to calculate pharmacokinetic parameters.

[0019] This invention selected 24 SPF-grade SD rats and randomly divided them into a blank control group, a single-dose Tongwei Xiaozhi liquid administration group, a multiple-dose Tongwei Xiaozhi liquid administration group (4.00 g medicinal material / kg), and an anal swelling model + multiple-dose Tongwei Xiaozhi liquid administration group (4.00 g medicinal material / kg), with 6 rats in each group. Rats in the single-dose Tongwei Xiaozhi liquid administration group were given Tongwei Xiaozhi liquid once a day for only 1 day; rats in the multiple-dose Tongwei Xiaozhi liquid administration group and the anal swelling model + multiple-dose Tongwei Xiaozhi liquid administration group were given Tongwei Xiaozhi liquid once a day for 7 days; rats in the blank control group were given the same volume of distilled water by gavage once a day for 7 days. Rats were fasted for 12 hours before the last administration, but water was allowed. For the anal swelling model + multiple-dose Tongwei Xiaozhi liquid administration group, the rat anal croton oil swelling model was established 1 hour before the last administration, based on previous pharmacodynamic studies.

[0020] Plasma samples were collected at different time points from the blank control group and the Tongwei Xiaozhi group after the last administration. Q-Orbitrap high-resolution liquid chromatography-mass spectrometry was used to analyze the blood-borne original components and metabolites of the Tongwei Xiaozhi drug. The results showed that jasmonic acid was not detected in the plasma of rats in the blank control group (who did not receive the drug), while jasmonic acid was detected in both the Tongwei Xiaozhi drug and the drug-containing plasma after administration. This indicates that jasmonic acid enters the bloodstream as its original component. The original component of jasmonic acid originates from plants and is synthesized from α-linolenic acid in the chloroplast membrane. Since chloroplasts are absent in animals, they cannot synthesize jasmonic acid themselves. Therefore, it can be determined that the blood-borne original component of jasmonic acid in the Tongwei Xiaozhi drug originates from the medicinal materials in the drug.

[0021] The Q-Orbitrap high-resolution liquid chromatography-mass spectrometry method was used to investigate the methodological characteristics of jasmonic acid plasma concentration in rats. The plasma concentration of jasmonic acid in Tongwei Xiaozhi solution was measured at different blood collection time points, and the time to peak concentration (t) was calculated using DAS 2.0 software. max Terminal elimination half-life t 1 / 2 Peak concentration C max Area under the curve (AUC) during drug administration0-t AUC 0-∞ Average dwell time MRT 0-t MRT 0-∞ To evaluate the pharmacokinetic characteristics of jasmonic acid under different administration routes and with or without disease.

[0022] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention uses Q-Orbitrap high-resolution liquid chromatography-mass spectrometry to identify 92 blood-entering components in Tongwei Xiaozhi medicine solution, including 17 prototype components and 75 metabolites. For the first time, jasmonic acid has been identified as one of the blood-entering prototype components of Tongwei Xiaozhi medicine solution.

[0023] 2. This invention optimizes the plasma sample pretreatment process for jasmonic acid, the original component entering the bloodstream, and establishes a highly specific, sensitive, and reproducible determination method. Method validation results show good linearity (R0). 2 =0.9960), intra-day and inter-day precision RSD <15%, accuracy RE >85%, extraction recovery and matrix effect RSD both <15%, and RSD <15% and RE >85% under all stability conditions.

[0024] 3. This invention systematically compared the pharmacokinetic behavior of jasmonic acid by administering it under single-dose, multiple-dose, and an anal swelling model. The results showed that jasmonic acid was rapidly absorbed (reaching peak concentration after 1 hour), metabolized slowly, and had a certain cumulative effect after multiple doses. Moreover, its pharmacokinetic parameters under the model state were not significantly different from those under the normal state (P>0.05). Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is the total ion chromatogram of the Tongwei Xiaozhi medicine solution of the present invention; the upper sub-graph, presented in black, is the total ion chromatogram of the Tongwei Xiaozhi medicine solution in negative ion mode, and the lower sub-graph, presented in red, is the total ion chromatogram of the Tongwei Xiaozhi medicine solution in positive ion mode.

[0027] Figure 2 The image shows the plasma total ion chromatogram of the blank control group in this invention; the upper sub-image, presented in black, is the plasma total ion chromatogram of the blank control group in negative ion mode, and the lower sub-image, presented in red, is the plasma total ion chromatogram of the blank control group in positive ion mode.

[0028] Figure 3 This is a plasma total ion flow map of the Tongwei Xiaozhi medicine solution group after administration according to the present invention; the upper sub-graph, presented in black, is the plasma total ion flow map of the Tongwei Xiaozhi medicine solution group after administration in negative ion mode, and the lower sub-graph, presented in red, is the plasma total ion flow map of the Tongwei Xiaozhi medicine solution group after administration in positive ion mode.

[0029] Figure 4 This is the Venn difference analysis diagram of the present invention; in the diagram, the yellow part in the upper middle circle represents 500 components of Tongwei Xiaozhi liquid that did not enter the bloodstream; the red part overlapping the upper middle circle and the lower left circle represents 12 components identical to Tongwei Xiaozhi liquid present in the plasma of the blank group; the blue part overlapping the upper middle circle and the lower right circle represents 17 components identical to Tongwei Xiaozhi liquid present in the plasma of the Tongwei Xiaozhi liquid group after administration, which are the 17 prototype components of Tongwei Xiaozhi liquid that enter the bloodstream; the cyan part overlapping the three circles represents 87 components present in the plasma of Tongwei Xiaozhi liquid and the blank group. The following components were present in the plasma of the Tongwei Xiaozhi liquid group after administration: the green part in the lower left circle indicates that 44 components in the plasma of the blank group did not come from Tongwei Xiaozhi liquid and did not enter the bloodstream after administration; the purple part in the lower right circle indicates that 75 components in the plasma of the Tongwei Xiaozhi liquid group were different from those in the blank group, which are 75 metabolites that entered the bloodstream after administration; the orange part where the lower left and lower right circles overlap indicates that 131 components were present in both the blank group plasma and the Tongwei Xiaozhi liquid group plasma after administration, and these 131 components were different from those in Tongwei Xiaozhi liquid.

[0030] Figure 5 This is the jasmonic acid standard curve of the present invention.

[0031] Figure 6 The following are plasma concentration-time curves of the present invention: A: Average plasma concentration-time curve of jasmonic acid after a single dose of Tongwei Xiaozhi medicine solution; B: Average plasma concentration-time curve of jasmonic acid after multiple doses of Tongwei Xiaozhi medicine solution; C: Average plasma concentration-time curve of jasmonic acid after multiple doses of Tongwei Xiaozhi medicine solution for anal swelling.

[0032] Figure 7 The chromatograms are specific blank rat plasma chromatograms for this invention; I is the total chromatogram, II is the jasmonic acid chromatogram, and III is the ibuprofen chromatogram.

[0033] Figure 8 Chromatograms of rat plasma containing 50 ng / mL jasmonic acid and 2.015 µg / L internal standard ibuprofen solution for the specific blank rat plasma of this invention are shown below; I is the total chromatogram, II is the jasmonic acid chromatogram, and III is the ibuprofen chromatogram.

[0034] Figure 9The chromatograms of 50µL plasma samples taken from rats 1h and 2h after oral administration of 4.00g of medicinal material / kg of Tongwei Xiaozhi medicine solution, which are specific to this invention, are shown below. I is the total chromatogram, II is the jasmonic acid chromatogram, and III is the ibuprofen chromatogram. Detailed Implementation

[0035] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0036] Example 1 S1. Preparation of Tongwei Hemorrhoid-Clearing Solution The applicant's previous research found that Tongwei Xiaozhi Liquid was significantly effective at a dose of 4.00 g raw drug / kg (equivalent to 6 times the human dose for rats based on body weight). Therefore, this study used 4.00 g raw drug / kg as the dosage and prepared Tongwei Xiaozhi Ointment with distilled water to make 0.20 g raw drug / mL / kg.

[0037] S2. Preparation of the test solution for Tongwei Xiaozhi medicine solution Take 100 µL of Tongwei Xiaozhi medicine solution, add 400 µL of 100% methanol solution, vortex mix for 10 min, centrifuge at 13000 rpm for 10 min, and take the supernatant for analysis.

[0038] S3. Preparation of jasmonic acid reference solution Accurately weigh an appropriate amount of jasmonic acid reference standard, dissolve it in 100% methanol to prepare a jasmonic acid reference standard stock solution, yielding a 2 mg / mL stock solution for later use. Take an appropriate amount of the stock solution and dilute it with 100% methanol to prepare standard curve solutions of 50, 10, 2, 0.5, and 0.1 ng / mL.

[0039] S4. Preparation of internal standard solution Take an appropriate amount of ibuprofen reference standard and add 100% methanol to prepare an ibuprofen internal standard solution of 2.015 µg / L.

[0040] S5. Preparation of the croton oil mixture as a molding agent Distilled water, pyridine, ether, and 6% croton oil ether solution were prepared in a volume ratio of 1:4:5:10.

[0041] S6. Grouping and Dosing Twenty-four SPF-grade SD rats were randomly divided into four groups: a blank control group, a single-dose Tongwei Xiaozhi liquid administration group, a multiple-dose Tongwei Xiaozhi liquid administration group, and an anal swelling model + multiple-dose Tongwei Xiaozhi liquid administration group. Each group consisted of six rats (half male and half female). Rats were acclimatized for 7 days and fasted for 12 hours before the experiment, but water was allowed. Each group of rats was administered 20 mL / kg of distilled water / drug via gavage. The single-dose Tongwei Xiaozhi liquid administration group received Tongwei Xiaozhi liquid once daily for one day only. The multiple-dose Tongwei Xiaozhi liquid administration group and the anal swelling model + multiple-dose Tongwei Xiaozhi liquid administration group received Tongwei Xiaozhi liquid once daily for 7 days. The blank control group received the same volume of distilled water via gavage once daily for 7 days. Before the last administration, the rats were fasted but allowed to drink water for 12 hours. For the anal swelling model + Tongwei Xiaozhi medicine multiple administration group, referring to the previous pharmacodynamic study, 1 hour before the last administration, the rats were lightly anesthetized with ether, and a cotton ball (4 mm in diameter) soaked in 16 uL of croton oil mixture was inserted into the rat anus for 30 seconds and slowly rotated 5 times to establish the rat anal croton oil swelling model.

[0042] S7. Sample Collection and Processing (1) Collection of plasma samples At 0.083, 0.5, 1, 2, 4, 12, and 24 h after the last administration of the drug to rats in each group, 1.0 mL of blood was collected from the fundus venous plexus of each rat and placed in a centrifuge tube containing heparin sodium. The collected blood samples were incubated at 4°C for 60 min and then centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was then collected and stored at -80°C. The method for collecting blank plasma was the same as that for collecting drug-containing plasma.

[0043] (2) Pretreatment of plasma samples Take 50 µL of plasma samples collected at 1 h and 2 h, mix well, add 150 µL of 100% methanol, vortex for 10 min, centrifuge at 13000 rpm for 10 min, collect the supernatant, dry under nitrogen, reconstitute the residue with 100 µL of 100% methanol, and incubate at 4 °C at 12000 r·min. -1 Centrifuge for 10 min, collect the supernatant, and use it as a sample for plasma component analysis.

[0044] S8. Analysis of Tongwei Xiaozhi Liquid and its Blood Components (1) Mass spectrometry conditions Ion source: Electrospray ionization source (ESI); Scanning mode: Positive and negative ion switching scan; Detection mode: Full mass / dd-MS2, where the Full mass resolution is 70,000 and the dd-MS2 resolution is 17,500; Scan range: 100.0–1500.0 m / z; Spray voltage: 3.2 kV (Positive, Negative); Capillary temperature: 300 ℃; Collision gas: High-purity argon, purity ≥99.999%; Collision energy (NCE): 30, 40, 60; Sheath gas: Nitrogen, purity ≥99.999%, 40 Arb; Auxiliary gas: Nitrogen, purity ≥99.999%, 15 Arb, 350℃; Data acquisition time: 30.0 min.

[0045] (2) Chromatographic conditions AQ-C18, 150×2.1mm, 1.8 µm, Welch; flow rate: 0.30 mL / min; Phase A: 0.1% formic acid / water solution; Phase B: methanol; oven temperature: 35℃; autosampler temperature: 10.0℃; autosampler injection volume: 5.00 µL. See Table 1 for chromatographic gradient details.

[0046] (3) Data Analysis Sample data were acquired using Q-Orbitrap high-resolution liquid chromatography-mass spectrometry. The obtained data were initially processed using Compound Discoverer version 3.3 (Thermo Fisher, USA) and then searched and compared using the mzCloud database.

[0047] The results showed that jasmonic acid was not detected in the plasma of rats in the blank control group (no drug administration), while it was detected in both Tongwei Xiaozhi liquid and drug-containing plasma after administration. This indicates that jasmonic acid enters the bloodstream as its original component. The original component of jasmonic acid originates from plants and is synthesized from α-linolenic acid in chloroplast membranes. Since animals do not have chloroplasts, they cannot synthesize jasmonic acid themselves. Therefore, it can be confirmed that the original component of jasmonic acid entering the bloodstream in Tongwei Xiaozhi liquid originates from the medicinal materials in the liquid.

[0048] The Tongwei Xiaozhi liquid sample successfully matched 942 compounds in the mzCloud database. Among them, 528 compounds had a comprehensive score greater than 60 under the mzCloud best match screening condition. The blank control group plasma sample matched 343 compounds in the mzCloud database. Under the mzCloud best match screening condition, 230 compounds had a comprehensive score greater than 60. The Tongwei Xiaozhi liquid group plasma sample, after administration, matched 392 compounds in mzCloud. Under the mzCloud best match screening condition, 266 compounds had a comprehensive score greater than 60. See details for the results. Figure 1 , Figure 2 and Figure 3 .

[0049] Using blank plasma as a control, a binary comparison was performed with drug-containing plasma. This revealed that the unique components of drug-containing plasma were the blood-entering components. Using blank solvent as a control, a binary comparison was performed with Tongwei Xiaozhi liquid to identify the unique components of the medicinal material. This was then compared with the blood-entering components of Tongwei Xiaozhi liquid. Components identical to those in Tongwei Xiaozhi liquid were the original components; all other components were metabolites. Based on key information such as retention time, precise molecular weight, and ion fragmentation, matching operations were performed with the platform's self-built library. Results were analyzed with an error within 6 mDa and a response value greater than 10,000. Validation was performed by comparing with control standards and searching databases such as PubChem and HMDB, as well as relevant literature. After removing duplicate values, a total of 92 blood-entering components were identified in Tongwei Xiaozhi liquid, including 17 original components and 75 metabolites. See details for further information. Figure 4 Tables 2 and 3. Figure 4In the diagram, the yellow portion in the upper center circle represents the 500 non-blood-entering components of Tongwei Xiaozhi Liquid; the red portion overlapping the upper center and lower left circles represents the 12 components identical to Tongwei Xiaozhi Liquid present in the plasma of the blank control group; the blue portion overlapping the upper center and lower right circles represents the 17 components identical to Tongwei Xiaozhi Liquid present in the plasma of the Tongwei Xiaozhi Liquid group after administration, which are the 17 prototype components of Tongwei Xiaozhi Liquid entering the bloodstream; the cyan portion overlapping the three circles represents the presence of 87 components in the blood plasma of Tongwei Xiaozhi Liquid, blank control group, and Tongwei Xiaozhi Liquid group. All components were present in the plasma after administration. The bluish-green portion in the lower left circle indicates that 44 components in the plasma of the blank group were not from Tongwei Xiaozhi liquid and did not enter the bloodstream after administration. The purple portion in the lower right circle indicates that 75 components in the plasma of the Tongwei Xiaozhi liquid group were different from those in the blank group, which are 75 metabolites that entered the bloodstream after administration. The orange portion where the lower left and lower right circles overlap indicates that the same 131 components were present in the plasma of both the blank group and the Tongwei Xiaozhi liquid group after administration, and these 131 components are different from those in the Tongwei Xiaozhi liquid.

[0050]

[0051] .

[0052] Example 2 S1. Investigation on plasma sample processing methods for detecting jasmonic acid content in Tongwei Xiaozhi liquid upon entering the bloodstream. Take 50 µL of plasma samples collected at 1 h and 2 h, mix well, and prepare three mixed plasma aliquots. Add 150 µL of anhydrous methanol, 75% methanol, and 50% methanol respectively, vortex to mix for 10 min, centrifuge at 13000 rpm for 10 min, collect the supernatant, dry under nitrogen, and reconstitute the residue with 100 µL of 100% methanol. Incubate at 4 ℃ at 12000 r·min -1 Centrifuge for 10 min, collect the supernatant, and use it as a sample for plasma component analysis.

[0053] The results showed that the jasmonic acid content in plasma extracted with anhydrous methanol was the highest, as detailed in Table 4.

[0054]

[0055] 100 µL of plasma samples were taken from each group at each time point, and jasmonic acid was extracted from the plasma using the method described above for extracting jasmonic acid with anhydrous methanol to prepare samples for pharmacokinetic analysis.

[0056] S2. Pharmacokinetic Analysis of Tongwei Hemorrhoid Relief Solution (1) Mass spectrometry conditions Ion source: Electrospray ionization source (ESI); Scanning mode: Positive and negative ion switching scan; Detection mode: Selective reaction monitoring (PRM); Resolution: 17500; Automatic gain control (AGC) target: 2e5; Spray voltage: 3.5kV Neg; Capillary temperature: 300 ℃; Collision gas: High-purity argon, purity ≥99.999%; Collision energy (NCE): 32; Sheath gas: Nitrogen, purity ≥99.999%, 40 Arb; Aux gas heater temp: Nitrogen, purity ≥99.999%, 350℃; Data acquisition time: 5 min.

[0057] (2) Chromatographic conditions Chromatographic column: Atlantis T3 100×2.1mm, 3 µm, Waters; flow rate: 0.6 mL / min; Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile; needle washing solution: methanol; column oven temperature: 35℃; autosampler temperature: 10.0℃; autosampler injection volume: 5.00 µL. Chromatographic gradient details are shown in Table 5.

[0058]

[0059] (3) Data Analysis The chromatograms of the indicators were collected and integrated using Xcalibur 4.0 (Thermo Fisher), and linear regression analysis was performed using weighted coefficients.

[0060] S3. Methodological Examination (1) Specificity examination Take 100 µL of blank plasma, 100 µL of blank plasma + 50 µL of 2.015 µg / L internal standard solution + 50 µL of 50 ng / mL jasmonic acid reference solution, and 50 µL of plasma samples taken 1 h and 2 h after oral administration of 4.00 g medicinal material / kg Tongwei Xiaozhi medicine solution. Mix the plasma samples and process them according to the method in step S1 of Example 2. Then, analyze the samples according to the detection conditions in steps (1) and (2) of step S2 of Example 2.

[0061] (2) Examination of linear relationships 100 µL of rat blank plasma was precisely measured and added to 10 µL of jasmonic acid reference solution of a series of concentrations in step S3 of Example 1. The solution was then processed and analyzed according to the method in step S2 of Example 2.

[0062] (3) Precision and accuracy assessment Take 100 µL of blank rat plasma and prepare three quality control samples with low, medium and high concentrations. Each concentration of sample is used in parallel with 6 replicates. The samples are processed and analyzed according to the method in step S2 of Example 2. Each sample is injected 3 times and continuously for 3 days.

[0063] (4) Investigation of extraction recovery rate and matrix effect Take 100 µL of blank rat plasma and prepare three quality control samples of low, medium and high concentrations. Each concentration of sample is prepared in parallel with six replicates. Extract the samples according to the processing method in step S7 of Example 1. Take another 100 µL of blank plasma and, except without adding jasmonic acid reference solution, operate in the same way until dried under nitrogen. The resulting blank matrix residue is reconstituted with low, medium and high concentrations of jasmonic acid reference solution, respectively. Take the corresponding low, medium and high concentrations of jasmonic acid reference solution and determine the peak area according to the detection conditions in step S2 of Example 2.

[0064] (5) Stability test Take 100 µL of blank rat plasma and prepare quality control samples of low, medium and high concentrations. Six samples of each concentration are paralleled. The samples are processed by three freeze-thaw cycles, 36 h at room temperature and 30 d frozen at -20 ℃. The samples are then processed and analyzed according to step S2 of Example 2.

[0065] (6) Processing of pharmacokinetic parameters Plasma samples from rats in each group were injected and analyzed at each time point according to the method in step S2 of Example 2. The blood drug concentration data were imported into DAS 2.0 software. A one-compartment model and a non-intravenous injection method were selected for statistical processing of pharmacokinetic parameters, and the time to peak concentration (t) was investigated. max Terminal elimination half-life t 1 / 2 Peak concentration C max Area under the curve (AUC) during drug administration 0-t AUC 0-∞ Average dwell time MRT 0-t MRT 0-∞ There are 7 parameters in total.

[0066] S4. Pharmacokinetic Methodology Investigation (1) Specificity examination Figures 7-9 The results showed that endogenous substances in plasma did not interfere with the determination of jasmonic acid and internal standard ibuprofen, indicating that the method has good specificity.

[0067] (2) Examination of linear relationships A standard curve was obtained by using the peak area of ​​jasmonic acid in plasma samples as the ordinate and the concentration of jasmonic acid as the abscissa, and regression was performed with weighted coefficients. The regression equation was then calculated, and R0 was obtained.2 =0.9960, indicating that jasmonic acid exhibits good linearity across the concentration range. See details for further results. Figure 5 The standard curve of jasmonic acid.

[0068] (3) Precision and accuracy assessment Intraday precision and interday precision were calculated, and accuracy was also calculated. The results showed that the relative standard deviation (RSD) of the method was <15%, and the accuracy (RE) was >85%, indicating that the method has good precision and accuracy.

[0069] (4) Investigation of extraction recovery rate and matrix effect Calculations showed that the extraction recovery rate and matrix effect RSD of each component at different mass concentrations were all <15%, indicating that the plasma samples extracted by this method can ignore the interference of endogenous substances.

[0070] (5) Stability test The results showed that the precision RSD values ​​of the index components under all conditions were <15%, and the precision RE values ​​were >85%, indicating that the determination results of each index component under the experimental conditions were stable and reliable.

[0071] S5. Pharmacokinetic Parameter Analysis (1) The mean plasma concentration time in rats in each treatment group and in all rats The blood drug concentration was calculated based on the standard curve equation, and the results were all within the standard curve range. A blood drug concentration-time curve was obtained by plotting time (X) on the x-axis and the blood drug concentration of jasmonic acid (Y) on the y-axis. Figure 6 As can be seen from the blood concentration-time curves, jasmonic acid was absorbed rapidly in rats in all treatment groups, reaching peak concentration after 1 hour. The metabolism was slow, and it was still detectable in vivo after 24 hours.

[0072] (2) Main pharmacokinetic parameters of jasmonic acid Based on the measured concentrations and corresponding collection times of each sample, the main pharmacokinetic parameters of jasmonic acid in rats of each treatment group were calculated using DAS2.0 according to the one-compartment model analysis method. 24 hours after a single administration of Tongwei Xiaozhi solution, jasmonic acid was not completely metabolized in vivo, resulting in a relatively long average residence time. This led to a certain accumulation of jasmonic acid in the body after multiple administrations. At the same blood collection time point, there was no statistically significant difference in the pharmacokinetic parameters of jasmonic acid in rats in the anal swelling model + Tongwei Xiaozhi solution multiple administration group compared to the Tongwei Xiaozhi solution multiple administration group (P>0.05). See Table 6 for detailed results.

[0073] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A pharmacokinetic analysis method for a hemorrhoid-relieving liquid, characterized in that, The concentration of jasmonic acid in plasma was determined using Q-Orbitrap high-resolution liquid chromatography-mass spectrometry. The mass spectrometry conditions were as follows: ion source: electrospray ionization source; scanning mode: positive and negative ion switching scan; detection mode: selected reaction monitoring. Resolution: 17500; Automatic gain control target: 2e5; Electrospray voltage: 3.5kV; Capillary temperature: 300 ℃; Collision gas: High-purity argon; Collision energy NCE: 32; Sheath gas: Nitrogen, 40 Arb; Chromatographic conditions: Column: Atlantis T3 100×2.1mm, 3 µm, Waters; Flow rate: 0.6 mL / min; Phase A: 0.1% formic acid aqueous solution; Phase B: Acetonitrile; Column oven temperature: 35℃; Injection volume: 5.00 µL; Gradient elution program: 0~2.0 min, 95% Phase A; 2.0~3.8 min, 5% phase A; 3.8~4.0 min, 5% phase A; 4.0~5.0 min, 95% phase A.

2. The pharmacokinetic analysis method according to claim 1, characterized in that, The procedure also includes analyzing the components of Tongwei Xiaozhi medication entering the bloodstream before determining the concentration of jasmonic acid in the plasma. The mass spectrometry conditions for analyzing these components are as follows: ion source: electrospray ionization source; scanning mode: positive and negative ion switching scan; detection mode: Full mass / dd-MS2, where the Full mass resolution is 70000 and the dd-MS2 resolution is 17500; scanning range: 100.0–1500.0 m / z; electrospray voltage: 3.2 kV; capillary temperature: 300 ℃; collision gas: high-purity argon; collision energy NCE: 30, 40, 60; sheath gas: nitrogen, 40 Arb; chromatographic conditions: AQ-C18, 150 × 2.1 mm, 1.8 µm, Welch; flow rate: 0.30 mL / min; phase A: 0.1% formic acid aqueous solution; phase B: methanol; column oven temperature: 35 ℃; injection volume: 5.00 mL / min. µL; gradient elution program: 1-5 min, 98% phase A; 5-10 min, 80% phase A; 10-15 min, 50% phase A; 15-20 min, 20% phase A; 20-27 min, 5% phase A; 27-28 min, 5% phase A; 28-30 min, 98% phase A.

3. The pharmacokinetic analysis method according to claim 1, characterized in that, The plasma treatment method is as follows: Take 100 µL of plasma and add 150 µL of anhydrous methanol, vortex mix for 10 min, centrifuge at 10000~15000 rpm for 5~15 min using a benchtop centrifuge, collect the supernatant, dry it, and redissolve the residue in 100 µL of 100% methanol, then centrifuge at 12000 r·min at 4 °C. -1 Centrifuge for 10 minutes and collect the supernatant.

4. The pharmacokinetic analysis method according to claim 1, characterized in that, The method for determining the concentration of jasmonic acid in plasma using Q-Orbitrap high-resolution liquid chromatography-mass spectrometry (LC-MS / MS) is as follows: Accurately weigh an appropriate amount of jasmonic acid reference standard, dissolve the jasmonic acid reference standard in 100% methanol to obtain a 2 mg / mL reference standard stock solution, and then take an appropriate amount of the 2 mg / mL reference standard stock solution and dilute it with 100% methanol to prepare a series of standard solutions with concentrations of 50, 10, 2, 0.5, and 0.1 ng / mL.

5. The pharmacokinetic analysis method according to claim 1, characterized in that, The concentration of jasmonic acid in plasma was determined using Q-Orbitrap high-resolution liquid chromatography-mass spectrometry. The internal standard solution was prepared by taking an appropriate amount of ibuprofen reference standard and adding 100% methanol to prepare an ibuprofen internal standard solution of 2.015 µg / L.

6. The pharmacokinetic analysis method according to claim 1, characterized in that, The plasma collection method is as follows: 1.0 mL of blood is collected from the venous plexus of the rat fundus and placed in a centrifuge tube containing heparin sodium. The collected sample is allowed to stand at 4°C for 60 min, and then centrifuged at 3000 r / min at 4°C for 10 min. The supernatant is then collected and stored at -80°C.

7. The pharmacokinetic analysis method according to claim 1, characterized in that, The method also includes calculating pharmacokinetic parameters after determining the concentration of jasmonic acid in plasma, including time to peak concentration, terminal elimination half-life, peak concentration, area under the curve, and mean residence time.

8. The pharmacokinetic analysis method according to claim 7, characterized in that, Pharmacokinetic parameters were calculated using a one-compartment model and non-intravenous injection.

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