Method for measuring multi-index content of components entering blood of Jinrong particles

Abstract

The invention discloses a multi-index content determination method for in-blood components of Jinrong particles. The method comprises the following steps: measuring an ion chromatogram by adopting a UPLC-Q-TOF-MS / MS analysis method; the chromatographic conditions are as follows: the flow rate is 0.3 to 0.5 mL / min; the column temperature is 20-30 DEG C; a mobile phase: a phase A is acetonitrile, a phase B is a 0.04-0.08 mmol / L ammonium formate aqueous solution, and the mobile phase comprises acetonitrile and the 0.04-0.08 mmol / L ammonium formate aqueous solution. According to the method, the contents of various main components after the Jinrong particles are absorbed into blood can be simultaneously measured, and the quality of the Jinrong particles can be more comprehensively controlled. The method for measuring the content of in-vivo and in-vitro components of the components is convenient and efficient, and a scientific foundation is laid for improving the overall quality standard of the Jinrong granules.

Description

Technical Field

[0001] The invention relates to a method for determining the content of drug components, in particular to a method for determining the multi-index content of blood-entering components of Jinrong granules. Background Art

[0002] Jinrong Granules is an innovative Chinese medicine for the treatment of breast hyperplasia. On April 12, 2005, it obtained the clinical approval of "Xiaopi Granules" (2005L01119) from the State Food and Drug Administration, and in December 2018, it was approved for marketing as an innovative Chinese medicine "Jinrong Granules" (National Medicine Approval No. Z20180002).

[0003] Jinrong Granules are made up of Epimedium, Cistanche, Curcuma, Salvia miltiorrhiza, Leonurus japonicus, Curcuma zedoaria, processed Polygonum multiflorum, Ligustrum lucidum, turtle shell, and oyster. It has the functions of tonifying the kidney and activating blood circulation, resolving phlegm and dispersing stagnation, and regulating Chong and Ren. It is used for breast hyperplasia, phlegm and blood stasis, and Chong and Ren disorders. Symptoms include breast pain and tenderness, chest and flank pain, depression and irritability, insomnia, fatigue, soreness of the waist and knees, pale red or cyanotic tongue or ecchymosis on the edges and tip of the tongue, white tongue coating, and stringy or slippery pulse.

[0004] Based on this, the main characteristic components detected by the present invention: icariin, baohuoside I, and magnolamine are the index components and main active ingredients specified in the pharmacopoeia of Epimedium; echinacoside is the index component and main active ingredient specified in the pharmacopoeia of Cistanche; tanshinone I, tanshinone IIA, and cryptotanshinone are the index components and main active ingredients specified in the pharmacopoeia of Salvia miltiorrhiza, and stachydrine is the index component and main active ingredient specified in the pharmacopoeia of Leonurus japonicus.

[0005] "Zhou Ruopeng, Deng Xiaojun. Determination of echinacoside and verbascoside in Jinrong Xiaopi Granules by HPLC [J]. Citation Edition: Medicine and Health, 2015, 1(4): 20-20." reported the determination of echinacoside and verbascoside in Jinrong Granules. "Zhou Ruopeng. Establishment of fingerprint spectrum of Jinrong Xiaopi Granules, 2015." reported the study of fingerprint spectrum of Jinrong Granules. In this paper, four common peaks of echinacoside, verbascoside, salvianolic acid B and icariin in Jinrong Granules were determined and the common peaks were attributed to medicinal materials. No quantitative analysis of the characteristic components representing the dose-effect relationship in the prescription was conducted. Jinrong Granules carried out the determination of icariin content in accordance with the "Standard of the State Drug Administration (YBZ00042018)". However, this single indicator quantification can no longer truly and comprehensively control the intrinsic quality of the preparation.

[0006] Therefore, in view of the current quality control problems of Jinrong Granules, it is urgent to invent an analytical method that can detect the representative components of Jinrong Granules, and on this basis establish a qualitative analysis method for the components absorbed into the blood and a method for determining the content of the main components, preliminarily clarify the material basis of the efficacy of Jinrong Granules, and provide a research method to solve the problem of the difficulty in monitoring trace components entering the blood. This has important scientific significance and practical value for improving the quality control of Jinrong Granules. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a method for determining the multi-index content of the blood-entering components of Jinrong Granules in view of the deficiencies in the existing technology, and to simultaneously determine the contents of multiple main components of Jinrong Granules after they are absorbed into the blood, so as to more comprehensively control the quality of Jinrong Granules.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0009] A method for determining the multi-index content of Jinrong granules entering blood, using a UPLC-Q-TOF-MS / MS analysis method to measure the ion current graph;

[0010] The chromatographic conditions include:

[0011] Column: C18

[0012] Flow rate: 0.3~0.5mL / min;

[0013] Column temperature: 20-30°C;

[0014] Mobile phase: Phase A is acetonitrile, phase B is 0.04-0.08 mmol / L ammonium formate aqueous solution, the mobile phase includes acetonitrile and 0.04-0.08 mmol / L ammonium formate aqueous solution;

[0015] The elution was performed using the following elution program:

[0016] 0-10min: 5%→15% mobile phase A, 95%→85% mobile phase B;

[0017] 10-15min: 15% → 35% mobile phase A, 85% → 65% mobile phase B;

[0018] 15-25 min: 35% → 65% mobile phase A, 65% → 35% mobile phase B;

[0019] 25-40min: 65%→95% mobile phase A, 35%→5% mobile phase B.

[0020] The present invention uses ultra-high performance liquid chromatography with higher resolution and shorter analysis time. When combined with mass spectrometry for analysis, information on chemical components without ultraviolet absorption can be obtained. Moreover, through the molecular feature extraction function, chemical components in drug serum that are masked by protein component ion peaks can be obtained, and the types and contents of components absorbed into the blood by Jinrong Granules can be quickly and efficiently determined.

[0021] The ingredients of Jinrong Granules are complex, including flavonoids and their glycosides, alkaloids, phenolic acids and phenylethanol glycosides. In order to achieve better separation of the components, gradient elution from large to small polarity was performed, and the total elution time was set according to the peak conditions. When the elution time reached 40 minutes, the baseline was basically smooth. The mobile phase B was an aqueous solution of ammonium formate. The first 10-15 minutes of elution were mainly phase B, and the mobile phase was highly acidic, which could preferentially separate the alkaloid compounds. The last 10-15 minutes were mainly phase A, and the acidity of the mobile phase was weakened. The acidic components of phenolic acids were detected during this time period. There are many glycoside components in the formula. Between 15-25 minutes, the mobile phase was neutral and low polarity. As the polarity of the mobile phase decreased, polysaccharide glycosides, disaccharide glycosides, and aglycone compounds eluted peaks in turn.

[0022] The purpose of adding ammonium formate salt to the aqueous phase is to adjust the pH value of the solution. Ammonium formate salt has good solubility, is fully compatible with the LC-MS method, and causes relatively little damage to the chromatographic column.

[0023] The UPLC-Q-TOF-MS / MS analysis method refers to Ultra Performance Liquid Chromatography-Quadrupole-Time of Flight-Mass Spectrometry (ULTRA PERFORMANCE LIQUID CHROMATography-Quadrupole-Time of Flight-Mass Spectrometry).

[0024] In a preferred embodiment of the present invention, the mass spectrometry detection conditions are as follows: the analysis mode is positive and negative ion mode, ESI ionization mode, and Agilent standard tuning solution ESI-L Low Concentration TuningMix (G1969-85000) is used to calibrate the accurate mass number before sample injection analysis;

[0025] Primary mass spectrometry scanning detection range: m / z 100~1700;

[0026] Solvent removal drying gas: nitrogen;

[0027] Temperature: 325℃;

[0028] Flow rate: 6.8L / min;

[0029] Sheath gas temperature: 350℃;

[0030] Capillary voltage: 4.0kv;

[0031] Fragment voltage: 150v.

[0032] In a preferred embodiment of the present invention, the UPLC-Q-TOF-MS / MS analysis method uses the Jinrong Granule drug serum sample solution as the sample solution.

[0033] In a preferred embodiment of the present invention, the method for preparing the Jinrong Granule drug serum sample solution comprises weighing Jinrong Granules to dissolve, administering the solution to rats by gavage, collecting blood, separating serum, precipitating protein, centrifuging, concentrating, re-dissolving, and filtering to obtain the solution.

[0034] In a preferred embodiment of the present invention, the dosage of the Jinrong granules is 2 to 4 times the clinical equivalent dosage.

[0035] The clinical equivalent dose is the relative drug concentration or dose that can cause an equivalent reaction. It reflects the relationship between the drug effect and the drug dose. The clinical equivalent dose is 0.8 to 1.2 g.

[0036] In a preferred embodiment of the present invention, the drug is administered to rats twice a day for 7 consecutive days.

[0037] In a preferred embodiment of the present invention, the specific method after separating the serum is: taking 2 ml of serum, precipitating protein with methanol, centrifuging, concentrating, re-dissolving, and filtering through a 0.22 μm filter membrane.

[0038] In a preferred embodiment of the present invention, the specifications of the chromatographic column are: 3.0×100 mm, 1.8 μm.

[0039] In a preferred embodiment of the present invention, in the UPLC-Q-TOF-MS / MS analysis method, the mixed reference blank serum solution includes icariin, baohuoside I, echinacoside, verbascoside, tanshinone I, tanshinone IIA, cryptotanshinone, terunguisu glycoside, magnolamine, stachydrine and salvianolic acid B.

[0040] In a preferred embodiment of the present invention, the preparation method of the mixed reference blank serum solution is to take 2-3 ml of blank serum, add 100 μl of the mixed reference solution, precipitate protein with methanol, concentrate, re-dissolve, and filter through a filter membrane;

[0041] In the mixed reference solution, the concentrations of icariin, baohuoside I, echinacoside, verbascoside, tanshinone I, tanshinone IIA, cryptotanshinone, teruncle glycoside, magnolamine, stachydrine, and salvianolic acid B are 0.4-0.5 mg / mL, 0.4-0.5 mg / mL, 0.5-0.6 mg / mL, 0.4-0.5 mg / mL, 0.4-0.5 mg / mL, 0.35-0.5 mg / mL, 0.35-0.5 mg / mL, 0.4-0.5 mg / mL, 0.4-0.5 mg / mL, 0.6-0.8 mg / mL, and 0.5-0.6 mg / mL, respectively.

[0042] In a preferred embodiment of the present invention, the method further comprises preparing a Jinrong granule sample solution: weighing 2-4 g of Jinrong granule dry extract powder, dissolving in methanol, ultrasonicating, centrifuging, filtering through a 0.22 μm microporous filter membrane, and placing in a sample injection bottle.

[0043] The specific conditions for centrifugation, concentration and ultrasonic treatment of the serum sample are:

[0044] Centrifugation conditions for protein removal: 5000 rpm, 10 min;

[0045] Ultrasonic conditions: 40-60KHz, 15min;

[0046] Reconstitution centrifugation conditions: 12000rpm; 5min.

[0047] The specific conditions for centrifugation and ultrasonic treatment of the Jinrong granule sample and the mixed reference solution are as follows:

[0048] Ultrasonic conditions: 40-60KHz, 15min;

[0049] Centrifugation conditions: 8000 rpm; 5 min.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1. The determination method provided by the present invention uses ultra-high performance liquid chromatography with higher resolution and shorter analysis time. When combined with mass spectrometry, it can obtain information on chemical components without ultraviolet absorption, and through the molecular feature extraction function, it can obtain chemical components in drug serum that are masked by protein component ion peaks, and quickly and efficiently determine the types and contents of components absorbed into the blood by Jinrong Granules;

[0052] 2. The method for determining the content of multiple components entering the blood provided by the present invention, wherein the standard component preparation has the corresponding components in Jinrong Granules, and the determination is performed by simultaneous detection using the UPLC-Q-TOF-MS method, and multiple index components are detected simultaneously in the same spectrum;

[0053] 3. Testing the components of Jinrong Granules absorbed into the blood will help the basic research on the efficacy of Jinrong Granules, achieve the basic quality evaluation of the efficacy of Jinrong Granules, and effectively control the quality of Jinrong Granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1a-1d The total ion current diagram of the Jinrong Granule sample solution and the total ion current diagram of the Jinrong Granule drug serum sample solution in Example 1 provided by the present invention

[0055] Figure 2a-2b is the total ion current diagram of the mixed reference solution in Example 1 provided by the present invention;

[0056] Figure 3a-3b It is an ion flow extraction diagram of the standard components of the Jinrong Granule sample solution and a total ion flow integral diagram of the mixed reference solution in Example 1 provided by the present invention;

[0057] Figure 4a It is an ion flow extraction diagram of the standard components in the Jinrong Granule drug serum sample solution and an ion flow extraction diagram of the mixed reference blank serum solution in Example 1 provided by the present invention.

[0058] The standard ingredients include: icariin, magnolamine, baohuoside I, echinacoside, stachydrine, tanshinone I, tanshinone IIA, and cryptotanshinone. DETAILED DESCRIPTION

[0059] The present invention is further described in detail with reference to the accompanying drawings and embodiments. The specific embodiments described here are only intended to illustrate the present invention, but not to limit the present invention.

[0060] 1. Instruments and equipment: Agilent 1290 / 6540UPLC-Q-TOF / MS chromatograph-mass spectrometer; Agilent ZORBAX Eclipse Plus C18 (3.0×100mm, 1.8μm); sonicator; S-23 high-speed desktop centrifuge; AUW120D electronic analytical balance; vacuum centrifugal concentrator.

[0061] The reference substance is the standard ingredient preparation: stachydrine hydrochloride (content is 98.81%), salidroside (content is 99.77%), magnolamine (content is 99.01%), echinacoside (content is 98.98%), verbascoside (content is greater than 98%), teruncifloside (content is 99.52%), salvianolic acid B (content is 94.53%), epimedin C (content is 98.19%), icariin (content is 98.47%), baohuoside I (content is 97.75%), rhamnoside (content is 98.83%), cryptotanshinone (content is 98.79%), tanshinone I (content is 99.37%), and tanshinone IIA (content is 99.53%).

[0062] Reagent materials: methanol (chromatographic grade), acetonitrile (mass spectrometry grade), ammonium formate (mass spectrometry grade), distilled water.

[0063] 2. Drug source: Jinrong granules dry paste (batch number: 170801-1) and volatile oil component inclusion compound (batch number: B-170807) are provided by Guangzhou Qiji Pharmaceutical Technology Co., Ltd.

[0064] Example 1

[0065] Step 1, Jinrong granule sample solution: weigh about 3g of Jinrong granule dry extract powder, add 30ml of methanol solution, dissolve by ultrasonic for 30min, let stand, take 2ml of the solution and centrifuge (8000rpm, centrifuge for 5min), filter the supernatant through a 0.22μm microporous filter membrane, and transfer to a sample injection bottle.

[0066] Step 2, mixed reference solution: accurately measure 10 mg of reference substance in a 25 ml volumetric flask, add 15 ml of methanol to ultrasonically dissolve, shake well, and adjust to volume to obtain reference solution. Take 100 μl of each reference solution, mix well, filter through a 0.22 μm filter membrane, and transfer to an injection bottle. The content of each characteristic component in the reference solution is: stachydrine 14.43 μg / ml, magnolia alkaloids 31.65 μg / ml, echinacoside 11.08 μg / ml, verbascoside 4.60 μg / ml, special privet glycoside 5.28 μg / ml, icariin 15.43 μg / ml, baohuoside I 33.33 μg / ml, cryptotanshinone 38.52 μg / ml, tanshinone I 28.68 μg / ml, tanshinone IIA 47.12 μg / ml.

[0067] Step 3, serum preparation: 20 female SPF SD rats were randomly divided into two groups: a blank group (Control) and a Jinrong granule group. Label and record body weight. The 20 rats were cultured for 3 days, and gavage was started on the 4th day (gavage was calculated according to the equivalent dose ratio), and gavage was continued for 7 days. Before the last administration, they fasted but could not drink water for 12 hours. One hour after the gavage, ketamine (Ketamine)-xylazine (Xylazine) (60mg / kg (K) + 10mg / kg (X)) was injected intraperitoneally for anesthesia. After anesthesia, blood was collected from the abdominal aorta, and the blood was centrifuged in a low-temperature centrifuge at 3000rpm, 4°C, and 5min. After centrifugation, the supernatant was taken and filtered through a 0.22μm filter membrane in a clean bench. The filtrate was transferred to a sterile centrifuge tube. The serum was marked according to the animal group and stored in a refrigerator.

[0068] Step 4, preparation of serum sample solution: ① Preparation of Jinrong Granule Drug Serum Sample Solution: Weigh 3 times the clinical equivalent dose of Jinrong Granules and dissolve it, gavage rats for 7 days, 2 times / day, collect blood, and separate serum. Take 2ml of serum, precipitate protein with methanol, centrifuge, concentrate, re-dissolve, filter with 0.22μm filter membrane, and place in a sample injection bottle. ② Preparation of mixed reference blank serum solution: Take 2ml of blank serum, add 100μl of the mixed reference solution described in step 2, precipitate protein with methanol, concentrate, re-dissolve, filter with 0.22μm filter membrane, and place in a sample injection bottle.

[0069] Step 5, using UPLC-Q-TOF-MS / MS method to detect the content of the reference solution and the test solution respectively;

[0070] Chromatographic conditions during detection:

[0071] Chromatographic column: Agilent ZORBAX Eclipse Plus C18 (3.0×100mm, 1.8μm)

[0072] Mobile phase composition: Phase A: acetonitrile (organic phase); Phase B: 0.05 mmol / L ammonium formate aqueous solution (aqueous phase).

[0073] Injection volume: 1 μL

[0074] Flow rate: 0.4mL / min

[0075] Selection of gradient elution program: The ingredients of Jinrong Granules are complex, including flavonoids and their glycosides, alkaloids, phenolic acids and phenylethanol glycosides. In order to achieve better separation of the components, gradient elution from large to small polarity was performed. The total elution time was set according to the peak conditions. When the elution time reached 40 minutes, the baseline was basically smooth. The mobile phase B was ammonium formate aqueous solution. The first 10-15 minutes of elution were mainly phase B, and the mobile phase was highly acidic, which could preferentially separate alkaloid compounds. The last 10-15 minutes were mainly phase A, and the acidity of the mobile phase was weakened. The acidic components of phenolic acids were detected during this time period. There are many glycoside components in the formula. Between 15-25 minutes, the mobile phase was neutral and low polarity. As the polarity of the mobile phase decreased, polysaccharide glycosides, disaccharide glycosides, and aglycone compounds eluted peaks in turn.

[0076] Gradient elution program settings: 0-10 min, 5%-15% A; 10-15 min, 15%-35% A; 15-25 min, 35%-65% A; 25-40 min, 65%-95% A

[0077] Mass spectrometry detection conditions:

[0078] Analysis mode: positive and negative ion analysis mode

[0079] Ionization method: electrospray ionization

[0080] Accurate mass calibration solution before injection analysis: Agilent standard tuning solution ESI-L Low Concentration Tuning Mix (G1969-85000)

[0081] Primary mass spectrometry scanning detection range: m / z 100~1700;

[0082] Solvent removal drying gas: nitrogen;

[0083] Temperature: 325℃;

[0084] Flow rate: 6.8L / min;

[0085] Sheath gas temperature: 350℃;

[0086] Capillary voltage: 4.0kv;

[0087] Fragment voltage: 150v

[0088] Refer to Figures 1-3, which are chromatograms of the Jinrong granule sample solution of this example.

[0089] Calculate the contents of 10 components in the test solution: calculate the contents of stachydrine, icariin, baohuoside I, echinacoside, verbascoside, tanshinone I, tanshinone IIA, cryptotanshinone, terunguiside and magnolamine in the test solution by using the standard curve method.

[0090] The calculation results are shown in Table 1:

[0091] Table 1 Determination results of the main components of Jinrong granules

[0092]

[0093] See Figure 4, which is a chromatogram of the serum sample solution of Jinrong Granules drug in this embodiment.

[0094] According to the ion flow extraction diagrams of the Jinrong Granules drug serum sample solution and the mixed reference blank serum solution, according to the reference concentration, the peak area of ​​the reference obtained and the peak area of ​​the corresponding standard component in the sample are detected, and the content of the corresponding standard component is calculated. The contents of the 8 components in the test solution are calculated separately: the contents of stachydrine, icariin, baohuoside I, echinacoside, tanshinone I, tanshinone IIA, cryptotanshinone, and magnolia alkaloids in the test solution are calculated using the standard curve method. Calculation formula: C serum sample = C reference * A serum sample / C reference; the content of each component in Jinrong Granules = C serum sample / C Jinrong Granules sample solution

[0095] Table 2 Results of determination of the main blood components of Jinrong granules

[0096]

[0097] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and inventive concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A method for determining the multi-index content of blood components of Jinrong granules, characterized in that: The ion chromatogram was measured by UPLC-Q-TOF-MS / MS analysis method; The chromatographic conditions include: Column: C18 Flow rate: 0.3~0.5mL / min; Column temperature: 20-30°C; Mobile phase: Phase A is acetonitrile, phase B is 0.04-0.08 mmol / L ammonium formate aqueous solution, the mobile phase includes acetonitrile and 0.04-0.08 mmol / L ammonium formate aqueous solution; The elution was performed using the following elution program: 0-10min: 5%→15% mobile phase A, 95%→85% mobile phase B; 10-15min: 15% → 35% mobile phase A, 85% → 65% mobile phase B; 15-25 min: 35% → 65% mobile phase A, 65% → 35% mobile phase B; 25-40min: 65%→95% mobile phase A, 35%→5% mobile phase B.

2. The method for determining the multi-index content of blood components of Jinrong Granules according to claim 1, characterized in that: The mass spectrometry detection conditions are as follows: the analysis mode is positive and negative ion mode, ESI ionization mode, and Agilent standard tuning solution ESI-L Low Concentration Tuning Mix (G1969-85000) is used to calibrate the accurate mass number before sample injection analysis; Primary mass spectrometry scanning detection range: m / z 100~1700; Solvent removal drying gas: nitrogen; Temperature: 325℃; Flow rate: 6.8L / min; Sheath gas temperature: 350℃; Capillary voltage: 4.0kv; Fragment voltage: 150v.

3. The method for determining the multi-index content of blood components of Jinrong Granules according to claim 1, characterized in that: In the UPLC-Q-TOF-MS / MS analysis method, the Jinrong Granule drug serum sample solution is used as the sample solution.

4. The method for determining the multi-index content of blood components of Jinrong Granules according to claim 3, characterized in that: The preparation method of the Jinrong granule drug serum sample solution comprises the following steps: weighing the Jinrong granules to dissolve, administering the drug to rats by gavage, collecting blood, separating serum, precipitating proteins, centrifuging, concentrating, re-dissolving and filtering to obtain the solution.

5. The method for determining the multi-index content of blood components of Jinrong Granules according to claim 4, characterized in that: The dosage of the Jinrong granules is 2 to 4 times the clinical equivalent dosage.

6. The method for determining the multi-index content of blood components of Jinrong Granules according to claim 4, characterized in that: Rats were given drugs by gavage twice a day for 7 consecutive days.

7. The method for determining the multi-index content of blood components of Jinrong Granules according to claim 4, characterized in that: The specific method after separating the serum is as follows: take 2 ml of serum, precipitate protein with methanol, centrifuge, concentrate, re-dissolve, and filter through a 0.22 μm filter membrane.

8. The method for determining the multi-index content of the blood components of Jinrong Granules according to any one of claims 1 to 7, characterized in that: Specifications of the chromatographic column: 3.0×100mm, 1.8μm.

9. The method for determining the multi-index content of the blood components of Jinrong Granules according to any one of claims 1 to 7, characterized in that: In the UPLC-Q-TOF-MS / MS analysis method, the mixed reference blank serum solution includes icariin, baohuoside I, echinacoside, verbascoside, tanshinone I, tanshinone II A, cryptotanshinone, terunguisu glycoside, magnolamine, stachydrine and salvianolic acid B.

10. The method for determining the multi-index content of blood components of Jinrong Granules according to claim 8, characterized in that: The preparation method of the mixed reference blank serum solution is to take 2-3 ml of blank serum, add 100 μl of the mixed reference solution, precipitate protein with methanol, concentrate, re-dissolve, and filter through a filter membrane; In the mixed reference solution, the concentrations of icariin, baohuoside I, echinacoside, verbascoside, tanshinone I, tanshinone IIA, cryptotanshinone, teruncle glycoside, magnolamine, stachydrine, and salvianolic acid B are 0.4-0.5 mg / mL, 0.4-0.5 mg / mL, 0.5-0.6 mg / mL, 0.4-0.5 mg / mL, 0.4-0.5 mg / mL, 0.35-0.5 mg / mL, 0.35-0.5 mg / mL, 0.4-0.5 mg / mL, 0.4-0.5 mg / mL, 0.6-0.8 mg / mL, and 0.5-0.6 mg / mL, respectively.

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