Qualitative detection method for internal and external components of Huangliver decoction liquid

Abstract

The invention belongs to the field of compound traditional Chinese medicine component detection, and particularly relates to a qualitative detection method for internal and external components of Huangliver decoction liquid. The ultra-high performance liquid chromatography-tandem mass spectrometry method for synchronously detecting the internal and external components of the Huangliver decoction liquid is established, and the method comprises the steps of pretreatment of a test sample and construction of the ultra-high performance liquid chromatography-tandem mass spectrometry method. According to the present invention, the 44 in vitro components and the 39 in vivo prototype components can be detected under the same chromatography-mass spectrometry condition, the 40 known compounds are determined, the pharmacodynamic material basis of the Huang liver decoction is preliminarily revealed, and the foundation is laid for the product development. The compound traditional Chinese medicine is researched as a whole, internal and external components of the Huangliver decoction are detected and qualitative, fundamental research on substances of the Huangliver decoction is promoted, and the blank of serum medicine chemical research of the Huangliver decoction is filled. Meanwhile, the detection method disclosed by the invention is short in required time, simple in pretreatment method and suitable for popularization.

Description

Technical Field

[0001] The invention belongs to the field of compound traditional Chinese medicine component detection, and specifically relates to a qualitative detection method for internal and external components of Huangganjian liquid. Background Art

[0002] Huanggan decoction is composed of 14 Chinese medicinal herbs, including Artemisia capillaris, Scutellaria baicalensis, Phellodendron chinense, Gentiana scabra, Poria cocos, Plantago asiatica, Rhubarb, Curcuma aromatica, Toosendan fruit, Angelica sinensis, Chinese yam, scorched malt, scorched hawthorn, and scorched Liushenqu. It has the effects of clearing heat, promoting dampness, and relieving jaundice. It is used for acute icteric hepatitis. Artemisia capillaris is the first choice for treating jaundice, clearing heat and promoting dampness, soothing the liver and promoting gallbladder, and relieving jaundice, and is the main medicine. The first group of ministerial medicines is Scutellaria baicalensis, Phellodendron chinense, and Gentiana scabra. Scutellaria baicalensis clears heat and dries dampness, and is good at clearing the heat of Banli. The heat of Banli first appears in the gallbladder, so Scutellaria baicalensis is good at clearing damp-heat in the liver and gallbladder; Phellodendron chinense clears the heat of the lower jiao, clears heat and dries dampness, and at the same time can clear and reduce the virtual heat and virtual fire of the kidney; Gentiana scabra is mainly in the liver and gallbladder meridians, is good at clearing the real fire of the liver and gallbladder, and is good at clearing damp-heat in the lower jiao. The three medicines are combined with Artemisia capillaris to enhance the effects of clearing heat and promoting gallbladder. The second group of ministerial drugs is Poria cocos, Plantago seed, and Rhubarb. Poria cocos and Plantago seed can promote water and conduct heat, leading heat to be discharged through urine. Among them, Poria cocos acts on the middle jiao, which can increase the downward flow of spleen dampness. Plantago seed acts on the lower jiao, where dampness is stagnant. Rhubarb can relieve heat and relieve constipation, clear dampness and heat, and is matched with Poria cocos and Plantago seed to discharge dampness and heat from urine and feces; the third group of ministerial drugs, Curcuma aromatica and Toosendan seed, both enter the liver meridian, and play the role of soothing the liver and promoting qi to relieve depression. There are two groups of adjuvant drugs. Dampness and heat are lurking in the human body and are easy to hurt the positive energy. Chinese yam can both strengthen the spleen and replenish qi, and can also strengthen the kidney. Liver and gallbladder fire can easily hurt yin and consume blood, and Chinese angelica is warm in nature, nourishing blood and activating blood circulation. The adjuvant drugs Chinese yam and Chinese angelica nourish qi and blood, prevent dampness and heat from hurting qi and blood, and make the whole prescription cool but not depressed. Damp heat blocks the middle jiao, weakens the transportation and transformation function, and causes abdominal distension. The adjuvant Jiao Sanxian invigorates the spleen and stimulates appetite, promotes qi and helps digestion. The combination of various medicines complements each other, giving full play to their strengths, and exerting the effects of clearing heat, promoting bile and relieving jaundice in many aspects.

[0003] The effective substances of Chinese medicine compound are the group of chemical components that exert therapeutic effects in the compound. They have the characteristics of component diversity and holistic effects. The research aims to reveal the connotation of Chinese medicine formula and optimize the compatibility, so as to provide a basis for improving efficacy and developing new drugs. Serum pharmacological chemistry believes that Chinese medicine is transported to the target site with blood as the medium to take effect, so the components entering the blood after administration can be used as its active ingredients. By analyzing the chemical substances inside and outside the body of Chinese medicine, the basic research on the effective substances of Chinese medicine compound can be promoted.

[0004] The determination of the content of the in vitro components of Huanggan decoction has been reported. "Identification and determination of the main components in Huanggan decoction" (Journal of Ningxia Medical University, 2020, 42(01): 104-108) disclosed the use of dual-wavelength high-performance liquid chromatography to simultaneously determine the contents of chlorogenic acid, baicalin, emodin and chrysophanol in Huanggan decoction. Among them, the YMC-Pack Pro C18 chromatographic column (250mm×4.6mm, 5μm); the detection wavelength of emodin and chrysophanol is 254nm, and the detection wavelength of chlorogenic acid and baicalin is 327nm; the mobile phase is acetonitrile (A)-0.1% phosphoric acid water (B) gradient elution: 0-8min, (A:B=18:82), 18-28min (A:B=85:15), 28-30min (A:B=18:82); flow rate: 1.0mL·min-1; injection volume: 20μL; column temperature: 35℃. This technical solution can quantitatively detect the content of chlorogenic acid, baicalin, emodin and chrysophanol. This technical solution can only detect the components of chlorogenic acid, baicalin, emodin and chrysophanol at the same time, and the number of components detected simultaneously is small, and it does not involve the determination of components in the body after entering the blood.

[0005] There are no reports on the qualitative detection of the in vivo and in vitro components of Huanggan decoction. The components of different compound prescriptions will change during the preparation process. In the process of decoction, the components of Chinese medicine will also undergo relatively complex chemical reactions, including isomerization, redox reaction, substitution reaction, etc., thereby producing new compounds or causing the original compounds to disappear, which in turn significantly affects the efficacy of Chinese medicine. The components in Chinese medicine can only produce effects after being absorbed into the blood. The medicinal components in the blood may be substances that have a direct effect on the human body, including prototype components, metabolites and physiologically active substances.

[0006] Therefore, how to intuitively reflect the in vitro components and the active ingredients absorbed into the blood after co-decocting of the complex Chinese medicine compound Huanggan Decoction is an urgent problem to be solved in the research on the material basis of the efficacy of Huanggan Decoction. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a method for qualitatively detecting the internal and external components of Huangganjian liquid.

[0008] The technical scheme of the present invention is as follows: a method for detecting the internal and external components of Huangganjian liquid, the detection method comprising ultra-high performance liquid chromatography-tandem mass spectrometry analysis, wherein a carbon 18 filler chromatographic column is used in the liquid chromatography conditions, the mobile phase A is a 0.1% formic acid aqueous solution, the mobile phase B is a 0.1% formic acid acetonitrile solution, and the gradient elution procedure is as follows:

[0009]

[0010] And in the process of gradient elution, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; the proportion of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of eluent.

[0011] Preferably, the chromatographic column is ACQUITY UPLC HSS T3, with specifications of 2.1 mm x 100 mm and 1.8 μm.

[0012] Preferably, the column temperature of the chromatographic column is 30-40°C.

[0013] Preferably, the flow rate of the mobile phase in the liquid chromatography is 0.2-0.4 mL·min -1 ,

[0014] Furthermore, the mass spectrometry analysis conditions include: electrospray ion source, positive and negative ion scanning, scanning mode FullMS / DD-MS 2 , Full ms resolution is 60000, dd-ms 2 The resolution is 15000, the scanning range m / z is 90~1300; the sheath gas pressure is 40-50arb, the auxiliary gas pressure is 15-25arb, the capillary temperature is 320℃, the vaporization temperature is 370℃, the spray voltage is 3.8kV for positive ion, 3.0kV for negative ion, and the collision energy is 20-60eV.

[0015] In some typical embodiments, the present invention provides a method for detecting components inside and outside Huangganjian liquid, comprising the following steps:

[0016] 1) Collection of serum samples: SD rats were fed with 17.11 g kg -1 The rats were given Huanggan decoction by intragastric administration twice a day for 3 consecutive days. Blood was collected from the orbital venous plexus of the rats 15, 45, 60, 90, and 120 minutes after the last administration. The blood collected at the five time points was collected into a centrifuge tube, allowed to stand for 30 minutes, centrifuged, and the upper serum was collected to obtain the administration serum sample.

[0017] 2) Preparation of drug-containing serum test samples: Take 200 μL of serum sample from the drug-containing serum group, add 800 μL of methanol, vortex mix for 1 min, let stand at -20°C for 30 min, centrifuge at 16000g at 4°C for 20 min, take the supernatant and vacuum dry, add 100 μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 16000g at 4°C for 15 min, and take the supernatant for analysis.

[0018] 3) The drug-containing serum test sample was subjected to ultra-high performance liquid chromatography-tandem mass spectrometry analysis, wherein a carbon 18-filled column was used in the liquid chromatography conditions, mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile solution, and the gradient elution procedure was as follows:

[0019]

[0020] And in the gradient elution process, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; wherein the proportion of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of the eluent;

[0021] The carbon 18 filler chromatographic column is ACQUITY UPLC HSS T3, with specifications of 2.1mm x 100mm, 1.8μm;

[0022] The column temperature of the chromatographic column is 30-40°C;

[0023] Preferably, the flow rate of the mobile phase in the liquid chromatography is 0.2-0.4 mL·min -1 ;

[0024] Mass spectrometry analysis conditions include: electrospray ion source, positive and negative ion scanning, scanning mode is Full ms / dd-ms 2 , Full ms resolution is 60000, dd-ms 2 The resolution is 15000, the scanning range m / z is 90~1300; the sheath gas pressure is 40-50arb, the auxiliary gas pressure is 15-25arb, the capillary temperature is 320℃, the vaporization temperature is 370℃, the spray voltage is 3.8kV for positive ion, 3.0kV for negative ion, and the collision energy is 20-60eV.

[0025] 4) The structural identification was carried out by matching with the standard spectrum database to obtain the analysis results of the internal and external components of Huangganjian liquid.

[0026] The present invention establishes a technical scheme for synchronously detecting the internal and external components of Huanggan decoction by ultra-high performance liquid chromatography tandem mass spectrometry, including the construction of the method of sample pretreatment and ultra-high performance liquid chromatography tandem mass spectrometry. Methanol is used to precipitate protein in the sample pretreatment, and 40% methanol is used to enrich the components. The present invention can detect 44 components in vitro and 39 prototype components in vivo under the same chromatographic mass spectrometry conditions, determine 40 known compounds, preliminarily reveal the pharmacological material basis of Huanggan decoction, and lay the foundation for product development.

[0027] Beneficial effects of the present invention:

[0028] (1) The present invention studies the compound Chinese medicine as a whole, detects and characterizes the internal and external components of Huanggan decoction, promotes the material basis research of Huanggan decoction, and fills the gap in the serum pharmacological chemistry research of Huanggan decoction.

[0029] (2) The detection method established by the present invention takes a short time, and ultra-high performance liquid chromatography tandem mass spectrometry detection can be completed in just 20 minutes. The pretreatment method is simple and suitable for promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 UHPLC-HRMS total ion chromatograms of Huanggan decoction. (A) BPC graphs of blank serum, drug-containing serum, and Huanggan decoction test samples. The chromatograms from top to bottom are Huanggan decoction, blank serum + Huanggan decoction, blank serum, and drug-containing serum. (B) Total ion BPC graphs of Huanggan decoction in positive and negative modes. DETAILED DESCRIPTION

[0031] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0032] Source of Huanggan decoction: Huanggan decoction is an in-hospital preparation of Ningxia Medical University General Hospital (Ningxia Medicine Preparation:

[0033] Z20200020000), has the effects of clearing away heat, promoting the flow of dampness and relieving jaundice. It is mainly used to treat acute and chronic icteric hepatitis.

[0034] Example 1: Plasma sample detection method

[0035] 1. Animal Dosing and Serum Sample Collection

[0036] Twelve SPF male SD rats, weighing (200±20)g, were purchased from Wuhan Yunkelong Animal Co., Ltd., with production license number SCXK(E)20180021. Before the experiment, they were adaptively fed in an animal room with a 12-h day and night cycle for 7 days, with free access to feed and drinking water. The animal experiment complies with ethical requirements (ethics number KYLL-2021-718). They were raised in accordance with the experimental animal operation instructions, with an ambient temperature of 20-25°C and a humidity of 50-60%. They were fasted for 12 hours before the experiment and had free access to water.

[0037] SD rats were randomly divided into a blank serum group and a drug-containing serum group, with 6 rats in each group. The drug-containing serum group was treated with 17.11 g·kg -1The dose was administered by gavage twice a day for 3 consecutive days. The blank serum group was given the same volume of normal saline. Fasting but not water was allowed for 12 hours before the last administration. Blood was collected from the orbital venous plexus of the rats 15, 45, 60, 90, and 120 minutes after the last administration. The blood collected from each rat at 5 time points was collected into a centrifuge tube, allowed to stand for 30 minutes, centrifuged, and the upper serum was taken and stored at -80°C for later use. The blank serum group was operated in the same way as above.

[0038] 2. Preparation of test samples

[0039] Yellow liver decoction test sample: Take 0.6 mL of yellow liver decoction, place it in a centrifuge tube (2 mL), add 0.4 mL of methanol solution, vortex mix, take 200 μL of the mixture, add 600 μl of 40% methanol aqueous solution to dilute, vortex mix, centrifuge at 16000g at 4℃ for 15 min, and take the supernatant for analysis.

[0040] Drug-containing serum test sample: Take 200 μL of serum sample from the drug-containing serum group, add 800 μL of methanol, vortex mix for 1 min, let stand at -20°C for 30 min, centrifuge at 16000g at 4°C for 20 min, take the supernatant and vacuum dry, add 100 μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 16000g at 4°C for 15 min, and take the supernatant for analysis.

[0041] Blank serum test sample: Take 200 μL of blank serum sample, add 800 μL of methanol, vortex mix for 1 min, let stand at -20 °C for 30 min, centrifuge at 16000g at 4 °C for 20 min, take the supernatant and vacuum dry, add 100 μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 16000g at 4 °C for 15 min, and take the supernatant for analysis.

[0042] Blank serum + yellow liver decoction sample: take 200 μL of blank serum, add 33.4 μL of yellow liver decoction test solution, add 800 μL of methanol, vortex mix for 1 min, let stand at -20℃ for 30 min, centrifuge at 16000g at 4℃ for 15 min, take the supernatant and vacuum dry, add 100 μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 16000g at 4℃ for 15 min, and take the supernatant for analysis.

[0043] 3. Liquid Chromatography and Mass Spectrometry Conditions

[0044] Liquid phase conditions: Thermo UHPLC vanquish ultra-high performance liquid chromatography system, ACQUITY UPLC HSS T3 column (2.1mm X 100mm, 1.8μm) was used for chromatographic experiments. The mobile phase was 0.1% formic acid in water (A) and the mobile phase was 0.1% formic acid in acetonitrile (B), the gradient elution program was: 0min, 5% B; 17min, 98% B; 17.2min, 5% B; 20min, 5% B; the flow rate was 0.3mL / min, and the column temperature was 35°C.

[0045] Mass spectrometry conditions: electrospray ion source, positive and negative ion scanning, scanning mode: Full ms / dd-ms 2 , Full ms resolution 60000, dd-ms 2 The resolution was 15000, and the scanning range was m / z 90 to 1300. The sheath gas pressure was 45 arb, the auxiliary gas pressure was 20 arb, the capillary temperature was 320°C, the vaporization temperature was 370°C, the spray voltage was 3.8 kV for positive ions and 3.0 kV for negative ions, and the collision energy gradient was 20, 40, and 60 eV.

[0046] 4. Data Processing

[0047] The raw data file in .raw format was imported into proteoWizard and converted into .mzXML format, and XCMS software was used for peak alignment, retention time correction and peak extraction. The data extracted by XCMS were then matched with the standard spectrum database for structural identification.

[0048] 5. Results of in vivo and in vitro component testing

[0049] Data were collected for the Huanggan decoction sample, blank serum + Huanggan decoction sample, blank serum sample, and drug-containing serum sample, and the peak shape of the chromatographic peaks with higher abundance in the ion flow graph was checked and confirmed by the secondary spectrum. The positive and negative ion graphs were marked with chromatographic peak numbers in numerical order. A total of 44 chromatographic peaks were marked in the Huanggan decoction, including 39 prototype components that entered the blood. The structures were identified by matching with the standard spectrum database, and 40 known compounds were determined.

[0050] The UHPLC-HRMS total ion chromatogram of yellow liver decoction is as follows: Figure 1 The results of the internal and external components of Huangganjian liquid are shown in Table 1.

[0051] Table 1 Chemical composition information of yellow liver decoction

[0052]

[0053]

[0054]

[0055] Table Notes: RT / min: retention time / min; Score: secondary mass spectrometry matching score (total score 1, the higher the score, the more reliable the identification result); NA: unknown compound.

Claims

1. A method for detecting the internal and external components of Huangganjian liquid, the detection method comprising ultra-high performance liquid chromatography-tandem mass spectrometry analysis, wherein a carbon 18 filler column is used in the liquid chromatography conditions, mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, and the gradient elution procedure is as follows: And in the process of gradient elution, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; the proportion of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of eluent.

2. The detection method according to claim 1, characterized in that: The chromatographic column is ACQUITY UPLC HSS T3, with specifications of 2.1 mm x 100 mm and 1.8 μm.

3. The detection method according to claim 1, characterized in that: The column temperature of the chromatographic column is 30-40°C.

4. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase in liquid chromatography is 0.2-0.4 mL min -1 .

5. The detection method according to claim 1, characterized in that: Mass spectrometry analysis conditions include: electrospray ion source, positive and negative ion scanning, scanning mode is Full ms / dd-ms 2 , Full ms resolution is 60000, dd-ms 2 The resolution is 15000, the scanning range m / z is 90~1300; the sheath gas pressure is 40-50arb, the auxiliary gas pressure is 15-25arb, the capillary temperature is 320℃, the vaporization temperature is 370℃, the spray voltage is 3.8kV for positive ion, 3.0kV for negative ion, and the collision energy is 20-60eV.

6. The detection method according to claim 1, characterized in that: The detection method comprises the following steps, 1) Collection of serum samples: SD rats were fed with 17.11 g kg -1 The rats were given Huanggan decoction by intragastric administration twice a day for 3 consecutive days. Blood was collected from the orbital venous plexus of the rats 15, 45, 60, 90, and 120 minutes after the last administration. The blood collected at the five time points was collected into a centrifuge tube, allowed to stand for 30 minutes, centrifuged, and the upper serum was collected to obtain the administration serum sample. 2) Preparation of drug-containing serum test samples: Take 200 μL of serum sample from the drug-containing serum group, add 800 μL of methanol, vortex mix for 1 min, let stand at -20°C for 30 min, centrifuge at 16000g at 4°C for 20 min, take the supernatant and vacuum dry, add 100 μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 16000g at 4°C for 15 min, and take the supernatant for analysis. 3) The drug-containing serum test sample was subjected to ultra-high performance liquid chromatography-tandem mass spectrometry analysis, wherein a carbon 18-filled column was used in the liquid chromatography conditions, mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was 0.1% formic acid acetonitrile solution, and the gradient elution procedure was as follows: And in the gradient elution process, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; wherein the proportion of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of the eluent; The carbon 18 filler chromatographic column is ACQUITY UPLC HSS T3, with specifications of 2.1mm x 100mm, 1.8μm; The column temperature of the chromatographic column is 30-40°C; Preferably, the flow rate of the mobile phase in the liquid chromatography is 0.2-0.4 mL·min -1 ; Mass spectrometry analysis conditions include: electrospray ion source, positive and negative ion scanning, scanning mode is Full ms / dd-ms 2 , Fullms resolution is 60000, dd-ms 2 The resolution was 15000, the scanning range m / z was 90-1300; the sheath gas pressure was 40-50arb, the auxiliary gas pressure was 15-25arb, the capillary temperature was 320°C, the vaporization temperature was 370°C, the spray voltage was 3.8kV for positive ions, 3.0kV for negative ions, and the collision energy was 20-60eV; 4) The structural identification was carried out by matching with the standard spectrum database to obtain the analysis results of the internal and external components of Huangganjian liquid.

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