Detection method for sijunzi decoction decoction and medicated serum

Abstract

The invention discloses a method for detecting sijunzi decoction and medicated serum. Chemical components of the sijunzi decoction and the medicated serum are qualitatively analyzed through a high performance liquid chromatography-mass spectrometry technology. The method comprises the following steps: preparing a water decoction according to the prescription proportion of the Sijunzi decoction, continuously dosing for 7 days through rat intragastric administration, collecting and treating serum, and separating and identifying chemical components in the water decoction and the serum through a Betasil C18 chromatographic column and gradient elution conditions in combination with electrospray ionization mass spectrometry in a negative ion mode. Experimental results show that 10 chemical components are detected in the water decoction; five chemical components are detected in the drug-containing serum. The method has the characteristics of high sensitivity and excellent separation effect, can accurately reveal the pharmacodynamic material basis and metabolic mechanism of the Sijunzi decoction, provides a scientific basis for modern research and quality control of traditional Chinese medicine compounds, and has wide application value.

Description

Technical Field

[0001] The invention relates to a detection method, and more particularly to a detection method for a decoction of Sijunzi decoction and a drug-containing serum. Background Art

[0002] Sijunzi Decoction is a classic Chinese medicine compound, which is composed of four herbs: Codonopsis pilosula, Atractylodes macrocephala, Poria cocos, and Licorice root. It is often used to treat symptoms such as spleen and stomach weakness, shortness of breath and fatigue, and has the effect of strengthening the spleen and replenishing qi. The pharmacodynamic material basis of traditional Chinese medicine compound and its in vivo metabolism have long been the focus of the field of Chinese medicine research. With the development of modern pharmacology and analytical chemistry technology, Chinese medicine serum pharmacochemistry has provided an important research method for studying the pharmacodynamic material basis and in vivo pharmacokinetics of traditional Chinese medicine compound.

[0003] TCM serum pharmacology is an applied science based on modern separation and analysis technology. Its core idea is to prepare drug-containing serum, combine fingerprints and modern analytical methods, analyze the chemical components in serum, including prototype drugs, metabolites and stress components, to infer the material basis and metabolic mechanism of the efficacy of TCM compound. Compared with traditional TCM chemical research methods, TCM serum pharmacology has significant advantages in sensitivity, separation effect and analysis efficiency, especially it can reveal the dynamic changes of drugs in the process of metabolism in the body, and provide key technical support for studying the compatibility rules and effective components of TCM compound.

[0004] Traditional Chinese medicine compound represented by Sijunzi Decoction has complex chemical composition and involves multiple biological processes, including intestinal flora metabolism, liver drug enzyme metabolism and transmembrane transport absorption. In this process, various chemical components interact with each other and generate active ingredients through metabolic transformation, thereby achieving therapeutic effects. However, the current research on the metabolic process of Sijunzi Decoction in vivo is still relatively limited.

[0005] Therefore, developing a detection method for Sijunzi Decoction decoction and drug-containing serum is of great significance for a deep understanding of its pharmacodynamic material basis and pharmacokinetic characteristics, and also provides a new technical path for the modern research of Chinese medicine compound prescriptions. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a method for detecting Sijunzi Decoction decoction and drug-containing serum, and the specific scheme is as follows:

[0007] A method for detecting Sijunzi Decoction decoction and drug-containing serum, the method comprising the following steps:

[0008] S1. Preparation of decoction of Sijunzi Decoction: According to the prescription of Sijunzi Decoction, 9 grams each of ginseng, Atractylodes macrocephala, and Poria cocos and 6 grams of roasted licorice root were used to prepare decoction:

[0009] S2. Preparation of drug-containing serum: Male SD rats were gavaged with concentrated Sijunzi decoction at a dose of 1 mL / 100 g for 7 consecutive days, once a day; the rats were centrifuged after standing, and the upper serum was taken, sterilized with a 0.22 μm microporous filter membrane, and stored at -80°C;

[0010] S3. Sample processing: Mix the drug-containing serum with methanol, extract by ultrasonication, centrifuge and take the supernatant, heat, concentrate and evaporate to dryness, add methanol to dissolve, filter through a 0.45 μm filter membrane and then sample and analyze;

[0011] S4. The analysis conditions are as follows:

[0012] Chromatographic conditions: Betasil C18 column 4.6 mm × 250 mm, 5 μm, mobile phase water and acetonitrile, flow rate 0.4 mL / min, injection volume 20 μL;

[0013] Mass spectrometry conditions: electrospray ion source ESI, negative ion mode, spray voltage 3.5 kV, capillary temperature 300 °C, full ion scan range m / z: 100-1200;

[0014] S5. HPLC-MS-MS was used to conduct qualitative analysis on the chemical components of Sijunzi Decoction decoction and drug-containing serum.

[0015] Furthermore, S1 also includes the following sub-steps:

[0016] S101: adding 10 times the amount of water to the four raw materials and soaking them for 0.5 hours, heating and boiling them for 1 hour, and filtering and separating the filtrate and the residue;

[0017] S102: Boil the residue with 6 times the amount of water, boil for 1 hour and filter;

[0018] S103: adding water to the residue and boiling it for 1 hour, then filtering it and combining the three filtrates;

[0019] S104: Concentrate to a drug concentration of 0.125 g / mL and store at 4°C.

[0020] Furthermore, the mice in S2 were fasted for more than 12 hours after the last administration, and blood was collected under anesthesia 2 hours after the last administration.

[0021] Furthermore, the centrifugal speed in S2 is 3000 rpm and the time is 10 min.

[0022] Furthermore, the gradient elution conditions are as follows:

[0023]

[0024] Furthermore, the column temperature of the chromatographic column is 35°C.

[0025] Furthermore, when the chemical components of the Sijunzi Decoction decoction and the drug-containing serum were qualitatively analyzed in S5, the detection wavelengths were 210nm, 240nm, and 280nm.

[0026] Furthermore, the test results showed 10 chemical substances in the decoction of Sijunzi Decoction, including 7 saponins and 3 flavonoids; and 5 metabolic components in the drug-containing serum.

[0027] Beneficial effects:

[0028] The present invention provides a method for detecting Sijunzi decoction decoction and drug-containing serum, and qualitatively analyzes the chemical components of Sijunzi decoction decoction and drug-containing serum by high performance liquid chromatography-mass spectrometry (HPLC-MS). The method comprises the following steps: preparing decoction according to the prescription ratio of Sijunzi decoction, collecting serum and processing serum after continuous oral administration to rats for 7 days, separating and identifying the chemical components in decoction and serum by Betasil C18 chromatographic column and gradient elution conditions, combined with electrospray ionization mass spectrometry in negative ion mode. The experimental results show that 10 chemical components are detected in the decoction, including 7 saponin compounds and 3 flavonoid compounds; 5 chemical components are detected in the drug-containing serum, including 3 prototype components ginsenoside Rb1, Re and glycyrrhizic acid and 2 metabolites glycyrrhetinic acid and glycyrrhizin glucuronic acid. This method has the characteristics of high sensitivity and excellent separation effect. It can accurately reveal the pharmacological material basis and metabolic mechanism of Sijunzi Decoction, provide a scientific basis for the modern research and quality control of Chinese herbal compound, and has wide application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the total ion current diagram of the decoction of Sijunzi Decoction.

[0030] Figure 2 This is the secondary mass spectrum of ginsenoside Rb1 in decoction.

[0031] Figure 3 This is the secondary mass spectrum of ginsenoside Re in decoction.

[0032] Figure 4 This is the secondary mass spectrum of ginsenoside Rg1 in decoction.

[0033] Figure 5 This is the secondary mass spectrum of glycyrrhizic acid in decoction.

[0034] Figure 6 This is the secondary mass spectrum of liquiritin in the decoction.

[0035] Figure 7 This is the tertiary mass spectrum of the liquiritin mass fragment 255.21.

[0036] Figure 8 This is the secondary mass spectrum of ginsenoside Rb2 in decoction.

[0037] Fig. 9 This is the secondary mass spectrum of ginsenoside Rd in decoction.

[0038] Fig.10 This is the secondary mass spectrum of ginsenoside Rg3 in decoction.

[0039] Fig.11 This is the secondary mass spectrum of glycyrrhizin in the decoction.

[0040] Fig.12 It is the tertiary mass spectrum of the glycyrrhizin fragment with a mass of 135.06.

[0041] Fig.13 This is the secondary mass spectrum of glycyrrhizin in the decoction.

[0042] Fig.14 It is the tertiary mass spectrum of the glycyrrhizic coumarin fragment with a mass of 309.22.

[0043] Fig.15 It is the total ion current of drug-containing serum.

[0044] Fig.16 This is the secondary mass spectrum of ginsenoside Rb1 in drug-containing serum.

[0045] Fig.17 This is a partial magnified image of ginsenoside Rb1 in the medicated serum.

[0046] Fig.18 This is the secondary mass spectrum of ginsenoside Re in drug-containing serum.

[0047] Fig.19 This is the secondary mass spectrum of glycyrrhizic acid in drug-containing serum.

[0048] Fig. 20 This is the secondary mass spectrum of glycyrrhetinic acid in drug-containing serum.

[0049] Fig.21 This is the secondary mass spectrum of glycyrrhizin glucuronic acid in drug-containing serum. DETAILED DESCRIPTION

[0050] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with examples. The examples are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0051] Example:

[0052] A method for detecting Sijunzi Decoction decoction and drug-containing serum, the specific steps are as follows:

[0053] 1. Experimental Materials

[0054] ①Drugs and reagents

[0055] All the medicines were purchased from Laocunrentang Pharmacy in Zhenjiang City: White Ginseng Slices, produced in Jilin, batch number 170908, Jingjiang Baishan Ginseng and Cordyceps Co., Ltd.; Poria cocos, produced in Anhui, batch number 180101, Bozhou Jibeier Chinese Medicine Pieces Co., Ltd.; Roasted Licorice, produced in Gansu, batch number 171128, Jiangsu Huahong Pharmaceutical Technology Co., Ltd.; Atractylodes macrocephala, produced in Anhui, batch number 171201, Anhui Huangtai Chinese Medicine Pieces Technology Co., Ltd.

[0056] Reagents:

[0057] Acetonitrile (chromatographic grade): Tiandi Co., Ltd., USA;

[0058] Purified water: Hangzhou Wahaha Group Co., Ltd., China.

[0059] ② Experimental instruments

[0060] Ultra-low temperature refrigerator: SANYO, Japan;

[0061] Ultracentrifuge: Jintan Medical Instrument Factory, Jiangsu Province;

[0062] Liquid chromatography-ion trap mass spectrometry equipped with electrospray ionization source (ESI source): ThermoFisher, USA.

[0063] ③ Experimental animals

[0064] Rats, male, weighing 260-300 g, were provided by the Experimental Animal Center of Jiangsu University.

[0065] 2. Experimental Methods

[0066] ①According to the proportion of drugs in the prescription of Sijunzi Decoction, take 9 grams each of ginseng, Atractylodes macrocephala, Poria cocos, and 6 grams of roasted licorice, put them in a 500ml beaker, add 10 times water and soak for 0.5 hours, put the beaker in a heating jacket, boil, and then decoct for 1 hour, filter, and separate the filtrate and residue. Add 6 times water to the residue, boil and decoct for 1 hour, filter to obtain the filtrate and residue. Add water to the residue and boil for 1 hour, filter to obtain the filtrate. Combine the three filtrates and place them in a beaker. Place the beaker in a heating jacket to concentrate the drug solution to 0.125g / ml. Store at 4℃. However, due to the excessive mucopolysaccharide in the drug solution during the experiment, the concentration process is very easy to fail, so the operation is repeated several times to obtain the decoction.

[0067] ② Preparation of drug-containing serum

[0068] Rats were given concentrated Sijunzi decoction by gavage, 1mL / 100g, once a day for seven consecutive days. The control group was given distilled water, and the dosage was 10 times the clinical adult dosage. The rats were fasted but not watered for more than 12 hours before blood collection. They were anesthetized 2 hours after the last administration, and blood was collected from the heart. The blood was allowed to stand for 30 minutes, centrifuged (3000 rpm) for 10 minutes, and the upper liquid, i.e. serum, was aspirated with a pipette. The serum was sterilized with a 0.22μm microporous filter membrane, marked, and stored at -80℃ for future use.

[0069] ③Sample processing

[0070] Take about 4 ml of drug-containing serum, add 20 ml of methanol, mix quickly and shake, extract in a closed ultrasonic manner for 15 minutes, centrifuge at 3500r / min for 10 minutes, obtain the supernatant and put it into a crucible for heating and concentration, evaporate to dryness, add 1 ml of methanol to dissolve, filter with a 0.45μm filter membrane, and analyze.

[0071] ④Analysis conditions

[0072] Chromatographic conditions: Betasil C18 (4.6mm×250mm, 5μm) column, mobile phase A: water, B: acetonitrile; flow rate: 0.4mL·min-1; injection volume 20μL; column temperature: 35℃; sample room temperature: 4℃; gradient elution conditions are shown in Table 2-1.

[0073] Mass spectrometry conditions: electrospray ion source (ESI); spray voltage: 3.5 kV; negative ion detection; sheath gas (N2) flow rate: 80 arb; auxiliary gas (N2) flow rate: 5 arb; capillary temperature: 300 °C; capillary voltage: -30 V. Full ion scanning mode was used, the scanning range was m / z: 100-1200, the collision gas was helium; the collision chamber energy was 35 V.

[0074] The gradient elution conditions were as follows:

[0075]

[0076] 3. Experimental results:

[0077] ①Analysis of the ingredients of Sijunzi Decoction

[0078] In this experiment, acetonitrile-water was used as the mobile phase for gradient elution. From the results, the chromatographic separation effect was not particularly good, but the experimental results were mainly based on the mass spectrum, and a good mass spectrum was obtained. According to the information found, the main chemical components of Sijunzi Decoction are saponins, flavonoids, polysaccharides and other compounds. The total ion flow chart obtained by mass spectrometry detection using the negative ion electrospray ionization source mode is highly characteristic and sensitive. HPLC-MS-MS was used to qualitatively analyze the chemical components of Sijunzi Decoction. The detection wavelengths were 210nm, 240nm, and 280nm, and the mass spectrum of ESI-MS was obtained, as shown below. Figure 1 shown.

[0079] The following table shows the chemical composition analysis data of Sijunzi Decoction:

[0080]

[0081] Figure 2 This is the secondary mass spectrum of ginsenoside Rb1 in decoction, such as Figure 2 As shown:

[0082] At the retention time of 120.16min, an ion peak with m / z of 1107.48 was obtained. According to the data, the molecular formula of ginsenoside Rb1 in ginseng is C 54 H 92 O 23 , molecular weight 1109.29, estimated m / z 1107.48 [MH] - The quasi-molecular ion peak of m / z 1107.48 was subjected to MS 2 Analysis revealed fragment peaks such as m / z945.20, 782.88, 620.68, and 459.80. Based on the elemental composition analysis, it can be inferred that the compound is ginsenoside Rb1.

[0083] Figure 3 This is the secondary mass spectrum of ginsenoside Re in decoction, such as Figure 3 As shown:

[0084] At the retention time of 107.85min, an ion peak of m / z 945.75 was obtained. According to the data, the molecular formula of ginsenoside Re in ginseng is C 48 H 82 O 18 , molecular weight 947.17, estimated m / z 945.75 [MH] - The quasi-molecular ion peak of m / z 945.75 was subjected to MS 2 Analysis revealed fragment peaks of m / z 783.95, 637.20, 474.88, and 390.08. Based on the elemental composition analysis, it can be inferred that the compound is ginsenoside Re.

[0085] Figure 4 This is the secondary mass spectrum of ginsenoside Rg1 in decoction, such as Figure 4 As shown:

[0086] At the retention time of 105.47min, an ion peak with m / z 800.06 was obtained. According to the data, the molecular formula of ginsenoside Rg1 in ginseng is C42H72O14, and the molecular weight is 801.01. It is speculated that m / z 800.06 [MH]- is its quasi-molecular ion peak. MS2 analysis of m / z 800.06 obtained fragment peaks such as m / z 637.52, 475.62, and 391.52. Based on the elemental composition analysis, it can be speculated that the compound is ginsenoside Rg1.

[0087] Figure 5 This is the secondary mass spectrum of glycyrrhizic acid in decoction, such as Figure 5 As shown:

[0088] At the retention time of 158.39 min, an ion peak with m / z of 821.62 was obtained. According to the data, the molecular formula of glycyrrhizic acid in licorice is C42H62O16, and the molecular weight is 822.93. It is estimated that m / z

[0089] 821.62[MH]- is its quasi-molecular ion peak. MS2 analysis of m / z 821.62 yields fragment peaks such as m / z803.72, 645.71, and 351.29. Based on elemental composition analysis, it can be inferred that the compound is glycyrrhizic acid. The structure of glycyrrhizic acid is as follows:

[0090]

[0091] Figure 6 This is the secondary mass spectrum of liquiritin in the decoction.

[0092] Figure 7 This is the tertiary mass spectrum of the liquiritin mass fragment 255.21, such as Figure 7 As shown:

[0093] At a retention time of 98.78 min, an ion peak with m / z of 417.31 was obtained. According to the literature, the molecular formula of liquiritin in licorice is C 21 H 22 O9, molecular weight 418, estimated m / z 417 [MH] - The quasi-molecular ion peak of m / z 417 was analyzed by MS 2 Analysis revealed fragment peaks such as m / z 255.21 and 135.06. MS analysis was performed on m / z 255.21. 3Analysis revealed a fragment peak of m / z 135.05. Based on the elemental composition analysis, it can be inferred that the compound is liquiritin.

[0094] Figure 8 This is the secondary mass spectrum of ginsenoside Rb2 in decoction, such as Figure 8 As shown:

[0095] At the retention time of 127.21min, an ion peak with m / z of 1077.45 was obtained. According to the data, the molecular formula of ginsenoside Rb2 in ginseng is C 53 H 90 O 22 , molecular weight 1079.27, estimated m / z 1077.45 [MH] - The quasi-molecular ion peak of m / z 1077.45 was analyzed by MS 2 Analysis revealed fragment peaks such as m / z 946.09, 783.20, 621.38, and 458.86. Based on the elemental composition analysis, it can be inferred that the compound is ginsenoside Rb2.

[0096] Fig. 9 is the secondary mass spectrum of ginsenoside Rd in decoction, such as Fig. 9 As shown:

[0097] At the retention time of 136.55min, an ion peak with m / z of 946.61 was obtained. According to the data, the molecular formula of ginsenoside Rd in ginseng is C 48 H 82 O 18 , molecular weight 947.12, estimated m / z 946.61[MH] - The quasi-molecular ion peak of m / z 946.61 was subjected to MS 2 Analysis revealed fragment peaks of m / z 783.78, 621.56, 459.02, and 375.24. Based on the elemental composition analysis, it can be inferred that the compound is ginsenoside Rd.

[0098] Fig.10 This is the secondary mass spectrum of ginsenoside Rg3 in decoction, such as Fig.10 As shown:

[0099] At the retention time of 160.68min, an ion peak with m / z of 783.33 was obtained. According to the data, the molecular formula of ginsenoside Rg3 in licorice is C 42 H 72 O 13 , molecular weight 785.01, estimated m / z 783.33 [MH] - The quasi-molecular ion peak of m / z 783.33 was analyzed by MS2 Analysis revealed fragment peaks such as m / z 620.90 and 458.76. Based on the elemental composition analysis, it can be inferred that the compound is ginsenoside Rg3.

[0100] Fig.11 This is the secondary mass spectrum of licorice in decoction

[0101] Fig.12 It is the tertiary mass spectrum of the licorice fragment mass 135.06, such as Fig.12 As shown:

[0102] At the retention time of 68.09 min, an ion peak with m / z of 255.22 was obtained. According to the data, the molecular formula of licorice in licorice is C 15 H 12 O4, molecular weight 256.25, estimated m / z 255.22 [MH] - The quasi-molecular ion peak of m / z 255.22 was subjected to MS 2 Analysis, m / z 135.06 and other fragment peaks were obtained, and MS 3 Analysis revealed a fragment peak of m / z 90.95. Based on the elemental composition analysis, it can be inferred that the compound is glycyrrhizin. The structural formula of glycyrrhizin is as follows:

[0103]

[0104] The structural formula of Licorice Coumarin is as follows:

[0105]

[0106] Fig.13 This is the secondary mass spectrum of glycyrrhizic coumarin in the decoction. Fig.14 It is the tertiary mass spectrum of the glycyrrhizic coumarin fragment with a mass of 309.22, such as Fig.13 , 14 As shown:

[0107] At a retention time of 98.26 min, an ion peak with m / z of 367.34 was obtained. According to the literature, the molecular formula of glycyrrhizic coumarin in licorice is C 21 H 20 O6, molecular weight 368, estimated m / z 367.34

[0108] [MH] - The quasi-molecular ion peak of m / z 367.34 was subjected to MS 2 Analysis revealed fragment peaks of m / z 309.22 and 297.25. Based on the elemental composition analysis, it can be inferred that the compound is glycyrrhizic coumarin. Figure 2-15 , as shown in 2-16.

[0109] According to the mass spectra obtained by LC-MS, a total of 10 chemical substances were inferred from the decoction of Sijunzi Decoction, including 7 saponins, ginsenosides Rb1, Re, Rg1, Rb2, Rd, Rg3, and glycyrrhizic acid, and 3 flavonoids, liquiritigenin, liquiritigenin, and glycyrrhizic coumarin, of which ginsenosides Rb1, Re, Rg1, and glycyrrhizic acid were used as indicator components of Sijunzi Decoction. Because the terpenes and organic acids in Poria and the lactones in Atractylodes macrocephala are insoluble in water, no related compounds were detected in the decoction of Sijunzi Decoction.

[0110] ②Analysis of the components of drug-containing serum

[0111] The drug-containing serum was gradient eluted with acetonitrile-water solution to eliminate the interference of the elution solvent on the experimental results, and a better mass spectrum was obtained. The detection wavelength was 210nm, 240nm, and 280nm. The mass spectrum of ESI-MS was shown in Figure 15. The following table shows the chemical composition analysis data of the drug-containing serum:

[0112]

[0113] Fig.16 This is the secondary mass spectrum of ginsenoside Rb1 in drug-containing serum. Fig.17 This is a partial enlarged picture of ginsenoside Rb1 in the drug-containing serum. Fig.16 , 17 As shown:

[0114] At the retention time of 87.69min, an ion peak with m / z 1107.08 was obtained. According to the data, the molecular formula of ginsenoside Rb1 in ginseng is C54H92O23, and the molecular weight is 1109.29. It is estimated that m / z 1107.08 [MH] - For its quasi-molecular ion peak, MS2 analysis of m / z1107.08 obtained fragment peaks such as m / z945.20, 782.88, 620.68, and 459.80. Based on the elemental composition analysis, it can be inferred that the compound is ginsenoside Rb1.

[0115] Fig.18 is the secondary mass spectrum of ginsenoside Re in drug-containing serum, such as Fig.18 As shown:

[0116] At the retention time of 74.43min, an ion peak with m / z of 945.51 was obtained. According to the data, the molecular formula of ginsenoside Re in ginseng is C48H82O18, and the molecular weight is 947.17. It is estimated that m / z is 945.51 [MH] - The quasi-molecular ion peak of m / z 945.51 was subjected to MS 2Analysis revealed fragment peaks of m / z 798.78, 636.88, and 474.40. Based on the elemental composition analysis, it can be inferred that the compound is ginsenoside Re.

[0117] Fig.19 is the secondary mass spectrum of glycyrrhizic acid in drug-containing serum, such as Fig.19 As shown:

[0118] At the retention time of 126.34min, an ion peak with m / z 821.72 was obtained. According to the data, the molecular formula of glycyrrhizic acid in licorice is C42H62O16, and the molecular weight is 822.93. It is speculated that m / z 821.72[MH]- is its quasi-molecular ion peak. MS2 analysis of m / z 821.72 obtained fragment peaks such as m / z 644.85 and 350.62. According to the elemental composition analysis, it can be speculated that the compound is glycyrrhizic acid.

[0119] Fig. 20 is the secondary mass spectrum of glycyrrhetinic acid in drug-containing serum, such as Fig. 20 As shown:

[0120] At the retention time of 102.17min, an ion peak of m / z 469.50 was obtained. Literature reports that licorice metabolites contain glycyrrhetinic acid, with a molecular formula of C30H46O4 and a molecular weight of 470. It is speculated that m / z 469.50 is its quasi-molecular ion peak. MS 2 Analysis revealed fragment peaks such as m / z 423.47. Based on elemental composition analysis, it was inferred that the compound was glycyrrhetinic acid, which was formed by the hydrolysis of glycyrrhizic acid to remove the sugar acid chain.

[0121] Fig.21 is the secondary mass spectrum of glucuronic acid of glycyrrhizin in drug-containing serum, such as Fig.21 As shown:

[0122] At the retention time of 61.53 min, an ion peak of m / z 255.27 was obtained. Literature reported that glycyrrhizin glucuronic acid existed in licorice metabolites, with a molecular weight of 256. It was speculated that m / z 255.27 was its quasi-molecular ion peak. MS analysis was performed on m / z 255.27. 2 The fragment peaks of m / z 237.36, 195.28, and 135.12 were obtained. Based on the elemental composition analysis, it was inferred that the compound was glycyrrhizin glucuronic acid.

[0123] According to the mass spectra obtained by LC-MS, three chemical prototype components were inferred from the drug-containing serum, ginsenoside Rb1, Re, and glycyrrhizic acid; and two transitional components, glycyrrhetinic acid and glycyrrhizin glucuronic acid. Glycyrrhetinic acid is formed by the hydrolysis of glycyrrhizic acid to remove the sugar acid chain, and glycyrrhizin glucuronic acid is also generated by the hydrolysis of glycyrrhizic acid.

[0124] According to HPLC-MS technology, the decoction of Sijunzi Decoction and the serum containing the drug were tested, and the total ion flow diagram and the fragmentation peaks of the mass spectrum were obtained. It was inferred that the decoction of Sijunzi Decoction contained 10 chemical substances, including 7 saponins, ginsenosides Rb1, Re, Rg1, Rb2, Rd, Rg3, and glycyrrhizic acid, and 3 flavonoids, liquiritin, liquiritigenin, and glycyrrhizic coumarin. Ginsenosides Rb1, Re, Rg1, and glycyrrhizic acid are the necessary chemical components of ginseng and licorice stipulated in the pharmacopoeia, and are also the 4 indicator components in Sijunzi Decoction. The serum containing the drug contained three prototype chemical components of ginsenosides Rb1, Re, and glycyrrhizic acid, and two transitional components of glycyrrhetinic acid and glycyrrhizin glucuronic acid, all of which were produced by the hydrolysis of glycyrrhizic acid. Since the terpenes and organic acids in Poria cocos and the lactones in Atractylodes macrocephala are insoluble in water, and the extraction method in this experiment was water extraction, the related compounds in Poria cocos and Atractylodes macrocephala were not detected in the end. This experiment confirmed the active ingredients in Sijunzi Decoction and their metabolism in the blood.

[0125] As a further improvement, the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting Sijunzi Decoction decoction and drug-containing serum, characterized in that: The detection method comprises the following steps: S1. Preparation of decoction of Sijunzi Decoction: According to the prescription of Sijunzi Decoction, 9 grams each of ginseng, Atractylodes macrocephala, and Poria cocos and 6 grams of roasted licorice root were used to prepare decoction: S2. Preparation of drug-containing serum: Male SD rats were gavaged with concentrated Sijunzi decoction at a dose of 1 mL / 100 g for 7 consecutive days, once a day; the rats were centrifuged after standing, and the upper serum was taken, sterilized with a 0.22 μm microporous filter membrane, and stored at -80°C; S3. Sample processing: Mix the drug-containing serum with methanol, extract by ultrasonication, centrifuge and take the supernatant, heat, concentrate and evaporate to dryness, add methanol to dissolve, filter through a 0.45 μm filter membrane and then sample and analyze; S4. The analysis conditions are as follows: Chromatographic conditions: Betasil C18 column 4.6 mm × 250 mm, 5 μm, mobile phase water and acetonitrile, flow rate 0.4 mL / min, injection volume 20 μL; Mass spectrometry conditions: electrospray ion source ESI, negative ion mode, spray voltage 3.5 kV, capillary temperature 300 °C, full ion scan range m / z: 100-1200; S5. HPLC-MS-MS was used to conduct qualitative analysis on the chemical components of Sijunzi Decoction decoction and drug-containing serum.

2. The method for detecting the decoction of Sijunzi Decoction and the drug-containing serum according to claim 1, characterized in that: S1 also includes the following sub-steps: S101: adding 10 times the amount of water to the four raw materials and soaking them for 0.5 hours, heating and boiling them for 1 hour, and filtering and separating the filtrate and the residue; S102: Boil the residue with 6 times the amount of water, boil for 1 hour and filter; S103: adding water to the residue and boiling it for 1 hour, then filtering it and combining the three filtrates; S104: Concentrate to a drug concentration of 0.125 g / mL and store at 4°C.

3. The method for detecting the decoction of Sijunzi Decoction and the drug-containing serum according to claim 1, characterized in that: In S2, the mice were fasted for more than 12 hours after the last administration, and blood was collected under anesthesia 2 hours after the last administration.

4. The method for detecting the decoction of Sijunzi Decoction and the drug-containing serum according to claim 1, characterized in that: The centrifugal speed in S2 was 3000 rpm and the time was 10 min.

5. The method for detecting Sijunzi Decoction decoction and drug-containing serum according to claim 1, characterized in that: The gradient elution conditions were as follows:

6. The method for detecting Sijunzi Decoction decoction and drug-containing serum according to claim 1, characterized in that: The column temperature was 35°C.

7. The method for detecting Sijunzi Decoction decoction and drug-containing serum according to claim 1, characterized in that: When the chemical components of the Sijunzi Decoction decoction and the drug-containing serum were qualitatively analyzed in S5, the detection wavelengths were 210nm, 240nm, and 280nm.

8. The method for detecting Sijunzi Decoction decoction and drug-containing serum according to claim 1, characterized in that: The test results showed 10 chemical substances in the decoction of Sijunzi Decoction, including 7 saponins and 3 flavonoids; and 5 metabolic components in the drug-containing serum.

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