Method for detecting chemical components of eight-component kidney-tonifying capsules

Abstract

The invention relates to the field of traditional Chinese medicine detection, in particular to a method for detecting chemical components of eight-component kidney-tonifying capsules. Through UPLC-Q-TOF-MS detection, 229 compounds in vitro of the eight-component kidney-tonifying capsule are characterized, 4 alkaloids, 4 quinic acids, 3 sterone, 3 anthraquinones, 3 oligosaccharides and 21 other compounds in the eight-component kidney-tonifying capsule are preliminarily classified and qualitatively determined by adopting the method, and 56 peaks are compared with a standard substance. The detection method provided by the invention can perform qualitative analysis on chemical components of the eight-seed kidney-tonifying capsule, and has guiding significance for subsequent research on in-vivo metabolism, content determination and anti-osteoporosis biological activity evaluation of the eight-seed kidney-tonifying capsule.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine detection, and in particular to a method for detecting the chemical components of Bazi Bushen Capsule. Background Art

[0002] Traditional Chinese medicine plays an important role in the prevention and treatment of diseases, and therefore has received increasing attention and recognition worldwide. The quality of traditional Chinese medicine directly affects the clinical efficacy and restricts the development of modernization of traditional Chinese medicine. However, the complexity of chemical composition and the ambiguity of pharmacological substances are huge obstacles to the quality control of traditional Chinese medicine. In the process of strategy exploration, Liu et al. proposed the concept of quality markers. This concept focuses on the measurability, specificity, transferability, effectiveness and traceability of quality markers of traditional Chinese medicine. The characterization of quality markers involves the integration of multiple properties, including component analysis, content determination, pharmacokinetics, network pharmacology, etc. There are a variety of plant secondary metabolites in traditional Chinese medicine, and components with different structures may have different biological activities. Therefore, the quality markers of traditional Chinese medicine need to consider the different structural types of different components, which is of great significance for the quality control of traditional Chinese medicine based on traditional efficacy. In addition, network pharmacology is increasingly being used to reveal the mechanism of action between active ingredients and disease therapeutic targets. At present, the use of network pharmacology to screen quality markers is mainly based on the degree of association between a single compound and a disease target, and the mode of action of multiple components of traditional Chinese medicine covering multiple targets in the form of a combination to exert pharmacological effects is not prominent. In addition, the metabolism of TCM ingredients in the body will lead to changes in their biological activity, and the in vivo process of TCM ingredients is also particularly important for determining the quality markers of TCM. It is well known that TCM prescriptions have the characteristics of multi-component, multi-target, and overall regulation, but there are currently few reports on mining TCM quality markers from a holistic perspective. Therefore, this study proposed a digital and holistic strategy to mine the quality markers of absorbable ingredients in Bazi Bushen Capsule in the form of different compositions to reflect the structural diversity and diversity of biological pathways.

[0003] Bazi Bushen Capsule (BZBS) is a traditional Chinese medicine compound, which is composed of 16 single herbs, including Cuscuta, Lycium, Schisandra, Cnidium, Rosa Laevigata, Rubus, Toosendan, Allium, Epimedium, Morinda, Cistanche, Rehmannia, Cyathula, Ginseng, Pilose Antler, and Hippocampus. It is clinically used to prevent and treat symptoms such as soreness of waist and knees, dizziness, tinnitus, and fatigue caused by insufficient kidney yang. Recent pharmacological studies have shown that Bazi Bushen Capsule can significantly improve the bone morphology of aged mice induced by D-galactose and NaNO2, and increase the mineral content in their bone tissue. In addition, Bazi Bushen Capsule can also improve the bone microstructure of the distal tibia in ovariectomized osteoporosis rats. A large number of literature reports show that a variety of single herbs in Bazi Bushen Capsule, such as Cuscuta, Lycium barbarum, Cnidium monnieri, Epimedium, Ginseng, Cistanche deserticola and Rehmannia root, can improve the bone microstructure parameters of ovariectomized osteoporosis model rats / mice. In summary, Bazi Bushen Capsule has a significant anti-osteoporosis effect. Bazi Bushen Capsule has a definite efficacy, but the quality control research of Bazi Bushen Capsule is seriously insufficient. Therefore, it is urgent to find the quality markers of Bazi Bushen Capsule to improve its quality control level.

[0004] It is worth noting that Bazibushen Capsule is a complex system composed of 16 single herbs, and its chemical composition has not been reported, which seriously hinders the quality control research of Bazibushen Capsule. Summary of the invention

[0005] In view of this, the present invention provides a method for detecting the chemical components of Bazi Bushen Capsule. The detection method characterizes 229 compounds in Bazi Bushen Capsule in vitro.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a method for detecting the chemical components of Bazi Bushen Capsule, comprising the following steps:

[0008] Take the contents of Bazi Bushen Capsule to prepare sample solution;

[0009] Take the standard sample to prepare the standard solution;

[0010] The sample solution and the standard solution were respectively taken for UPLC-Q-TOF-MS detection to determine the chemical components of the Bazi Bushen Capsule.

[0011] In some specific embodiments of the present invention, the detection comprises chromatography and mass spectrometry;

[0012] The mobile phase of the chromatographic analysis includes: A is a 0.1% (v / v) formic acid solution, and B is a 0.1% (v / v) formic acid acetonitrile solution.

[0013] In some specific embodiments of the present invention, the flow rate of the chromatography is 0.4 ml / min; and / or

[0014] The injection volume of the chromatographic analysis was 1 μL.

[0015] In some specific embodiments of the present invention, the gradient elution conditions of the chromatographic analysis are: 0-3.5 min, 2%-15% B; 3.5-12.5 min, 15%-36% B; 12.5-19.5 min, 36%-55% B; 19.5-23.5 min, 55%-80% B; 23.5-26 min, 80%-100% B; 26-28 min, 100% B; 28-29 min, 100%-2% B; 29-30 min, 2% B.

[0016] In some specific embodiments of the present invention, the chromatographic analysis uses an ACQUITY UPLC BEH C18 chromatographic column (2.1 mm×100 mm, 1.7 μm), and the temperature of the chromatographic column is 35°C.

[0017] In some specific embodiments of the present invention, the mass spectrometry analysis uses an electrospray ion source (ESI), positive and negative ion mode detection, the acquisition method is FastDDA, the scanning range is m / z 50 to 1500Da, the capillary spray voltage ion is 3.0kV (ESI+) and 2.1kV (ESI-), and the cone voltage is 35V.

[0018] In some specific embodiments of the present invention, the source temperature of the mass spectrometry analysis is 140° C., the desolvation gas temperature is 500° C., the desolvation gas flow rate is 1000 L / h, and the cone gas flow rate is 50 L / h.

[0019] In some specific embodiments of the present invention, the low-mass end energy of the dual dynamic collision energy of the mass spectrometry analysis is set to 6-30 eV, and the high-mass end energy is set to 40-90 V; when the TIC intensity rises to above 5000 intensity / second, it automatically switches to MS / MS mode, and switches back to MS patrol scan after five MS / MS scans; the MS and MS / MS patrol scan times are both set to 0.2 seconds.

[0020] In some specific embodiments of the present invention, the sample solution is prepared by: dissolving the contents of the Bazi Bushen Capsule with methanol, ultrasonically extracting, centrifuging, filtering, and collecting the supernatant to obtain the sample solution;

[0021] The preparation method of the standard solution is: dissolving the standard in methanol, centrifuging, filtering, collecting the supernatant, and obtaining the standard solution.

[0022] In some specific embodiments of the present invention, the sample solution is prepared by taking the contents of the Bazi Bushen Capsule, dissolving and extracting it with a 70% methanol solution, ultrasonically treating it for 30 minutes to obtain a 50 mg / ml sample solution, centrifuging the sample solution at 13200 rpm for 10 minutes, filtering it, and collecting the supernatant.

[0023] The preparation method of the standard solution is as follows: dissolving the standard in methanol, centrifuging at 13200 rpm for 10 minutes, filtering, and collecting the supernatant.

[0024] In a second aspect, the present invention also provides a method for identifying the contents of Bazi Bushen Capsule, wherein the sample to be tested is tested according to the detection method described in the present invention and identified according to the following standards:

[0025] (I), chemical components including lignans, prenyl flavonoids, coumarins, ginsenosides, flavonols, phenylethanoid glycosides, triterpenes, iridoids, organic acids, spermidines, phospholipids, resin glycosides, nucleotides, alkaloids, quinic acid, sterones, anthraquinones, oligosaccharides and 21 other compounds; and / or

[0026] (II), chemical composition as shown in Table 1;

[0027] If the test result meets the standard, it is the content of Bazi Bushen Capsule;

[0028] If the test results do not meet the standards, they are not the contents of the Bazi Bushen Capsule.

[0029] The present invention characterizes 229 compounds in vitro and in plasma of Bazi Bushen Capsule by UPLC-Q-TOF-MS detection, including 34 lignans, 29 prenyl flavonoids, 26 coumarins, 18 ginsenosides, 16 flavonols, 12 phenylethanoid glycosides, 11 triterpenes, 10 cyclohexene ether terpenes, 10 organic acids, 9 spermidines, 7 phospholipids, 5 resin glycosides, 4 nucleotides, 4 alkaloids, 4 quinic acids, 3 sterones, 3 anthraquinones, 3 oligosaccharides and 21 other compounds. The above method is used to preliminarily classify and characterize the 4 alkaloids, 4 quinic acids, 3 sterones, 3 anthraquinones, 3 oligosaccharides and 21 other compounds in Bazi Bushen Capsule, and 56 peaks thereof are compared with standard products. The detection method provided by the present invention can perform qualitative analysis on the chemical components of Bazi Bushen Capsule, and has guiding significance for subsequent research on in vivo metabolism, content determination, and anti-osteoporosis biological activity evaluation of Bazi Bushen Capsule. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0031] Figure 1 Shows the base peak intensity (BPI) chromatograms of BZBS capsules in (A) positive ion mode and (B) negative ion mode; wherein, (A) shows the positive ion mode; (B) shows the negative ion mode;

[0032] Figure 2 The chemical structures of the identified and annotated components in BZBS capsules are shown; (A), (B), and (C) represent different components, respectively; red: compared with the reference standard;

[0033] Figure 3 The main degradation pathways of lignans are shown;

[0034] Figure 4 The main cleavage pathways of prenylated flavonoids are shown;

[0035] Figure 5 The main fragmentation pathways of coumarin are shown;

[0036] Figure 6 Show the main fragmentation pathways of ginsenosides;

[0037] Figure 7 The main fragmentation pathways of flavonols are shown;

[0038] Figure 8 The main cleavage pathways of phenylethanoid glycosides are shown;

[0039] Fig. 9 The main cleavage pathways of iridoid glycosides are shown;

[0040] Fig.10 Shown is the main cleavage pathway of spermidine. DETAILED DESCRIPTION

[0041] The present invention discloses a method for detecting the chemical components of Bazi Bushen Capsule. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] The present invention preliminarily classified and characterized 229 compounds in the contents of Bazi Bushen Capsule based on ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), and the chromatographic parameters are as follows:

[0043] Chromatographic system: Waters ACQUITY UPLC I-Class Plus system

[0044] Chromatographic column: ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm);

[0045] Column temperature: 35 °C;

[0046] Mobile phase: A is 0.1% (v / v) formic acid solution, B is 0.1% (v / v) formic acid acetonitrile solution;

[0047] Flow rate: 0.4ml / min;

[0048] Injection volume: 1 μL;

[0049] Gradient elution conditions: 0-3.5min, 2%-15% B; 3.5-12.5min, 15%-36% B; 12.5-19.5min, 36%-55% B; 19.5-23.5min, 55%-80% B; 23.5-26min, 80%-100% B; 26-28min, 100% B; 28-29min, 100%-2% B; 29-30min, 2% B;

[0050] Mass spectrometer: Waters Xevo G2-XS QTOF

[0051] Mass spectrometry conditions: electrospray ion source (ESI), positive and negative ion mode detection; acquisition method Fast DDA; scanning range m / z 50-1500Da; capillary spray voltage ion 3.0kV (ESI+) and 2.1kV (ESI-); cone voltage 35V; source temperature 140℃; desolvation gas temperature 500℃; desolvation gas flow rate 1000L / h, cone gas flow rate 50L / h; dual dynamic collision energy low mass end energy set to 6-30eV, high mass end energy set to 40-90V; automatically switch to MS / MS mode when TIC intensity rises to more than 5000 intensity / second, and switch back to MS inspection scan after five MS / MS scans; MS and MS / MS inspection scan times are both set to 0.2 seconds;

[0052] Sample and standard solution preparation method: Take 2g of the contents of Bazi Bushen Capsule, use 40ml of 70% methanol solution to dissolve and extract, use ultrasonic treatment for 30min to obtain 50mg / ml sample solution, centrifuge the sample solution at 13200rpm for 10 minutes, filter, and the supernatant is the injection solution.

[0053] The raw materials and reagents used in the method for detecting the chemical components of the Bazi Bushen Capsule provided by the present invention can all be purchased from the market.

[0054] The present invention will be further described below in conjunction with embodiments:

[0055] Example 1 Chemical composition analysis and sample preparation of Bazi Bushen Capsule

[0056] 1.1 Sample pretreatment

[0057] Weigh 2g of the contents of Ba Zi Bushen Capsule and 16 ingredients (Cuscuta, Lycium, Schisandra, Cnidium, Rosa Laevigata, Rubus, Toosendan, Allium, Epimedium, Morinda, Cistanche, Rehmannia, Cyathula, Ginseng, Pilose Antler, Hippocampus), add 40mL 70% methanol-water solution, and ultrasonically extract for 30 minutes to obtain a sample solution with a concentration of 50mg / mL. In order to summarize the characteristic ions of each structural type, 56 chemical standard substances were dissolved in methanol to obtain a standard solution. Finally, centrifuge at 13200rpm for 10 minutes, and take 1μL of the supernatant for UPLC / Q-TOF-MS analysis.

[0058] 1.2 UPLC-Q-TOF-MS conditions

[0059] The samples were measured on a Waters ACQUITY UPLC I-Class Plus system equipped with an autosampler, an online degasser, a binary solvent delivery system, and a Waters Xevo G2-XS Q TOF with an ESI interface. Chromatographic separation was performed on an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) at a column temperature of 35 ° C. The mobile phases included 0.1% (v / v) formic acid in water (phase A) and 0.1% (v / v) formic acid in acetonitrile (phase B). Flow rate: 0.4 mL / min; elution gradient: 0-3.5 minutes, 2%-15% B; 3.5-12.5 minutes, 15%-36% B; 12.5-19.5 minutes, 36%-55% B; 19.5-23.5 minutes, 55%-80% B; 23.5-26 minutes, 80%-100% B; 26-28 minutes, 100% B; 28-29 minutes, 100%-2% B; 29-30 minutes, 2% B.

[0060] In positive and negative ESI modes, comprehensive mass spectrometric information was obtained using Fast-DDA mode. The optimal conditions were set as follows: mass range 50-1500Da; capillary voltage 3.0kV (ESI +) and 2.1kV (ESI-); cone voltage was 35V; ion source temperature was 140℃; desolvation temperature was 500℃; cone backflush gas flow rate was 50L / h; desolvation gas flow rate was 1000L / h; low channel collision energy was 6-30eV, and high channel collision energy was 40-90V; when the TIC intensity exceeded 5000intensity / s, it automatically switched to MS / MS mode and switched back to MS scan after five MS / MS scans; the MS and MS / MS survey scan times were both set to 0.2. In one MS scan, the five ions with the highest response were automatically selected for MS / MS. The mass accuracy was calibrated using a leucine-enkephalin solution internal standard (m / z556.2771[M+H] in positive ion mode) + , m / z 554.2615 [MH]-) in negative ion mode. LockSpray TM The flow rate was 5 μL / min.

[0061] 1.3 Data processing

[0062] Data acquisition was performed using Waters Masslynx4.2 mass spectrometer control platform and MZ mine2.5.3 data processing software. Total Ion Chromatogram (TIC) of the analyzed samples was acquired in Fast-DDA mode.

[0063] Example 2 Characterization of multiple component types in Bazi Bushen Capsule

[0064] The present invention adopts the method of Example 1 to preliminarily classify and characterize 229 ingredients in Bazi Bushen Capsule, including 34 lignans, 29 isopentenyl flavonoids, 26 coumarins, 18 ginsenosides, 16 flavonols, 12 phenylethanoid glycosides, 11 triterpenes, 10 cyclohexene ether terpenes, 10 organic acids, 9 spermidines, 7 phospholipids, 5 resin glycosides, 4 nucleotides, 4 alkaloids, 4 quinic acids, 3 sterones, 3 anthraquinones, 3 oligosaccharides and 21 other compounds. The above method is used to preliminarily classify and characterize 4 alkaloids, 4 quinic acids, 3 sterones, 3 anthraquinones, 3 oligosaccharides and 21 other compounds in Bazi Bushen Capsule, and 56 peaks thereof are compared with standard products. Figure 2 Chemical structures of identified and resolved components in Bazi Bushen Capsule. Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 and Fig.10The main fragmentation pathways (characteristic ions and neutral losses) of lignans, prenylflavones, coumarins, ginsenosides, flavonols, phenylethanoid glycosides, iridoids, and spermidines were shown. The detailed mass information extracted from Bazi Bushen Capsule is shown in Table 1 , respectively.

[0065] 2.1 Lignans

[0066] A total of 34 lignans were identified in Bazi Bushen Capsule, all of which were from Schisandra chinensis. When methoxy groups are present in lignans, one or more methyl groups or residues will undergo neutral losses. Therefore, the neutral losses of lignans usually include CH2 (14Da), CH3 (15Da), OCH2 (30Da), and OCH3 (31Da). Some side chain groups containing ester bonds on the lignan ring will also break the chain to form neutral losses of C5H6O (82Da) or C7H4O (104Da). In addition, the characteristic ions of lignans include m / z415.21, m / z384.20, m / z401.20, m / z399.18, m / z415.17, and m / z368.16 in positive ion mode. The main fragmentation pathways of lignans can be seen in Figure 3 . For example, peaks 157 and 174 show [M+H]+ and [M+Na]+ ions at m / z433.2221 and m / z455.2039, respectively. Their empirical molecular formulas are both considered to be C24H32O7. After comparison with the SIRIUS software database, schisandrin is consistent with its molecular formula. In the MS / MS spectrum, the characteristic ion [M+H-H2O]+ at m / z415.2134 is generated by the loss of H2O. In addition, m / z400.1883[M+H-H2O-CH3]+, m / z384.1948[M+H-H2O-OCH3]+, m / z369.1703

[0067] [M+H-H2O-OCH3-CH3]+,m / z353.1720[M+H-H2O-2OCH3]+ and m / z338.1516

[0068] The characteristic ions of [M+H-H2O-2OCH3-CH3]+ were generated by the loss of methyl and methoxy groups. Therefore, peak 157 was identified as schisandrin by comparison with the reference standard. Peak 174 was tentatively characterized as a schisandrin isomer. Other lignans were inferred based on similar fragmentation pathways and identified by comparison with the reference standard, SIRIUS software database, and MS2LDA.

[0069] 2.2 Prenylated flavonoids

[0070] A total of 29 prenylated flavonoids were detected from Epimedium. In positive ion mode, prenylated flavonoids easily lost glucose, rhamnose and xylose molecules, generating typical fragment ions of m / z 369, 313, 355 and 299. In addition, neutral losses of methyl (CH3, 15Da), carbonyl CO (28Da) and isopentenyl (C4H8, 56Da) of some prenylated flavonoids were observed. Figure 4 The main fragmentation pathways of prenylated flavonoids were shown. Seven compounds were confirmed by alignment in the SIRIUS software database and comparison with reference standards, including epimedinA (peak 102), epimedinB (peak 103), epimedinC (peak 104), icariin (peak 106), icariside I (peak 150), icariside II (peak 165) and icaritin (peak 202). Peak 99 produced a protonated molecular ion m / z 839.2971 [M+H]+ (same as peak 102) with a molecular formula of C39H50O20. Then, characteristic ions were generated at m / z 677.2448 [M+H-Glc]+, m / z 531.1866 [M+H-Glc-Rha]+, m / z 369.1338

[0071] [M+H-2Glc-Rha]+ and m / z313.0711[M+H-2Glc-Rha-Isopentenyl]+ appeared, indicating the loss of glucose, rhamnose and isopentenyl groups. Therefore, it was preliminarily identified as epimedin A isomer. Based on the summarized characteristic ions and neutral loss (MS2LDA), other isopentenyl flavonoids were identified.

[0072] 2.3 Coumarin

[0073] In this study, 26 coumarins in Bazi Bushen Capsule were identified. They are mainly derived from Cnidium monnieri. In the MS / MS spectrum (positive ion mode), neutral loss of carbonyl CO (28Da), methoxy (30Da), methyl (15Da) and characteristic ions of m / z189.06, m / z 151.08, m / z131.05, m / z203.11 and m / z147.04 can be usually detected. The main fragmentation paths of coumarins are shown in Figure 5 Peak 182 has a quasi-molecular ion (C 15 H 16 O3). The product ion is m / z189.0579[M+H-C4H8] + 、m / z159.0448[M+H-C4H8-CH2O] +, m / z131.0514[M+H-C4H8-CH2O-CO] + and m / z 103.0553 [M+H-C4H8-CH2O-2CO] + This corresponds to the consecutive loss of isopentenyl (C4H8, 56Da), methoxy (CH2O, 30Da) and carbonyl (CO, 28Da) groups in the MS / MS spectrum. Therefore, peak 182 was characterized as osthole and further confirmed by comparison with the reference standard. Peak 74 at m / z 203.0341 (C 11 H6O4) a quasi-molecular ion [M+H] + In the MS / MS spectrum, CO (28 Da) and CO2 (44 Da) disappeared successively, and the characteristic fragment ion m / z 175.0390 [M+H-CO] was observed. + 、m / z147.0444[M+H-2CO] + 、m / z119.0462[M+H-3CO] + 、m / z131.0497

[0074] [M+H-CO-CO2] + Therefore, peak 74 was identified as xanthotoxol. Based on these similar fragmentation patterns and online database comparison, other coumarins were also preliminarily identified, and the fragment ions are shown in Table 1.

[0075] 2.4 Ginsenosides

[0076] A total of 20 ginsenosides were identified from Bazi Bushen Capsule. The main types of ginsenosides are protopanaxatriol, protopanaxadiol and oleanolic acid. The characteristic ions of ginsenoside types are relatively stable, as follows: (1) Protopanaxatriol, m / z 425.38, m / z 407.37 and m / z 325.11 (ESI + ); m / z 621.44, m / z 459.39 and m / z 323.10 (ESI - ); (2) protopanaxadiol, m / z 441.37 and m / z 423.36 (ESI + ); m / z 637.43 and m / z 475.38 (ESI - ); (3) oleanolic acid, m / z 439.36 (ESI +); m / z569.39, m / z523.38 and m / z455.36 (ESI-). Most ginsenosides contain at least one sugar chain, which is generally connected to monosaccharides or oligosaccharides such as glucose (Glc), rhamnose (Rha), arabinose (Ara) and xylose (Xyl) in different substitution patterns. Neutral losses of 162Da, 146Da and 132Da can be observed in the MS / MS spectrum. The monosaccharide or oligosaccharide fragments of most ginsenosides are similar. The main fragmentation pathways of ginsenosides are shown in Figure 6 For example, peaks 91 and 116 yield [M+Na] at m / z 823.4819 and m / z 845.4902, respectively. + and [M+HCOOH-H] - ions. Their molecular formula is C 42 H 72 O 14 .m / z

[0077] 621.4368[M+H-Glc-H2O] + 、m / z441.3730[M+H-2Glc-2H2O] + 、m / z

[0078] 423.3631[M+H-2Glc-3H2O] + and m / z 405.3533 [M+H-2Glc-4H2O] + The characteristic ions are generated by the continuous loss of Glc and H2O. Therefore, by comparison with the reference standard, peak 91 was identified as ginsenoside Rg1. By comparison in the SIRIUS software database, peak 116 was preliminarily identified as ginsenoside Rf or ginsenoside Rg7. Based on similar fragmentation pathways, other ginsenosides in Bazi Bushen Capsule were also preliminarily identified.

[0079] 2.5 Characteristics of other types of ingredients

[0080] In addition to the four main types mentioned above, there are 16 flavonols (from Cuscuta, Lycium, Rubus, Toosendan and Epimedium), 12 phenylethanoid glycosides (from Cistanche and Rehmannia), 11 triterpenoids (from Rosaceae plants Rosa laevigata, Toosendan and Rubus), 10 cycloterpenes (from Cistanche, Morinda officinalis and Rehmannia), 10 organic acids, 9 spermidines (from Lycium, 7 phospholipids, 5 resin glycosides (from Cuscuta), 4 nucleotides (extracted from seahorse and deer antler), 4 alkaloids (extracted from Cuscuta, Lycium and Epimedium), 4 quinic acids, 3 steroids (extracted from Cyathula, seahorse and deer antler), 3 anthraquinone compounds (from seahorse and deer antler), and 3 oligosaccharides (from Morinda officinalis and Rehmannia). Figure 7 , Figure 8 , Fig. 9 , Fig.10 The main fragmentation pathways of flavonols, phenylethanoid glycosides, iridoids and spermidine are shown. Peaks 88 and 96 show the quasi-molecular ion m / z 271.1084 (C 15 H 14 N2O3). Fragment ion m / z 225.10 [M+H-CO-H2O] + 、m / z 197.11[M+H-2CO-H2O] + and m / z 180.08 [M+H-2CO-H2O-NH3] + , corresponding to the loss of CO, H2O and NH3, respectively. Therefore, by comparison with the reference peak, peak 88 was determined to be cuscutamine. Peak 96 was preliminarily identified as an isomer of cuscutamine. Peaks 38 and 40 appeared [MH] at m / z 785.2494. - ions. Their empirical molecular formulas are all considered to be C 35 H 46 O 20 [MH-Caffeoyl] at m / z 623.2180 - The characteristic ion is caused by the loss of the caffeoyl group. Then, m / z 161.0245 [Caffeoyl-H] is generated. - and m / z 133.2092[Caffeoyl-H-CO] - Therefore, by comparison with the reference standard, peak 40 was identified as echinacoside. Peak 38 was preliminarily identified as an echinacoside isomer. Mass analysis of peak 32 in positive ion mode revealed that its quasi-molecular ion peak was m / z 798.3644 ([M+H] + ,C 37 H 55 N3O 16). In the MS / MS spectrum, the product ion of peak 32 is m / z 636.3098 [M+H-Glc] + 、m / z 474.2599[M+H-2Glc] + 、m / z222.1133[C 12 H 16 NO3] + and m / z 165.0555 [C9H9O3] + Peak 32 was identified as lycibarbarspermidine M by comparison with similar fragmentation pathways in spermidine using the SIRIUS software database. Peak 69 appeared at m / z 521.3098 and m / z 565.3013 with [M+H] + and [M+HCOOH-H] - Ion, molecular formula C 29 H 44 O8, in the MS / MS spectrum, due to the continuous loss of H2O, produced m / z 503.30 [M+H-H2O] + ,m / z 485.29[M+H-2H2O] + ,m / z467.28[M+H-2H2O] + The product ion was confirmed to be cyasterone by comparison with the reference standard.

[0083] It is worth noting that through intuitive analysis, the main structural types of the ingredients of Bazi Bushen Capsule with high mass spectrometry response are lignans in Schisandra chinensis, prenylflavones in Epimedium, coumarins in Cnidium monnieri, ginsenosides in Ginseng, etc. In addition, hyperin and cuscutamine in Cuscuta chinensis also have high mass spectrometry responses.

[0084] Table 1. Characterization of chemical components of Bazi Bushen Capsule

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] Cuscutae Semen, Lycium barbarum, Schisandrae Chinensis Fructus, Cnidii Fructus, Rosae Laevigatae Fructus, Rubi Fructus, Toosendan Fructus, Semen allii tuberosi, Epimedium, Morindae Officinalis Radix, Cistanches Herba, Rehmanniae Radix, Cyathulae Radix, Ginseng Radix Et Rhizoma, Deer Antler, Hippocampus

[0097] *: Confirmed by reference material

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting the chemical components of Bazi Bushen Capsule, characterized in that: The steps include: Take the contents of Bazi Bushen Capsule to prepare sample solution; Take the standard sample to prepare the standard solution; The sample solution and the standard solution were respectively taken for UPLC-Q-TOF-MS detection to determine the chemical components of the Bazi Bushen Capsule.

2. The detection method according to claim 1, characterized in that The detection includes chromatography and mass spectrometry; The mobile phase of the chromatographic analysis includes: A is a 0.1% (v / v) formic acid solution, and B is a 0.1% (v / v) formic acid acetonitrile solution.

3. The detection method according to claim 2, characterized in that The flow rate of the chromatographic analysis is 0.4 ml / min; and / or The injection volume of the chromatographic analysis was 1 μL.

4. The detection method according to claim 2 or 3, characterized in that: The gradient elution conditions of the chromatographic analysis are: 0-3.5 min, 2%-15% B; 3.5-12.5 min, 15%-36% B; 12.5-19.5 min, 36%-55% B; 19.5-23.5min, 55%-80%B; 23.5-26min, 80%-100%B; 26-28min, 100%B; 28-29min, 100%-2%B; 29-30min, 2%B.

5. The detection method according to any one of claims 2 to 4, characterized in that: The chromatographic analysis used an ACQUITY UPLC BEH C18 chromatographic column, and the temperature of the chromatographic column was 35°C.

6. The detection method according to claim 2, characterized in that: The mass spectrometry analysis adopts an electrospray ion source, positive ion and negative ion mode detection, the acquisition method is Fast DDA, the scanning range is m / z 50-1500Da, the capillary spray voltage ion is 3.0kV (ESI+) and 2.1kV (ESI-), and the cone voltage is 35V.

7. The detection method according to any one of claims 2 to 6, characterized in that: The source temperature of the mass spectrometry analysis was 140° C., the desolvation gas temperature was 500° C., the desolvation gas flow rate was 1000 L / h, and the cone gas flow rate was 50 L / h.

8. The detection method according to any one of claims 2 to 7, characterized in that: The dual dynamic collision energy of the mass spectrometry analysis is set to 6 to 30 eV at the low mass end and 40 to 90 eV at the high mass end; when the TIC intensity rises to more than 5000 intensity / second, it automatically switches to MS / MS mode, and switches back to MS inspection scanning after five MS / MS scans; The scan time for both MS and MS / MS was set to 0.2 s.

9. The detection method according to any one of claims 1 to 8, characterized in that: The sample solution is prepared by dissolving the contents of the Bazi Bushen Capsule with methanol, ultrasonically extracting, centrifuging, filtering, and collecting the supernatant to obtain the sample solution; The preparation method of the standard solution is: dissolving the standard in methanol, centrifuging, filtering, collecting the supernatant, and obtaining the standard solution.

10. A method for identifying the contents of Bazi Bushen Capsule, characterized in that: Take the sample to be tested and test it according to the detection method according to any one of claims 1 to 9, and identify it according to the following standards: (I), chemical components including lignans, prenyl flavonoids, coumarins, ginsenosides, flavonols, phenylethanoid glycosides, triterpenes, iridoids, organic acids, spermidines, phospholipids, resin glycosides, nucleotides, alkaloids, quinic acid, sterones, anthraquinones, oligosaccharides and 21 other compounds; and / or (II), chemical composition as shown in Table 1; If the test result meets the standard, it is the content of Bazi Bushen Capsule; If the test results do not meet the standards, they are not the contents of the Bazi Bushen Capsule.

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