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Qualitative and quantitative analysis of the chemical constituents in Mahuang-Fuzi-Xixin decoction based on high performance liquid chromatography combined with time-of-flight mass spectrometry and triple quadrupole mass spectrometers: Qualitative and quantitative analysis
Abstract
High performance liquid chromatography coupled with time-of-flight mass spectrometry (HPLC-TOF/MS) and high performance liquid chromatography - triple quadrupole mass spectrometry (HPLC-QQQ/MS/MS) were utilized to clarify the chemical constituents of Mahuang-Fuzi-Xixin Decoction. There are 52 compounds, including alkaloids, amino acids and organic acids were identified or tentatively characterized by their characteristic high resolution mass data by HPLC-QQQ/MS/MS. In the subsequent quantitative analysis, 10 constituents, including methyl ephedrine, aconine, songrine, fuziline, neoline, talatisamine, chasmanine, benzoylmesaconine, benzoylaconine and benzoylhypaconine were simultaneously determined by HPLC-QQQ/MS/MS with multiple reaction monitoring (MRM) mode. Satisfactory linearity was achieved with wide linear range and fine determination coefficient (r > 0.9992). The relative standard deviations (RSD) of inter-and intra-day precisions were less than 3% .This method was also validated by repeatability, stability and recovery with RSD less than 3% respectively. A high sensitive and efficient method was established for chemical constituents studying, including identification and quantification of Mahuang-Fuzi-Xixin decoction.
... SPF Male BALB/c mice (16-18 g) were purchased from the Jinan Pengyue Experimental Animal Breeding Company Limited. After three days acclimation, all mice were randomly assigned to two groups: KYDS mice (model group) were established by intraperitoneal injection of estradiol benzoate (8 mg/kg, Ningbo Second Hormone Factory, China) for 7 d as previously described [21,23] and normal control mice (normal group) were intraperitoneally injected with normal saline. e physical signs in mice were observed, and spontaneous activity, swimming time, rectal temperature, and body weight were monitored. ...
... In this study, we established a KYDS mouse model with a classic method by intraperitoneally injecting estradiol benzoate as previously described [21,23] and combined with influenza A (H1N1) virus infection to imitate high-risk patients who were infected with influenza A virus. e KYDS mice and normal mice were separately infected with the influenza A (H1N1) virus through nose dropping and tracheal injection. ese data manifested that the KYDS virus group (MI and MD) had a significant decrease in body weights and rectal temperatures compared with normal control mice (Figures 2(a) and 2(b)). ...
Objective. Influenza virus poses a major threat to human health and has serious morbidity and mortality which commonly occurs in high-risk populations. Pharynx and larynx of the upper respiratory tract mucosa is the first defense line against influenza virus infection. However, the ability of the pharynx and larynx organ to eliminate the influenza pathogen is still not clear under different host conditions. Methods. In this study, a mouse model of kidney yang deficiency syndrome (KYDS) was used to mimic high-risk peoples. Two different methods of influenza A (H1N1) virus infection by nasal dropping or tracheal intubation were applied to these mice, which were divided into four groups: normal intubation (NI) group, normal nasal dropping (ND) group, model intubation (MI) group, and model nasal dropping (MD) group. The normal control (NC) group was used as a negative control. Body weight, rectal temperature, and survival rate were observed every day. Histopathologic changes, visceral index, gene expressions of H1N1, cytokine expressions, secretory IgA (SIgA) antibodies of tracheal lavage fluids in the upper respiratory tract, and bronchoalveolar lavage fluids were analyzed by ELISA. Results. The MD group had an earlier serious morbidity and mortality than the others. MI and NI groups became severe only in the 6th to 7th day after infection. The index of the lung increased significantly in NI, MI, and MD groups. Conversely, indices of the thymus and spleen increased significantly in NC and ND groups. H&E staining showed severe tissue lesions in MD, MI, and NI groups. H1N1 gene expressions were higher in the MD group compared with the MI group on the 3rd day; however, the MD group decreased significantly on the 7th day. IL-6 levels increased remarkably, and SIgA expressions decreased significantly in the MD group compared with the NC group. Conclusions. SIgA secretions are influenced directly by different conditions of the host in the pharynx and larynx in the upper respiratory tract mucosa. In the KYDS virus disease mode, SIgA expressions could be inhibited severely, which leads to serious morbidity and mortality after influenza A virus infection. The SIgA expressions of the pharynx and larynx would be an important target in high-risk populations against the influenza A virus for vaccine or antiviral drugs research.
1. Introduction
Influenza is a highly infectious disease that causes a worldwide public health problem in millions of peoples per year. The most common influenza A (H1N1) virus can cause seasonal infections with significant morbidity and mortality in elderly and high-risk adults all around the world [1–5]. It remains unclear why these high-risk populations have serious difficulties in the resolution of influenza virus infection and what factors affect the infectious outcome.
Kidney yang deficiency syndrome (KYDS) is one of the classical syndrome patterns in traditional Chinese medicine (TCM), and it reflects a constitutional tendency of elder peoples with weakness in the knees and lumbar regions, fatigue, difficulty in urination, enuresis, female sterility, reduction of ear functions, and tooth impairment [6–8]. It is the most popular syndrome that occurs in more than sixty-year-olds [6, 9–11]. The influenza virus could cause up to 90% mortality in this age group [12–15], and these susceptible individuals belong to high-risk peoples with influenza infection, which is mostly similar to the KYDS according to the principles of TCM [10, 16]. When an epidemiological investigation was conducted in 2137 healthy elderly above 60-year-old residents, the results showed that the incidence rate of kidney deficiency was 78.80% [16], and the kidney deficiency syndrome prevalence in participants showed an increasing trend with increasing age and deteriorating health status [17]. Another epidemiological study on 2,067 adults aged >60 years revealed that 45.33% suffered from KYDS, showing that KYDS is the predominant TCM syndrome in high-risk populations [18] and these elderly groups are at increased risk for serious flu complications. Therefore, a key challenge is how to reduce the high mortality in susceptible population infected with the influenza virus.
The mucous layer in the upper respiratory tract (pharynx and larynx) is the first innate barrier of defense against influenza virus infection. The virus replication is restricted by the first innate immune line [19, 20]. The defensive functional differences in the mucosal immunity of the pharynx and larynx between normal population and high-risk population are still unknown. As the influenza viral infections mostly have upper respiratory symptoms with sore throat or discomfort, we speculated that the mucosal immunity of the pharynx and larynx may play an important role against influenza virus infection in the initial stage. In order to test this hypothesis, we designed a special experiment by separate infection normal control mice and KYDS mice (by injecting estradiol benzoate intraperitoneally [21, 22]) combined with influenza A (H1N1) virus through nose dropping and tracheal injection, respectively. This experiment would be conductive to reveal the primary mechanism to combat viral infections in the mucosal immunity of the pharynx and larynx in the KYDS mice model.
2. Materials and Methods
2.1. Experimental Design
This study was approved by the Ethic Committee of Shandong University of Traditional Chinese Medicine, and all animals received humane care in compliance with the Chinese Animal Protection Act and the National Research Council Criteria. SPF Male BALB/c mice (16–18 g) were purchased from the Jinan Pengyue Experimental Animal Breeding Company Limited. After three days acclimation, all mice were randomly assigned to two groups: KYDS mice (model group) were established by intraperitoneal injection of estradiol benzoate (8 mg/kg, Ningbo Second Hormone Factory, China) for 7 d as previously described [21, 23] and normal control mice (normal group) were intraperitoneally injected with normal saline. The physical signs in mice were observed, and spontaneous activity, swimming time, rectal temperature, and body weight were monitored. All the animals were housed at 21–23°C in a 12 h light/12 h dark cycle, and environmental humidity was controlled between 60 and 70%.
After the KYDS mice model was finished, these model animals were randomly divided into two groups: model intubation (MI) group and model nasal dropping (MD) group. Normal group animals were divided into three groups: normal control (NC) group, normal intubation (NI) group, and normal nasal dropping (ND) group.
2.2. Virus
Mouse-adapted influenza A virus (A/FM/1/47, H1N1) was kindly donated by the Institute of Basic Medicine, Shandong Academy of Medical Sciences. The virus was amplified in the allantoic cavity of embryonated eggs at 36°C for 48 h with a hemagglutination titer of 1 : 320 and then stored at −80°C. All tests were performed in class II biosafety cabinets [21].
2.3. Visualized Method for Tracheal Intubation
We used mouse-sized speculum, a plastic incisor loop, and a rodent work stand (Hallowell Engineering and Manufacturing Corporation, USA) for the visualization procedure of tracheal intubation. The mouse-sized speculum is attached with an otoscope and has a side cut-away portion that is conducive to the ET tube and a mouse ET tube introducer passing through the trachea [24].
2.4. Tracheal Infection with Influenza A Virus (H1N1)
The intubation method was adapted from the operation guidelines by Hallowell EMC and previous literature [25–27]. Mice were anesthetized intraperitoneally (i.p.) with 500 mg·kg⁻¹ tribromoethanol solution in the biosafety cabinet. Mice were placed on the work stand, and a clear view of the trachea was obtained with the otoscope and attached mouse speculum. The NI group and the MI group mice were injected with 5 μL of viral suspension containing a hemagglutination titer of 1 : 320 of the influenza A/FM/1/47 (H1N1) virus per mouse through the ET tube (1.22 mm OD; 23.5 mm length). We have tested that the ET tube length position was just located in the upper respiratory tract below the oropharyngeal structure. The NC group mice were injected with the same volume normal saline.
2.5. Nasal Dropping Infection with Influenza A Virus (H1N1)
As a common nasal dropping infection method, the ND group and the MD group were anaesthetized intraperitoneally (i.p.) with 500 mg·kg⁻¹ tribromoethanol solution and intranasally inoculated with 5 μL and the same titer of the influenza A/FM/1/47 (H1N1) virus, and then they ate food and drank water freely.
2.6. Serum and Tissue Preparation
On the 14th day, the whole blood was collected from the mice orbit and serum was later separated by centrifugalization, and then mice were sacrificed by cervical dislocation. The thymus, lung, and spleen were isolated and weighed for the organ index calculation. After harvesting, little amount of the right part of the lung samples was immediately submerged in RNAstore Reagent (Tiangen, China) at a dilution ratio of 1 : 10 (w/v) and then stored at 4°C. The left part of the lung tissue was taken, fixed in 10% formalin solution as histological specimens, and observed under the microscope to distinguish the pathological changes.
2.7. Cytometric Bead Array for Cytokine Measurements
Cytokine serum levels were determined using commercially available kits, including the mouse inflammation kit cytometric bead array (CBA; BD Biosciences Pharmingen, USA), to quantify IL-6, MCP-1, IL-12p70, and TNF-alpha. The CBA immunoassay uses 7.5 μm polystyrene microbeads assembled in distinct fluorescent sets, unique on their type-4 fluorescence intensity (FL-4). Each microbead is coupled to the monoclonal antibody (MAb) against a given cytokine. Following incubation with the test sample, the bead-captured cytokines were detected by the direct immunoassay using a “detection cocktail” of distinct MAbs labeled with type-2 fluorescence, phycoerythrin-PE (FL-2). Data acquisition and analysis was performed in a dual-laser C6™ flow cytometer (BD Biosciences Pharmingen, San Jose, CA, USA), using the BD Bioscience CBA software. The fluorescently labeled particles in the BD CBA immunoassay are designed to be excited by the 488 nm and 532 nm lasers on the BD flow cytometer [28].
2.8. Total RNA Extraction
Total RNA was isolated from 10 to 20 mg tissues using RNAprep Pure Tissue Kit (Tiangen, China) following the manufacturer’s instructions. Yield and purity of RNA were determined by the Quawell 5000 spectrophotometer (Quawell Technology, USA). RNA samples with an absorbance ratio OD 260/280 between 1.8 and 2.0 were used for further analysis. RNA integrity was assessed using agarose gel electrophoresis.
2.9. Reverse Transcription cDNA Synthesis
The first-strand cDNA was synthesized from 2 μg of total RNA with random hexamer oligonucleotide primers using a 20 μl reverse transcription system (FastQuant RT kit with gDNase, Tiangen, China) by incubation at 42°C for 3 min to protect the total RNA from genomic DNA interference. FastQuant RT Enzyme was used for reverse transcription at 42°C for 15 min. Then, cDNA was stored at −20°C for future use. For qPCR analysis, each cDNA sample was diluted 10 times with nuclease-free water.
2.10. Real-Time PCR
Real-time PCRs were conducted in Bio-Rad CFX Connect Real-Time System. For each reaction, the 20 μL mixture contained 2 μL of cDNA, 6 pmol each of the forward and reverse primers, and 10 μL of 2 × SuperReal PreMix Plus with SYBR Green I (Tiangen, China). The amplification program was as follows: 95°C for 15 min, 40 cycles at 95°C for 10 s, and 60°C for 32 s. After amplification, a thermal denaturing cycle was added to derive the dissociation curve of the PCR product to verify amplification specificity. Half-quantification of the genes of interest were normalized to Grcc10 and expressed as fold increases over the negative control for each treatment at each time point, as previously described. The primers are as follows: Grcc10 primers: forward 5′- GCGGAGGTGATTCAAGCG -3′ and reverse 5′- TGACCAGGCGGGCAA ACT -3′; influenza A (H1N1) virus M gene Primers: forward 5′-CTGAGAAGCAGATACTGGGC-3′ and reverse 5′-CTGCATTGTCTCCGAAGAAAT-3′.
2.11. Measurement of Secretory IgA Levels
In order to collect bronchoalveolar lavage fluids (BALF) and tracheal lavage fluids (TLF), these animals were divided into six groups: normal control group-intubation (NC-I), normal control group-nasal drop (NC-D), normal intubation (NI) group, normal nasal drop (ND) group, model intubation (MI) group, and model nasal drop (MD) group. On the 14th day, tracheal lavage fluids and bronchoalveolar fluids of 5–7 animals in each group were collected and subjected to the enzyme-linked immunosorbent assay (ELISA) to determine secretory IgA [29, 30].
2.12. Data Analysis
Data are expressed as mean ± SEM. The statistical analysis of two groups data is calculated by Student’s t-test. For multiple groups, one-way ANOVA analysis with the LSD test is used to compare means. Analysis involved the use of SPSS 22; statistical significance is considered at and . For survival studies, a log-rank (Mantel–Cox) test involved the use of GraphPad Prism (GraphPad 5.0 Software).
3. Results
3.1. Establishment of KYDS Mice Model
In order to mimic the high-risk population, we established a KYDS mice model by injecting estradiol benzoate for seven days as previously described; the body weight of the KYDS group was significantly lower than normal control mice from the 3rd day to the 7th day () (Figure 1(a)). The rectal temperature of the KYDS group decreased significantly compared with the normal group for six days () (Figure 1(b)). The spontaneous activity and swimming time of KYDS mice were also obviously lower than the control group (; ) (Figures 1(c) and 1(d)). These results showed that the KYDS mice model was established successfully [21, 22].
(a)
... They were both well-known Chinese herbal medicines that were frequently used in combination to enhance therapeutic efficacy. Danhong Injection (DHI) was a Chinese Material Medical standardized product made from aqueous extracts of DS and HH with the raw material dose ratio of 3:1, which was used to nourish liver and kidney, activate blood and resolve stasis for cardiovascular and cerebrovascular diseases in the clinic, such as acute myocardial infarction, cerebral infarction, hypertension, coronary heart disease, and ischemia-reperfusion injury [7,8]. The main pharmacological active components of Dan-Hong herb pair (DH) are phenolic acids, diterpenes, and flavonoids, for example, salvianolic acid B, danshensu, protocatechuic aldehydrate, and safflor yellow [9,10]. ...
... MS quantification covered an extensive range of chemical constituents irrespective of their UV absorption. Therefore, it can ensure a more sensitive and more common quantitative analysis for the complex chemical system [8,15]. Furthermore, the contents of main constituents in both herbs were first comparatively analyzed before and after the compatibility of DS and HH. ...
A sensitive, reliable and powerful ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry method was developed for simultaneous quantification of the 15 main bio-active components including phenolic acids and flavonoids within 13 min for the first time. The proposed method was first reported and validated by good linearity (r(2) > 0.9975), limit of detection (1.12-7.01 ng/mL), limit of quantification (3.73-23.37 ng/mL), intra- and inter-day precisions (RSD ≤ 1.92%, RSD ≤ 2.45%), stability (RSD ≤ 5.63%), repeatability (RSD ≤ 4.34%), recovery (96.84-102.12%) and matrix effects (0.92-1.02). The established analytical methodology was successfully applied to comparative analysis of main bio-active components in the herb pair Danshen-Honghua and its single herbs. Compared to the single herb, the content of most flavonoid glycosides were remarkable increases in their herb pair, and main phenolic acids were decreased, conversely. The content changes of the main components in the herb pair supported the synergistic effects on promoting blood circulation and removing blood stasis. The results could provide a scientific basis and reference for the quality control of Danshen-Honghua herb pair and the drug interactions based on variation of bio-active components in herb pairs. This article is protected by copyright. All rights reserved.
... Scan modes of triple quadrupole include full-scan mode, product ion scan mode, parent ion scan mode, neutral loss scan mode, selected ion scan mode, and multiple reactions monitoring scan mode [12,13]. LC-QQQ-MS is mainly used for quantification [12][13][14][15][16][17][18][19][20][21][22][23][24], as shown in Table 3. Some studies are used for qualitation [25,26]. ...
... All the authors declare that they have no competing interests. [12] Protopine and so forth Jitai tablet + + [13] Rutin R1 and so forth Actinidia valvata leaves + + [14] Neobavaisoflavone and so forth Psoralea corylifolia L. + + [15] -Eclipta prostrata L. + + [16] Naringin and so forth Chaihu-shu-gan-san + + [17] Psoralen and so forth Bushen zhuanggu formula + + [18] Berberine and so forth Huang lian jie du decoction + + [19] Fuziline and so forth Mahuang-fuzi-xixin decoction + + [20] Rutin Ginkgo biloba L. + + [21] Liquiritin and so forth Gan-sui-ban-xia decoction plus-minus gansui and gancao drug combination + + [22] (−)-Epigallocatechin and so forth Ginkgo biloba leaves + + [23] Pedunculoside and so forth ...
With increasingly improved separation of complex samples and detection of unknown material capabilities, liquid chromatography coupled with mass spectrometry (LC-MS) has been widely used in traditional Chinese medicine (TCM) research. This article describes the principles of liquid chromatography (LC) and mass spectrometry (MS) and their advantages and disadvantages in qualitative and quantitative analysis of TCM. We retrieved research literatures about the application of LC-MS in TCM published during the past five years at home and abroad. To better guide the analysis of TCM, this review mainly focuses on the applications category of LC-MS, how often different kinds of LC-MS are used, and the qualitative and quantitative ability of various LC-MS in the study of TCM.
... Thus, four alkaloids from ephedrae herba were identified. Meanwhile, proline and diisobutyl phthalate were also 2020 0327-HSS T3-HSBD-neg 2020 0327-HSS T3-HSBD-2 [24,25] . ...
Huashi Baidu prescription (HSBDF), recommended in the Guideline for the Diagnosis and Treatment of Novel Coronavirus (2019-nCoV) Pneumonia (On Trials, the Seventh Edition), was clinically used to treat severe corona virus disease 2019 (COVID-19) with cough, blood-stained sputum, inhibited defecation, red tongue etc. symptoms. This study was aimed to elucidate and profile the knowledge on its chemical constituents and the potential anti-inflammatory effect in vitro. In the study, the chemical constituents in extract of HSBDF were characterized by UPLC-Q-TOF/MS in both negative and positive modes, and the pro-inflammatory cytokines were measured by enzyme-linked immunosorbent assays (ELISA) to determine the effects of HSBDF in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The results showed that a total of 217 chemical constituents were tentativedly characterized in HSBDF. Moreover, HSBDF could alleviate the expression levels of IL-6 and TNF-α in the cell models, indicating that the antiviral effects of HSBDF might be associated with regulation of the inflammatory cytokines production in RAW264.7 cells. We hope that the results could be served as the basic data for further study of HSBDF on anti-COVID-19 effect.
... Ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS) operated in multiple reaction monitoring (MRM) mode is an effective quantification method owing to its well-known high sensitivity and specificity, which could avoid the interference from the background matrix [7][8][9]. It has been successfully utilized to quantify bioactive components in the complex systems [10][11][12][13][14][15]. In the current study, therefore, an UHPLC-QQQ-MS method was developed to simultaneously quantify 43 bioactive components in the Chinese herbal spirit samples produced by year 2014 and 2018, and their concentrations in different peoduction years were also compared. ...
Background
The Chinese medicinal wine made from herbal medicines became prevalent among Chinese people. The Chinese herbal spirit is composed of several herbal extracts, and has the certain health functions, such as anti-fatigue and immune regulation. The quality evaluation of Chinese herbal spirit is greatly challenged by the enormous and complex components with great structural diversity and wide range of concentration distribution.
Methods
An ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) with multiple reaction monitoring (MRM) method was developed to simultaneously determine forty-three bioactive components in the Chinese herbal spirits produced by year 2014 and 2018.
Results
Quantitative results showed that 11 components, i.e.., puerarin ( 5 ), purpureaside C ( 7 ), daidzin ( 8 ), echinacoside ( 9 ), acteoside ( 15 ), epimedin B ( 22 ), epimedin C ( 23 ), icariin ( 24 ), eugenol ( 27 ), chikusetsusaponin iva ( 30 ) and Z-ligustilide ( 40 ), significantly decreased along with the increasing years of storage, while 5 compounds, i.e.., geniposidic acid ( 1 ), protocatechuic acid ( 2 ), crustecdysone ( 14 ), daidzein ( 18 ) and icariside I ( 35 ), were basically stable in all samples across the years.
Concusion
The established method allowing to simultaneously determined 43 components with wide structural diversity and trace amounts will facilitate the quality control research of Chinese herbal spirits.
... The precise retention time (Rt) and molecular mass were obtained by mass spectrometry. A total of 38 alkaloids were identified using the method described in section 2.4.5 (Zhang et al., 2018Zhao et al., 2018;Xu et al., 2017;Sun et al., 2016a;Samanbay et al., 2018;Chen et al., 2017;Csupor et al., 2008;Gao et al., 2018;Zhu et al., 2012;Shim et al., 2003;Shyaula et al., 2016). The results are shown in Table 2. ...
Radix aconiti lateralis (Fuzi) is widely used in China as a traditional Chinese medicine for the treatment of asthenia, pain and inflammation. However, its toxic alkaloids often lead to adverse reactions. Currently, most of the toxicity studies on Fuzi are focused on the heart and nervous system, and more comprehensive toxicity studies are needed. In this study, based on the previous reports of Fuzi hepatotoxicity, serum pharmacochemistry and network toxicology were used to screen the potential toxic components of Heishunpian(HSP), a processed product of Fuzi, and to explore the possible mechanism of HSP-induced hepatotoxicity. The results obtained are expressed based on the toxicological evidence chain (TEC). It was found that 22 potential toxic components screened can affect Th17 cell differentiation, Jak-STAT signaling pathway, glutathione metabolism, and other related pathways by regulating AKT1, IL2, F2, GSR, EGFR and other related targets, which induces oxidative stress, metabolic disorders, cell apoptosis, immune response, and excessive release of inflammatory factors, eventually inducing liver damage in rats. This is the first study on HSP-induced hepatotoxicity based on the TEC concept, providing references for further studies on the toxicity mechanism of Fuzi.
... Mahuang-Fuzi-Xixin decoction (MFX) was first prescribed in Treatise on Febrile Diseases and has efficiency in the treatment of migraine, asthma, rheumatoid arthritis, and MDD [35][36][37][38]. Studies show that MFX has good anti-inflammatory and immunosuppressive effect, as well as antioxidant effect [39,40], which may be related to its clinical antidepressive effect. MFX composed of Radix Aconiti Lateralis, Ephedrae, and Asarum were mixed at the ratio of 3 : 2 : 1. Radix Aconiti Lateralis polysaccharide and alkaloids, which are the virtual components, have pharmacological action in anti-inflammation, antidepression, antiepileptic, and analgesic [41][42][43][44]. ...
Evidence suggests that inflammation and neurogenesis play an important role in major depressive disorder (MDD). Mahuang-Fuzi-Xixin decoction (MFX), as the traditional Chinese prescription, has been widely applied for asthma, migraine, and MDD in clinics. However, the effects of MFX on the potential mechanism in MDD are still unclear. Hence, the present study is aimed at exploring whether the antidepressive effect of MFX is connected to the anti-inflammatory and promoting neurogenesis. Besides, lipopolysaccharide (LPS) of Gram-negative bacteria can induce depressive-like behaviors. We demonstrated that administration of MFX corrected the depressive-like behaviors in LPS-induced mice and significantly decreased the expression of IL-1 β in the hippocampus. LPS injection induced a significant increase in the levels of NLRP3, cleaved caspase-1 p20, and ASC in the hippocampus, as well as Trx-interacting protein (TXNIP), and MFX could reverse this change. What is more, treatment of MFX increased the level of doublecortin (DCX), brain-derived neurotrophic factor (BDNF), and tropomyosin-related kinase receptor B (TrkB) in the hippocampus which means that MFX could promote the neurogenesis. In conclusion, the study indicates that MFX relieves a depressive-like state in LPS-induced mice through the inhibition of the NLRP3 inflammasome and the enhancement of the neurogenesis pathway.
... It can also provide isotopic abundances and the elemental composition of fragment ions which are greatly valuable to the structural analysis of ingredients [12,13]. Furthermore, ultraperformance liquid chromatography coupled with tandem mass spectrometry (UPLC-QqQ-MS/MS) in the multiplereaction monitoring mode (MRM) has been developed as a convenient and time-saving method for quantitative analysis of various compounds because of the remarkable separation effect of UPLC and the high sensitivity of tandem mass spectrometry [14][15][16][17][18]. erefore, UPLC-Q/TOF-MS/MS and UPLC-QqQ-MS/MS are suitable for qualitative and quantitative analysis of bile acids in Bile Arisaema. ...
Ultrahigh-performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight tandem mass spectrometry (Q/TOF-MS) in the MS/MS mode and UPLC coupled with triple quadrupole mass spectrometry (QqQ-MS) using the multiple reaction monitoring (MRM) mode were used to make a qualitative and quantitative analysis of twelve bile acids in Bile Arisaema. The fragmentation pathway of twelve bile acids was proposed. The quantification method showed a good linearity over a wide concentration range ( R² > 0.99), repeatability (RSD < 4.12%), stability (RSD < 4.25%), precision (RSD < 4.06%), and recovery (95.36–102.15%). Content of twelve compounds in Bile Arisaema varied significantly depending on region. Chemometric methods, hierarchical clustering analysis (HCA), and principal components analysis (PCA) were successfully used to optimize the fermentation time of the Bile Arisaema. The results suggested that the Bile Arisaema could complete fermentation in 15 days. The possible processing mechanism of Bile Arisaema promoted the transformation of conjugated bile acids into free bile acids in fermentation.
... Mass spectrometric and chromatographic detection identified 52 compounds, including alkaloids, amino acids and organic acids. The main constituents are methyl ephedrine, aconine, songrine, fuziline, neoline, alatisamine, chasmanine, benzoylmesaconine, benzoylaconine, and benzoylhypaconine (Sun et al., 2016). Experiments showed that MFX decoction significantly depressed the expression of IL-6, MCP-1 and TNF-α, and markedly increased expression of IL-10 in serum, indicating the effect of reducing inflammation and increasing antioxidant activities (Tang et al., 2015;Rong et al., 2016). ...
Importance: The incidence of Bradyarrhythmias is high among the population. However, at early stages of the disease, it cannot always get enough attention and is lack of safe and effective therapies, until it is serious enough to resort to pacemaker implantation. Traditional Chinese Medicine (TCM) has a long history of treating Bradyarrhythmia, with a lot of formulas being widely used in clinical practice. While the effectiveness and the underlying mechanisms of these formulas have not yet been clearly identified.
Objective: To evaluate the effectiveness of some common TCM formulas in treating patients with Bradyarrhythmia and to summarize the current evidence as to their mechanisms.
Data Sources: Relevant studies were identified by searching for papers published from January 2000 to August 2017 in Pubmed; EMBASE; the Cochrane Library (Cochrane Central Register of Controlled Trials); the China National Knowledge Internet; and the China biology medicine, Wanfang, and VIP databases. The following medical subject heading (MeSH) terms were included for Pubmed search and adapted for other databases as needed-“Medicine, Chinese Traditional,” “Bradycardia.”
Study Selection: Randomized clinical trials investigating treatment outcomes in Bradyarrhythmia patients with one of the six TCM formulas (Shenxian-shengmai oral liquid, Shensong Yangxin capsule, XinBao pill, Mahuang-Fuzi-Xixin decoction, Zhigancao decoction and Shengmai injection).
Data Extraction and Synthesis: Two independent reviewers performed the data extraction and assessed study quality. A meta-analysis was performed to calculate risk ratio (RR) and 95% confidence index (CI) using random-effects and fixed-effects model.
Results: A total of 121 clinical trials with 11138 patients were included. Of the six TCM formulas, SXSM (RR:1.33, 95% CI 1.27 to 1.39, P < 0.00001), SSYX (RR:1.52, 95% CI 1.40 to 1.66, P < 0.00001), XB can be more effective than common treatment (RR 1.18, 95% CI 1.11 to 1.26, P < 0.00001), as well as placebo (RR 5.33, 95% CI 2.88-9.87, P < 0.00001), but less effective than TCM dialectical therapy (RR:0.75, 95% CI 0.68 to 0.82, P < 0.00001). Compared to the control group, MFX (RR:1.30, 95%CI 1.23 to 1.37, P < 0.00001), ZGC (RR:1.35, 95%CI 1.23 to 1.48, P < 0.00001), SMI (RR:1.36, 95%CI 1.21 to 1.52, P < 0.00001) can be more effective. The overall quality of the included trials were relatively low, with the limitations of small sample size, inadequate descriptions in randomization, allocation concealment and blinding methods.
Conclusions and Relevance: There are evidence that some TCM formulas might help to relieve Bradyarrhythmias. But with the relatively low quality of the clinical trials and mechanism studies, we still need more high-quality researches to verify the conclusions.
... Among them, the TOF mass analyzer, with high sensitivity and mass accuracy of the analysis, is suitable for structural analysis of unknown compounds. 40,41 Analysis and identication of herbal medicines by spectrum has been accepted as an effective identication method by most countries, which identies herbs by different wavelengths and penetrability of ultraviolet, uorescence, and infrared radiation. Aer the discovery of the near-infrared (NIR) region in 1800, NIR spectroscopy sprung up, reformed in the early 1950s, and was used in the 1970s. ...
Natural products are the most representative form of conventional therapy as compared to any other traditional or alternative medicine systems. They have numerous active components, either primary or secondary metabolites, which are associated with the diverse, intricate, and distinct characteristics of natural products and result in various pharmacological effects in clinic. However, some problems are associated with research on herb quality, which is the core of the drug industry, and restrict the development of this field to a certain extent. Quality-markers (Q-markers), a novel concept for quality assessment, open up a new avenue for promotion of healthy development of traditional medicine industry and improvement of the quality standard system to enhance traditional medicine or product quality standards. In this study, we first summarized the main factors affecting the quality of traditional medicines and natural products and importance for safety and then presented the concept of background and relevant factors of Q-markers. Moreover, the modern science technology and related methods used to identify the chemical composition have been discussed. Especially, based on the systematic analysis and discussion of the basic properties and clinical features of natural products, we have discussed new trends and effective strategies for identifying relevant Q-markers from herbs and probed the future research directions and challenges.
... ng.mL −1 and 0.05~824.74 ng.mL −1 (shown in Table 1 29,30 ). ...
Lepidium meyenii (Maca), originated from Peru, has been cultivated widely in China as a popular health care food. However, the chemical and effective studies of Maca were less in-depth, which restricted its application seriously. To ensure the quality of Maca, a feasible and accurate strategy was established. One hundred and sixty compounds including 30 reference standards were identified in 6 fractions of methanol extract of Maca by UHPLC-ESI-Orbitrap MS. Among them, 15 representative active compounds were simultaneously determined in 17 samples by UHPLC-ESI-QqQ MS. The results suggested that Maca from Yunnan province was the potential substitute for the one from Peru. Meanwhile, the neuroprotective effects of Maca were investigated. Three fractions and two pure compounds showed strong activities in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced zebrafish model. Among them, 80% methanol elution fraction (Fr5) showed significant neuroprotective activity, followed by 100% part (Fr6). The inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was a possible mechanism of its neuroprotective effect.
... The MS/MS device is well suited for quantitative determination of known compounds in the selected reaction monitoring mode. 30 In addition, TOF mass analyzers (eg, QTOF/MS) providing high sensitivity and mass accuracy are suitable for the structural elucidation of unknown compounds, 31 and are particularly useful for the analysis of HM. 32 ...
Herbal medicine (HM) has made a major contribution to the drug discovery process with regard to identifying products compounds. Currently, more attention has been focused on drug discovery from natural compounds of HM. Despite the rapid advancement of modern analytical techniques, drug discovery is still a difficult and lengthy process. Fortunately, mass spectrometry (MS) can provide us with useful structural information for drug discovery, has been recognized as a sensitive, rapid, and high-throughput technology for advancing drug discovery from HM in the post-genomic era. It is essential to develop an efficient, high-quality, high-throughput screening method integrated with an MS platform for early screening of candidate drug molecules from natural products. We have developed a new chinmedomics strategy reliant on MS that is capable of capturing the candidate molecules, facilitating their identification of novel chemical structures in the early phase; chinmedomics-guided natural product discovery based on MS may provide an effective tool that addresses challenges in early screening of effective constituents of herbs against disease. This critical review covers the use of MS with related techniques and methodologies for natural product discovery, biomarker identification, and determination of mechanisms of action. It also highlights high-throughput chinmedomics screening methods suitable for lead compound discovery illustrated by recent successes.
Fangji Huangqi Tang (FHT) is a traditional prescription frequently utilized in clinical practice, with a wide range of clinical applications and good therapeutic effects. Quality control of FHT is difficult because Chinese medicine compounds usually contain a vast array of components characterized by significant structural diversity. A quick and accurate method to determine the content of active constituents in FHT was essential, by which the purpose of quality control and efficacy assessment could be achieved. A method utilizing UHPLC‐QqQ‐MS technology in multiple reaction monitoring (MRM) mode was established to quantify 14 bioactive components in FHT simultaneously. These analytes included tetrandrine, fangchinoline, calycosin, calycosin‐7‐glucoside, medicarpin, formononetin, atractylenolide I, atractylenolide II, atractylenolide III, liquiritigenin, isoliquiritigenin, liquiritin, isoliquiritin, and glycyrrhizic acid. And to our knowledge, the content of calycosin, medicarpin, formononetin, and atractylenolide II in FHT was reported for the first time in this paper. The method was thoroughly validated for stable and reliable application regarding specificity, linearity, precision, stability, repeatability, and accuracy. The established method allowed the simultaneous determination of 14 bioactive components with diverse structures and trace amounts in FHT, ultimately achieving the quality control and assessment of FHT for its safe and appropriate clinical use.
Introduction
Frankincense is used for analgesic, tumor‐suppressive, and anti‐inflammatory treatments in Traditional Chinese Medicine but poses toxicological concerns. Vinegar processing is a common technique used to reduce the toxicity of frankincense.
Objective
This study aimed to investigate the chemical composition and quality evaluation of raw and vinegar‐processing frankincense by multiple UPLC‐MS/MS techniques. Additionally, we purposed refining the vinegar processing technique and identifying potentially harmful ingredients in the raw frankincense.
Methodology
Sub‐chronic oral toxicity studies were conducted on raw and vinegar‐processing frankincense in rats. The composition of frankincense was identified by UPLC‐Q‐TOF‐MS/MS. Chemometrics were used to differentiate between raw and vinegar‐processing frankincense. Potential chemical markers were identified by selecting differential components, which were further exactly determined by UPLC‐QQQ‐MS/MS. Moreover, the viability of the HepG2 cells of those components with reduced contents after vinegar processing was assessed.
Results
The toxicity of raw frankincense is attenuated by vinegar processing, among which vinegar‐processing frankincense (R40) (herb weight: rice vinegar weight = 40:1) exhibited the lowest toxicity. A total of 83 components were identified from frankincense, including 40 triterpenoids, 37 diterpenoids, and 6 other types. The contents of six components decreased after vinegar‐processing, with the lowest levels in R40. Three components, specifically 3α‐acetoxy‐11‐keto‐β‐boswellic acid (AKBA), 3α‐acetoxy‐α‐boswellic acid (α‐ABA), and 3α‐acetoxy‐β‐boswellic acid (β‐ABA), inhibited the viability of HepG2 cells. The processing of frankincense with vinegar at a ratio of 40:1 could be an effective method of reducing the toxicity in raw frankincense.
Conclusion
Our research improves understanding of the toxic substance basis and facilitates future assessments of frankincense quality.
Background
Radix Aconiti Lateralis (Fuzi), a mono‐herbal preparation of Aconitum herbs in the genus Aconitum , is commonly used in traditional Chinese medicine (TCM) to treat critical illnesses. The curative effect of Fuzi is remarkable. However, the toxic effects of Fuzi are still a key clinical focus, and the substances inducing nephrotoxicity are still unclear. Therefore, this study proposes a research model combining “ in vitro and in vivo component mining‐virtual multi‐target screening‐active component prediction‐literature verification” to screen potential nephrotoxic substances rapidly.
Method
The UHPLC‐Q‐Exactive‐Orbitrap MS analysis method was used for the correlation analysis of Fuzi's in vitro–in vivo chemical substance groups. On this basis, the key targets of nephrotoxicity were screened by combining online disease databases and a protein–protein interaction ( PPI ) network. The computer screening technique was used to verify the binding mode and affinity of Fuzi's components with nephrotoxic targets. Finally, the potential material basis of Fuzi‐induced nephrotoxicity was screened.
Results
Eighty‐one Fuzi components were identified. Among them, 35 components were absorbed into the blood. Based on the network biology method, 21 important chemical components and three potential key targets were screened. Computer virtual screening revealed that mesaconine, benzoylaconine, aconitine, deoxyaconitine, hypaconitine, benzoylhypaconine, benzoylmesaconine, and hypaconitine may be potential nephrotoxic substances of Fuzi.
Conclusions
Fuzi may interact with multiple components and targets in the process of inducing nephrotoxicity. In the future, experiments can be designed to explore further. This study provides a reference for screening Fuzi nephrotoxic components and has certain significance for the safe use of Fuzi.
Quality control of Chinese medicines (CMs) is a difficult work because of their intrinsic complexity. UPLC-MS is at the forefront of this crucial challenge due to its prominent performance in qualitative and quantitative analysis. This chapter aims to explain UPLC-MS-based methodologies in the field of quality control for CMs, covering the basic principles of various instruments, and their specific applications in the characterization of chemical composition, screening, and quantification of chemical markers. Meanwhile, a description of some universal or integrated strategies (e.g., fingerprints, chemical markers’ knock-out, multivariate statistical analysis and quantitative analysis of multi-components by single marker) are also provided.
Objectives
This study was designed to investigate the pharmacological activity and therapeutic mechanism of Mahuang Xixin Fuzi decoction (MXFD) on migraine.
Methods
Migraine model rats induced by nitroglycerin were established, and then orally administered with MXFD for 7 days. Blood and urine samples were collected to identify differential metabolites with metabolomics. To integrate the findings from network pharmacology and metabolomics analysis, the metabolites and targets related to MXFD therapy for migraine were filtered.
Key findings
MXFD was found to alleviate the symptoms of migraines in rats. After treatment with MXFD, nine metabolites were found to be regulated and returned to normal levels. MXFD acted directly on nine key targets including MAOB, MAOA, ADRB1, ADRB2, ADRB3, ADORA2A, ADORA2B, DRD5, and HTR4 and regulated two out of nine metabolites, namely deoxycholic acid and 5-methoxyindoleacetate.
Conclusions
The study found that MXFD can alleviate migraines through multitarget and multicomponent interaction networks.
Mahuang-Fuzi-Xixin Decoction (MFXD) is widely used in the treatment of asthma, however, the functional components in the decoction targeting beta2-adrenoceptor (β2-AR) remain unclear. Herein, we immobilized the haloalkane dehalogenase (Halo)-tagged β2-AR on the 6-chlorocaproic acid-modified microspheres. Using the affinity stationary phase, the interactions of four ligands with the receptor were analyzed by stepwise frontal analysis. The association constants were (4.75±0.28)×104 M-1 for salbutamol, (2.93±0.15)×104 M-1 for terbutaline, (1.23±0.03)×104 M-1 for methoxyphenamine, 5.67±0.38)×104 M-1 for clorprenaline at high-affinity binding site, and (2.73±0.05)×103 M-1 at low-affinity binding site. Theseassociation constants showed the same rank order as the radioligand binding assay, demonstrating that immobilized β2-AR had capacity to screen bioactive compounds binding to the receptor while stepwise frontal analysis could predict their binding affinities. Application of the immobilized receptor in analysis of MFXD by chromatographic method revealed that ephedrine, aconifine, karakoline, and chasmanine were the bioactive compounds targeting β2-AR. Among them, ephedrine and chasmanine exhibited association constants of (2.94±0.02)×104 and (4.60±0.15)×104 M-1 to the receptor by stepwise frontal analysis. Molecular docking analysis demonstrated that ephedrine, chasmanine, and the other two compounds interact with β2-AR through the same pocket involving the key amino acids such as Asn312, Asp113, Phe289, Trp286, Tyr316, and Val114. As such, we reasoned that the four compounds dominate the therapeutic effect of MFXD against asthma through β2-AR mediating pathway. This work shed light on the potential of immobilized β2-AR for drug discovery and provided a valuable methodology for rapid screening.
Li‐Zhong‐Xiao‐Pi (LZXP) granule is effective for treating gastric precancerous lesions (GPL) in traditional Chinese medicine. However, the active compounds of LZXP and their potential therapeutic mechanism in GPL remained unclarified. The purpose of this study is to investigate the chemical composition and potential targets of LZXP, which is clinically applied in the treatment of GPL. Based on the accurate masses, ion fragments, and literature data, a total of 128 compounds were identified in the LZXP sample using ultra‐performance liquid chromatography and quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) in both positive and negative ion modes, and 28 of these compounds were exactly determined by comparison with authentic reference standards. Meanwhile, 11 typical components were quantified via UPLC during a 24‐min period. The linearity, accuracy, stability and recovery of the method were all proven. Through the network pharmacological analysis, six chemicals (quercetin, 4'‐hydroxywogonin, sinensetin, 5, 7, 8, 3', 4'‐pentamethoxyflavanone, 8‐gingerdione and quercetin) were identified as the active ingredients, and five LZXP targets (AKT1, CYP1B1, PTGS2, MMP9, and EGFR) were found to be the crucial molecules in the treatment of GPL. This study provides a systematic and applicable method for the rapid screening and identification of the chemical constituents from LZXP, and an effective understanding the mechanism of LZXP in the treatment of GPL.
Ethnopharmacological relevance:
Mahuang Xixin Fuzi Decoction (MXF), as a classical prescription of traditional Chinese medicine (TCM), has been used to treat the immunocompromised individuals infected with influenza A virus (IAV).
Aim of the study:
The study aims to explore the regulatory of MXF on inflammation and secretory immunoglobulin A (SIgA) antibodies immune response in BALB/c-nude mice infected with IAV.
Materials and methods:
The BALB/c-nude mice were infected with IAV, then different dosages of MXF were orally administrated to the mice. The weight, rectal temperature, spontaneous activity, spleen index, lung index, pathological changes of lung tissues, and the relative mRNA expression level of H1N1 M gene were measured for the purpose of valuing the antiviral effect of MXF. The expression levels of cytokines in lungs and immunoglobulin A (IgA) in serum of BALB/c-nude mice were determined with Cytometric Bead Array System (CBA). SIgA in bronchoalveolar lavage fluids (BALF) was detected with Enzyme-linked Immunosorbent Assay (ELISA). The mRNA and protein expression levels of B cell activating factor (BAFF), chemokine receptors 10 (CCR10), and polymeric immunoglobulin receptor (pIgR) in the lung tissues, which are related to the secretion of SIgA, were determined by using RT-PCR and Western blot.
Results:
MXF could alleviate the clinical features and reduce the severity of viral lung lesions, including improving the body weight, rectal temperature and spontaneous activity of nude mice infected with IAV, increasing spleen index, decreasing lung index, alleviating pathological damage, and decreasing the relative expression level of H1N1 M gene. Levels of pro-inflammatory cytokines, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), interleukin-12p70 (IL-12p70), and interleukin-17A (IL-17A) were also significantly decreased after treatment with MXF. Interferon-γ (IFN-γ), an antiviral cytokine, was significantly up-regulated in high dose MXF (3.12 g/kg) group. Moreover, after MXF treatment, the expressions of SIgA in BALF and IgA in serum were both at relatively low levels. And the mRNA and protein expressions of BAFF, CCR10, and pIgR were significantly decreased after treatment with MXF.
Conclusions:
MXF has obviously protective effects on BALB/c-nude mice infected with IAV by inhibiting virus replication, calming inflammatory cytokine storm, and regulating SIgA immune response weakly.
Ethnopharmacological relevance
Mahuang Xixin Fuzi Decoction (MXF), as a classical prescription of traditional Chinese medicine (TCM), has been used to treat the symptoms of fever, nasal congestion and headache in elderly people for almost a thousand years.
Aim of the study
The purpose of this study was to evaluate the effects and possible mechanisms of MXF on thermal stimulation-induced mouse cardiac myocytes (MCM) cell apoptosis.
Materials and methods
The apoptosis of the MCM cell model was induced by a PCR-calculated temperature control system with a gradual heating pattern at 43 °C for one hour. The cytotoxic effects were determined using real-time cell analyzer (RTCA) technology. Annexin V-FITC/7-AAD staining, and JC-1 fluorescence were used to assess apoptosis. Specific substrates, enzyme-linked immunosorbent assays (ELISAs), and Western blotting were used to identify proteins in the mitochondrial-mediated pathway. The identification of chemical components in the mouse heart was performed by ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry analysis.
Results
MXF inhibited apoptosis through the mitochondrial-mediated signaling pathway, including ameliorating MMP reduction, blocking mitochondrial Cyt C release, reducing Bax levels and increasing Bcl-2 levels, suppressing caspase-9 and caspase-3 activation in cytoplasmic fractions. Moreover, the components of MXF that act on the heart are mainly ephedra alkaloids and aconitine alkaloids.
Conclusions
The findings demonstrated that MXF treatment markedly reduced MCM cell apoptosis induced by thermal stimulation, which may be ascribed to the mitochondrial-mediated signaling pathway.
As a well‐known traditional Chinese medicine formula, the chemical constituents of Shengxian Decoction still remain unclear due to its complexity. In this study, a multidimensional strategy based on ultra‐performance liquid chromatography coupled with ion mobility spectrometry quadrupole time‐of‐flight mass spectrometry and informatics UNIFI™ platform was applied to achieve rapid and comprehensive identification of the complex composition of Shengxian Decoction. Data‐independent acquisition, fast data‐directed analysis, and high‐definition MSE were used to obtain more and cleaner mass spectrum information. As a result, a total of 120 compounds including 74 saponins, 17 flavonoids,7 cinnamic acid derivatives, 8 triterpenoids and 14 others were identified or tentatively characterized by high‐resolution molecular mass, fragment ions, and collision cross‐section values. Furthermore, high‐definition MSE was used to identify six pairs of co‐eluting isomers that could not be detected from conventional data‐independent acquisition and data‐independent acquisition. This research strategy has a certain potential for the analysis of other Compound formulae and lays the foundation for the study of traditional Chinese medicine efficacy. This article is protected by copyright. All rights reserved
Introduction:
Aconitum spp. are prime medicinal plants rich in alkaloids and have been used as the main constituents of traditional medicine in India and China. The whole plant can be toxic and creates pathophysiological conditions inside the human body. Therefore, simultaneous quantification of alkaloids within plant parts and herbal medicines associated with this genus is essential for quality control.
Objective:
We aimed to develop and validate methods using ultra-high-performance liquid chromatography-diode array detector-quadrupole time-of-flight ion mobility mass spectrometry (UHPLC-DAD-QTOF-IMS) and to develop an analytical strategy for the identification and quantification of alkaloid compounds (aconitine, hypaconitine, mesaconitine, aconine, benzoylmesaconitine, benzoylaconine, bulleyaconitine A, and deoxyaconitine) from Aconitum heterophyllum.
Methodology:
We developed a simultaneous identification and quantification method for eight alkaloids using UHPLC-DAD-QTOF-IMS. The method was validated as per International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines and also in IMS mode.
Results:
The developed method has good linearity (r2 = 0.997-0.999), LOD (0.63-8.31 μg/mL), LOQ (0.63-2.80 μg/mL), recovery (86.01-104.33%), reproducibility, intra- and inter-day variability (<3.25%), and stability. Significant qualitative and quantitative variations were found among different plant parts (flower, leaf, stem, root, and tuber) and five market products of A. heterophyllum. Furthermore, a total of 21 metabolites were also profiled based on the fragmentation pattern of MS2 using the validated method.
Conclusion:
An appropriate mobile phase using acetonitrile and water in a gradient elution gave a satisfactory chromatographic separation of eight Aconitum alkaloids with their adjacent peaks. Therefore, this method could provide a scientific and technical platform for quality control assurance.
Background
High content image (HCI), an automatic imaging and analysis system, provides a fast drug screening method by detecting the subcellular distribution of protein in intact cells.
Objective
This study established the first standardized HCI platform for lipopolysaccharide (LPS)-induced RAW264.7 macrophages to screen anti-inflammatory compounds by measuring nuclear factor-κB (NF-κB) nuclear translocation.
Method
The influence of the cell passages, cell density, LPS induction time and concentration, antibody dilution, serum, dimethyl sulfoxide and analysis parameters on NF-κB nuclear translocation and HCI data quality was optimized. The BAY-11-7085, the positive control for inhibiting NF-κB and Western blot assay were separately employed to verify the stability and reliability of the platform. Lastly, the effect of BHA on NO release, iNOS expression, IL-1β, IL-6, and TNF-α mRNA in LPS-induced RAW264.7 cells was detected.
Results
The optimal conditions for measuring NF-κB translocation in LPS-induced RAW264.7 cells by HCI were established. Cells that do not exceed 22 passages were seeded at a density of 10 k cells/well and pretreated with compounds following 200 ng/mL LPS for 40 min. Parameters including nuclear area of 65 μm2, cell area of 80 μm2, collar of 0.9 μm and sensitivity of 25% were recommended for image segmentation algorithms in the analysis workstation. Benzoylhypaconine from aconite was screened for the first time as an anti-inflammatory candidate by the established HCI platform. The inhibitory effect of benzoylhypaconine on NF-κB translocation was verified by Western blot. Furthermore, benzoylhypaconine reduced the release of NO, inhibited the expression of iNOS, decreased the mRNA levels of IL-1β, IL-6, and TNF-α.
Conclusion
The established HCI platform could be applied to screen anti-inflammatory compounds by measuring the NF-κB nuclear translocation in LPS-induced RAW264.7 cells.
Qingfei Paidu (QFPD) granules have played a critical role during the Coronavirus Disease 2019 (COVID-19) in China. However, worldwide acceptance has been a problem because of the complex ingredients and unique theory of treatment. In this study, high-performance liquid chromatography (HPLC)-Q Exactive Orbitrap-mass spectrometry (MS) and the Orbitrap traditional Chinese medicine library (OTCML) were used to investigate the chemical constituents of QFPD granules. By comparing retention times, masses, isotope ion patterns, and MS² profiles, 108 compounds were putatively identified using the OTCML combined with manual verification, including 12 alkaloids, 49 flavonoids, 13 terpenoids, 14 phenylpropanoids, 4 phenolic acids, 5 phenols, and 11 other phytochemicals. Of these compounds, 17 were confirmed using reference standards. In addition, representative compounds of these different chemical types were used as examples to analyze the fragmentation pathways and characteristic product ions. Moreover, 20 herbs within the QFPD granules were also identified to establish the sources of these chemical components. This is the first rapid profiling of the chemical constituents of QFPD granules using HPLC-Q Exactive Orbitrap-MS and yields valuable information for further quality control and mechanistic studies of QFPD granules.
Respiratory viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV)-1, SARS-CoV-2, influenza A viruses, and respiratory syncytial virus, pose a serious threat to society. Based on the guiding principles of “holism” and “syndrome differentiation and treatment”, traditional Chinese medicine (TCM) has unique advantages in the treatment of respiratory virus diseases owing to the synergistic effect of multiple components and targets, which prevents drug resistance from arising. According to TCM theory, there are two main strategies in antiviral treatments, namely “dispelling evil” and “fu zheng”. Dispelling evil corresponds to the direct inhibition of virus growth and fu zheng corresponds to immune regulation, inflammation control, and tissue protection in the host. In this review, current progress in using TCMs against respiratory viruses is summarized according to modern biological theories. The prospects for developing TCMs against respiratory viruses is discussed to provide a reference for the research and development of innovative TCMs with multiple components, multiple targets, and low toxicity.
Production of minor crop varieties often requires intensive pesticide use, which raises serious concerns over food safety and human health. Chaenomeles speciosa (Sweet) Nakai as one of the representative of this kind of crops is therefore used for investigating the residue behaviour of fenpropathrin and emamectin benzoate, a synthetic pyrethroid and macrocyclic lactone widely used as an insecticide, respectively, from cultivation to C. speciosa postharvest processing. Results showed that the degradation trends of those selected insecticides in C. speciosa followed first-order kinetics with an average half-life (t1/2) of 3.7-4.1 days and a dissipation rate of 97% over 14 days. The terminal residues of fenpropathrin and emamectin benzoate at 120 and 3 g a.i./ha were below the U.S Environmental Protection Agency (FAD, 1.00 mg/kg) and European Union (EU, 0.01 mg/kg) maximum residue limits (MRLs) in papaya species, respectively, when measured 14 days after the final application, which suggested that the use of these insecticides was safe for humans. Postharvest processing procedure resulted in a |90 % reduction of the insecticides. Moreover, the hazard quotient (HQ) for C. speciosa decoction (with processing factors) indicated an acceptable risk for human consumption. These findings provide the scientific evidence of reasonable application and risk assessment of the selected pesticide residues in C. speciosa.
Background
HuaTanJiangQi capsule (HTJQ) is a classical Chinese medicine compound preparation, mainly used for clinically treating and improving the Chronic Obstructive Pulmonary Disease (COPD) in China.
Objective
To establish a rapid and efficient analytical method for the identification and characterization of chemical constituents in HTJQ based on ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS).
Methods
UPLC-QTOF-MS was used to rapidly separate and identify the chemical constituents of HTJQ via gradient elution system. The accurate mass data of the protonated and deprotonated molecules and fragment ions was detected in positive and negative ion modes. Compounds of HTJQ can be identified and assigned by analyzing accurate mass measurements and ion fragmentation mechanisms and comparing them with the chemical compositions database.
Results
A total of 61 compounds in HTJQ were separated and identified, including 14 flavonoids, 16 organic acids, 4 isothiocyanic acids, 8 butyl phthalides, 2 alkaloids, 10 terpenoids, 4 methoxyphenols and furanocoumarins, 3 other compounds. The chemical compounds of HTJQ were identified and elucidated comprehensively for the first time.
Conclusions
A rapid, accurate, and efficient UPLC-QTOF-MS method has been developed for the identification of the chemical components and applied to simultaneously evaluate the quality and effectiveness of the HTJQ.
Highlights
Systematic identification of chemical constituents in HTJQ can provide a scientific and reasonable basis for the application of HTJQ in the clinical treatment of COPD.
Ethnopharmacological relevance
Aconiti Lateralis Radix Praeparata (the Chinese name is Fuzi, FZ), the lateral or daughter root of Aconitum carmichaelii Debx. (Ranunculaceae), is a controversial traditional Chinese medicine (TCM) that is universally distributed and applied in many countries, such as China, Japan, Korea, and India. FZ can be used to treat various diseases, including rheumatic fever, rheumatism, painful joints, syncope, collapse, bronchial asthma, some endocrinal disorders, etc. However, quality control and assessment of FZ are challenging due to its obvious and high toxicological risks, and only its processed products are allowed to be used clinically according to the relative safety regulations. Consequently, it is necessary to analyze the whole chemical composition and the dynamic changes of FZ before and after processing. Addressing the changes in the chemical substance of raw and processed products is a way to reduce toxicity.
Aim of the study
In this article, the whole chemical composition of FZ is analyzed, the differences between raw and processed FZ are evaluated, and possible factors that influence the reduced toxicity of processed FZ are explained from the perspective of its chemical composition using qualitative and quantitative analysis methods.
Materials and methods
A novel strategy of multiple data collection and processing based on ultra-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) method in the positive ion mode, together with Global Natural Product Social Molecular Networking (GNPS) and multivariate statistical analysis, was established to systematically identify the chemical constituents of FZ and comprehensively investigate the chemical markers that can be used to differentiate FZ processed with vinegar and honey from its raw product. Combined with the qualitative analysis results, 12 components, including 8 chemical marker compounds and 4 toxicity components, were quantitatively analyzed by using high-performance liquid chromatography equipped with triple-quadrupole mass spectrometry (HPLC-MS/MS).
Results
Using the molecular networking (MN) analysis method, a total of 145 compounds were identified, of which 13 were identified using reference compounds. Seventy seven chemical markers were also detected between raw and processed FZ. The identification results of the chemical markers were also verified by orthogonal partial least squares discriminant analysis (OPLS-DA). The quantitative results indicated that the contents of 12 important components all decreased, especially diester-diterpenoid alkaloids (DDAs), after processing.
Conclusion
The decrease of toxicity of FZ after processing is closely related to the changes in its chemical composition. The method developed in this study is a comprehensive analysis technique for quality assessment of FZ, and this study provides a useful and quick strategy to characterize chemical compounds of TCM and explore the different chemical markers between raw and processed Chinese herbal medicine.
A natural product is any organic phytochemical that is synthesized by a living organism. Today, the growing interest in the characterization and analysis of natural products of plant, bacterial or fungi origin is unquestionable due to their potential applications as new drugs, functional foods, nutraceuticals or bioactive compounds. Natural products have been studied for the prevention of many diseases, including cancer. More than 800 new approved drugs in the last years were of natural origin. Analytical methodologies for the isolation, identification, and characterization of natural products are on demand.
Liquid chromatography (LC) separation methodologies are nowadays among the most employed strategies to address the analysis and characterization of natural products. The combination of these LC techniques with low-resolution mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS), either using ion trap (IT), linear ion trap (LIT), and triple quadrupole (QqQ) instruments, are the most frequently proposed methodologies for the determination of plant-based natural products. Of great importance in this field is the combination of liquid chromatography techniques with high-resolution mass spectrometry (LC-HRMS), mainly using time-of-flight and Orbitrap mass analyzers. The high-resolution power and accurate mass determination achieved with these techniques are crucial tools for the characterization and identification of new natural products in very complex matrices such as plants with a huge number of phytochemicals with grate differences in properties, structures, and concentration levels. Obviously, due to the high number of chemical data obtained by these techniques, chemometrics becomes an indispensable tool for the correct data processing. In this chapter, the role of liquid chromatography, mass spectrometry and chemometrics for the analysis and characterization of plant natural products will be addressed. Coverage of all kinds of applications in such an important field is beyond the scope of the present contribution, so the chapter will focus on the most relevant applications published in the last years.
Introduction
Jian‐Pi‐Yi‐Shen pill (JPYSP) is a Chinese medicine formula developed for the treatment of anaemic patients with chronic kidney disease (CKD).
Objective
To investigate the chemical profile of JPYSP in the treatment of renal anaemia.
Methods
A method coupling ultra‐performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐Q‐TOF‐MS/MS) was established to characterise the chemical constituents present in JPYSP. Subsequently, a high‐performance liquid chromatography method coupled with triple‐quadrupole tandem mass spectrometry (HPLC‐QQQ‐MS/MS) was developed to quantify the major constituents from the identified compounds related to the treatment of CKD and anaemia.
Results
A total of 71 compounds were tentatively identified from JPYSP, including saponins, flavonoids, sesquiterpenoids, coumarins, phenylpropanoids, anthranones, anthraquinones, tannins, phenolic acids and others. Amongst them, 12 compounds (i.e. astragaloside IV, calycosin, calycosin 7‐O ‐glucoside, salvianolic acid A, rosmarinic acid, rhein, liquiritin, formononetin, atractylenolide I, dioscin, tanshinone IIA, and acteoside) were further quantified simultaneously by HPLC‐QQQ‐MS/MS.
Conclusion
The newly developed approach is suitable for the chemical profiling analysis and quality control of JPYSP, and could lead to additional pharmacodynamic studies involving the components of JPYSP.
This review paper covers the forensic-relevant literature in controlled substances from 2016 to 2019 as a part of the 19th Interpol International Forensic Science Managers Symposium. The review papers are also available at the Interpol website at: https://www.interpol.int/content/download/14458/file/Interpol%20Review%20Papers%202019.pdf.
Talatisamine, as the efficacy ingredient of Aconitum, was known as a novel specific blocker for the delayed rectifier K+ channels in rat hippocampal neurons. In this study, a rapid, selective and reproducible UPLC-MS/MS separation method was established and fully validated for the quantitative determination of talatisamine levels in ICR (Institute of Cancer Research) mouse blood. A total of 24 healthy male ICR mice were divided into four groups that was administered talatisamine via intravenous at a dose of 1 mg/kg and oral administration of three doses (2, 4, 8 mg/kg). All blood samples were protein precipitate by using acetonitrile with an internal standard (IS) deltaline. The effective chromatographic separation was carried out through an UPLC BEH C18 analytical column (2.1 mm × 50 mm, 1.7 μm) with an initial mobile phase that consisted of acetonitrile and 10 mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid) with a gradient elution pumped at a flow rate of 0.4 mL/min. Also, an electrospray ionization (ESI) was applied to quantify the talatisamine in the positive ions mode. The method validation demonstrated good linearity over the range of 1-1000 ng/mL (r2 ≥ 0.9993) for talatisamine in mouse blood with a lower limit of quantification (LLOQ) at 1 ng/mL. The accuracy values of the method were within 89.4% to 113.3%, and the matrix effects were between 103.2% and 106.3%. The mean extraction recoveries for talatisamine obtained from four concentrations of QC blood samples were exceeded 71.7%, and the relative standard deviation (RSD) both of intra- and inter-day precision values for replicate quality control samples did not exceed 15% respectively for all analytes during the assay validation. This method was successfully applied to the evaluation of the pharmacokinetic of talatisamine, regardless of intragastric or intravenous administration in mice. Based on the pharmacokinetics data, the bioavailability of talatisamine in mice was >65.0% after oral administration, exhibiting an excellent oral absorption.
Current evidence shows that herbal medicines could be beneficial for the treatment of various diseases. However, the complexities present in chemical compositions of herbal medicines are currently an obstacle for the progression of herbal medicines, which involve unclear bioactive compounds, mechanisms of action, undetermined targets for therapy, non‐specific features for drug metabolism, etc. To overcome those issues, metabolomics can be a great to improve and understand herbal medicines from the small‐molecule metabolism level. Metabolomics could solve scientific difficulties with herbal medicines from a metabolic perspective, and promote drug discovery and development. In recent years, mass spectrometry‐based metabolomics was widely applied for the analysis of herbal constituents in vivo and in vitro. In this review, we highlight the value of mass spectrometry‐based metabolomics and metabolism to address the complexity of herbal medicines in systems pharmacology, and to enhance their biomedical value in biomedicine, to shed light on the aid that mass spectrometry‐based metabolomics can offer to the investigation of its active ingredients, especially, to link phytochemical analysis with the assessment of pharmacological effect and therapeutic potential. © 2019 Wiley Periodicals, Inc. Mass Spec Rev
Snake bile is one of the most expensive traditional Chinese medicines (TCMs). However, due to the complicated constitutes of snake bile and the poor ultraviolet absorbance of some trace bile acids (BAs), effective analysis methods for snake bile acids were still unavailable, making it difficult to solve adulteration problems. In present study, ultrahigh-performance liquid chromatography with triple quadrupole linear ion trap mass spectrometry (UHPLC-QqQ-MS/MS) was applied to conduct a quantitative analysis on snake BAs. The mass spectrometer was monitored in the negative ion mode, and multiple-reaction monitoring (MRM) program was used to determine the contents of BAs in snake bile. In all, 61 snake bile from 17 commonly used species of three families (Elapidae, Colubridae and Viperidae), along with five batches of commercial snake bile from four companies, were collected and detected. Nine components, Tauro-3α,12α-dihydroxy-7-oxo-5β-cholenoic acid (T1), Tauro-3α,7α,12α,23R-tetrahydroxy-5β-cholenoic acid (T2), taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), cholic acid (CA), Tauro-3α,7α-dihydroxy-12-oxo-5β-cholenoic acid (T3), and Tauro-3α,7α,9α,16α-tetrahydroxy-5β-cholenoic acid (T4) were simultaneously and rapidly determined for the first time. In these BAs, T1 and T2, self-prepared with purity above 90%, were first reported with their quantitative determination, and the latter two (T3 and T4) were tentatively determined by quantitative analysis multi-components by single marker (QAMS) method for roughly estimating the components without reference. The developed method was validated with acceptable linearity (r(2)≥0.995), precision (RSD＜6.5%) and recovery (RSD＜7.5%). It turned out that the contents of BAs among different species were also significantly different; T1 was one of the principle bile acids in some common snake bile, and also was the characteristic one in Viperidae and Elapidae; T2 was the dominant components in Enhydris chinensis. This quantitative study of BAs in snake bile is a remarkable improvement for clarifying the bile acid compositions and evaluating the quality of snake bile.
The herb-pair, Astragali Radix (AR) and Curcumae Rhizoma (CR), often occurs in traditional herbal prescriptions used for cancer treatment in Asian areas. In clinical application, the AR-CR herb pair was often produced by different preparation methods or with raw materials from different sources, which raised a challenge for quality control of the herb-pair medicines. In this paper, ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry method (UPLC-QQQ-MS) was applied for the first time to simultaneously determine 17 main bioactive components for quality control of AR-CR herb pair. The chromatographic separation was studied on an ACQUITY UPLC BEH C18 column (100mm×2.1mm, 1.7μm) with a mobile phase composed of 0.1% aqueous formic acid and acetonitrile using a gradient elution in 12min. The proposed method was optimized and validated by good linearity (r(2)>0.9970), limit of detection (0.33-10.78ng/mL), limit of quantification (0.81-2.54ng/mL), intra- and inter-day precisions (RSD≤3.64%, RSD≤5.68%), stability (RSD≤4.29%), repeatability (RSD≤5.98%), recovery (90.20-107.60%). The established method was successfully applied to comparative analysis of main bioactive components in AR-CR herb pair and its single herbs, and quality evaluation of different batches of clinical dispensing granules. Compared to the single herb, the content of most liposoluble constituents such as curcumenol, curdione, isocurcumenol, furanodienone, curcumol, and germacrone were remarkable increased in their herb pair, suggesting mixed preparation produced synergistic effects on promoting the extraction of bioactive ingredients. This study is the first time to report the rapid and simultaneous analysis of 17 compounds in AR-CR herb pair by UPLC-QQQ-MS, and provides a feasible method for holistic quality control of preparations containing AR-CR herb-pair.
Herbal preparation has recently increased health awareness and its efficacy is brought about by the multi-constituents. Therefore, the application of mass spectrometry (MS) in herbal preparation has been dramatically growing due to the improved detection capabilities. It offered high sensitivity, high-throughput and selectivity, has better accuracy, and can be utilized to quantitative, qualitative and imaging. High-throughput screening method integrated with MS platform is an excellent tool to analyse the multi-components of herbal preparation in the post-genomic era. For providing an up-to-date overview of MS application on the HM-derived compounds, the papers published in the latest years involving quantitative and qualitative analysis of chemical constituents and MS platform integrated with high-throughput screening method are summarized in this review. Examples of the application of MS in the HM sciences focusing on chemical analysis are highlighted, and new developments and future prospects are also highlighted.
An offline two-dimensional system combining a rat cardiac muscle cell membrane chromatography time-of-flight mass spectrometry (CMC-TOF/MS) with a high Performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF/MS) was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis ofthe analysis of the first dimension, retention components of the urine sample were collected into 30 fractions (one fraction per minute). Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine (Songorine, 14-benzoylneoline, Deoxyaconitine, etc.) exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the System suggest that the CMC can be applied to in vivo study.
Abstract Background Metabolomic studies are targeted at identifying and quantifying all metabolites in a given biological context. Among the tools used for metabolomic research, mass spectrometry is one of the most powerful tools. However, metabolomics by mass spectrometry always reveals a high number of unknown compounds which complicate in depth mechanistic or biochemical understanding. In principle, mass spectrometry can be utilized within strategies of de novo structure elucidation of small molecules, starting with the computation of the elemental composition of an unknown metabolite using accurate masses with errors
Aconitum coreanum (Lèvl.) Rapaics (Guanbaifu in Chinese) is a widely used, centuries-old Chinese herb. A preparative high-speed counter-current chromatography (HSCCC) coupled with evaporative light scattering detection (ELSD) method was employed for isolation and purification of alkaloids from the crude extract of Aconitum coreanum (Lèvl.) Rapaics using ethyl acetate-n-butanol-methanol-0.2 m HCl (7:2:2:7, v/v) as a two-phase solvent system. Six alkaloids, including GFO, GFQ, GFZ, hetisinone, hetisine and GFAA, were obtained in one-step separation. The purity of these compounds was 97.6, 93.8, 91.8, 91.9, 96.2 and 91.1%, respectively.
This paper summarized the chemical constituents and pharmacological activities of Gynostemma pentaphyllum based on systematic literature research. G. pentaphyllum mainly contains dammarane-type saponins and a total of 165 gynostemma saponins were obtained from 1976 to 2011.In recent years, some new gypenosides were isolated and identified. Two hundred and one gypenosides were classified and summarized in this paper. In addition, G. pentaphyllum also contains a variety of other chemical components including flavonoids, polysaccharides, amino acids, and trace elements, which are worth further research. The pharmacological effects of G. pentaphyllum including antitumor, regulating blood lipid, hypoglycemic, liver protection, anti-senility and improving immunity effects were summarized. This review will provide some information for further research of G. pentaphyllum.
The plants of genus Solanum L. contain alkaloids, flavonoids, steroids, and other various chemical constituents which exhibit protective activity, antifungal, antiviral, antitumor, and anti-oxidant activities. This paper reviews the research survey of the chemical constituents and biological activities of the plants of Solanum L. from domestic and foreign over the last 20 years, and provides the relative reference for the further development of the plants in Solanum L. © 2016, Editorial Office of Chinese Traditional and Herbal Drugs. All right reserved.
Objective: To analyze the changes of chemical ingredients before and after compatibility of Aconiti Lateralis Radix Praeparata (ALRP) and Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (GRRPM), and to explore the possible mechanism of toxicity attenuation of their compatibility. Methods: The chemical ingredients in the decoction of ALRP and the decoction of compatibility of ALRP and GRRPM were comparatively researched by HPLC-MS. Single decoction of ALRP and compound decoction of compatibility of ALRP and GRRPM were prepared, and their HPLC-MS fingerprints were respectively established and determined by Q-TOF/MS under the same condition. Results: Twenty ingredients and their structures were identified from single decoction of ALRP; 32 ingredients and their structures were indentified from the decoction of ALRP and GRRPM, among them 4 from GRRPM, 28 from ALRP. And the alkaloid categories and contents of ALRP were significantly different before and after the compatibility with GRRPM. Conclusion: The compatibility with GRRPM could change the alkaloid composition in ALRP, which provides the experimental evidences for the toxicity attenuation mechanism of compatibility of ALRP and GRRPM.
In the present UPLC/Q-TOF-MS study, an ultra performance liquid chromatography coupled with time-of-fight mass spectrometry (UPLC/Q-TOFMS) based chemical profiling approach was used to evaluate chemical constitution between co-decoction and mixed decoction of radix aconiti and pinellia praeparata. Two different kinds of decoctions, namely radix aconiti and pinellia praeparata co-decoction: water extract of mixed two herbs, and radix aconiti and pinellia praeparata mixed decoction: mixed water extract of each individual herbs, were prepared. Batches of these two kinds of decoction samples were subjected to UPLC/Q-TOF-MS analysis, the datasets of tR-m/z pairs, ion intensities and sample codes were processed with orthogonal partial least squared discriminant analysis (OPLS-DA) to holistically compare the difference between these two kinds of decoction samples. The results of positive ion mode showed significant difference between two kinds of decoction samples, the content of hypaconitine, mesaconitine, aconitine, deoxyaconitine, 10-OH-mesaconitine, 10-OH-aconitine increased, while the content of mesaconine, dehydrated benzoylmesaconine, dehydrated benzoylhypaconine, benzoylaconine, benzoylhypaconine, 10-OH-benzoylmesaconine decreased in samples of co-decoction of radix aconiti and pinellia praeparata. The content of diester-diterpenoid alkaloids increased, while the content of monoester-diterpenoid alkaloids decreased, which may be basis of chemical composition in combination of radix aconiti and pinellia praeparata.
To investigate the spectrum-effect relationship between the fingerprint in plasma of Heishunpian (black-processed pieces of Aconit Lateralis Radix Praeparata) and its pharmacodynamic parameters (blood pressure and heart rate) after ig administration to rats, and to elucidate its effective substances. Ultra performance liquid chromatography with quadrupole and time-of-flight mass spectrometry (UPLC-QTOF/MS) was used for fingerprint profiling by analyzing plasma samples obtained after ig administration of the ethanol extract from Heishunpian to rats. Meanwhile, the blood pressure and heart rate of heart-failure rats were continuously monitored and recorded. The relationship between the absorbed fingerprint and pharmacodynamic parameters of Heishunpian was established by correlation analysis with partial least square regression (PLSR) method. A total of 13 alkaloids were detected in rat plasma, among which hypaconine, benzoylmesaconitine, benzoylhypaconitine, and 3, 13-deoxybenzoylaconitine were found to be significantly and positively related to the pharmacodynamic data with VIP > 1. Hypaconine, benzoylmesaconitine, benzoylhypaconitine, and 3, 13-deoxybenzoylaconitine are potential effective substances with anti-heart failure effects of Heishunpian. ©, 2015, Chinese Traditional and Herbal Drugs. All right reserved.
Aconite poisoning continues to be a major type of poisoning caused by herbal drugs in many countries. Nevertheless, despite its toxic characteristics, aconite is used because of its valuable therapeutic benefits. The aim of the present study was to determine the distribution of toxic alkaloids in tissues of aconite roots through chemical profiling. Three species were studied, all being used in traditional Chinese Medicine (TCM) and traditional Indian medicine (Ayurveda), namely: Aconitum carmichaelii, Aconitum kusnezoffii and Aconitum heterophyllum. Laser micro-dissection was used for isolation of target microscopic tissues, such as the metaderm, cortex, xylem, pith, and phloem, with ultra-high performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS) employed for detection of metabolites. Using a multi-targeted approach through auto and targeted LC-MS/MS, 48 known compounds were identified and the presence of aconitine, mesaconitine and hypaconitine that are the biomarkers of this plant was confirmed in the tissues. These results suggest that the three selected toxic alkaloids were exclusively found in A. carmichaelii and A. kusnezoffii. The most toxic components were found in large A. carmichaelii roots with more lateral root projections, and specifically in the metaderm, cork and vascular bundle tissues. The results from metabolite profiling were correlated with morphological features to predict the tissue specific distribution of toxic components and toxicity differences among the selected species. By careful exclusion of tissues having toxic diester diterpenoid alkaloids, the beneficial effects of aconite can still be retained and the frequency of toxicity occurrences can be greatly reduced. Knowledge of tissue-specific metabolite distribution can guide users and herbal drug manufacturers in prudent selection of relatively safer and therapeutically more effective parts of the root. The information provided from this study can contribute towards improved and effective management of therapeutically important, nonetheless, toxic drug such as Aconite.
Aim
The aim of this work was to establish a specific and sensitive method to comprehensively investigate and compare chemical constituents of Fuzi-Gancao herb pair (FG), consisting of Aconitum carmichaelii Debeaux (Fuzi, Chinese) and Roast Radix Glycyrrhizae (Glycyrrhiza glabra L., Gancao, in Chinese) and Fuzi alone to explore the underlying interaction mechanism of FG.
Method
An ultra-fast liquid chromatography-ion trap/time-of-flight mass spectrometry (UFLC/MS-IT-TOF) method using diazepam as internal standard was developed for the identification and semi-quantitative analysis of the phytochemical constituents of Fuzi and FG. Chromatographic separation was achieved on a UFLC column using a gradient program with 40 mmol·L−1 ammonium acetate and acetonitrile as the mobile phase.
Results
Fifty-one of the sixty compounds, including forty-five C19-diterpenoid alkaloids and six C20-diterpenoid alkaloids were tentatively identified in the extracts of Fuzi and FG through accurate mass measurements and fragmentation patterns. Comparing the contents of these alkaloids in these two extracts, it was found that the diester-diterpenoid alkaloids (DDAs) and the alkylolamine-diterpenoid alkaloids (ADAs) were increased, while the monoester-diterpenoid alkaloids (MDAs) were decreased in the extracts of FG.
Conclusion
This work provided comprehensive information for the quality control of Fuzi preparations, and the further investigation on the compatibility mechanisms of FG
Da-Huang-Fu-Zi-Tang (DHFZT) is a crucial TCM formula commonly used for the treatment of acute pancreatitis in Chinese clinical application. Our previous work found that DHFZT could act against pancreatic injury in rats with severe acute pancreatitis (SAP).
The goal of this paper was to study the underlying correlations between the chemical spectra and the protective effect of DHFZT on pancreatic acinar cell to reveal the real bioactive compounds in DHFZT.
The fingerprint chromatograms of rat serum after oral administration of DHFZT were established by UHPLC-ESI-Q-TOF-MS technique. At the same time, the model of anti-acute pancreatitis on cells was established by adding 10(-7)mol/L cerulein to AR42J cell line, and the protective effects of the serum on pancreatic acinar cell from injury was evaluated by detecting the efficacy of amylase. Then, the spectrum-effect relationships between UHPLC fingerprints and anti-acute pancreatitis activities were evaluated using canonical correlation analysis (CCA) statistical method. The chromatogram separation was performed on a C18 reversed phase UHPLC column (2.1mm×100mm, 3.5μm, Agilent), the column temperature was set at 35°C. The mobile phase consisted of 0.1% formic acid and acetonitrile with gradient elution. The serum samples were analyzed both in negative and positive ion mode. The mother and productive ions were scanned within the mass range of m/z 100-1200 and 50-1200, respectively. A thorough analysis of a great deal of information of the constituents in the rat serum was undertaken. The structure identification of the detected compounds was achieved by using high resolution MS values as well as the MS/MS fragments.
18 peaks in rat serum after oral administration of DHFZT were detected within only 30min recorded chromatograms. The structure of the 18 compounds were then given out, of which 10 were the original form of compounds absorbed from DHFZT, 8 were the metabolites of the compounds existed in rat serum. According to the CCA results, talatisamine, rhein glucoside, rhein isomer methylation, hypaconine, hydroxyl-chrysophanol, emodin glucuronide conjugation, and chrysophanol glucuronide conjugation were finally found to be the main anti-acute pancreatitis components in DHFZT.
The model presented in this paper successfully discovered the spectrum-effect relationships of DHFZT, which showed a representative way to discover the primary active ingredients from the complicated herbal drugs.
The aim of this study was to elucidate the structure of a potential ageing marker for Cava sparkling wine. In order to clarify the structure of this compound, NMR spectroscopy and hyphenated UHPLC-DAD-MS/MS techniques were used. We identified the hitherto unknown compound as 5-hydroxymethyl-2-furfuraldehyde (5-HMF). This is the first time that this compound has been reported in sparkling wines. A survey, based on the analysis of 80 commercial sparkling wines, showed that 5-HMF is present between 0.25 and 12.81mg/L, and is compared with those reported for other types of wine. Hypothetical origin of 5-HMF in Cava sparkling wine is discussed.
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed which, with sample preparation using a commercially available kit, allows rapid quantitation of 39 chloroformate-derivatised amino acids (AAs), polyamines (PAs) and dipeptides (DPs) in complex biological matrices. Lower limits of quantitation (LOQ) were 20-150nM for putrescine, spermine, spermidine, cadaverine, agmatine, and below 5μM for all analytes. Responses were linear for all analytes between 0.5 and 50μM. Quantitative measurements of all 39 metabolites were achieved within a 15min runtime. The method was evaluated with a Pseudomonas aeruginosa cell extract study (n=24) and a larger human urine study (n=308). Batch effects were observed in the urine study and an investigation of instrument and sample stability showed a wave-like pattern in the MS responses. Both the run order and inter-batch variation were successfully corrected by normalising to pooled urine quality control data. Thus, this method should be suitable for diverse biological matrices and for large as well as small sample sets.
Objective: To rapidly identify the chemical components in a traditional Chinese herb Xiaochaihu decoction by high performance liquid chromatography-time of flight mass spectrometry (HPLC-TOF/MS). Methods: An Agilent ZORBAX SB-C 18 column (100 mmX3.0 mm,3.5 μm) was used for rapid separation and identification of chemical components in Xiaochaihu decoction, with a mobile phase of methanol and 0.1% formic acid aqueous solvent in gradient elution. The flow rate was 0.6 ml/min and the injection volume was 4 μl. The detector was time-of-flight mass spectrometer with an ESI ion source. Scanning mass range was m/z 200-1 500. Results: Thirty-eight chemical compounds were identified from Xiaochaihu decoction in one run. Conclusion: Thirty-eight chemical compounds have been identified in Xiaochaihu decoction by HPLC-TOF/MS in one run, which paves a way for further understanding the metabolism and mechanism of the chemical compounds in Xiaochaihu decoction.
Determination of fourteen alkaloids, toxic Aconitum alkaloids, aconitine, mesaconitine, jesaconitine, hypaconitine and deoxyaconitine, and their hydrolysis products, benzoylaconines and aconines, have been established using capillary liquid chromatography (LC) fast atom bombardment mass spectrometry (FAB-MS) with a frit interface. Protonated molecular ions were observed as base peaks in the FAB-MS for these fourteen alkaloids. All the alkaloids were simultaneously quantified with linear gradient LC elution by solvent mixture of acetonitrile and 0.3% trifluoroacetic acid using selected ion monitoring of the protonated molecular ions. The calibration curves of these alkaloids were linear in injection amounts ranging from 5 to 500 pg, and their detection limits were 1 pg per injection (S/N=3). Solid-phase extraction using Sep-Pak Plus PS-1 was also investigated to clean-up and concentrate alkaloids in blood and urine samples, and showed satisfactory recoveries. This capillary LC–frit-FAB-MS method enables determination of low levels of Aconitum alkaloids in blood and urine samples, coupled with solid-phase extraction.
To evaluate the correlativity between volatile oils of Mahuang Fuzi Xixin decoction and its major constituted herbs in composition and proportion.
The chemical compositions of volatile oils obtained by wet distillation from Mahuang Fuzi Xixin decoction and its major constituted herbs (Herba Ephedrae, processed Radix Aconiti Lateralis, Herba Asari) were analyzed by GC-MS.
44 volatile components of Mahuang Fuzi Xixin decoction were identified, which mainly derived from its constituted herbs Mahuang and Xixin. 68, 8, 39 volatile components were respectively identified from volatile oils of Herba Epherae, processd Radix Aconiti Lateralis and Herba Asari. There were apparent changes in composition and proportion between volatile oils of Mahuang Fuzi Xixin decoction and its major constituted herbs.
There are apparent changes not only in quantity but also in quality between decoction and its major constituted herbs. Meanwhile any changes in composition may lead to a change in efficacy.
A high-performance liquid chromatography with diode-array detection coupled to time-of-flight mass spectrometry (HPLC/DAD/TOFMS) method was established to clarify the chemical composition of Sini decoction (SND) and rat plasma after oral administration of SND. With dynamic adjustment of fragmentor voltage in TOFMS, an efficient transmission of the ions was achieved to obtain the best sensitivity for providing the molecular formula for each analyte and abundant fragment ions for structural information. By accurate mass measurements within 5 ppm error for each molecular ion and subsequent fragment ions, 53 compounds including diterpenoid alkaloids, flavonoids, triterpenoids and gingerol-related compounds were identified in SND. Major compounds identified from SND were further assigned in the three individual herbs. After oral administration of SND, 33 compounds and five metabolites in rat plasma were detected and identified by comparing and contrasting the compounds measured in SND with those in the plasma samples by HPLC/DAD/TOFMS. The results provided helpful chemical information for further pharmacology and active mechanism research on SND.
Studies of aconitine-type alkaloids in the Chinese herb Aconitum Carmichaeli were performed by HPLC/ESIMS/MS(n) and FTICR/ESIMS in positive ion mode. The characteristic fragmentation pathways in the MS(n) spectra were summarized based on previously published research literature and further study. According to the fragmentation pathways of mass spectrometry, results from the analysis of standard compounds and reports from literature, 111 compounds were identified or deduced in a total of 117 found compounds in A. Carmichaeli. In the 11 monoester-diterpenoid alkaloids (MDA), 10 diesterditerpenoid alkaloids (DDA) and 81 lipo-alkaloids, the novel alkaloids including 1 MDA, 2 DDA and 48 lipo-alkaloids were detected. In addition, 1 DDA, 7 lipo-alkaloids and 2 alkaloids with small molecular weights that possess C19-norditerpenoid skeleton were reported in A. Carmichaeli for the first time.
To estabilish a rapid and sensitive LC-ESI-ITMSn method for the identification of ephedrine and its main metabolites in rat urine.
After optimizing the detection condition of LC-ESI-ITMSn chromatography and mass spectrometry by using a standard ephedrine, the ionization and cleavage rules of ephedrine in ESI-MS and ESI-MSn modes were summarized, and then serving as the basis for the metabolite analysis of ephedrine in rat urine. Rat urine samples of 0-48 h were collected after ig 10 mg x kg(-1) ephedrine, then the samples were purified through C18 solid-phase extraction cartridge. The purified samples were analyzed by LC-ESI-ITMSn.
The structures of ephedrine metabolites were elucidated according to the changes of the molecular weights of the metabolites (deltaM) and their cleavage pattern in ESI-ITMSn. As a result, three phase I metabolites and the parent drug ephedrine were identified existing in rat urine, but no phase II metabolites were found.
The LC-ESI-ITMSn method is rapid and highly sensitive and sepecific, it is suitable for the identification of ephedrine and its metabolites in rat urine.
Amino acids in biological fluids have previously been shown to be detectable using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with perfluorinated acids as ion-pairing agents. To date, these studies have used precursor mass, retention time and tandem mass spectrometry (MS/MS) to identify and quantify amino acids. While this is a potentially powerful technique, we sought to adapt the method to time-of-flight (TOF)MS. A new application of a recently described liquid chromatographic separation method was coupled with TOFMS to employ accurate mass for qualitative identification; resulting in additional qualitative data not available with standard single quadrupole data. In the current study, we evaluated 25 physiological amino acids and one dipeptide that are routinely quantified in human plasma. Accuracy and precision of the method was evaluated by spiking human plasma with a mix of the 25 amino acids; in addition, the inclusion of a cation-exchange cleanup step was evaluated. The calibration curves were linear over a range from 1.56 to 400 microM. The dynamic range was found to be within physiological levels for all amino acids analyzed. Accuracy and precision for most of the amino acids was between 80-120% spike recovery and <10% relative standard deviation (RSD). The LC/MS technique described in this study relies on mass accuracy and is suitable for the quantitation of free amino acids in plasma.
Blocking specific K+ channels has been proposed as a promising strategy for the treatment of neurodegenerative diseases. Using a computational virtual screening approach and electrophysiological testing, we found four Aconitum alkaloids are potent blockers of the delayed rectifier K+ channel in rat hippocampal neurons. In the present study, we first tested the action of the four alkaloids on the voltage-gated K+, Na+ and Ca2+ currents in rat hippocampal neurons, and then identified that talatisamine is a specific blocker for the delayed rectifier K+ channel. External application of talatisamine reversibly inhibited the delayed rectifier K+ current (IK) with an IC50 value of 146.0+/-5.8 microM in a voltage-dependent manner, but exhibited very slight blocking effect on the voltage-gated Na+ and Ca2+ currents even at the high concentration of 1-3 mM. Moreover, talatisamine exerted a significant hyperpolarizing shift of the steady-state activation, but did not influence the steady state inactivation of IK and its recovery from inactivation, suggesting that talatisamine had no allosteric action on IK channel and was a pure blocker binding to the external pore entry of the channel. Our present study made the first discovery of potent and specific IK channel blocker from Aconitum alkaloids. It has been argued that suppressing K+ efflux by blocking IK channel may be favorable for Alzheimer's disease therapy. Talatisamine can therefore be considered as a leading compound worthy of further investigations.
Experimental comparison study on mice's acute toxicity of different composition in asarum
- Li R.
Study on cardiac effect of processing of aconite root
- Zhang Y. Q.
Study on effective components from roots of Aconitum carmichaeli through cell membrane chromatography and HPLC-TOF/MS method
- Y. Cao
- J. Jing
- D. Y. Lv
Mass spectrometric behavior analysis of aconitine
- Chen Y. J.
Simultaneous determination of five ephedrine alkaloids in Ephedrae Herba by HPLC‐UV
- Chen Y.
The ESI‐MS study on the fragmentation pathways of the aconitine, mesaconitine, hypaconitine and their hydrolyzates
- Li Z.
The change of the main chemical constituents and anti inflammatory activity of Mahuang‐Gancao before and after compatibility
- Meng X. Y.
Analysis on chemical constituents of sini decoction by RRLC‐TOF/MS
- Tan G. G.
Studies on the chemical constituents and bioactivities of Aconiti Lateralis Radix Praeparata. China academy chinese medical sciences
- K H Wu
Basic study on effective substance and application of Aconitum decoction
- Yu C. C.