[Synthesis and tissue distribution of bufadienolides in Bufo bufo gargarizans].

This study explored the biosynthesis of bufadienolides(BDs) in Bufo bufo gargarizans to solve the dilemma of the decreasing resources of B. bufo gargarizans and provide a theoretical basis for the sustainable utilization of the resources. Ultra-high performance liquid chromatography-Orbitrap-mass spectrometry(UHPLC-Orbitrap-MS) was employed to detect the synthesis sites of BDs in B. bufo gargarizans, and the results were verified by desorption electrospray ionization-mass spectrometry imaging(DESI-MSI) and homogenate incubation experiments. BDs in B. bufo gargarizans had the highest content in the liver and the highest concentration in the gallbladder, in addition to the parotid gland and skin, which suggested that the liver could synthesize BDs. The results of DESI-MSI also showed that BDs were mainly enriched in the liver rather than the immature parotid gland. The incubation experiment of liver homogenates demonstrated the liver of B. bufo gargarizans had the ability to synthesize BDs. This study showed that the liver was a major organ for the synthesis of BDs in B. bufo gargarizans during metamorphosis, development, and growth, which provided strong theoretical support for the biosynthesis of BDs and the sustainable utilization of B. bufo gargarizans resources.

[Evaluation of efficacy of Dihuang Baoyuan Granules for diabetes mellitus and composition and in vivo distribution analysis based on UHPLC-LTQ-Orbitrap MS].

Dihuang Baoyuan Granules is a prescription endorsed by HU Tianbao, a renowned and elderly Chinese medicine practitioner from Beijing, and has demonstrated definite clinical efficacy. The composition of this prescription is intricate as it includes 7 distinct herbal medicines. This study aims to analyze the chemical composition of Dihuang Baoyuan Granules, evaluate its efficacy in the treatment of diabetes and analyze the distribution of the drug components in the plasma, liver, and kidney after administration. The findings will serve as a reference for future research on pharmacodynamic substances of this prescription. UHPLC-LTQ-Orbitrap MS was employed to analyze the main chemical components of Dihuang Baoyuan Granules. A Waters ACQUITY Premier HSS T3 column(2.1 mm×100 mm, 1.8 µm) was used for chromatographic separation with 0.1% formic acid(A)-acetonitrile(B) as the mobile phases in a gradient elution at a flow rate of 0.3 mL·min~(-1). Electrospray ionization(ESI) source was used to acquire data in positive and negative ion modes. Furthermore, a rat model of diabetes mellitus was established by feeding with a high-sugar high-fat diet, and injection with streptozocin at a dose of 35 mg·kg~(-1), and the modeled rats were then administrated with Dihuang Baoyuan Granules. The fasting blood glucose, hemoglobin A1c, and other relevant indicators were measured, and the substances present in the plasma, liver, and kidney were identified. By reference to quasi-molecular ions, MS/MS fragment ions, MS spectra of reference substances, and compound information in available reports, 191 components were identified in Dihuang Baoyuan Granules, including 29 alkaloids, 24 flavonoids, 22 organic acids, 16 amino acids, 12 terpenes, 11 steroid saponins, 9 sugars, 8 phenylethanoid glycosides, 8 nucleosides, 2 phenylpropanoids, and 49 others compounds. Eighty-three chemical components were identified in rat plasma, 109 in the liver, and 98 in the kidney. Component identification and characterization of Dihuang Baoyuan Granules in vitro and in vivo provide efficacy information and guidance for the basic research on the pharmacodynamic substances and further clinical application of this prescription.

[Tissue distribution of Qingfei Paidu Decoction based on HPLC-MS/MS].

The tissue distribution of Qingfei Paidu Decoction was studied by HPLC-MS/MS in vivo. Hypersil GOLD C_(18) column(2.1 mm×50 mm, 1.9 µm) was used for gradient elution with acetonitrile as the mobile phase A and 0.1% formic acid solution as the mobile phase B. High-resolution liquid chromatography-mass spectrometry in both positive and negative ion scanning mode and multiple response monitoring(MRM) mode was employed to analyze the behaviors of the active components of Qingfei Paidu Decoction in diffe-rent tissues. The results showed that 19, 9, 17, 14, 22, 19, 24, and 2 compounds were detected in plasma, heart, liver, spleen, lung, kidney, large intestine, and brain, respectively. The compounds belonged to 8 groups, covering 14 herbs in the prescription. After administration with Qingfei Paidu Decoction, the compounds were rapidly distributed in various tissues, especially in the lung, liver, large intestine, and kidney. The majority of the compounds displayed secondary distribution. This study comprehensively analyzed the distribution rules of the main active components in Qingfei Paidu Decoction and provided a basis for the clinical application.

[Systematic comparison of two kinds of Bufonis Venenum derived from different Bufo gargarizans subspecies based on metabolomics and antitumor activity].

This paper compared the differences between two kinds of Bufonis Venenum produced by Bufo gargarizans gargarizans and B. gararizans andrewsi, and verified the rationality of the market value orientation of Bufonis Venenum based on the zebrafish mo-del. Twenty batches of Bufonis Venenum from Jiangsu province, Hebei province, Liaoning province, Jilin province, and Liangshan, Sichuan province, including B. gargarizans gargarizans and B. gararizans andrewsi, were collected. The UHPLC-LTQ-Orbitrap-MS combined with principal component analysis was used to compare the differences between two kinds of Bufonis Venenum. According to the limiting conditions of VIP>1, FC<0.5 or FC>2.0, and peak total area ratio>1%, 9 differential markers were determined, which were cinobufagin, cinobufotalin, arenobufagin, resibufogenin, scillaredin A, resibufagin, 3-(N-suberoylargininyl)-arenobufagin, 3-(N-suberoylargininyl)-marinobufagin, and 3-(N-suberoylargininyl)-resibufogenin. The content of 20 batches of Bufonis Venenum was determined according to the Chinese Pharmacopoeia(2020 edition) by high-performance liquid chromatography, and the 2 batches of Bufonis Venenum, CS7(8.99% of total content) and CS9(5.03% of total content), with the largest difference in the total content of the three quality control indexes of the Chinese Pharmacopoeia(bufalin, cinobufagin, and resibufogenin) were selected to evaluate their anti-liver tumor activity based on the zebrafish model. The tumor inhibition rates of the 2 batches were 38.06% and 45.29%, respectively, proving that only using the quality control indexes of the Chinese Pharmacopoeia as the value orientation of Bufonis Venenum market circulation was unreasonable. This research provides data support for the effective utilization of Bufonis Venenum resources and the establishment of a rational quality evaluation system of Bufonis Venenum.

[Study on biomarkers of acteoside in treating puromycin aminonucleoside nephropathy in young rats based on non-targeted urine metabolomics technology].

This study aims to reveal the endogenous metabolic characteristics of acteoside in the young rat model of purinomycin aminonucleoside nephropathy(PAN) by non-targeted urine metabolomics and decipher the potential mechanism of action. Biochemical indicators in the urine of rats from each group were determined by an automatic biochemical analyzer. The potential biomarkers and related core metabolic pathways were identified by ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) combined with principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). MetaboAnalyst 5.0 was used to establish the receiver operating characteristic(ROC) curve for evaluating the clinical diagnostic performance of core metabolites. The results showed that acteoside significantly decreased urinary protein-to-creatinine ratio in PAN young rats. A total of 17 differential metabolites were screened out by non-targeted urine metabolomics in PAN young rats and they were involved in phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. Thirtten differential metabolites were screened by acteoside intervention in PAN young rats, and they were involved in phenylalanine metabolism and arginine and proline metabolism. Among them, leucylproline and acetophenone were the differential metabolites that were significantly recovered after acteoside treatment. These pathways suggest that acteoside treats PAN in young rats by regulating amino acid metabolism. The area under the curve of two core biomarkers, leucylproline and acetophenone, were both greater than 0.9. In summary, acteoside may restore amino acid metabolism by regulating endogenous differential metabolites in PAN young rats, which will help to clarify the mechanism of acteoside in treating chronic glomerulonephritis in children. The characteristic biomarkers screened out have a high diagnostic value for evaluating the treatment of chronic glomerulonephritis in children with acteoside.

[Clinical application value of Huanglian Jiedu Pills in improving syndrome of excess heat and fire toxin based on phase â ¡ clinical trial study on plasma ATP, 4-HNE, and ACTH levels].

A randomized, double-blind, placebo-controlled, multi-center phase Ⅱ clinical trial design was used in this study to recruit subjects who were in line with the syndrome of excess heat and fire toxin, and were diagnosed as recurrent oral ulcers, gingivitis, and acute pharyngitis. A total of 240 cases were included and randomly divided into a placebo group and a Huanglian Jiedu Pills group. The clinical efficacy of Huanglian Jiedu Pills in treating the syndrome of excess heat and fire toxin was evaluated by using the traditional Chinese medicine(TCM) syndrome scale. Enzyme-linked immunosorbent assay(ELISA) was used to determine and evaluate the levels of adenosine triphosphate(ATP), 4-hydroxynonenal(4-HNE), and adrenocorticotropic hormone(ACTH) in plasma of the two groups before and after administration and to predict their application value as clinical biomarkers. The results showed that the disappearance rate of main symptoms in the Huanglian Jiedu Pills group was 69.17%, and that in the placebo group was 50.83%. The comparison between the Huanglian Jiedu Pills group and the placebo group showed that 4-HNE before and after administration was statistically significant(P<0.05). The content of 4-HNE in the Huanglian Jiedu Pills group decreased significantly after administration(P<0.05), but that in the placebo group had no statistical significance and showed an upward trend. After administration, the content of ATP in both Huanglian Jiedu Pills group and placebo group decreased significantly(P<0.05), indicating that the energy metabolism disorder was significantly improved after administration of Huanglian Jiedu Pills and the body's self-healing ability also alleviated the increase in ATP level caused by the syndrome of excess heat and fire toxin to a certain extent. ACTH in both Huanglian Jiedu Pills group and placebo group decreased significantly after administration(P<0.05). It is concluded that Huanglian Jiedu Pills has a significant clinical effect, and can significantly improve the abnormal levels of ATP and 4-HNE in plasma caused by the syndrome of excess heat and fire toxin, which are speculated to be the effective clinical biomarkers for Huanglian Jiedu Pills to treat the syndrome of excess heat and fire toxin.

[Component profile analysis of Sanhan Huashi Formula based on UHPLC-LTQ-Orbitrap-MS, GC-MS, and UHPLC-QQQ-MS/MS].

This study comprehensively analyzed the active components of Sanhan Huashi Formula using qualitative and quantitative mass spectrometry techniques, laying the foundation for understanding its pharmacological substance basis. UHPLC-LTQ-Orbitrap-MS and GC-MS technologies were used to analyze and identify the volatile and non-volatile components in Sanhan Huashi Formula. UHPLC-QQQ-MS/MS technology was used to simultaneously determine the content of 27 major active components in the formula. The results showed that 308 major chemical components were identified in Sanhan Huashi Formula, among which 60 compounds were identified by comparing with reference standards, mainly including alkaloids, flavonoids, coumarins, triterpenoid saponins, amino acids, and nucleosides. GC-MS technology preliminarily identified 52 volatile compounds, with γ-eudesmol and ß-eudesmol as the main components. The quantitative results demonstrated good linearity(r>0.99) for the 27 active components, indicating the stability, simplicity, and reliability of the established method. Among them, amygdalin, nodakenin, arecoline, ephedrine, and pseudoephedrine had relatively high content and were presumably the main pharmacologically active substances. In conclusion, this study systematically and comprehensively characterized the major chemical components and patterns in Sanhan Huashi Formula, providing a basis for understanding its pharmacological mechanisms and clinical applications.

[Comprehensive evaluation system for quality of Chinese medicinal decoction pieces based on "experience-ingredients-activity-electronic sensing"].

Quality evaluation of Chinese medicinal decoction pieces is vital for the development of the downstream industries, and is an important channel for implementing the strategy of "higher quality, higher price, and priority for the high quality" for traditional Chinese medicine. At the moment, the quality of Chinese medicinal decoction pieces is mainly evaluated based on chemical component examination. Considering the weak preliminary research foundation and poor research conditions, traditional experience-based evaluation is undervalued in the quality rating of Chinese medicinal decoction pieces. However, traditional experience is a summary of the quality of Chinese medicinal materials based on clinical experience, which thus can be a potential basis for the quality evaluation of the decoction pieces. It is a challenge in the evaluation of Chinese medicinal decoction pieces to objectify the traditional experience-based evaluation from multiple aspects such as chemistry, effect, and characterization via modern techniques. Therefore, this study developed the "experience-ingredients-activity-electronic sensing" evaluation system for Chinese medicinal decoction pieces on the basis of experience-based assessment, chemical ingredients that can truly reflect the traditional experience, biological effect assessment, and electronic sensory evaluation, which is expected to quantify the traditional experience of quality evaluation of Chinese medicinal decoction pieces via chemistry, biology, and sensory simulation. The evaluation system can serve as a reference for clinical experience-based quality evaluation of Chinese medicinal decoction pieces.

[Biomarkers related to cognitive dysfunction in APP/PS1 mice based on non-targeted metabonomics and intervention mechanism of Huanglian Jiedu Decoction].

Through the non-targeted metabonomics study on endogenous substances in APP/PS1 transgenic mice, this paper aimed to discover biomarkers related to APP/PS1 mice with cognitive dysfunction, and find targets of Huanglian Jiedu Decoction(HLJDD) in the treatment of Alzheimer's disease(AD) and its mechanism. The brain tissue and serum metabolic mass spectrometry of mice were analyzed by ultra-high performance liquid chromatography-Orbitrap mass spectrometry(UPLC-Orbitrap MS). Through partial least squares-discriminant analysis(PLS-DA) and orthogonal partial least squares-discriminant analysis(OPLS-DA), the metabolic data of the normal group, the model group, the high-dose and low-dose HLJDD groups, and the berberine group were compared and analyzed to screen out potential biomarkers, and the relevant metabolic pathways were constructed with the help of the Kyoto Encyclopedia of Genes and Genomes(KEGG) database. Forty-five potential endogenous metabolites were identified, including 13 in brain and 35 in serum, among which leukotriene B4, tyrosine, and adenosine were expected to be differential metabolites related to cognitive function. HLJDD recalled 22 differential metabolites, and the pathways mainly involved in aminoacyl-tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, pantothenic acid and coenzyme A biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, and arachidonic acid metabolism. These pathways suggested that the main mechanism of HLJDD in the intervention of AD was to inhibit central and peripheral inflammation, and regulate energy metabolism, fatty acid metabolism, and amino acid metabolism. HLJDD has a certain effect on the improvement of cognitive dysfunction, and regulates relative pathways by recalling endogenous differential metabolites, which helps to further discover the biomarkers of AD and clarify the intervention mechanism of HLJDD in the treatment of AD.

[Determination of neohesperidin and naringin in Qingfei Paidu Granules by RP-HPLC and their transfer rates in preparation process].

The present study established an RP-HPLC method for simultaneous determination of two active components in Qingfei Paidu Granules and investigated the transfer rates of neohesperidin and naringin in the preparation process to provide references for improving the quality control standard and production of Qingfei Paidu Granules.RP-HPLC was performed on a YMC Triart C_(18) column(4.6 mm×150 mm, 5 µm)with column temperature of 30 ℃, acetonitrile(A) and 0.2% phosphoric acid solution(B) as mobile phases for gradient elution at a flow rate of 1.0 mL·min~(-1) and detection wavelength of 284 nm.Good linearity was observed for naringin at 0.10-1.0 µg(R~2=0.999 9) and neohesperidin at 0.12-1.2 µg(R~2=0.999 9).The average recovery of naringin was 99.52% with an RSD of 1.2%, and that of neohesperidin was 100.8% with an RSD of 1.2%.The transfer rates of naringin and neohesperidin between medicinal materials, extracts, concentrates, and granules were measured by this method.The average transfer rate of naringin from medicinal materials to granules was 54.89%±4.38%, and that of neohesperidin was 57.63%±5.88%.The process from medicinal materials to extracts was presumedly the key link affecting the whole preparation process.The established method is simple and sensitive and can be adopted for the quality control of Qingfei Paidu Granules.Meanwhile, it can be used to investigate the transfer rate of neohesperidin and naringin in the preparation of Qingfei Paidu Granules, and further improve the quality control standard of Aurantii Fructus Immaturus in Qingfei Paidu Granules.

[Determination and comparison of 10 active components in different parts of Loropetalum chinensis based on ultra-high performance liquid chromatography-tandem mass spectrometry].

To establish a method for the simultaneous determination of ellagic acid, quercetin, gallic acid, kaempferol, myricetin, tiliroside, salidroside, isoquercetin, chlorogenic acid, and quinic acid in the leaves, flowers, fruits, and roots of Loropetalum chinensis by ultra-performance liquid chromatography-tandem mass spectrometry, and provide references for the development and utilization of L. chinensis resources. The analysis was performed on the chromatographic column ACQUITY UPLC HSS T3(2.1 mm×100 mm, 1.8 µm) with a gradient mobile phase of acetonitrile-0.2% formic solution at the flow rate of 0.3 mL·min~(-1). Column temperature was 30 ℃ and injection volume was 2 µL. Multiple reactive ion monitoring mode(MRM) was used in the negative ion ionization mode of electrospray ion source. The 10 active components had a good linear relationship, and the established method was stable, simple, and accurate. The 10 active components existed in different parts of L. chinensis, with significant different content. The main components in different parts of L. chinensis were polyphenols, with the highest content, followed by flavonoids. The content of 10 active components was generally high in flowers. Among them, the content of quinic acid was the highest, reaching 22.539 1 mg·g~(-1). This study elucidates the differences of active components in the same part and the different parts of L. chinensis, thereby providing basis for the research on the pharmacodynamic substances of L. chinensis and references for the comprehensive development and utilization of L. chinensis resources.

[Study on anti-inflammatory activity and mechanism of indolealkylamines in toad skin on LPS-activated neutrophils].

Indolealkylamines(IAAs) are the main hydrophilic substances in toad skin, mainly including free N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine, dehydrobufotenine, and binding bufothionine. In this study, the LPS-activated neutrophils were used to investigate the structure-activity relationship and anti-inflammatory mechanism of the above-mentioned five monomers from the toad skin in vitro. The neutrophils were divided into the control group, model group(1 µg·mL~(-1) LPS), positive drug group(100 µg·mL~(-1) indometacin), as well as the low-(50 µg·mL~(-1)), medium-(100 µg·mL~(-1)) and high-dose(200 µg·mL~(-1)) free N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine, dehydrobufotenine, and binding bufothionine groups. The levels of IL-6, TNF-α and IL-1ß in the neutrophil supernatant of each group was measured by enzyme-linked immunosorbent assay(ELISA) after LPS stimulation, followed by the detection of apoptosis in each group after Annexin V/PI staining. The protein expression levels of caspase-3, Bax, Bcl-2, beclin1, LC3-I, and LC3-Ⅱ were assayed by Western blot. The results showed that IAAs reduced the excessive secretion of inflammatory cytokines caused by LPS compared with the model group. Besides, the activity of each free IAAs(N-methyl-5-hydroxytryptamine, bufotenine, bufotenidine and dehydrobufotenine), especially bufotenine, was stronger than that of the binding bufothionine. As revealed by Annexin V/PI staining, LPS delayed the early apoptosis of neutrophils compared with the control group, while bufotenine promoted the apoptosis of neutrophils in a dose-dependent manner, which might be related to the elevated expression of apoptosis-related protein Bax/Bcl-2. In addition, LPS activated the autophagy pathways in neutrophils. This study confirmed the efficacy of IAAs in reducing the secretion of inflammatory cytokines in neutrophils induced by LPS for the first time. For instance, bufotenine exerts the anti-inflammatory effect possibly by inducing the apoptosis of neutrophils.

[Identification of chemical constituents of Xiaoer Chiqiao Qingre Granules based on UHPLC-LTQ-Orbitrap-MS/MS and network pharmacology analysis].

This study aimed to qualitatively analyze the chemical components in Xiaoer Chiqiao Qingre Granules(XRCQ) by UHPLC-LTQ-Orbitrap-MS/MS and identify its material basis. The absorbed components in plasma were combined for exploring the potential action mechanism by integrated network pharmacology. ACQUITY UPLC HSS T3(2.1 mm×100 mm, 1.8 µm) column and mobile phase system of 0.1% formic acid solution(A)-acetonitrile(B) were used for gradient elution, followed by high resolution liquid chromatography-mass spectrometry in both positive and negative ion scanning modes. According to the precise relative molecular mass and MS/MS fragment ions, a total of 124 chemical components were identified in XRCQ by the comparison with references and literature reports, among which 29 compounds were completely confirmed by comparison with reference substances. Then, the main absorbed components of XRCQ in plasma were also analyzed and clarified by UHPLC-LTQ-Orbitrap-MS/MS. BATMAN-TCM and SwissTargetPrediction were used for target prediction of absorbed components in plasma. Following the plotting of association network with Cytoscape 3.8.2, the core targets were subjected to GO and KEGG pathway enrichment analysis and a component-target-pathway network was constructed. A total of eight main targets of XRCQ against fever in children were obtained together with eight absorbed components in plasma, including glycyrhydinic acid, hesperidin, emodin, reticuline, daidzein, magnolignan C, magnolignan A, and magnolaldehyde D. It was inferred that XRCQ might improve alimentary system abnormality, inflammatory response, oxidative stress, and endocrine disorder through tumor necrosis factor, PI3 K-AKT, and other signaling pathways. The present study comprehensively expounded the chemical profiles of XRCQ and the main absorbed components in plasma and predicted the potential mechanism of XRCQ based on integrated network pharmacology, which has provided certain theoretical reference for the clinical application of XRCQ.

[Application of MALDI-mass spectrometry imaging in spatial distribution of secondary metabolites in medicinal plants -- a case study of Lepdium meyenii root].

Matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI) is a novel technique for in-situ distribution of various substances in tissue without labeling. This technique is increasingly applied to the study of medicinal plants owing to its high spatial resolution and its potential of in-situ analysis in small molecules. In this study, the structural information and their fragmentation patterns of the midazole alkaloids(1,3-dibenzyl-4,5-dimethylimidazolium chloride and 1,3-dibenzyl-2,4,5-trimethylimi-dazolium chloride) and benzylglucosinolate in the medicinal plant Maca(Lepdium meyeni) root were analyzed by ultra-high-performance liquid phase combined with LTQ-Orbitrap mass spectrometry(UHPLC-HR-MS). The localization of these active ingredients in the cross-sections of Maca root was performed by MALDI-MSI. These results demonstrated that the two types of imidazole alkaloids had a similar distributed pattern. They were located more in the cortex and the periderm than those in the medulla of a lateral root, while the localization of benzylglucosinolate was concentrated in the center of the root rather than in the cortex and the periderm. The precise spatial distribution of various secondary metabolites in tissue provides an important scientific basis for the accumulation of medicinal plant active ingredients in tissues. In addition, this imaging method is a promising technique for the rapid evaluation and identification of the active ingredients of traditional Chinese medicine in plant tissues, as well as assisting the research on the processing of medicinal plants.

[Identification and attribution of chemical constituents of Qingfei Paidu Decoction based on UHPLC-LTQ-Orbitrap-MS technology].

UHPLC-LTQ-Orbitrap-MS was developed for the identification of chemical constituents in Qingfei Paidu Decoction, which will clarify its material basis. ACQUITY UHPLC HSS T3 chromatography column(2.1 mm×100 mm, 1.8 µm) was used with 0.1% formic acid(B)-acetonitrile(A) as the mobile phase in gradient elution. The decoction was detected by high-resolution liquid chromatography-mass spectrometry equipped with an ESI ion source in positive and negative mode. Based on the accurate mass measurements, retention time, mass fragmentation patterns combined with comparison of reference and literature reports, a total of 87 major compounds including 43 flavonoids, 9 alkaloids, 4 triterpenoid saponins, 1 sesquiterpene, 2 coumarins, 10 phenolic acids and 18 other compounds were tentatively screened and characterized. UHPLC-LTQ-Orbitrap-MS was employed to comprehensively elucidate the chemical components in Qingfei Paidu Decoction, which basically covered 20 Chinese medicines except gypsum in Qingfei Paidu Decoction. These collective results provide a scientific basis for further research on the quality control standard of Qingfei Paidu Decoction.

A Newly Discovered Phenylethanoid Glycoside from Stevia rebaudiana Bertoni Affects Insulin Secretion in Rat INS-1 Islet ß Cells.

The tea-like beverage Stevia rebaudiana Bertoni (Stevia) is popular in China because it reduces blood glucose and has a sweet taste. In this work, a comprehensive quality assessment of Stevia led to the discovery of five phenylethanoid glycosides, namely steviophethanoside (1), cuchiloside (2), salidroside (3), icariside D (4), and tyrosol (5). Of them, compound 1 is a novel compound. Mass spectrometry and NMR spectroscopy were employed to confirm the absolute configuration. A hydrolytic step with 4 N TFA at 95 °C for 4 h was used to confirm the monosaccharides. In addition, Discovery Studio 4.0 was used to predict the ADME and toxicity activity of compound 1. The results suggested that compound 1 was biocompatible and had poor toxicity, which was verified by rat INS-1 islet ß cells through an MTT assay. Meanwhile, a significant stimulatory effect on INS-1 cells was observed, which indicated a hypoglycemic effect of compound 1. This is the first report that describes a natural, novel, and hypoglycemic phenylethanoid glycoside in Stevia.

[Pharmacokinetics of active ingredients of Pudilan Xiaoyan Oral Liquid in rats and effect on liver drug-metabolizing enzymes].

This study is aimed to establish a method for the determination of baicalin,baicalin and purpurin in the plasma of rats after oral administration of Pudilan Xiaoyan Oral Liquid( PDL) by using liquid chromatography-mass spectrometry( LC-MS),analyze the pharmacokinetics of three components in rats,and investigate the effects of PDL on drug-metabolizing enzymes in rat liver. C18 column was used for liquid chromatography separation,with acetonitrile-water( containing 0. 2% formic acid) as the mobile phase for gradient elution. The mass spectrometry was detected by electrospray ion source( ESI) under multi-reaction monitoring mode( MRM),as well as positive and negative ion alternating mode. Plasma sample collection was performed by using an automatic blood collection meter for small animals. The pharmacokinetic parameters were calculated by Win Nonlin software. The total protein concentration of rat liver microsomes and the total enzyme content of CYP450 were determined by BCA method and spectrophotometry respectively. The methodological study in terms of linear range,recovery rate,precision and sample stability,was used to confirm that the LC-MS analysis method established in this experiment was simple,exclusive,accurate and reliable,and can meet the requirement of determining the content of baicalin,oroxindin and corynoline in plasma after PDL administration in rats. The drug-time curve showed that baicalin and oroxindin had a bimodal phenomenon,and the pharmacokinetic parameters indicated that baicalin,oroxindin and corynoline in PDL had certain drug-like properties. After 7 consecutive days of PDL administration,the rat liver coefficient,total liver microparticle protein and CYP450 enzyme content were increased,but there was no significant difference,indicating that PDL was less likely to develop drug-drug interaction based on CYP enzyme. The results of this experiment can provide reference for the research on in vivo efficacy and drug interaction of PDL as well as on its clinical application.

[Lipid metabolism study of sodium norcantharidate in LO2 hepatocytes based on lipidomics].

In order to find the endogenous potential biomarkers of in vitro hepatic injury caused by NCTD-Na and elucidate the mechanism of hepatic injury of NCTD-Na,ultra-high performance liquid chromatography coupled quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS) was used for lipidomics detection.Multivariate statistical analysis was used to study the endogenous lipid metabolic changes of human normal liver cells LO2 injury after the treatment with sodium norcantharidate(NCTD-Na).The results showed that the half maximal inhibitory concentration(IC50) of NCTD-Na was 0.034 mmol·L-1.A total of 280 differential metabolites were found between the control group and the low-dose group,with VIP > 2.0 and P<0.05.At the same time,a total of 273 differential metabolites were found between the control group and the high-dose group,with VIP > 2.0 and P<0.05.Cell metabolite profiles showed clear separation among control group,the low-dose group and the high-dose group,and 111 differential metabolites were found,with VIP > 2.0,P<0.05,RSD<30% and in a dose-dependent manner.It was found that most of the above differential metabolites were lipid metabolites after the analysis of simple preparnation methods and database search.A total of 32 potential biomarkers were identified,including 3 phosphatidylcholine(PC),5 lysophosphatidylcholine(Lyso PC),3 ceramide(Cer),1 sphingomyelin(SM),1 phosphatidylethanolamine(PE),10 lysophosphatidylethanolamine(LysoPE),4 diacylglycerol(DG),1 Phosphatidic acid(PA),1 lysophosphatidic acid(Lyso PA),1 phosphatidyl glycerol(PG),1 fatty acid hydroxy fatty acid(FAHFA) and 1 phosphatidylserine(PS).The changes of PCs,Cers,SM,PE and DGs were closely related liver protection,DNA methylation and self-repair in hepatocytes,apoptosis,methylation and detoxification of carcinogens,as well as lipid peroxides production process.Also,they had impact on the proliferation of hepatocytes,differentiation and gene transcription disorders.Cells stimulated by NCTD-Na could promote the production of PA as well as the synthesis and catabolism of FAHFA in a variety of ways.The levels of Lyso PCs,LysoPEs and Lyso PA were correlated with PCs,PE and PA;PE and PS might have valgus during apoptosis,triggering phagocytosis.

[Identification and attribution of chemical compounds of Pudilan Antiphlogistic Oral Liquid].

UPLC-ESI-Orbitrap-MS/MS was used to analyze,identify and attribute the chemical constituents in Pudilan Antiphlogistic Oral Liquid. The analysis was performed on an Agilent Eclipse XDB-C18(4.6 mm × 150 mm,3.5 µm) with a gradient mobile phase of methanol-0.1% formic solution system at the flow rate of 0.5 m L·min-1. The sample volume was 2 µL. The column temperature was30 ℃. The high-resolution orbitrap mass spectrometry was used as detector,with electrospray ion source in both positive and negative models,and the MS scanning ranged between m/z 50 and 2 000. Based on the analysis of mass spectrometry and literature reports,79 compounds were confirmed,including 30 alkaloids,28 organic acids,18 flavonoids and 3 coumarins. Finally,39 compounds,such as rutin,esculetin,gallic acid,caffeic acid,cichoric acid,were identified from Taraxacum mongolicum; 11 compounds,such as baicalin,baicalein,apigenin,chrysin,oroxylin A,were identified from Scutellaria baicalensis; 13 compounds,such as arginine,proline,hypoxanthine,epigoitrin,indirubin,were identified from Isatis indigotica; and 18 compounds,such as dehydrocheilanthifoline,oxysanguinarine,corynoline,protopine,spallidamine,were identified from Corydalis bungeana. After the analysis of chemical model and attribution,the contents of some compounds were high in Pudilan Antiphlogistic Oral Liquid,such as baicalin,wogonoside,baicalein,wogonin,apigenin,chrysin,skullcapflavonⅡ,oroxylin A,cichoric acid,chlorogenic acid,caffeic acid,esculetin,dehydrocheilanthifoline,dihydrosanguinarine,protopine,corynoline and indirubin. The established method is simple,accurate,rapid,sensitive and reproducible,and thus suitable for the qualitative identification and quantitative determination of Pudilan Antiphlogistic Oral Liquid,which lays a foundation for the systematic quality control and the establishment of whole-course traceability system of active ingredients.

[Pharmacodynamics of Huanglian Jiedu decoction in Alzheimer's disease (AD) model rats and effect on improvement of inflammation microenvironment in brain].

To study the pharmacokinetics of active ingredients (alkaloids, iridoids and flavonoids) in Huanglian Jiedu decoction (HLJDD) in Alzheimer's disease (AD) model rats, and investigate its mechanism in treatment of AD. All of rats were divided into normal control group (n=6), shame operation group (n=6) and model group (n=12). Rats in shame operation group received daily subcutaneous injection of D-galactose (D-gal 50 mg·kg⁻¹) for a total of 45 d to induce subacute aging model. Based on the operation of shame operation group, the rats in model group were given with an injection of Aß25₋35 (4.0 g·L⁻¹)-ibotenic acid (2.0 g·L⁻¹) into the nucleus basalis magnocellularis. Then rats in model group were divided into HLJDD one day administration group (n=6) and HLJDD one week group (n=6). The plasma concentration of alkaloids, iridoid and flavonoids was determined by liquid chromatography tandem mass spectrometry (LC-QQQ-MS) at different time points. The levels of seven inflammatory factors (MIP-2, IL-1ß, IL-2, IL-8, IL-10, IL-13, TNF-α) in cerebrospinalfluid (CSF) were measured by Bio-Plex multi-factor detection technology. Iridoids in AD model rats, with high bioavailability, were easily absorbed and eliminated. The plasma concentration of alkaloid was lower, and the AUC (area under the curve) was higher in HLJDD one week group than that in HLJDD one day group. The plasma concentration-time curves of flavonoids showed obvious bimodal phenomena. After the gastric administration of HLJDD, the inflammatory factors in CSF of AD rats demonstrated a callback trend, including IL-1ß/IL-10 (P<0.05) with significant difference. The pharmacokinetic behaviors of iridoids, alkaloids and flavonoids (41 compounds) in AD model rats were fundamentally elucidated, and HLJDD can improve the central inflammatory status of AD rats by regulating the levels of inflammatory factors.