Systematic screening and structural characterization of dipeptides using offline 2D LC-LTQ-Orbitrap MS:A case study of Cordyceps sinensis / 药物分析学报

Cordyceps sinensis(C.sinensis)is a widely used and highly valuable traditional Chinese medicine.Several dipeptides have been detected in C.sinensis,but current scientific knowledge of its chemical makeup remains limited.In this study,an improved approach that integrates offline two-dimensional liquid chromatography(2D LC)separation,precursor ion list,library screening,and diagnostic ion filtering was established to systematically screen and characterize dipeptides in C.sinensis.Offline 2D LC integrating hydrophilic interaction LC and reverse phase separations was established to eliminate interference and identify the target dipeptides.A library containing the potential 400 dipeptides was created,and a precursor ion list with all theoretical precursor ions was adopted to trigger the MS/MS scan with high sensitivity.To identify dipeptides,the type and connection sequence of amino acids were determined according to the product ions.Ile and Leu residues were differentiated for the first time according to the characteristic ion at m/z 69.07.Ultimately,170 dipeptides were identified or tentatively characterized from C.sinensis,and most are reported for the first time in this species herein.In addition,the identified dipeptides were also applied for discrimination among the three Cordyceps species,and 11 markers were identified.The obtained results provide a deeper understanding of the chemical basis of C.sinensis.

An integrated strategy for comprehensive characterization of metabolites and metabolic profiles of bufadienolides from Venenum Bufonis in rats / 药物分析学报

Comprehensive characterization of metabolites and metabolic profiles in plasma has considerable sig-nificance in determining the efficacy and safety of traditional Chinese medicine(TCM)in vivo.However,this process is usually hindered by the insufficient characteristic fragments of metabolites,ubiquitous matrix interference,and complicated screening and identification procedures for metabolites.In this study,an effective strategy was established to systematically characterize the metabolites,deduce the metabolic pathways,and describe the metabolic profiles of bufadienolides isolated from Venenum Bufonis in vivo.The strategy was divided into five steps.First,the blank and test plasma samples were injected into an ultra-high performance liquid chromatography/linear trap quadrupole-orbitrap-mass spectrometry(MS)system in the full scan mode continuously five times to screen for valid matrix compounds and metabolites.Second,an extension-mass defect filter model was established to obtain the targeted precursor ions of the list of bufadienolide metabolites,which reduced approximately 39％of the interfering ions.Third,an acquisition model was developed and used to trigger more tandem MS(MS/MS)fragments of precursor ions based on the targeted ion list.The acquisition mode enhanced the acquisition capability by approximately four times than that of the regular data-dependent acquisition mode.Fourth,the acquired data were imported into Compound Discoverer software for identification of metabolites with metabolic network prediction.The main in vivo metabolic pathways of bufadienolides were elucidated.A total of 147 metabolites were characterized,and the main biotransformation reactions of bufadienolides were hydroxylation,dihydroxylation,and isomerization.Finally,the main prototype bufadienolides in plasma at different time points were determined using LC-MS/MS,and the metabolic profiles were clearly identified.This strategy could be widely used to elucidate the metabolic profiles of TCM preparations or Chinese patent medicines in vivo and provide critical data for rational drug use.

Deep chemical identification of phytoecdysteroids in Achyranthes bidentata Blume by UHPLC coupled with linear ion trap-Orbitrap mass spectrometry and targeted isolation / 中国天然药物

Achyranthes bidentata Blume is widely used as a traditional Chinese medicine with the effects of nourishing the liver and kidneys and strengthening muscles and bones. In this work, a rapid and simple strategy was developed for characterizing phytoecdysteroids by ultra-high-performance liquid chromatography coupled with liner ion trap-Orbitrap mass spectrometry using electrospray ionization in the negative mode. As a result, 47 phytoecdysteroids were unambiguously or tentatively characterized. Among them, seven known compounds were identified according to the reference standards along with molecular formula, retention time and fragmentation patterns, while others were mostly potential new compounds. Through targeted isolation, the structures of three new compounds were determined by NMR spectra, which were consistent with LC-MS characterization. The present study provides an efficient method to deeply characterize phytoecdysteroids.

Identification of chemical constituents of Xiaochengqi Decoction by UPLC-Q-TOF/Fast DDA combined with UNIFI software / 中国中药杂志

Based on the combination of ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF) and Waters UNIFI software, the chemical constituents of the classic prescription Xiaochengqi Decoction were qualitatively analyzed and identified. The UPLC conditions are as follows: Acquity HSS T3 reverse phase column(2.1 mm ×100 mm, 1.8 μm), column temperature of 30 ℃, mobile phase of 0.1% formic acid aqueous solution(A)-acetonitrile(B), and flow rate of 0.3 mL·min~(-1). High-resolution MS data of Xiaochengqi Decoction were collected in ESI~(+/-) modes by Fast DDA. The structures of the chemical constituents were tentatively characterized or identified by UNIFI software according to the retention time of reference standards and characteristic fragment ions in MS profile, and literature data. A total of 233 components in Xiaochengqi Decoction were identified, with 93 from wine-processed Rhei Radix et Rhizoma, 104 from bran-processed Aurantii Fructus Immaturus, and 36 from ginger-processed Magnoliae Officinalis Cortex. These 233 components included anthraquinones, flavonoids, lignans, alkaloids, coumarins, and phenylethanoid glycosides. The result provided experimental evidence for the further study on establishment of quality standard and product development of the formula.

Analysis of chemical constituents in classical prescription Danggui Buxue Decoction based on RP-Q-TOF-MS and HILIC-Q-TOF-MS / 中国中药杂志

The chemical constituents of classical prescription Danggui Buxue Decoction were analyzed by reversed-phase(RP) chromatography and hydrophilic interaction chromatography(HILIC) coupled with quadrupole time-of-flight mass spectrometry. RP separation of Danggui Buxue Decoction was performed on ACQUITY UPLC HSS T3(2.1 mm×100 mm, 1.8 μm), while HILIC separation was on Waters BEH Amide(2.1 mm×100 mm, 1.7 μm). Mass spectrometry(MS) data were acquired in both negative and positive ion modes. Chemical constituents of Astragali Radix and Angelicae Sinensis Radix were searched from Reaxys and thus the in-house library was established. MS data were further analyzed by MassLynx 4.1 combined with in-house library, HMDB, Reaxys, and comparison with reference substances. In conclusion, a total of 154 compounds were identified and characterized: 16 saponins, 44 flavonoids, 10 phthalides, 7 phenylpropanoids, 15 bases and the corresponding nucleosides, 30 oligosaccharides, and 32 other compounds. Among them, 65 compounds were detected by HILIC-MS/MS. This study provides experimental evidences for the material basis research, quality control, and preparation development of Danggui Buxue Decoction and a reference method for comprehensive characterization of Chinese medicine decoctions typified by classical prescriptions.

Insight into chemical basis of traditional Chinese medicine based on the state-of-the-art techniques of liquid chromatography-mass spectrometry

Traditional Chinese medicine (TCM) has been an indispensable source of drugs for curing various human diseases. However, the inherent chemical diversity and complexity of TCM restricted the safety and efficacy of its usage. Over the past few decades, the combination of liquid chromatography with mass spectrometry has contributed greatly to the TCM qualitative analysis. And novel approaches have been continuously introduced to improve the analytical performance, including both the data acquisition methods to generate a large and informative dataset, and the data post-processing tools to extract the structure-related MS information. Furthermore, the fast-developing computer techniques and big data analytics have markedly enriched the data processing tools, bringing benefits of high efficiency and accuracy. To provide an up-to-date review of the latest techniques on the TCM qualitative analysis, multiple data-independent acquisition methods and data-dependent acquisition methods (precursor ion list, dynamic exclusion, mass tag, precursor ion scan, neutral loss scan, and multiple reaction monitoring) and post-processing techniques (mass defect filtering, diagnostic ion filtering, neutral loss filtering, mass spectral trees similarity filter, molecular networking, statistical analysis, database matching, etc.) were summarized and categorized. Applications of each technique and integrated analytical strategies were highlighted, discussion and future perspectives were proposed as well.

Chemical profiling of Huashi Baidu prescription, an effective anti-COVID-19 TCM formula, by UPLC-Q-TOF/MS / 中国天然药物

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.

Simple and robust differentiation of Ganoderma species by high performance thin-layer chromatography coupled with single quadrupole mass spectrometry QDa / 中国天然药物

In this study, a high performance thin-layer chromatography/single quadrupole mass spectrometry QDa (HPTLC-QDa) method for robust authentication of Ganoderma lucidum, a popular and valuable herbal medicine, has been developed. This method is simple and practical, which allows direct generation of characteristic mass spectra from the HPTLC plates automatically with the application of in situ solvent desorption interface. The HPTLC silica gel plates were developed with toluene-ethyl formate-formic acid (5 : 5 : 0.2, V/V) and all bands were transferred to QDa system directly in situ using 80% methanol with 0.1% formic acid as desorption solvent. The acquired HPTLC-QDa spectra showed that luminous yellow band b3, containing ganoderic acid B/G/H and ganodeneric acid B, the major active components of Ganoderma, could be found only in G. lucidum and G. lucidum (Antler-shaped), but not in G. sinense and G. applanatum. Moreover, bands b13 and b14 with m/z 475/477 and m/z 475/491/495, respectively, could be detected in G. lucidum (Antler-shaped), but not in G. lucidum, thus allowing simple and robust authentication of G. lucidum with confused species. This method is proved to be simple, practical and reproducible, which can be extended to analyze other herbal medicines.

Plant metabolomics for studying the effect of two insecticides on comprehensive constituents of Lonicerae Japonicae Flos / 中国天然药物

Pesticides' overuse and misuse have been reported to induce ingredient variations in herbal medicine, which is now gaining attention in the medicinal field as a form of alternative medicine. To date, available studies on pesticide-induced ingredient variations of herbal medicine are limited only on a few compounds and remain most others unexamined. In this study, a plant metabolomics-based strategy was performed to systematically explore the effects of two frequently used insecticides on the comprehensive constituents of Lonicerae Japonicae Flos (LJF), the flower buds of Lonicera japonica Thunb. Field trials were designed on a cultivating plot of L. japonica with controls and treatments of imidacloprid (IMI) and compound flonicamid and acetamiprid (CFA). Unbiased metabolite profiling was conducted by ultra-high performance liquid chromatography/quadrupole-Orbitrap mass spectrometer. After data pretreatment by automatic extraction and screening, a data matrix of metabolite features was submitted for statistical analyses. Consequently, 29 metabolic markers, including chlorogenic acids, iridoids and organic acid-glucosides were obtained and characterized. The relative quantitative assay was subsequently performed to monitor their variations across flowering developments. This is the first study that systematically explored the insecticide-induced metabolite variations of LJF while taking into account the inherent variability of flowering development. The results were beneficial for holistic quality assessment of LJF and significant for guiding scientific use of pesticides in the large-scale cultivation.

Active constituents and mechanisms of Respiratory Detox Shot, a traditional Chinese medicine prescription, for COVID-19 control and prevention: Network-molecular docking-LC-MS analysis / 中西医结合学报

OBJECTIVE@#Lung-toxin Dispelling Formula No. 1, referred to as Respiratory Detox Shot (RDS), was developed based on a classical prescription of traditional Chinese medicine (TCM) and the theoretical understanding of herbal properties within TCM. Therapeutic benefits of using RDS for both disease control and prevention, in the effort to contain the coronavirus disease 2019 (COVID-19), have been shown. However, the biochemically active constituents of RDS and their mechanisms of action are still unclear. The goal of the present study is to clarify the material foundation and action mechanism of RDS.@*METHODS@#To conduct an analysis of RDS, an integrative analytical platform was constructed, including target prediction, protein-protein interaction (PPI) network, and cluster analysis; further, the hub genes involved in the disease-related pathways were identified, and the their corresponding compounds were used for in vitro validation of molecular docking predictions. The presence of these validated compounds was also measured in samples of the RDS formula to quantify the abundance of the biochemically active constituents. In our network pharmacological study, a total of 26 bioinformatic programs and databases were used, and six networks, covering the entire Zang-fu viscera, were constructed to comprehensively analyze the intricate connections among the compounds-targets-disease pathways-meridians of RDS.@*RESULTS@#For all 1071 known chemical constituents of the nine ingredients in RDS, identified from established TCM databases, 157 passed drug-likeness screening and led to 339 predicted targets in the constituent-target network. Forty-two hub genes with core regulatory effects were extracted from the PPI network, and 134 compounds and 29 crucial disease pathways were implicated in the target-constituent-disease network. Twelve disease pathways attributed to the Lung-Large Intestine meridians, with six and five attributed to the Kidney-Urinary Bladder and Stomach-Spleen meridians, respectively. One-hundred and eighteen candidate constituents showed a high binding affinity with SARS-coronavirus-2 3-chymotrypsin-like protease (3CL), as indicated by molecular docking using computational pattern recognition. The in vitro activity of 22 chemical constituents of RDS was validated using the 3CL inhibition assay. Finally, using liquid chromatography mass spectrometry in data-independent analysis mode, the presence of seven out of these 22 constituents was confirmed and validated in an aqueous decoction of RDS, using reference standards in both non-targeted and targeted approaches.@*CONCLUSION@#RDS acts primarily in the Lung-Large Intestine, Kidney-Urinary Bladder and Stomach-Spleen meridians, with other Zang-fu viscera strategically covered by all nine ingredients. In the context of TCM meridian theory, the multiple components and targets of RDS contribute to RDS's dual effects of health-strengthening and pathogen-eliminating. This results in general therapeutic effects for early COVID-19 control and prevention.

Profiling and identification of aqueous extract of Cordyceps sinensis by ultra-high performance liquid chromatography tandem quadrupole-orbitrap mass spectrometry / 中国天然药物

Characterization of aqueous extract in traditional Chinese medicine (TCM) is challenging due to the poor retention of the analytes on conventional C columns. This study presents a systematic characterization method based on a rapid chromatographic separation (8 min) on a polar-modified C (Waters Cortecs T3) column of aqueous extract of Cordyceps sinensis. UHPLC-HRMS method was used to profile components in both untargeted and targeted manners by full MS/PIL/dd-MS acquisition approach. The components were identified or tentatively identified by reference standards comparison, fragmentation rules elucidation and available databases search. A total of 91 components, including 10 nucleobases, 20 nucleosides, 39 dipeptides, 18 amino acids and derivatives and 4 other components, were characterized from the aqueous extract of C. sinensis. And this was the first time to systematically report the presence of nucleosides and dipeptides in C. sinensis, especially for modified nucleosides. The chemical basis inquiry of this work would be beneficial to mechanism exploration and quality control of C. sinensis and related products. Meanwhile, this work also provided an effective solution for characterization of aqueous extract in TCM.

Three new alkaloids isolated from the stem tuber of Pinellia pedatisecta / 中国天然药物

The present study was designed to determine the chemical constituents of the stem tuber of Pinellia pedatisecta. The chemical constituents were isolated and purified by various chromatographic techniques, and their structures were elucidated on the basis of physicochemical properties and spectral data. Three new alkaloids (compounds 1, 2, and 3) were obtained and identified as 9-((5-methoxypyridin-2-yl)methyl)-9H-purin-6-amine (1), 4-(2-(2, 5-dioxopyrrolidin-1-yl)ethyl)phenyl acetate (2), and N-(9-((5-methoxypyridin-2-yl)methyl)-9H-purin-6-yl)acetamide (3). These compounds were evaluated for their cytotoxicity against human cervical cancer HeLa cells. Compounds 1 and 3 significantly inhibited the proliferation of HeLa cells with IC values being 3.02 ± 0.54 and 7.16 ± 0.62 μmol·L, respectively.

Profiling and identification of chemical components of Shenshao Tablet and its absorbed components in rats by comprehensive HPLC/DAD/ESI-MS analysis / 中国天然药物

Shenshao Tablet (SST), prepared from Paeoniae Radix Alba (PRA) and total ginsenoside of Ginseng Stems and Leaves (GSL), is a traditional Chinese medicine (TCM) preparation prescribed to treat coronary heart disease. However, its chemical composition and the components that can migrate into blood potentially exerting the therapeutic effects have rarely been elucidated. We developed an HPLC/DAD/ESI-MS approach aiming to comprehensively profile and identify both the chemical components of SST and its absorbed ingredients (and metabolites) in rat plasma and urine. Chromatographic separation was performed on an Agilent Eclipse XDB C column using acetonitrile/0.1% formic acid as the mobile phase. MS detection was conducted in both negative and positive ESI modes to yield more structure information. Comparison with reference compounds (t, MS), interpretation of the fragmentation pathways, and searching of in-house database, were utilized for more reliable structure elucidation. A total of 82 components, including 21 monoterpene glycosides, four galloyl glucoses, two phenols from PRA, and 55 ginsenosides from GSL, were identified or tentatively characterized from the 70% ethanolic extract of SST. Amongst them, seven and 24 prototype compounds could be detectable in the plasma and urine samples, respectively, after oral administration of an SST extract (4 g·kg) in rats. No metabolites were observed in the rat samples. The findings of this work first unveiled the chemical complexity of SST and its absorbed components, which would be beneficial to understanding the therapeutic basis and quality control of SST.

Three new alkaloids isolated from the stem tuber of Pinellia pedatisecta / 中国天然药物

The present study was designed to determine the chemical constituents of the stem tuber of Pinellia pedatisecta. The chemical constituents were isolated and purified by various chromatographic techniques, and their structures were elucidated on the basis of physicochemical properties and spectral data. Three new alkaloids (compounds 1, 2, and 3) were obtained and identified as 9-((5-methoxypyridin-2-yl)methyl)-9H-purin-6-amine (1), 4-(2-(2, 5-dioxopyrrolidin-1-yl)ethyl)phenyl acetate (2), and N-(9-((5-methoxypyridin-2-yl)methyl)-9H-purin-6-yl)acetamide (3). These compounds were evaluated for their cytotoxicity against human cervical cancer HeLa cells. Compounds 1 and 3 significantly inhibited the proliferation of HeLa cells with IC values being 3.02 ± 0.54 and 7.16 ± 0.62 μmol·L, respectively.

Profiling and identification of chemical components of Shenshao Tablet and its absorbed components in rats by comprehensive HPLC/DAD/ESI-MS analysis / 中国天然药物

Shenshao Tablet (SST), prepared from Paeoniae Radix Alba (PRA) and total ginsenoside of Ginseng Stems and Leaves (GSL), is a traditional Chinese medicine (TCM) preparation prescribed to treat coronary heart disease. However, its chemical composition and the components that can migrate into blood potentially exerting the therapeutic effects have rarely been elucidated. We developed an HPLC/DAD/ESI-MS approach aiming to comprehensively profile and identify both the chemical components of SST and its absorbed ingredients (and metabolites) in rat plasma and urine. Chromatographic separation was performed on an Agilent Eclipse XDB C column using acetonitrile/0.1% formic acid as the mobile phase. MS detection was conducted in both negative and positive ESI modes to yield more structure information. Comparison with reference compounds (t, MS), interpretation of the fragmentation pathways, and searching of in-house database, were utilized for more reliable structure elucidation. A total of 82 components, including 21 monoterpene glycosides, four galloyl glucoses, two phenols from PRA, and 55 ginsenosides from GSL, were identified or tentatively characterized from the 70% ethanolic extract of SST. Amongst them, seven and 24 prototype compounds could be detectable in the plasma and urine samples, respectively, after oral administration of an SST extract (4 g·kg) in rats. No metabolites were observed in the rat samples. The findings of this work first unveiled the chemical complexity of SST and its absorbed components, which would be beneficial to understanding the therapeutic basis and quality control of SST.

A feasible, economical, and accurate analytical method for simultaneous determination of six alkaloid markers in Aconiti Lateralis Radix Praeparata from different manufacturing sources and processing ways / 中国天然药物

Aconiti Lateralis Radix Praeparata (Fuzi) is a commonly used traditional Chinese medicine in clinic for its potency in restoring yang and rescuing from collapse. Aconiti alkaloids, mainly including monoester-diterpenoidaconitines (MDAs) and diester-diterpenoidaconitines (DDAs), are considered to act as both bioactive and toxic constituents. In the present study, a feasible, economical, and accurate HPLC method for simultaneous determination of six alkaloid markers using the Single Standard for Determination of Multi-Components (SSDMC) method was developed and fully validated. Benzoylmesaconine was used as the unique reference standard. This method was proven as accurate (recovery varying between 97.5%-101.8%, RSD 0.999 9) over the concentration ranges, and subsequently applied to quantitative evaluation of 62 batches of samples, among which 45 batches were from good manufacturing practice (GMP) facilities and 17 batches from the drug market. The contents were then analyzed by principal component analysis (PCA) and homogeneity test. The present study provided valuable information for improving the quality standard of Aconiti Lateralis Radix Praeparata. The developed method also has the potential in analysis of other Aconitum species, such as Aconitum carmichaelii (prepared parent root) and Aconitum kusnezoffii (prepared root).

Anti-proliferation activity of terpenoids isolated from Euphorbia kansui in human cancer cells and their structure-activity relationship / 中国天然药物

Euphorbia kansui is a commonly used traditional Chinese medicine for the treatment of edema, pleural effusion, and asthma, etc. According to the previous researches, terpenoids in E. kansui possess various biological activities, e.g., anti-virus, anti-allergy, antitumor effects. In this work, twenty five terpenoids were isolated from E. kansui, including thirteen ingenane- and eight jatrophane-type diterpenoids (with two new compounds, kansuinin P and Q) and four triterpenoids. Eighteen of them were analyzed by MTS assay for in vitro anticancer activity in five human cancer cell lines. Structure-activity relationship for 12 ingenane-type diterpenoids in colorectal cancer Colo205 cells were preliminary studied. Significant anti-proliferation activities were observed in human melanoma cells breast cancer MDA-MB-435 cells and Colo205 cells. More than half of the isolated ingenane-type diterpenoids showed inhibitory activities in MDA-MB-435 cells. Eight ingenane- and one jatrophane-type diterpenoids possessed much lower IC values in MDA-MB-435 cells than positive control staurosporine. Preliminary structure-activity relationship analysis showed that substituent on position 20 was important for the activity of ingenane-type diterpenoids in Colo205 cells and substituent on position 3 contributed more significant biological activity of the compounds than that on position 5 in both MDA-MB-435 and Colo205 cells.

Nano-LC-ESI MS/MS analysis of proteins in dried sea dragon Solenognathus hardwickii and bioinformatic analysis of its protein expression profiling / 中国天然药物

The sea dragon Solenognathus hardwickii has long been used as a traditional Chinese medicine for the treatment of various diseases, such as male impotency. To gain a comprehensive insight into the protein components of the sea dragon, shotgun proteomic analysis of its protein expression profiling was conducted in the present study. Proteins were extracted from dried sea dragon using a trichloroacetic acid/acetone precipitation method and then separated by SDS-PAGE. The protein bands were cut from the gel and digested by trypsin to generate peptide mixture. The peptide fragments were then analyzed using nano liquid chromatography tandem mass spectrometry (nano-LC-ESI MS/MS). 810 proteins and 1 577 peptides were identified in the dried sea dragon. The identified proteins exhibited molecular weight values ranging from 1 900 to 3 516 900 Da and pI values from 3.8 to 12.18. Bioinformatic analysis was conducted using the DAVID Bioinformatics Resources 6.7 Gene Ontology (GO) analysis tool to explore possible functions of the identified proteins. Ascribed functions of the proteins mainly included intracellular non-membrane-bound organelle, non-membrane-bounded organelle, cytoskeleton, structural molecule activity, calcium ion binding and etc. Furthermore, possible signal networks of the identified proteins were predicted using STRING (Search Tool for the Retrieval of Interacting Genes) database. Ribosomal protein synthesis was found to play an important role in the signal network. The results of this study, to best of our knowledge, were the first to provide a reference proteome profile for the sea dragon, and would aid in the understanding of the expression and functions of the identified proteins.

Agglutinin isolated from Arisema heterophyllum Blume induces apoptosis and autophagy in A549 cells through inhibiting PI3K/Akt pathway and inducing ER stress / 中国天然药物

Arisaema heterophyllum Blume is one of the three medicinal plants known as traditional Chinese medicine Rhizoma Arisaematis (RA). RA has been popularly used to treat patients with convulsions, inflammation, and cancer for a long time. However, the underlying mechanisms for RA effects are still unclear. The present study was designed to determine the cytotoxicity of agglutinin isolated from Arisema heterophyllum Blume (AHA) and explore the possible mechanisms in human non-small-cell lung cancer A549 cells. AHA with purity up to 95% was isolated and purified from Arisaema heterophyllum Blume using hydrophobic interaction chromatography. AHA dose-dependently inhibited the proliferation of A549 cells and induced G phase cell cycle arrest. AHA induced apoptosis by up-regulating pro-apoptotic Bax, decreasing anti-apoptotic Bcl-2, and activating caspase-9 and caspase-3. In A549 cells treated with AHA, the PI3K/Akt pathway was inhibited. Furthermore, AHA induced increase in the levels of ER stress markers such as phosphorylated eukaryotic initiation factor 2α (p-eIF2α), C/EBP-homologous protein (CHOP), inositol-requiring enzyme 1α (IRE1α), and phosphorylated c-Jun NH-terminal kinase (p-JNK). AHA also induced autophagy in A549 cells. Staining of acidic vesicular organelles (AVOs) and increase in the levels of LC3II and ATG7 were observed in AHA-treated cells. These findings suggested that AHA might be one of the active components with anti-cancer effects in Arisaema heterophyllum Blume. In conclusion, cytotoxicity of AHA on cancer cells might be related to its effects on apoptosis and autophagy through inhibition of PI3K/Akt pathway and induction of ER stress.

Development of specific and quantitative methods for the quality control of the polysaccharides from sea-tangle and sargassum / 中国天然药物

Polysaccharides from numerous traditional Chinese medicines have been proven as the bioactive ingredients and are hence used as the quality control markers. However, the assessment criteria always show a poor specificity, due to the lack of systematic comparison among the analogous herbs. In the present study, two similar materials, namely sea-tangle and sargassum, were selected as the model herbs to develop more specific methods for quality control. Two well-established methods, determination of the total polysaccharides content and monosaccharides composition analysis, were both employed. Based upon the quantitative results, the evaluation criteria of the polysaccharides contents of not less than 2.0% and 1.7% were proposed for sea-tangle and sargassum, respectively. Nine identical monosaccharide derivatives appeared on the HPLC chromatograms of the hydrolysis and derivatized solutions of the two drugs. Principal component analysis and orthogonal partial least squares discriminant analysis using the peak areas of monosaccharides derivatives as the variables were performed, and the results indicated that mannuronic acid and xylose with the opposite concentrations in the two drugs were the differential components. A discriminative criterion using the peak area ratio of these two monosaccharides derivatives was proposed for the qualitative identification. In conclusion, a more specific and quantitative quality control method was developed for sea-tangle and sargassum.