Rapid screening and evaluation of natural antioxidants from leaf, stem, and root of Artemisia argyi by online liquid microextraction combined with HPLC-based antioxidant assay system coupled with calibration quantitative analysis.

To reveal the utilization value of leaf, stem, and root of Artemisia argyi, a rapid online liquid microextraction combined with a high-performance liquid chromatography coupled with 2,2-nitrogen-di (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt antioxidant assay system was established for analysis of antioxidants in the leaf, stem, and root of A. argyi, and a calibration quantitative method of antioxidant activity with equivalent chlorogenic acid was proposed. Thirty-three positive peaks were identified; among them, 12 compounds were found that possess good antioxidant activity including eleven organic acids (components 2-4, 8, 11-14, 17, 19, and 21) and one flavonoids (component 22). The proposed calibration quantitative method avoided the influence of content of compound and compared the extent of radical scavenging capacity of five antioxidant compounds, which were ranked as follow: 3,5-dicaffeoylquinic acid > 3,4-dicaffeoylquinic acid ≈ 4,5-dicaffeoylquinic acid > 1,4-dicaffeoylquinic acid > chlorogenic acid. In conclusion, this study provided composition and biological potential for the future development of the leaf, stem, and root of A. argyi. It is believed that the online liquid microextraction combined with high-performance liquid chromatography based antioxidant assay system can be widely used for the rapid screening of natural antioxidant components in the different parts of natural products.

Identification and quantification of nine compounds in Fangwen Jiuwei decoction by liquid chromatography-mass spectrometry.

Fangwen Jiuwei Decoction is a traditional Chinese medicine preparation for the treatment of pneumonia developed by Shenzhen Bao'an Chinese Medicine Hospital, which shows remarkable clinical responses. Qualitative and quantitative analyses of the main active compounds are crucial for the quality control of traditional Chinese medicine prescription in clinical application. In this study, we identified nine active compounds essential for the pharmacological effects of Fangwen Jiuwei Decoction based on the analysis of the Network Pharmacology and relevant literature. Moreover, these compounds can interact with several crucial drug targets in pneumonia based on molecular docking. We applied high-performance liquid chromatography-tandem mass spectrometry method was established these nine active ingredients' qualitative and quantitative detections. The possible cleavage pathways of nine active components were determined based on secondary ions mass spectrometry. The results of high-performance liquid chromatography-tandem mass spectrometry were further validated, which show a satisfactory correlation coefficient (r > 0.99), recovery rate (≥93.31%), repeatability rate (≤5.62%), stability (≤7.95%), intra-day precision (≤6.68%), and inter-day precision (≤9.78%). The limit of detection was as low as 0.01 ng/ml. In this study, we established a high-performance liquid chromatography-tandem mass spectrometry method to qualitatively and quantitatively analyze the chemical components in the Fangwen Jiuwei Decoction extract.

Determination of six core components from Mahuang Xuanfei Zhike syrup in rat plasma and tissues by UPLC-MS/MS: Application to a pharmacokinetics and tissue distribution study.

Mahuang Xuanfei Zhike (MXZ) syrup, a Chinese patent medicine, has been widely used in the clinical treatment of cough. However, there is no reported method for the quantitative analysis of the effective components of MXZ syrup in biological samples. In this study, the effective components of MXZ syrup were screened by network pharmacology and molecular docking technology. A sensitive and rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established to test the active components of MXZ syrup in rat plasma and tissue homogenates, including ephedrine, amygdalin, chlorogenic acid, harpagoside, forsythin and forsythoside A. Chromatographic separation was performed on a Waters Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) and the mass analysis was conducted using a Waters Xevo TQ mass spectrometer using multiple reaction positive and negative ion simultaneous monitoring mode. The results showed that the linearity ranged from 0.3 to 409.4 ng/ml. The extraction recoveries were all <8.33%, and the matrix effects were all <8.45, which met the requirements. The pharmacokinetic and tissue distribution results indicated that the main active components of MXZ syrup were absorbed quickly and eliminated slowly in vivo, and there may be a reabsorption process.

Identification of Chinese Herbal Compounds with Potential as JAK3 Inhibitors.

The Janus kinases (JAKs) consist of four similar tyrosine kinases and function as key hubs in the signaling pathways that are implicated in both innate and adaptive immunity. Among the four members, JAK3 is probably the more attractive target for treatment of inflammatory diseases because its inhibition demonstrates the greatest immunosuppression and most profound effect in the treatment of such disorders. Although many JAK3 inhibitors are already available, certain shortcomings have been identified, mostly acquired drug resistance or unwanted side effects. To discover and identify new promising lead candidates, in this study, the structure of JAK3 (3LXK) was obtained from the Protein Data Bank and used for simulation modeling and protein-ligand interaction analysis. The ~36,000 Chinese herbal compounds obtained from TCM Database@Taiwan were virtually screened by AutoDock Vina docking program and filtered with Lipinski's Rules and ADME/T virtual predictions. Because of high occurrence of fake hits during docking, we selected 12 phytochemicals which have demonstrated modulating JAKs expressions among the top 50 chemicals from docking results. To validate whether these compounds are able to directly mediate JAK3 kinase, we have investigated the inhibitory activity using enzymatic activity assays, western blot, and HEK 293 cell STAT5 transactivity assays. The molecular analysis included docking and molecular dynamics (MD) simulations in order to investigate structural conformations and to explore the key amino acids in the interaction between JAK3 kinase and its putative ligands. The results demonstrated that Cryptotanshinone, Icaritin, and Indirubin exhibited substantial inhibitory activity against JAK3 kinase in vitro. The results also provide binding models of the protein-ligand interaction, detailing the interacting amino acid residues at the active ATP-binding domains of JAK3 kinase. In conclusion, our work discovered 3 potential natural inhibitors of JAK3 kinase and could provide new possibilities and stimulate new insights for the treatment of JAK3-targeted diseases.

Helicteric Acid, Oleanic Acid, and Betulinic Acid, Three Triterpenes from Helicteres angustifolia L., Inhibit Proliferation and Induce Apoptosis in HT-29 Colorectal Cancer Cells via Suppressing NF-κB and STAT3 Signaling.

Colorectal cancer (CRC) is one of the most common malignancies and most frequent cause of cancer death worldwide. The activation of both NF-κB and STAT3 signaling and the crosstalk between them play an important role in colorectal tumor. Helicteres angustifolia L. is a type of commonly used Chinese medicinal herb and possesses a wide variety of biological activities. In the present study, we investigate the effects of three triterpenes from H. angustifolia (HT) such as helicteric acid (HA), oleanic acid (OA), and betulinic acid (BA), on inhibiting CRC progression. Our results showed that HT extracts could decrease proliferation and induce apoptosis in HT-29 colorectal cancer cells. Moreover, HT extracts could suppress LPS-triggered phosphorylation of IKK, IκB, and NF-κB, attenuate IL-6-induced phosphorylation of JAK2 and STAT3, and suppress the expression of c-Myc, cyclin-D1, and BCL-xL, the downstream gene targets of NF-κB and STAT3. Therefore, HT extracts showed potent therapeutic and antitumor effects on CRC via inhibiting NF-κB and STAT3 signaling.

[Study on Triterpenoids from Helicteres angustifolia by High-Speed Countercurrent Chromatography].

Objective: To develop a method for separation and purification of triterpenoids from Helicteres angustifolia by highspeed countercurrent chromatography( HSCCC). Methods: The ethyl acetate extract of Helicteres angustifolia were separated by two-step HSCCC. The structures of the target compounds were identified by ESI-MS and1H-NMR and characterized by FTIR. Results: Six compounds were isolated and prepared from ethyl acetate extract of Helicteres angustifolia,and identified as methyl helicterilate( 1),methyl helicterate( 2),helicterilic acid( 3),helicteric acid( 4),betulic acid( 5),oleanolic acid( 6). The purity of the chemical constituents were determined by HPLC,and the mass fraction was more than 95. 0%,respectively. Conclusion: This method is suitable for the separation and preparation of triterpenoids from Helicteres angustifolia,which is rapid,high purity and high yield.

[Study on HPLC Fingerprint of Solanum nigrum Fruit].

Objective: To establish the HPLC fingerprint method of Solanum nigrum fruit for its identification and quality evaluation. Methods: The analysis was carried out by Agilent ZORBAX SB-C18column( 150 mm × 4. 6 mm,5 µm),and eluted with mobile phase containing of acetonitrile-0. 3% phosphoric acid in a gradient mode. The temperature of column was 25 ℃,the flow rate was 1. 0m L / min, the detection wavelength was set at 205 nm, the injection volume was 10 µL. The similarity evaluation system for chromatographic fingerprint of TCM( version 2004A) was used to calculate the similarity degree of the HPLC fingerprint of Solanum nigrum fruit. . Results: The HPLC fingerprint of ten batches Solanum nigrum fruit was established with twelve common peaks. Two characteristic peaks included solasonine and solamargine were confirmed. Conclusion: The results indicate that establishing HPLC fingerprint of Solanum nigrum fruit can provide more comprehensive reference for identification and quality evaluation of Solanum nigrum fruit

[Study on Chemical Constituents of Petroleum Ether Fraction from Rubus alceaefolius].

OBJECTIVE: To investigate the chemical constituents of Rubus alceaefolius. METHODS: Nine compounds were isolated and purified from the petroleum ether extract of 95% alcohol extract of Rubus alceaefolius by repeated column chromatography on silica, Sephadex LH-20 and structurally identified by spectral analysis. RESULTS: The compounds were identified as chrysophanol(1), physcion (2), ß-sitosterol(3), 3-oxotirucalla-7, 24-dien-21-oic acid(4), myricadiol(5), 19-α-hydroxy-3-acetyl-ursolic acid(6), N-benzoylphenylalaninyl-N-benzoylphenylalaninate(7), aurantiamide acetate(8) and euscaphic acid(9). CONCLUSION: Compounds land 4~8 are isolated from this plant for the first time, and compounds 4 - 8 are found in plants of Rubus genus for the first time.

[HPLC Fingerprints of Clerodendrum lindleyi].

OBJECTIVE: To establish the HPLC fingerprint of Clerodendrum lindleyi in order to provide the basis for its quality standard. METHODS: The chromatographic fingerprint was obtained with Angilent Zorbax C18 (250 mm x 4.6 mm, 5 µm) column and gradiently eluted with acetonitrile-0.1% phosphoric acid solution. The column temperature was maintained at 35 degrees C. The flow rate was 1.0 mL/min and the detection wavelength was 327 nm. RESULTS: HPLC fingerprint of Clerodendrum lindleyi was established and 21 common peaks from 11 batches of samples were found. CONCLUSION: The method has good precision, stability and repeatability, which can provide reliable basis for quality evaluation of Clerodendrum lindleyi.

[Chemical constituents from roots of Rubus parvifolius].

OBJECTIVE: To investigate the chemical constituents from the roots of Rubus parvifolius. METHODS: The chemical constituents were isolated by silica gel column chromatography, Sephadex LH-20, as well as preparative high performance liquid chromatography. Their structures were identified on the basis of physicochemical properties and NMR analysis. RESULTS: Six comounds were isolated and identified as 3-O-Acetyl-11α, 12α-epoxy-oleanan-28,ß-olide( I ) ,3-O-Acetyl-pomolic acid( II ), Ursolic acid( Ill),Ursolic acid acetate (1V ), Euscaphic acid ( V) and ß-Sitosterol ( VI). CONCLUSION: Compounds I , II and IV are isolated from Rubus parvifolius for the first time.

[Optimization extraction conditions of triphyllin A in Pronephrium triphyllum].

OBJECTIVE: To optimize the extraction conditions for triphyllin A in Pronephrium triphyllum. METHODS: The content of triphyllin A in the Pronephrium triphyllum was determined by HPLC. Concentration and volume of the alcohol,time and times of the extraction were assayed by orthogonal test to detect their influences on the extraction rate of triphyllin A in the Pronephrium triphyllum. RESULTS: Alcohol volume and extraction times had significant influence on the process (P < 0.05) while alcohol concentration and extraction time had no effect. The optimal extraction conditions were as follows:50 fold 60% alcohol, extraction for 3 times and 50 min for each time. CONCLUSION: The extration rate of triphyllin A is higher,and the process can be used for the development and production of Pronephrium triphyllum.

[Study on the chemical constituents from the aerial parts of Medicago sativa and their hypolipidemic activity].

OBJECTIVE: To research the chemical constituents from the aerial parts of Medicago sativa. METHODS: Various column chromatographies were emoployed to isolate and purify the constituents. Their structures were elucidated by spectral analysis (IR, UV, MS, 1H-NMR, 13C-NMR) and chemical evidence. RESULTS: Two constituents were obtained and identified as soysaponin I (I), azukisaponin V (II). CONCLUSION: These two compounds are isolated from the arial parts of Mesicago sativa for the first time. Moreover,the compounds are found to have hypolipidemic activity.

Studies on the toxicity of Aristolochia manshuriensis (Guanmuton).

AIMS: (1) To study the acute and chronic toxicity of stem of Aristolochia manshuriensis (AMA Guanmuton) which is a Chinese medicinal herb in order to provide basis for safe clinical use. (2) To investigate the possibility of reducing toxicity of the herb combined with Rhizoma Coptidis (Huanglian). METHODS: The 70% ethanol extract of the herb was fed to mice via gastric tube for 8 weeks. The blood was collected to assess liver and renal functions. The tissue samples of the liver, kidney and bladder were collected from executed animals for pathology examination. RESULTS: The LD50 with a 95% average trustable probability (P=0.95) of AMA from Hanzhong (HZ) is 29.2+/-3.71 g/kg. The weight of animals in the treatment group at a dose of 4 g/kg raw drug, equivalent to 40 times of normal human dose in clinical prescription, remained the same as the control group (P>0.05). On pathological examination, there were no morphological changes under light microscope in the liver and bladder. For the kidney, the renal toxicity was significantly reduced after using ethanol extract of R. coptidis to process HZ AMA in that there were no interstitial inflammation, formation of hyaline cast or regeneration of renal tubules.