UPLC-MS Analysis, Quantification of Compounds, and Comparison of Bioactivity of Methanol Extract and Its Fractions from Qiai (Artemisia argyi Lévl. et Van.).

The Artemisia argyi Lévl. et Van. growing in the surrounding areas of Qichun County in China are called Qiai (QA). Qiai is a crop that can be used both as food and in traditional folk medicine. However, detailed qualitative and quantitative analyses of its compounds remain scarce. The process of identifying chemical structures in complex natural products can be streamlined by combining UPLC-Q-TOF/MS data with the UNIFI information management platform and its embedded Traditional Medicine Library. For the first time, 68 compounds in QA were reported by the method in this study. The method of simultaneous quantification of 14 active components in QA using UPLC-TQ-MS/MS was reported for the first time. Following a screening of the activity of QA 70% methanol total extract and its three fractions (petroleum ether, ethyl acetate, and water), it was discovered that the ethyl acetate fraction enriched with flavonoids such as eupatilin and jaceosidin had the strongest anti-inflammatory activity, while the water fraction enriched with chlorogenic acid derivatives such as 3,5-di-O-caffeoylquinic acid had the strongest antioxidant and antibacterial activity. The results provided the theoretical basis for the use of QA in the food and pharmaceutical industries.

A Biflavonoid-Rich Extract from Selaginella doederleinii Hieron. against Throat Carcinoma via Akt/Bad and IKKß/NF-κB/COX-2 Pathways.

Selaginella doederleinii Hieron. is a common pharmacological plant, and this folk herbal medicine and its complex preparations have been widely used for the treatment of throat carcinoma (TC) and several associated complications in traditional Chinese medicine. This study was aimed at investigating the specific anti-throat carcinoma impacts and potential mechanisms of a biflavonoid-rich extract from S. doederleinii (SD-BFRE). The phytochemical profiling of SD-BFRE was performed by HPLC-ESI-QTOF-MS and UPLC-PDA, and the detailed pharmacological effects and mechanisms were respectively evaluated in vitro and in vivo. MTT assay, the Transwell assay and flow cytometry were performed to evaluate the abilities of SD-BFRE on inhibiting cell infiltrative growth in TC cells (Hep-2 and FaDu) in in vitro experiments. In vivo experiments used Hep-2 tumor-bearing nude mice to evaluate the anti-TC effect of SD-BFRE. Western blotting was used to explore the potential apoptotic pathway of TC cells. Here, we found that SD-BFRE exhibited anti-proliferation and pro-apoptotic effects in TC cells. Mechanistic studies have identified that SD-BFRE can suppress the activity of IKKß and IκB-α kinase and then down-regulate the effector proteins of NF-κB/COX-2 signaling. Moreover, SD-BFRE induced apoptosis partly by regulating the Akt/Bad/caspase signaling pathway. Taken together, this study firstly demonstrated that SD-BFRE exerted its anti-TC effects by way of IKKß/NF-κB/COX-2 and Akt/Bad pathways and might represent a potential chemotherapeutic agent for throat carcinoma.

Full-Length Transcriptomic Sequencing and Temporal Transcriptome Expression Profiling Analyses Offer Insights into Terpenoid Biosynthesis in Artemisia argyi.

Artemisiae argyi Folium is a traditional herbal medicine used for moxibustion heat therapy in China. The volatile oils in A.argyi leaves are closely related to its medicinal value. Records suggest that the levels of these terpenoids components within the leaves vary as a function of harvest time, with June being the optimal time for A. argyi harvesting, owing to the high levels of active ingredients during this month. However, the molecular mechanisms governing terpenoid biosynthesis and the time-dependent changes in this activity remain unclear. In this study, GC-MS analysis revealed that volatile oil levels varied across four different harvest months (April, May, June, and July) in A. argyi leaves, and the primarily terpenoids components (including both monoterpenes and sesquiterpenes) reached peak levels in early June. Through single-molecule real-time (SMRT) sequencing, corrected by Illumina RNA-sequencing (RNA-Seq), 44 full-length transcripts potentially involved in terpenoid biosynthesis were identified in this study. Differentially expressed genes (DEGs) exhibiting time-dependent expression patterns were divided into 12 coexpression clusters. Integrated chemical and transcriptomic analyses revealed distinct time-specific transcriptomic patterns associated with terpenoid biosynthesis. Subsequent hierarchical clustering and correlation analyses ultimately identified six transcripts that were closely linked to the production of these two types of terpenoid within A. argyi leaves, revealing that the structural diversity of terpenoid is related to the generation of the diverse terpene skeletons by prenyltransferase (TPS) family of enzymes. These findings can guide further studies of the molecular mechanisms underlying the quality of A. argyi leaves, aiding in the selection of optimal timing for harvests of A. argyi.

A biflavonoid-rich extract from Selaginella moellendorffii Hieron. induces apoptosis via STAT3 and Akt/NF-κB signalling pathways in laryngeal carcinoma.

Selaginella moellendorffii Hieron. (SM), a perennial evergreen plant, has been used in the treatment of acute infectious hepatitis, thoracic and hypochondriac lumbar contusions, systemic oedema and thrombocytopaenia. However, the role of a biflavonoid-rich extract from SM (SM-BFRE) in anti-larynx cancer has rarely been reported. In this study, the in vitro and in vivo anti-laryngeal cancer activity and potential mechanisms of SM-BFRE were investigated. An off-line semipreparative liquid chromatography-nuclear magnetic resonance protocol was carried out to determine six biflavonoids from SM-BFRE. In vitro, MTT assay revealed that SM-BFRE inhibited the proliferation of laryngeal carcinoma cells. A wound healing assay indicated that SM-BFRE suppressed the migration of laryngeal cancer cells. Hoechst 33 258 and Annexin V-FITC/PI double staining assays were performed and verified that SM-BFRE induced apoptosis in laryngeal carcinoma cells. The Hep-2 bearing nude mouse model confirmed that SM-BFRE also exhibited anticancer effect in vivo. In addition, Western blot analysis demonstrated that SM-BFRE exerted its anti-laryngeal cancer effect by activating the mitochondrial apoptotic pathway and inhibiting STAT3 and Akt/NF-κB signalling pathways. All results suggested that SM-BFRE could be considered as a potential chemotherapeutic drug for laryngeal cancer.

Liquid chromatography with mass spectrometry and NMR spectroscopy based discovery of cytotoxic principles from Daphne tangutica Maxim.

An ethyl acetate extract from the barks of the ethnic Chinese medicine Daphne tangutica Maxim. exhibited antihepatocellular carcinoma activity against HepG2 and Hep3B cell lines. By using high-performance liquid chromatography based activity profiling in combination with offline liquid chromatography with mass spectrometry and NMR analysis, we rapidly identified ten major components of the extract, including seven active principles, coumarins (1-4) and biscoumarins (7, 8, 10), along with three inactive flavonoids (5, 6, 9). This study demonstrated that our combined protocol can be used as an important strategy for chemical profiling, dereplication as well as the identification of bioactive compounds from herbal medicines.

Amplexicaule A exerts anti-tumor effects by inducing apoptosis in human breast cancer.

Chemotherapy is the main treatment for patients with breast cancer metastases, but natural alternatives have been receiving attention for their potential as novel anti-tumor reagents. Amplexicaule A (APA) is a flavonoid glucoside isolated from rhizomes of Polygonum amplexicaule D. Don var. sinense Forb (PADF). We found that APA has anti-tumor effects in a breast cancer xenograft mouse model and induces apoptosis in breast cancer cell lines. APA increased levels of cleaved caspase-3,-8,-9 and PARP, which resulted from suppression of MCL-1 and BCL-2 expression in the cells. APA also inactivated the Akt/mTOR pathway in breast cancer cells. Thus, APA exerts a strong anti-tumor effect on breast cancer cells, most likely through induction of apoptosis. Our study is the first to identify this novel anti-tumor compound and provides a new strategy for isolation and separation of single compounds from herbs.

Development and Characterization of Polymorphic Microsatellite Markers for Sedum sarmentosum (Crassulaceae) and Their Cross-Species Transferability.

Sedum sarmentosum is an important Chinese medicinal herb that exhibits anti-inflammatory, anti-angiogenic and anti-nociceptive properties. However, little is known about its genetic background. The first set of 14 microsatellite markers were isolated and characterized for S. sarmentosum using an SSR-enriched library. Fourteen polymorphic microsatellite markers were acquired with satisfactory amplifications and a polymorphic pattern in 48 S. sarmentosum individuals. The number of alleles ranged from 3 to 15. The observed and expected heterozygosities varied from 0.0833 to 0.8750 and 0.2168 to 0.9063, respectively. Two loci showed significant departure from the Hardy-Weinberg equilibrium. Cross-species amplification was carried out in other Sedum species. High rates of cross-species amplification were observed. The transferability value ranged from 85.7% in S. lineare to 64.3% in S. ellacombianum. These markers will be valuable for studying the genetic variation, population structure and germplasm characterization of S. sarmentosum and related Sedum species.

In Vivo Evaluation of the Antiasthmatic, Antitussive, and Expectorant Activities and Chemical Components of Three Elaeagnus Leaves.

The leaf of Elaeagnus lanceolata and Elaeagnus henryi as well as Elaeagnus pungens has been documented as an effective herb for the treatment of asthma and chronic bronchitis in traditional clinical medicine. This study was aimed at evaluating the antiasthmatic, antitussive, and expectorant activities of the water extracts from the three plants in vivo and analyzing their chemical components by HPLC-DAD. At the medium and high doses, the water extracts of three Elaeagnus leaves significantly prolonged the preconvulsive time (P < 0.01) in guinea pigs, lengthened the latent period of cough (P < 0.01) and decreased the cough frequency caused by aqueous ammonia in mice (P < 0.01), and enhanced tracheal phenol red output in mice (P < 0.01). There were no significant differences in the pharmacological actions between the three Elaeagnus leaves. Moreover, there was more similarity on overlap peaks in the range of retention time from 10 to 40 min by HPLC and many peaks that belonged to flavonoids compounds. It suggested that the main constituents of the three Elaeagnus leaves were flavonoid for the pharmacological activities. These effects were the important evidence for the traditional use of E. henryi leaf and E. lanceolata leaf as well as E. pungens to treat asthma and chronic bronchitis.

[Study on Identification of Three Medicinal Plant Leaves from Elaeagnus Genus by Infrared Spectroscopy].

OBJECTIVE: To study and identify the three species of dry medicinal plant leaves trom Elaeagnus genus (E. pungens, E. lanceolata and E. henryi) by Infrared Spectroscopy(IR). METHODS: Fourier transform infrared spectroscopy and second derivative infrared spectroscopy were used to study and compare the characteristics of leaves of three Elaeagnus medicinal plants. RESULTS: The IR spectra and second derivative infrared spectra of the three Elaeagnus plants leaves were similar on the whole, the intensity or ratio of intensity of some absorption peaks still had certain distinctions, and the differences of the second derivative infrared spectra were more obvious. There were only slight differences between large and small leaf type of samples of Elaeagnus lanceolata; the differences of the plant leaves of one species collected in different harvest periods were far smaller than those of others belonging to the same genus. CONCLUSION: IR can be relatively reliably used for identification of the three Elaeagnus leaves.

Determination of rutin and isoquercetin contents in Hibisci mutabilis Folium in different collection periods by HPLC.

Hibisci mutabilis Folium (HMF), the dried leaf of Hibiscus mutabilis (Malvaceae), is commonly used in traditional Chinese medicine. This article aimed to establish a high-performance liquid chromatography method for the determination of rutin and isoquercitrin contents in the HMF, and to compare the content variation in all the samples, including nearly withered yellow leaf, in different collection periods, so as to provide research basis for the quality evaluation and determination of the optimal collection period. A reversed-phase HPLC separation method was employed, with a BDS Hypersil C18 column (4.6 m × 250 mm, 5 µm), under the following conditions: acetonitrile-0.3% phosphoric acid (15:85, v/v) solution as the mobile phase, flow rate 1.0 mL/min at 30°C and detection wavelength 254 nm. The calibration curves for rutin and isoquercitrin were linear over the range of 1.5-48 and 0.25-8 µg/mL, and the average recoveries were 99.92 and 100.45% (RSD: 2.39% and 2.11%, respectively)]. Based on the analysis results, it was found that contents of rutin and isoquercitrin in HMF (mature green leaf) harvested in different periods had significant difference, and reached the highest in mid-December. It was also found that the contents of the two components in the mature green leaf were much higher than those in the nearly withered leaf from the same collection period. In conclusion, the results indicated that the HPLC method was easy-to-operate and precise, and could be applied for the determination of rutin and isoquercitrin contents in the HMF. The experimental data also showed that early winter should be the most suitable collection period for HMF.

Studies on quantitative determination of total alkaloids and berberine in five origins of crude medicine "Sankezhen".

The roots of Berberis plants are widely used as a traditional Chinese medicine called "Sankezhen", having the activities of antibacterial and anti-inflammatory, and the ingredients are alkaloids. This work aims to study and compare the total alkaloids and individual alkaloid (berberine) contents in roots and stems from five origins of Berberis plants (Berberis soulieana Schneid., B. henryana Schneid., B. triacanthophora Fedde, B. gagnepainii Schneid. and B. bergmanniae Schneid.) and provides some references for resource and quality evaluation of the medicine. Acid dye colorimetry and high-performance liquid chromatography were used in the determination. The results showed that the contents for the total alkaloids in root and stem samples were in the range of 1.60-4.72% and 0.76-2.70%, while those of the berberine were 0.70-2.92% and 0.23-1.07%. With higher contents of the total alkaloids and berberine, the roots of B. soulieana, B. gagnepainii and B. bergmanniae were good sources of "Sankezhen". Meanwhile, the contents were also high in stems of the three plants, indicating that the stems were likely to be alternative sources of "Sankezhen" after further research. As the results of precision, stability and recovery tests shown, the methods were simple, rapid and reliable, and provided valuable basis for quality evaluation and new resource investigation of "Sankezhen".

[HPLC characteristic fingerprints of sedi linearis herba and sedi herba].

OBJECTIVE: To study HPLC characteristic fingerprint of Sedum lineare from different harvest periods, and to compare with its related species Sedum sarmentosum. METHODS: The HPLC fingerprints of Sedum lineare from different collecting periods were established and compared with Sedum sarmentosum by the same detection method. RESULTS: Hyperin, isoquercitrin and astragaloside were identified from the HPLC fingerprint of Sedum lineare. The fingerprint of Sedum lineare growing in the same area but different environment were basically identical; while there were remarkable differences of Sedum lineare growing in the same place but from different harvest periods, with the area of most common peaks changing from little to great, and slightly different peak number. The HPLC fingerprint of the two Sedum species had four common peaks, but could be distinguished from each other. The optimal harvest period of these two species should be full-bloom stage. CONCLUSION: The established method can provide reference for identification and quality analysis of Sedum lineare.

Antidiabetic Effect of Methanolic Extract from Berberis julianae Schneid. via Activation of AMP-Activated Protein Kinase in Type 2 Diabetic Mice.

We have investigated the antidiabetic effect and mechanism of methanolic extract of Berberis julianae Schneid. (BJSME) in STZ induced Type 2 diabetes mellitus mice. T2DM mice were induced by high fat diet and low dose streptozotocin (STZ). BJSME was orally administrated at the doses of 60, 120, and 240 mg/kg/d, for 21 days. Metformin was used as positive control drug. Food intake, body weight, plasma glucose, oral glucose tolerance test, insulin tolerance test, insulin, and blood-lipid content were measured. The effects of BJSME on the glucose transporter 4 (GLUT4) translocation in L6 myotubes and the GLUT4 protein expression in skeletal muscle as well as phosphorylation of the AMP-activated protein kinase (AMPK) in liver and muscle were examined. In vitro and in vivo results indicate that BJSME increased GLUT4 translocation by 1.8-fold and BJSME significantly improved the oral glucose tolerance and low density lipoprotein cholesterol (LDL-C) of serum and reduced body weight, glucose, and other related blood-lipid contents. The BJSME treatment also stimulated the phosphorylation of AMPK. Thus, BJSME seems to possess promising beneficial effects for the treatment of T2DM with the possible mechanism via stimulating AMPK activity.

Simultaneous determination by HPLC of quercetin and kaempferol in three Sedum medicinal plants harvested in different seasons.

A high-performance liquid chromatography method was established for the fast quantification of quercetin and kaempferol in three Sedum crude medicines: Sedi Herba (Sedum sarmentosum Bunge.), Sedi Linearis Herba (Sedum lineare Thunb.) and Sedi Emarginati Herba (Sedum emarginatum Migo.). The column used was a YMC-pack ODS-A (250 × 4.6 mm, 5 µm), the mobile phase was a solution of methanol-0.4% phosphoric acid (47:53) with a flow rate of 1.0 mL/min at 35°C and the detection wavelength was 360 nm. The calibration curves for quercetin and kaempferol were linear over the range of 0.01-0.62 µg for quercetin and 0.02-0.78 µg for kaempferol, and the average recoveries were 99.72% [relative standard deviation (RSD): 1.63% and 99.50% (RSD: 1.16%), respectively]. In conclusion, the method established in this paper is accurate and repeatable. It can be used for the determination of quercetin and kaempferol, controlling the quality of the three crude drugs. Furthermore, the experimental data showed that the best harvest season for the three Sedum medicinal species should be the full-bloom period between the end of April and the beginning of May.

Relaxant effect of 1-butanol fraction from Elaeagnus pungens leaf through inhibiting L-type Ca2+ channel on guinea pig tracheal smooth muscle.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaf of Elaeagnus pungens thunb. (Family Elaeagnaceae) has been documented as an effective herb for the treatment of asthma and chronic bronchitis in traditional Chinese medicine. In the past years, only a few of preliminary studies reported the chemical constituents and pharmacology effects of the herb, but their action on the tracheal relaxation has not been investigated. AIM OF THE STUDY: To investigate the relaxing effect and mechanism of the extracts from Elaeagnus pungens leaves on guinea pig tracheal smooth muscle and bronchi smooth muscle cells. MATERIALS AND METHODS: Four fractions of different polarities from Elaeagnus pungens leaves were tested to the tracheal strips on the resting tension or pre-contracted by histamine (20 µM) and acetylcholine (20 µM). Inhibitory effects of the 1-butanol fraction (400mg/ml) on cumulative histamine and acetylcholine (0.2-20 µM) induced contraction were measured. In order to determine the mediators on the 1-butanol fraction effect, the relaxing effect of the 1-butanol fraction was evaluated in the absence and presence of ß-adrenoceptor antagonists (1 µM propranolol), K(+) channels-blockers (4-aminopyridine (2mM), tetraethylammonium chloride (5mM) or glibenclamide (10 µM)), the cyclooxygenase inhibitor (indomethacin, 10 µM), nitric oxide synthase inhibitor (Nω-nitro-L-arginine methyl ester, 100 µM) or L-type Ca(2+) channel inhibitor (nifedipine, 1 µM). Moreover, [Ca(2+)]i in bronchi smooth muscle cells was analyzed by measuring the fluorescence intensity with confocal system. RESULTS: 1-Butanol fraction induced the highest relaxant effect among four fractions of different polarities from Elaeagnus pungens leaves, and significantly relaxed the tracheal strip in the concentration-dependent manner on the resting tension and pre-contracted by histamine phosphate and acetylcholine. It also produced an unparallel rightward shift of the cumulative concentration-response curve of histamine or acetylcholine. Furthermore, the relaxant effect of 1-butanol fraction was not affected by propranolol, glibenclamide, tetraethylammonium chloride, 4-aminopyridine, indomethacin and Nω-nitro-L-arginine methyl ester. However, 1-butanol fraction-induced relaxation decreased after adding nifedipine. It also concentration-dependently inhibited CaCl2-induced contraction in the Ca(2+)-free, 60mM K(+)-containing solution. Additionally, [Ca(2+)]i in the BSMCs significantly reduced after administration of the 1-butanol fraction. CONCLUSIONS: The 1-butanol fraction from Elaeagnus pungens leaves resulted in a relaxation in the non-precontracted and pre-contracted tracheal strips. The relaxant effect was not related to K(+) channels, NO, cGMP or ß-adrenoceptors, but related to the inhibition of Ca(2+) influx through L-type Ca(2+) channels.

Identification of four Sedum plant medicines by fourier transform infrared spectra.

BACKGROUND: Sedum sarmentosum bunge (SSB)., S. lineare Thunb. (SLT), S. erythrostictum migo. (SEM), and S. aizoon L. (SAL) were four widely used Chinese traditional drugs or ethnic drugs, which were easy to be confused with each other. OBJECTIVE: This study aimed at developing a rapid and accurate method to identify the four Sedum plant medicines with very similar appearances and close relationships. MATERIALS AND METHODS: The herbal medicines employed here were SSB, SLT, SEM, and SAL collected in different places and seasons. Through comparing the infrared (IR) spectra of their 70% ethanol extracts, the results showed that the IR spectra of the four plant medicines possessed not only some common characteristics but also certain notable distinctions, such as shapes, numbers, positions, intensity, and ratios of the absorbing peaks. RESULTS: By fourier transform infrared (FT-IR) spectroscopy, the four medicines could be effectively differed, their habitats could be judged preliminarily, and the genetic relationships of the original plants of the four medicines could also be estimated to some extent. CONCLUSION: The application of FT-IR spectroscopy in crude medicine authentication and quality evaluation deserved to be further emphasized.

Quantitative determination of total and individual flavonoids in stems and leaves of Buddleja davidii and Buddleja albiflora.

BACKGROUND: Buddleja davidii and B. albiflora are two different original plants of the famous crude medicine "Diaoyangchen". MATERIALS AND METHODS: An ultraviolet-visible spectrophotometric method and a HPLC method were used for the determination of total and individual flavonoids (luteolin and apigenin) contents from their stems and leaves for the first time. RESULTS: From the comparative evaluation, remarkable differences in flavonoids contents were observed between different origins and different parts of the samples. And content of specific flavonoid did not correspond to the total flavonoids contents in Buddleja davidii and Buddleja albiflora. CONCLUSION: With a better accuracy and precision, the methods had been proved simple, rapid, and reliable for quantitative determination of the total flavonoids and luteolin and apigenin in the two phytomedicines. Furthermore, our present study will pave the way of guidelines for the differentiation and standardization and exploitation of individual parts of this herb material.

[Comparative identification research on different extracts of Dai Medicine "Pokou" and its processed product by FTIR].

OBJECTIVE: To study identification methods of different extracts of Dai Medicine "Pokou" (the rhizome of Homalomena gigantea Engl. ) and its processed product made by immersing it in water, and provide reference for identification of the drug in further researches and applications. METHODS: FTIR technique was used for identifying the features of different extracted parts of this crude drug and its processed product. RESULTS: Compared with the crude drug, the petroleum ether-extracted parts of processed product turned out to have no obvious distinction in the FTIR. There was a large difference in the ethyl acetate-extracted parts, and the n-butanol-extracted parts also had certain discrepancy. A preliminary analysis was made on the chemical fundamentals which caused the changes in the FTIR before and after the drug's processing. CONCLUSION: The results provide an infrared spectral identification method for the drug and its applications.

Determination of total flavonoids in three Sedum crude drugs by UV-Vis spectrophotometry.

A simple, rapid UV-Vis spectrophotometry method for the determination of total flavonoids in Sedum sarmentosum Bunge., S. lineare Thunb., and S. erythrostictum Migo. was developed, with a good linearity, precision, and stability. The detection wavelength was set at 500 nm, and an extraction solvent was optimized. Through the comparative study of multiple samples of the three plant drugs, their collected seasons and the habitats can be preliminarily ascertained, which may help to control the quality of the medicines to some extent.

[Comparative identification on Dai medicine "Pokou" and its processing product].

OBJECTIVE: To research identification methods of the Dai Medicine "Pokou" (the rhizome of Homalomena gigantea) and its processing product, and provide basis for identification of the drug in further research and application. METHODS: Macroscopic, microscopic observation and TLC and FTIR techniques were used to authenticate this raw medicine and its processing product. RESULTS: There were certain differences in the macroscopic features. The TLC result and infrared spectra of the samples had also obvious differences. The methods for identification of this raw medicine and its processing product were established, The detailed tissue and powder of this medicine were drawn. CONCLUSION: The results provided the basis for identification of the medicine and establishment of its quality standard.