Antiviral activity of mangiferin from the rhizome of Anemarrhena asphodeloides against herpes simplex virus type 1

Objective:To evaluate the antiviral activity of pure compounds against herpes simplex virus type 1(HSV-1)from the rhizome of Anemarrhena asphodeloides.Methods:Bioassay-guided isolation was conducted to separate the active compound and its chemical structure was elucidated by spectral analysis.In vitro antiviral efficacy of active compound was detected by Cell Counting Kit-8 assay,plaque reduction assay,and fluorescence observation.RT-PCR was used to determine the viral load and the cytokine-related gene expression after HSV-1 infection.In vivo study was also conducted to further determine antiviral efficacy of an active compound against HSV-1.Results:An active compound was isolated and elucidated as mangiferin.Mangiferin significantly inhibited the replication of HSV-1 in Vero cells with a half maximal inhibitory concentration(IC_(50))of 64.0 mg/L.Time-of-addition and time-of-removal assays demonstrated that mangiferin could effectively inhibit the replication of HSV-1 in the early stage(8 h).UL12,UL42,and UL54 gene expression levels of HSV-1 in the 64 mg/L mangiferin-treated group were markedly reduced as compared with the HSV-1 group(P<0.01).Fluorescence observation showed that mangiferin attenuated the mitochondrial damage maintainingΔΨm induced by HSV-1 in Vero cells.The expression of inflammatory factors TNF-α,IL-1β,and IL-6 was remarkably increased in the virus-infected group as compared with that in the normal group(P<0.05),the levels of these inflammatory factors dropped after treatment with mangiferin.Mangiferin significantly decreased the viral load and attenuated the HSV-1-induced up-regulation of TNF-α,IL-1β,and IL-6.The relative protection rate of HSV-1-infected mice could reach up to55.5%when the concentration of mangiferin was 4 g/kg.Conclusions:Mangiferin exhibits promising antiviral activity against HSV-1 in vitro and in vivo and could be a potential antiviral agent for HSV-1.

Inhibitory Effects of Saponins From Anemarrhena asphodeloides Bunge on the Growth of Vascular Smooth Muscle Cells

Objective To investigate the effects of saponins from Anemarrhena asphodeloides Bunge （SAaB） （Botanical Name： Anemarrhena Asphodeloidis Rhizoma） on the growth of vascular smooth muscle cells （VSMCs）. Methods Cell proliferation was measured by a newly developed cell proliferation reagent, WST-1. Cell apoptosis was assayed by flow cytometry through detecting annexin V. Nitric oxide production was evaluated using confocal laser scanning microscopy with diaminofluorescein diacetate （DAF-2, DA）. Cell aldose reductase （AR） activity, as well as the effect of Epalrestat and interleukin-1β were also explored. Results WST assay showed that cell proliferation induced by serum was significantly inhibited by SAaB （P〈0.01）. Flow cytometry analysis revealed that SAaB could enhance apoptotic rate of VSMCs （P〈0.01）. Nitric oxide production was significantly enhanced after administration of SAaB and interleukin-Iβ Moreover, AR activity of VSMCs was also remarkably inhibited by both SAaB and Epalrestat （P〈 0.01）. Conclusion SAaB can inhibit proliferation and enhance apoptosis of VSMCs. It may protect vascular cells by inhibiting VSMC proliferation and augmenting apoptotic rate of VSMCs via NO-dependent pathway.

Two new saponins from Anemarrhena asphodeloides Bge.

Two new saponins 3-O-β-d-glucopyranosyl (1 → 2)-β-d-mannopyranosyl sarsasapogenin, named timosaponin A IV(1) and (5β, 25S)-26-O-β-d-glucopyranosyl-furost-20(22)-en-3,26-diol-3-O-β-d-glucopyranosyl (1 → 4) glucopyranosyl (1 → 2)-β-d-galacopyranoside, named timosaponin B IV(2), were isolated by silica gel column chromatography and preparative HPLC from Anemarrhena asphodeloides Bge. Their structures were elucidated by chemical characters and spectroscopic analysis.

Simultaneous determination of steroidal saponins in Anemarrhena asphodeloides Bge. by ultra high-performance liquid chromatography/ quadrupole time-of-flight mass spectrometry

The objective of the research is to develop a quantitative method by ultra high-performance liquid chromatography/ quadrupole time-of-flight mass spectrometry （UHPLC/Q-TOF MS） for the analysis of seven major steroidal saponins （timosaponin N, timosaponin El, timosaponin BII, timosaponin B, anemarrhenasaponin I, anemarrhenasaponin A2, and timosaponin AIII） in Anemarrhena asphodeloides Bge. The complete separation of these seven steroidal saponins was achieved within 18 min with an ACQUITY UPLC HSS T3 column using an acetonitrile-water （contain 0.1% formic acid） gradient system. The limits of quantitation （LOQ）, 0.18-0.75 ng/pL for seven steroidal saponins, were determined experimentally. The limits of detection （LOD） were found to be 0.05-0.22 ng/μL for these saponins. The correlation coefficients （r2） for calibration curves varied from 0.9902 to 0.9979. This method showed good repeatability for the quantification of these saponins in rhizomes ofA. asphodeloides, with intra-day and inter-day variations of less than 5.0% for seven steroidal saponins. The recoveries of seven steroidal saponins were from 97.13% to 101.98%. The validated method was successfully applied to quantifying seven steroidal saponins in various sources ofA. asphodeloides （different collecting places or processing methods） and Zhimu concentrate-granules （ZMCG）.

Two new steroidal saponins isolated from Anemarrhena asphodeloides

Two new steroidal saponins,named timosaponin P(1) and timosaponin Q(2),were isolated from the rhizome parts of Anemarrhena asphodeloides Bunge using various chromatographic methods.Their structures and absolute configurations were elucidated by a combination of spectroscopic and spectrometric data,including 1D,2D NMR,HR-ESI-MS and ECD calculations,and this is the first time the absolute configuration of C-23 of steroidal saponin was confirmed by ECD calculations.

Screening potential α-glucosidase inhibitors from Anemarrhena asphodeloides using response surface methodology coupled with grey relational analysis

Objective: To screen potential α-glucosidase inhibitors from Anemarrhena asphodeloides.Methods: Response surface methodology employing Box-Behnken design was used to optimize conditions for the extraction of α-glucosidase inhibitory active compounds from A. asphodeloides. The powders(20.0 g) of A. asphodeloides were extracted under the optimized conditions. The extract was applied to a D-101 macroporous resin column. It was eluted with ethanol and water to give six fractions. Compounds from the active fraction were identified by UPLC-Q-TOF-MS. The structure-activity relationship was discussed based on grey relational analysis.Results: The optimum extraction conditions were as follows: ethanol concentration, 100%; extraction temperature, 51 ℃; and solvent to solid ratio, 23 mL/g. It indicated that the active compounds were concentrated into 80% ethanol fraction. Twenty five steroid saponins from 80%ethanol fraction were identified by UPLC-Q-TOF-MS. Peaks 19 and 23 were tentatively identified as new structures. The predicted α-glucosidase inhibitory activities of the compounds were 7 > 2 > 1 > 22 > 23 > 3 > 9 > 21 > 24 > 4 > 13 > 8 > 14 > 16 > 17 > 25 > 6 > 19.Conclusion: The fraction eluted by 80% ethanol showed the best inhibitory activity. After analyzing the data of UPLC-Q-TOF-MS, 25 steroid saponins were tentatively identified in this fraction.

Exploring the catalytic function and active sites of a novel C-glycosyltransferase from Anemarrhena asphodeloides

Anemarrhena asphodeloides is an immensely popular medicinal herb in China,which contains an abundant of mangiferin.As an important bioactive xanthone C-glycoside,mangiferin possesses a variety of pharmacological activities and is derived from the cyclization reaction of a benzophenone C-glycoside(maclurin).Biosyntheti-cally,C-glycosyltransferases are critical for the formation of benzophenone C-glycosides.However,the benzo-phenone C-glycosyltransferases from Anemarrhena asphodeloides have not been discovered.Herein,a promiscuous C-glycosyltransferase(AaCGT)was identified from Anemarrhena asphodeloides.It was able to catalyze efficiently mono-C-glycosylation of benzophenone,together with di-C-glycosylation of dihydrochalcone.It also exhibited the weak O-glycosylation or potent S-glycosylation capacities toward 12 other types of flavonoid scaffolds and a simple aromatic compound with–SH group.Homology modeling and mutagenesis experiments revealed that the glycosylation reaction of AaCGT was initiated by the conserved residue H23 as the catalytic base.Three critical residues H356,W359 and D380 were involved in the recognition of sugar donor through hydrogen-bonding interactions.In particular,the double mutant of F94W/L378M led to an unexpected enzy-matic conversion of mono-C-to di-C-glycosylation.This study highlights the important value of AaCGT as a potential biocatalyst for efficiently synthesizing high-value C-glycosides.

KT和NAA对知母试管苗生长发育的影响

探讨了激动素(KT)和萘乙酸(NAA)对知母(Anemarrhena asphodeloides)分蘖生长发育和生理生化指标的影响。结果表明,知母分蘖生长发育的最佳培养基为MS+KT 4 mg/L+NAA 0.1 mg/L,此培养基条件下,知母分蘖的总叶绿素含量、可溶性总糖含量和可溶性蛋白质含量最高;而低浓度的KT与高浓度的NAA组合下知母试管苗总叶绿素含量、可溶性总糖含量和可溶性蛋白质含量较低。

知母内生真菌多样性及其对浅表感染真菌的体外拮抗活性

分析药用植物知母Anemarrhena asphodeloides根、茎、叶中内生真菌的多样性及其次级代谢产物对浅表感染真菌的抑菌活性,为寻找皮肤真菌病的潜在药物奠定基础。从河北省太行山采集到5株健康的野生知母,采用组织块法分离根、茎、叶的内生真菌;结合形态学和分子生物学方法分类鉴定内生真菌;并分别采用菌丝生长抑制速率法和琼脂扩散法筛选对5种浅表感染真菌具有抑制活性的菌株。本研究从知母中共分离获得438株内生真菌,隶属于23个属。根、茎、叶的真菌定殖率分别为99.33%、74.00%和47.33%,其中根部的优势类群为镰刀菌属Fusarium(60.29%)和棘壳孢属Setophoma(33.97%);茎部的优势类群为镰刀菌属(91.50%)和粘帚霉属Clonostachys(7.19%);叶部的优势类群为镰刀菌属(35.53%)和链格孢属Alternaria(14.47%)。香农多样性指数显示,叶中的物种多样性最高,其次是根和茎。对30株代表性菌株发酵液的抑菌实验结果表明:对犬小孢子菌Microsporum canis、红色毛癣菌Trichophyton rubrum、黏膜毛孢子菌Trichosporon mucoides、糠秕马拉色菌Malassezia furfur和白色念珠菌Candida albicans具有抑制活性的菌株分别占总数的96.67%、100.00%、100.00%、3.33%和3.33%,其中菌株ZMR35对4种浅表感染真菌均具较强的抑制活性,菌株ZML33则对犬小孢子菌、红色毛癣菌、黏膜毛孢子菌的抑制率均高于70%,经分子鉴定两者分别属于黑团孢属Periconia和曲霉属Aspergillus。知母蕴含较为丰富的内生真菌资源,其中还有较高比例菌株的发酵产物对浅表感染真菌表现出良好的拮抗活性,这为浅表感染真菌病药物的筛选提供了潜在资源库。

Network Pharmacology Investigation in the Mechanism of Radix Pseudostellariae-Rhizoma Anemarrhenae Therapy for Diabetes Mellitus

Objective:To explore the targets and signaling pathways of Radix Pseudostellariae-Rhizoma Anemarrhenae treating diabetes mellitus by network pharmacology and expound its mechanism.Methods:The TCMSP and SymMap databases were used to screen out the active ingredients and targets of Radix Pseudostellariae-Rhizoma Anemarrhenae.The GenCards database was used to screen the predicted targets of diabetes mellitus.Two targets were mapped.The Cytoscape 3.7.2 software was used to construct the network diagram of Chinese medicine-active ingredient-target and screen out the core ingredient and target.The GO,KEGG pathway enrichment analysis was carried out by OmicShare and David.Results:According to the screening criteria,23 active ingredients of Radix Pseudostellariae-Rhizoma Anemarrhenae and 146 potential targets were obtained.138 common targets were obtained related with diabetes mellitus,including 5 key components and 37 core targets.The enrichment of biological process in GO is mainly related to the reaction to organic substances,the response of cells to chemical stimulation,the reaction to chemical substances,etc..The molecular function is mainly related to the enzyme binding,the same protein binding,and the activity of nuclear receptor.The cell composition is mainly associated with the membrane raft,membrane microdomain,membrane region,etc..The KEGG pathway enrichment is mainly concentrated in TNF,Toll-like receptors,NOD-like receptors,insulin resistance and type II diabetes.Conclusion:Radix Pseudostellariae-Anemarrhena asphodeloides plays the anti-inflammatory,antioxidant,hypoglycemic,and insulin-resistance improving role through multiple targets and pathways,and therefore achieves the effect on diabetes mellitus.

菝葜皂苷元对高脂饮食诱导认知障碍大鼠的影响

本研究旨在探究知母源性菝葜皂苷元对高脂饮食诱导认知障碍大鼠认知功能的影响,并进一步研究其潜在的作用机制。通过使用Morris水迷宫测试、Nissl染色、酶联免疫吸附试验等方法,本研究发现运动前菝葜皂苷元补充显著降低了高脂饮食诱导的大鼠逃避潜伏期,增加了穿越平台的次数,减轻了海马神经元损伤。此外,菝葜皂苷元补充也抑制了海马神经炎症,减少了IL-1β、IL-6和TNF-α的水平。NF-κB的激活是高脂饮食导致的神经炎症的关键,而菝葜皂苷元能够降低NF-κB的磷酸化水平,从而抑制了炎症反应。此外,本研究还观察到菝葜皂苷元能够下调BACE1的表达,减少Aβ1-42的生成,为改善认知功能提供了分子基础。综上所述,本研究结果强调了运动前菝葜皂苷元在改善高脂饮食诱导认知障碍中的潜在应用和机制。

Fingerprint analysis of Zhimu-Huangbai herb pair and simultaneous determination of its alkaloids, xanthone glycosides and steroidal saponins by HPLC-DAD-ELSD

AIM: To develop and validate a high performance liquid chromatography(HPLC) coupled with diode array and evaporative light scattering detectors(DAD-ELSD) method for the quantitative determination and fingerprint analysis of ten active constituents in three chemical classes(namely, xanthone glycosides, steroidal saponins, and alkaloids) in Zhimu-Huangbai herb pair(ZB). METHOD: Chromatographic separation was performed on a Diamonsil C18 column(4.6 mm × 250 mm, 5 μm, Dikma) by gradient elution using acetic acid in acetonitrile solution at a flow rate of 1.0 mL·min–1 at 260 nm. The drift tube temperature of ELSD was set to 60 ℃ and nebulizer gas pressure was 4.0 Bar. Method validation was performed to assure its linearity, limits of detection and quantification, precision, repeatability, stability, and accuracy. RESULTS: The HPLC-DAD-ELSD method allowed the quantification of ten compounds(phellodendrine, jatrorrhizine, palmatine, berberine, neomangiferin, mangiferin, timosaponin E-I, timosaponin B-II, timosaponin B, and timosaponin A-III), and was successfully applied to fingerprint analysis for ten batches of ZB samples. CONCLUSION: This was the first time to apply the combination of DAD and ELSD for the simultaneous determination of ten active ingredients in ZB. The results showed that the combination of quantitative analysis for marker ingredients and chemical fingerprint for the TCM herb pair provides a potentially powerful, widely introduced, and internationally accepted strategy for assessment of complex TCM formulas.

Hypnotic Effects of A Novel Anti-Insomnia Formula on Drosophila Insomnia Model

Objective： To assess the biological effects of the six-herb mixture Anti-lnsomia Formula （AIF） extract using caffeine-induced insomnia Drosophila model and short-sleep mutants. Methods： Caffeine- induced insomnia wild-type Drosophila and short-sleep mutant flies minisleep （runs） and Hyperkinetic Y（Hk Y） were used to assess the hypnotic effects of the AIF in vivo. The night time activity, the amount of night time sleep and the number of sleep bouts were determined using Drosophila activity monitoring system. Sleep was defined as any period of uninterrupted behavioral immobility （0 count per minute） lasting 〉 5 min. Night time sleep was calculated by summing up the sleep time in the dark period. Number of sleep bouts was calculated by counting the number of sleep episodes in the dark period. Results： AIF at the dosage of 50 mg/mL, effectively attenuated caffeine-induced wakefulness （P〈0.01） in wild-type Canton-S flies as indicated by the reduction of the sleep bouts, night time activities and increase of the amount of night time sleep. AIF also significantly reduced sleeping time of short-sleep Hk y mutant flies （P〈0.01）. However, AIF did not produce similar effect in mns mutants. Conclusion： AIF might be able to rescue the abnormal condition caused by mutated modulatory subunit of the tetrameric potassium channel, but not rescuing the abnormal nerve firing caused by Shaker gene mutation. This study provides the scientific evidence to support the use of AIF in Chinese medicine for promoting sleep quality in insomnia.