In vitro antiviral activity of Phyllanthus orbicularis extracts against herpes simplex virus type 1

The antiviral activity of butanol- and acetic acid-soluble fractions, prepared from the leaves and stems of Phyllanthus orbicularis H.B.K., has been investigated against acyclovir-sensitive or -resistant herpes simplex virus type 1 (HSV-1) strains, using human foreskin fibroblast (HFF) and green ape kidney (Vero) cell lines. Both fractions showed antiviral selectivity indexes (SI) from 10.3 to 22.8, while their extracellular virucidal activities reached SI values ranging from 371 to 1,040. Time-addition experiments suggested that the active compounds present in the studied fractions acted on early steps of the virus replication cycle.     

Effect of the Potent Antiviral 1‐Cinnamoyl‐3,11‐Dihydroxymeliacarpin on Cytokine Production by Murine Macrophages Stimulated with HSV‐2

The limonoid 1‐cinnamoyl‐3,11‐dihydroxymeliacarpin (CDM) isolated from leaf extracts of Melia azedarach L, has potent antiherpetic effect in epithelial cells. Since Meliacine, the partially purified extract source of CDM, has therapeutic effect on murine genital herpes, the potential use of CDM as microbicide against herpetic infections was studied here. To determine the cytotoxic effect of CDM, the MTT assay and acridine orange staining of living cells were performed. The antiherpetic action of CDM was measured by plaque reduction assay, and the immunomodulatory effect was determined by measuring the cytokine production using a bioassay and ELISA method. The results presented here showed that CDM inhibited Herpes Simplex Virus type 2 (HSV‐2) multiplication in Vero cells but did not affect its replication in macrophages which were not permissive to HSV infection. In macrophages, levels of TNF‐α, IFN‐γ, NO, IL‐6 and IL‐10 were increased by CDM used alone or in combination with HSV‐2. Besides, CDM not only synergized TNF‐α production combined with IFN‐γ, but also prolonged its expression in time. Results indicate that CDM inhibits HSV‐2 multiplication in epithelial cells and also increases cytokine production in macrophages, both important actions to the clearance of infecting virus in the mouse vagina. Copyright © 2013 John Wiley & Sons, Ltd.     

Modulation of rat and human cytochromes P450 involved in PhIP and 4-ABP activation by an aqueous extract of Phyllanthus orbicularis

Phyllanthus orbicularis HBK (Euphorbiaceae) is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antimutagenic effects against hydrogen peroxide and some promutagenic aromatic amines (AAs), in addition to its antiviral properties. In this paper, antimutagenesis of this extract against two carcinogenic AAs, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (4-ABP) has been studied. Liver microsomal fractions from both induced rats and humans were used to metabolise both procarcinogenic compounds in the Salmonella assay. The plant extract was effective in reducing the mutagenesis of these AAs, activated by both kinds of fractions. The optimal antimutagenic effect was obtained when both AAs were metabolised by human enzymes, with an almost total reduction of 4-ABP mutagenesis and a decrease of about 75% of PhIP mutagenicity. Mutagenicity of both AAs, activated by induced rat fraction, was only decreased by about 50%. Inhibition by plant extract of alkoxyresorufin O-dealkylation activities, dependent on CYP1A, of both fractions was determined. In accordance with the results obtained, the inhibition or modulation of CYP1A subfamily activities, and possibly of CYP1A2, is thought to be the main mechanism of antimutagenesis of the aqueous extract of Phyllanthus orbicularis against 4-ABP and PhIP.     

Hippomanin A from acetone extract of Phyllanthus urinaria inhibited HSV-2 but not HSV-1 infection in vitro

Phyllanthus urinaria Linnea (Euphorbiaceae) is a commonly used traditional medicinal plant in oriental countries and has been reported to possess various biological activities. Previously, the acetone extract and some pure compounds from P. urinaria were found to suppress herpes simplex virus (HSV). In this study, another two pure compounds were isolated from acetone extract of P. urinaria and were tested for their in vitro anti-HSV-1 and HSV-2 activities. The results showed that hippomanin A impeded HSV-2 but not HSV-1 infection. Corilagin, however, inhibited neither HSV-1 nor HSV-2 replication. The similarity between corilagin and hippomanin A in structure, but difference in antiviral activity, therefore, merit further investigation.     

Formula Optimization of the Jiashitang Scar Removal Ointment and Antiinflammatory Compounds Screening by NF‐κB Bioactivity‐guided Dual‐luciferase Reporter Assay System

Inflammation plays a role in scar formation; therefore, decreasing inflammation benefits scar removal. Jiashitang scar removal ointment (JST) is a commercially available traditional Chinese medicinal formulation. It is composed of extracts from Carthamus tinctorius L. (Car), Rheum officinale Baill. (Rhe), Salvia miltiorrhiza Beg. (Sal), and Panax notoginseng (Burk.) F. H. Chen (Pan), which are all herbs with potent antiinflammatory activities. Our aims are to optimize the formula of JST and to elucidate its antiinflammatory active components. Response surface methodology was applied to optimize proportions of the four herb extracts. The antiinflammatory effects were evaluated using in vitro and in vivo models. To screen for active components in this formula, a bioactivity‐based ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry analysis was performed. After optimization, the antiinflammatory effects of the new formula were significantly superior to the original one. Screening identified 13 active ingredients: a series of saffiomin, emodin, salvianolic acid, tanshinone, and triterpenoid saponin derivatives. These active ingredients were predicted to exert nuclear factor‐κB inhibiting effects through MAPK, PI3K/AKT, and NIK‐IKK pathways. In conclusion, the original formula was successfully optimized with more potent antiinflammatory activity. These methods can be applied to researches of other formulas. Copyright © 2014 John Wiley & Sons, Ltd.     

应用ITS2条形码鉴定岭南药材余甘子及其混淆中药品种

目的：本研究应用ITS2序列作为DNA条形码,建立余甘子及其混淆品分子鉴定方法。方法：收集8种共17份余甘子及其混淆品植物样本,提取基因组DNA,扩增ITS2基因并双向测序。所得序列采用Codon Code Aligner进行拼接,同时,从GenBank数据库中获3条相关ITS2序列用于比较分析。利用MEGA 6.06软件分析其种内及种间遗传距离,并构建系统进化树。结果：余甘子ITS2序列长度为208bp,与其他各物种种间变异位点较多,遗传距离较大,为0.174-0.823。聚类树结果显示,余甘子基原植物独聚一支,与其他混淆品能够容易区分。结论：本研究采用ITS2序列能够有效鉴别余甘子及其混淆品基原植物,可为保证临床用药真实安全提供技术支撑。     

Mechanistic Evidence of Viscum schimperi (Viscaceae) Antihyperglycemic Activity: From a Bioactivity‐guided Approach to Comprehensive Metabolite Profiling

Diabetes mellitus is possibly the world's largest growing metabolic disorder. Effective treatment of diabetes is increasingly dependent on active constituents of medicinal plants capable of controlling hyperglycemia as well as its secondary complications. Viscum schimperi Engl. is a plant growing in Saudi Arabia and known for its antidiabetic activity. The potential antidiabetic activity of its methanol extract as well as its chloroform, n‐butanol, and the remaining water fractions was evaluated in streptozotocin‐induced diabetic rats at two dose levels. The antidiabetic activity was assessed through the determination of fasting blood glucose level, insulin levels, area under the curve (AUC) in oral glucose tolerance test, glucose absorption in isolated rat gut assay, and glucose uptake by psoas muscle. Moreover, large‐scale untargeted metabolite profiling of methanol extract was performed via UPLC‐PDA and qTOF‐MS (ultra‐performance liquid chromatography photodiode array detection and quadrupole time‐of‐flight mass spectrometry) respectively, to explore its chemical composition and standardization of its extract. Multivariate statistical analysis including principal component analysis and orthogonal projection to latent structures discriminant analysis was used to determine bioactives in its fractions. In conclusion, oleanane triterpenes and O‐caffeoyl quinic acid conjugates were the major compounds that might account for antihyperglycemic effect of the plant. Copyright © 2015 John Wiley & Sons, Ltd.     

Effects of Ilex paraguariensis A. St. Hil. (yerba mate) on herpes simplex virus types 1 and 2 replication

The antiherpes effects of the crude extract obtained from Ilex paraguariensis leaves (yerba mate) and their purified fractions were investigated. The most active fraction was selected and assayed to determine the viral multiplication steps upon which it acted. In order to detect the major components of this fraction, thin layer chromatography (TLC) analysis was performed. The antiviral activity was evaluated against HSV-1 and HSV-2 by a viral plaque number reduction assay (IC(50) ) and the cytotoxicity by a MTT assay (CC(50) ). According to the obtained results, all tested samples showed antiherpes activity at noncytotoxic concentrations, and the ethyl acetate fraction was the most active (SI = CC(50) /IC(50) = 188.7 and 264.7 for HSV-1 and HSV-2, respectively). The results also demonstrated that this fraction exerts antiviral activity by the reduction of viral infectivity, the inhibition of virus entry into cells and cell-to-cell virus spread, as well as by the impaired levels of ICP27, ICP4, gD and gE proteins of HSV-1. The TLC analysis showed that this fraction contains monodesmosidic triterpenoid saponins, matesaponin-1 (a bidesmosidic one), caffeic and chlorogenic acids and rutin, which suggests that they could act synergistically and be responsible for the detected antiherpes activity.     

New Activities for Isolated Compounds from Convolvulus austro‐aegyptiacus as Anti‐ulcerogenic,Anti‐Helicobacter pylori and Their Mimic Synthesis Using Bio‐guided Fractionation

Bio‐guided fractionation of the total alcoholic extract of Convolvulus austro‐aegyptiacus was screened for its anti‐ulcerogenic activity, using an absolute‐ethanol‐induced ulcer model at 500 and 1000 mg/kg doses. Two compounds were isolated from the butanol extract of C. austro‐aegyptiacus and identified by ¹H and ¹³C nuclear magnetic resonance as scopoletin and scopolin. The isolated compounds (50 mg/kg) showed a remarkable anti‐ulcerogenic activity because they exhibited control‐ulcer protection by 16.7% and 90.8%, respectively. The acute toxicity study showed that the extract is highly safe; the median lethal dose (LD₅₀₎ was more than 4000 mg/kg. Moreover, the obtained results were confirmed by the sub‐chronic toxicity because the rats that have been administered 1000 mg/kg of the extract for 15 consecutive days showed no alteration in the liver and kidney functions. Copyright © 2015 John Wiley & Sons, Ltd.     

叶下珠复方对人肝癌HePG2细胞体外增殖抑制及诱导凋亡作用

目的观察叶下珠复方对人肝癌HePG2细胞体外增殖的抑制作用并探讨其作用机制。方法采用四甲基偶氮唑蓝比色法(MTT)和生长曲线测定法,比较不同浓度的叶下珠复方在不同作用时间对体外培养的HePG2细胞增殖的影响;同时应用流式细胞术、荧光显微镜和电镜对HePG2细胞的凋亡率及形态学进行观察。结果叶下珠复方对HePG2细胞增殖有抑制作用,在一定范围内,叶下珠复方的浓度越大、作用时间越长,对HePG2的抑制作用越强,叶下珠复方浓度为500μg/mL作用72h时,其抑制率达93.58%,IC50为240μg/mL。不同浓度的叶下珠复方均具有一定程度诱导HePG2细胞凋亡的作用。结论叶下珠复方在体外能一定程度抑制肝癌细胞增殖,其机制可能与诱导肝癌HePG2细胞凋亡有关。     

余甘子加工沉淀固废物中活性成分鉴定与提取工艺优化

目的:研究余甘子鲜汁沉淀物的化学组成与资源化物质鞣花酸的提取纯化工艺。方法:收集余甘子鲜汁沉淀物,采用能谱仪、傅里叶变换红外光谱仪和超高效液相色谱与串联四极杆飞行时间质谱联用技术（UPLC-Q-TOF-MS）分析余甘子鲜汁沉淀物的化学成分;以鞣花酸含量为指标,分别采用醇提法、酶提法、碱溶酸沉法对鞣花酸进行提取纯化,且通过单因素实验优化筛选提取工艺。结果:红外分析表明,余甘子鲜汁沉淀物具有C、O元素,苯环、酚羟基等基本结构;UPLC-Q-TOF-MS结果表明,沉淀物的主要成分为鞣花酸、邻苯二甲酸二丁酯等,且鞣花酸占比最高,为39.91%;提取工艺优化结果显示,碱溶酸沉法为较优工艺,进一步优化后得到最佳工艺参数为料液比1:40、NaOH浓度2%、超声提取时间20 min,该条件下得到的提取物中鞣花酸含量为47.38%,纯度为94.22%。扫描电镜观察表明,鞣花酸提取物粉末中有大量柱状结晶,推测为鞣花酸分子平面聚合形成。结论:余甘子鲜汁沉淀物中鞣花酸天然丰度高,可作为鞣花酸天然来源途径之一。所得到的鞣花酸提取纯化工艺步骤简单、可操作性强、成本较低、含量及纯度较高,适宜用于余甘子鲜汁固废物中鞣花酸的富集。     

Pain Modulation by Lignans (Phyllanthin and Hypophyllanthin) and Tannin (Corilagin) Rich Extracts of Phyllanthus amarus in Carrageenan‐induced Thermal and Mechanical Chronic Muscle Hyperalgesia

The current study was aimed at evaluating the antihyperalgesic effects of lignans (phyllanthin and hypophyllanthin) and tannin (corilagin) rich three standardized extracts of Phyllanthus amarus in a model of chronic musculoskeletal inflammatory pain. Three percent carrageenan injected in the gastrocnemius muscle produced hyperalgesia to mechanical and heat stimuli ipsilaterally, which spreads to the contralateral side within 7 to 9 days. To investigate the effects on chronic thermal and mechanical hypersensitivity, three extracts of P. amarus in three doses (100, 200, and 400 mg/kg) were administered to animals intraperitoneally from 14th day to 22nd day after intramuscular injection of carrageenan. It was observed that intraperitoneal administrations of Phyllanthus extracts showed antihyperalgesic activity, as they elevated thermal and mechanical threshold, which was supported by histopathological observations along with reduction in prostaglandin E2 (PGE2) concentration. In conclusion, we strongly suggest that the observed antihyperalgesic and antiinflammatory effects of P. amarus in current pain model are mediated via spinal or supraspinal neuronal mechanisms, mainly by inhibition of PGE2. Modulation of chronic muscular inflammation may be due to presence of phytoconstituents like phyllanthin, hypophyllanthin, and corilagin, which offers a promising means for treatment of chronic muscle pain. Copyright © 2015 John Wiley & Sons, Ltd.     

Sanguisorbae Radix Protects Against 6‐Hydroxydopamine‐induced Neurotoxicity by Regulating NADPH Oxidase and NF‐E2‐related Factor‐2/Heme Oxygenase‐1 Expressions

6‐Hydroxydopamine (6‐OHDA) produces neuronal cell damage by generating reactive oxygen species (ROS). The major mechanisms of protection against ROS‐induced stress are inhibiting expression of ROS generating genes such as NADPH oxidase (NOX) and increasing expression of endogenous antioxidant genes such as heme oxygenase‐1 (HO‐1). This study investigated whether a standardized Sanguisorbae Radix extract (SRE), a medical herb commonly used in Asian traditional medicine, has a protective effect on 6‐OHDA‐induced cell toxicity by regulating ROS in SH‐SY5Y cells. SRE at 10 and 50 µg/mL significantly reduced 6‐OHDA‐induced cell damage dose dependently in the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and by Hoechst 33342 staining. SRE increased the B‐cell lymphoma 2 (Bcl‐2)/Bcl‐2‐associated X ratio and decreased cytochrome C release and caspase‐3 activity. SRE also abolished 6‐OHDA‐induced ROS by inhibiting NOX expression and by inducing HO‐1 expression via NF‐E2‐related factor‐2 activation. Taken together, these results demonstrate that SRE has protective effects against 6‐OHDA‐induced cell death by regulating ROS in SH‐SY5Y cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Anti‐Ulcerative Colitis Activity of Compounds from Euphorbia granuleta Forssk

The aim of the present study was to evaluate the anti‐ulcerative colitis (UC) activity of the total alcohol extracts of Euphorbia granuleta Forssk. (Euphorpiaceae), isolate and identify the active compounds that could be responsible for the activity, in addition to determination of the possible mechanism of action. Six compounds were isolated and identified from this plant: three phenolic compounds (kampferol, kampferol‐3‐glucoside and kampferol‐3‐galactoside) in addition to three steroidal compounds (1‐ethoxypentacosane, heptacosan‐1‐ol and β‐sitosterol). Three compounds (heptacosan‐1‐ol, β‐sitosterol and kampferol‐3‐galactoside) were found to be responsible for the anti‐UC activity of E. granuleta extract. The anti‐UC activity of these compounds may be explained by reducing the pro‐inflammatory cytokine tumor necrosis factor‐alpha (TNF‐α), in addition to reduction of colonic malondialdehyde (MDA) contents. No side effects were reported on liver and kidney functions. The active compounds reduced both serum TNF‐α and mucosal MDA levels. Copyright © 2013 John Wiley & Sons, Ltd.     

Inhibition of Inducible Nitric Oxide Synthase and Cyclooxygenase‐2 Expression by Phenolic Compounds from Roots of Rhododendron mucronulatum

The roots of Rhododendron mucronulatum Turzaninov have been used in Oriental traditional medicine for the treatment of dysuria, fever, increase of digestive activity and tonics in China and Korea. Activity guided isolation of the roots of Rhododendron mucronulatum Turzaninov has led to the isolation of three flavonoids, one flavan 3‐ol and one proanthocyanidin. Chemical investigation of the 80% Me₂CO extract from the roots of Rhododendron mucronulatum led to the isolation and identification of five compounds: taxifolin (1), taxifolin 3‐O‐β‐d ‐glucopyranoside (2), quercetin 3‐O‐α‐l ‐arabinofuranoside (3), (‐)‐epicatechin (4), procyanidin B‐3 (5). To investigate the antioxidative and antiinflammatory effects of these compounds, their 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activities and the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in LPS‐stimulated HaCaT cells were also quantified by western blotting and their end products, nitric oxide (NO) and prostaglandin E₂ (PGE₂), respectively. Compounds (1–5) showed potent DPPH radical scavenging compared with positive controls (l ‐ascorbic acid). Also, compounds 1 and 2 dose‐dependently inhibited the expressions of inflammatory mediators, NO and PGE₂, suggesting they are promising candidates as antiinflammatory agents. Copyright © 2011 John Wiley & Sons, Ltd.     

Inhibitory Effects of (2′R)‐2′,3′‐dihydro‐2′‐(1‐hydroxy‐1‐methylethyl)‐2,6′‐bibenzofuran‐6,4′‐diol on Mushroom Tyrosinase and Melanogenesis in B16‐F10 Melanoma Cells

(2′R)‐2′,3′‐Dihydro‐2′‐(1‐hydroxy‐1‐methylethyl)‐2,6′‐bibenzofuran‐6,4′‐diol (DHMB) is a natural compound extracted from Morus notabilis. It was found that DHMB acts as a competitive inhibitor against mushroom tyrosinase with a Ki value of 14.77 μM. Docking results further indicated that it could form strong interactions with one copper ion with a distance of 2.7 Å, suggesting the mechanism of inhibition might be due to chelating copper ions in the active site. Furthermore, melanin production in B16‐F10 murine melanoma cells was significantly inhibited by DHMB in a concentration‐dependent manner without cytotoxicity. The results of western blotting also showed that DHMB decreased 3‐isobuty‐1‐methxlzanthine‐induced mature tyrosinase expression. Taken together, these findings indicated that DHMB may be a new promising pigmentation‐altering agent for agriculture, cosmetic, and therapeutic applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Effects of Penta‐O‐galloyl‐β‐D‐glucose on Human Neutrophil Function: significant Down‐Regulation of L‐selectin Expression

Penta‐O‐galloyl‐β‐D‐glucose (PGG) occurrs in high concentrations in medicinal herbs such as Rhus chinensis, Paeonia suffruticosa, Acer truncatum and Terminalia chebula, which demonstrate anti‐inflammatory activity. We investigated the effect of PGG on stimulated and non‐stimulated neutrophils in processes which included reactive oxygen species generation (ROS), metalloproteinase‐9 and interleukin‐8 secretion (IL‐8), β₂ integrin (CD11b) and L‐selectin (CD62L) expression and apoptosis. In concentrations of 5 μM–20 μM, PGG demonstrated statistically significant inhibition of ROS generation, IL‐8 secretion and β₂ integrin expression in stimulated neutrophils. The inhibition of L‐selectin expression by PGG resulted in prevention in neutrophils’ endothelial attachment. The result obtained may explain the anti‐inflammatory activity of this compound and underline the contribution of PGG in the activity of PGG rich plant extracts. Copyright © 2012 John Wiley & Sons, Ltd.     

Brassinin,a phytoalexin in cruciferous vegetables,suppresses obesity‐induced inflammatory responses through the Nrf2‐HO‐1 signaling pathway in an adipocyte‐macrophage co‐culture system

The aim of this study was to investigate the effect of brassinin (BR), a phytoalexin found in plants belonging to the Brassicaceae family, on the obesity‐induced inflammatory response and its molecular mechanism in co‐culture of 3T3‐L1 adipocytes and RAW264.7 macrophages. BR effectively suppressed lipid accumulation by down‐regulating the expression of adipogenic factors, which in turn, were regulated by early adipogenic factors such as CCAAT‐enhancer‐binding protein‐β and Kruppel‐like factor 2. Production of inflammatory cytokines and reactive oxygen species, induced by adipocyte‐conditioned medium, was significantly decreased in BR‐treated cells. This effect of BR was more prominent in contact co‐culture of adipocytes and macrophages with a 90% and 34% reduction in IL‐6 and MCP‐1 levels, respectively. BR also restored adiponectin expression, which was significantly reduced by culturing adipocytes in macrophage‐conditioned medium. In the transwell system, BR increased the protein levels of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) and its target molecule, hemoxygenase‐1 (HO‐1), by 55%–93% and 45%–48%, respectively, and also increased Nrf2 translocation into the nucleus. However, knockdown of Nrf2 or HO‐1 in RAW264.7 cells restored this BR‐mediated inhibition of IL‐6 and MCP‐1 production. These results indicated that BR inhibited obesity‐induced inflammation via the Nrf2‐HO‐1 pathway.