Strong Specific Inhibition of UDP‐glucuronosyltransferase 2B7 by Atractylenolide I and III

Drug‐metabolizing enzymes inhibition‐based drug–drug interaction remains to be the key limiting factor for the research and development of efficient herbal components to become clinical drugs. The present study aims to determine the inhibition of uridine 5′‐diphospho‐glucuronosyltransferases (UGTs) isoforms by two important efficient herbal ingredients isolated from Atractylodes macrocephala Koidz, atractylenolide I and III. In vitro recombinant UGTs‐catalysed glucuronidation of 4‐methylumbelliferone was used to determine the inhibition capability and kinetics of atractylenolide I and III towards UGT2B7, and in silico docking method was employed to explain the possible mechanism. Atractylenolide I and III exhibited specific inhibition towards UGT2B7, with negligible influence towards other UGT isoforms. Atractylenolide I exerted stronger inhibition potential than atractylenolide III towards UGT2B7, which is attributed to the different hydrogen bonds and hydrophobic interactions. Inhibition kinetic analysis was performed for the inhibition of atractylenolide I towards UGT2B7. Inhibition kinetic determination showed that atractylenolide I competitively inhibited UGT2B7, and inhibition kinetic parameter (Ki) was calculated to be 6.4 μM. In combination of the maximum plasma concentration of atractylenolide I after oral administration of 50 mg/kg atractylenolide I, the area under the plasma concentration‐time curve ration AUC_i/AUC was calculated to be 1.17, indicating the highly possible drug–drug interaction between atractylenolide I and drugs mainly undergoing UGT2B7‐catalysed metabolism. Copyright © 2015 John Wiley & Sons, Ltd.     

Evaluation of nitric oxide scavenging activity, in vitro and ex vivo, of selected medicinal plants traditionally used in inflammatory diseases

Steroidal and non-steroidal antiinflammatory drugs, despite their various side effects, are in great demand worldwide. Alternatively, herbal formulations provide relief to a large percentage of the population suffering from inflammatory diseases. Therefore, such practices need to be rationalized through a mechanistic approach. Thus, four traditional medicinal plants, namely Ventilago madraspatana Gaertn., Rubia cordifolia Linn., Lantana camara Linn. and Morinda citrifolia Linn. were selected for a study on the inhibition of nitric oxide (NO*), a key mediator in the phenomenon of inflammation, signifying the presence of effective antiinflammatory constituents therein. Plant samples were extracted with different solvents for evaluation of their inhibitory activity on NO* produced in vitro from sodium nitroprusside, and in LPS-activated murine peritoneal macrophages, ex vivo. Further, the inhibition of NO* synthesis was correlated with the reduction of iNOS protein expression through Western blot. Notable NO* scavenging activity was exhibited in vitro by some extracts of V. madraspatana, R. cordifolia and L. camara (IC(50) < 0.2 mg/mL). Most of them showed marked inhibition (60%-80%), ex vivo, at a dose of 80 microg/mL without appreciable cytotoxic effect on the cultured macrophages. Immunoblot analysis confirmed that the modulatory effect of the samples had occurred through suppression of iNOS protein.     

白术内酯I对免疫性肝损伤的保护作用

目的:研究白术内酯Ⅰ对卡介苗(BCG)联合脂多糖(LPS)诱导的免疫性肝损伤的保护作用,并探讨其可能的机制。方法:昆明种雄性小鼠随机分为正常对照组、模型组、阳性对照药联苯双酯组和白术内酯Ⅰ低、中、高剂量组(60,120,240mg.kg-1),每组12只。BCG联合LPS诱导免疫性肝损伤,比较各组小鼠的肝脏、脾脏系数,检测小鼠血清丙氨酸氨基转移酶(ALT)和门冬酸氨基转移酶(AST)的含量、肝匀浆液中丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-px)的含量以及血浆和肝组织中肿瘤坏死因子α(TNF-α),NO,诱导型一氧化氮合酶(iNOS)含量,HE染色切片观察肝组织病理学变化。结果:白术内酯Ⅰ与联苯双酯均可显著降低免疫性肝损伤小鼠增加的肝脏指数、脾脏指数(P<0.05),改善肝脏组织病理学变化和肝脏组织病理学分级,减轻BCG联合LPS所致肝损伤的炎症反应;对免疫性肝损伤中肝匀浆MDA产生和GSH-px水平有明显改善作用。结论:白术内酯I对免疫性肝损伤具有显著的保护作用,并且在实验剂量范围内呈显著的剂量依赖性。抑制NO,iNOS,TNF-α等炎症介质的释放或过强表达可能是其主要的作用机制。     

Inhibition of nitric oxide synthase expression in activated microglia and peroxynitrite scavenging activity by Opuntia ficus indica var. saboten

Activated microglia by neuronal injury or inflammatory stimulation overproduce nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) such as superoxide anion, resulting in neurodegenerative diseases. The toxic peroxynitrite (ONOO-), the reaction product of NO and superoxide anion further contributes to oxidative neurotoxicity. A butanol fraction obtained from 50% ethanol extracts of Opuntia ficus indica var. saboten (Cactaceae) stem (SK OFB901) and its hydrolysis product (SK OFB901H) inhibited the production of NO in LPS-activated microglia in a dose dependent manner (IC50 15.9, 4.2 microg/mL, respectively). They also suppressed the expression of protein and mRNA of iNOS in LPS-activated microglial cells at higher than 30 microg/mL as observed by western blot analysis and RT-PCR experiment. They also inhibited the degradation of I-kappaB-alpha in activated microglia. Moreover, they showed strong activity of peroxynitrite scavenging in a cell free bioassay system. These results imply that Opuntia ficus indica may have neuroprotective activity through the inhibition of NO production by activated microglial cells and peroxynitrite scavenging activity.     

Immunomodulatory effect of Glossogyne tenuifolia in murine peritoneal macrophages and splenocytes

Glossogyne tenuifolia Cass., a medicinal plant native to Taiwan, is traditionally used as an anti-inflammatory remedy. Oleanolic acid and luteolin-7-glucoside have been previously identified as active components of Glossogyne tenuifolia in the murine macrophage-like cell line, RAW264.7. Current study investigates the effect and mechanism of the ethanol extract of Glossogyne tenuifolia (GT) and its major constituents on the release of inflammatory mediators in activated elicited murine peritoneal macrophages and splenocytes. Our results showed that GT (up to 0.15 mg/ml) inhibited the production of proinflammatory mediators, TNF-alpha, IL-1beta, IL-6, nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in LPS-activated macrophages, and IFN-gamma in PHA-activated splenocytes. GT also inhibited LPS-activated murine iNOS and COX-2 promoter activities in transiently transfected RAW264.7 cells. The major constituents, oleanolic acid and luteolin-7-glucoside, as well as its aglycone, luteolin, inhibited the release of NO, PGE(2), TNF-alpha and IL-1beta in activated peritoneal macrophages. However, only luteolin-7-glucoside and luteolin were able to reduce IFN-gamma release in PHA-stimulated splenocytes. To further investigate the possible mechanisms that interfere with LPS- and PHA-signaling, this study focused on nuclear factor-kappaB activation signaling pathways. Our results demonstrate that GT (0.075-0.15 mg/ml) treatment reduces nuclear factor-kappaB (NF-kappaB) DNA binding activity, as demonstrated by electrophoretic mobility shift assay (EMSA). Collectively, the results suggest that GT inhibits proinflammatory mediator synthesis in activated murine peritoneal macrophages and splenocytes, in part through NF-kappaB-dependent pathways.     

Anti-inflammatory activities of constituents isolated from Phyllanthus polyphyllus

Four compounds, including one benzenoid, 4-O-methylgallic acid (1), together with three arylnaphthalide lignans, namely phyllamyricin C (2), justicidin B (3) and diphyllin (4) were isolated from the whole plants of Phyllanthus polyphyllus L. (Euphorbiaceae). This was the first isolation report of compounds 1-4 from this plant species. The in vitro inhibitory effects of these compounds were evaluated on the production of nitric oxide (NO) and cytokines (tumor necrosis factor (TNF)-alpha and interleukin (IL)-12), from LPS/IFN-gamma activated murine peritoneal macrophages. The results indicated that the 50% inhibition concentration (IC(50)) values of NO production from activated peritoneal macrophages by compounds 1-4 were 100, 25, 12.5 and 50 microM, respectively. In parallel, these dilutions were approximately inhibited in a similar manner to that observed for cytokines (TNF-alpha, and IL-12) production. On the other hand, at 100 microM concentration compounds 3 and 4 showed 50% inhibition of NO production from peritoneal macrophages that had been pre-activated with LPS/IFN-gamma for 24h, whereas compounds 1 and 2 inhibited only about 20 and 10%, respectively. These results support the use of this plant for the treatment of inflammatory diseases in oriental traditional medicine.     

Inhibition of lipopolysaccharide and interferon-gamma-induced expression of inducible nitric oxide synthase and tumor necrosis factor-alpha by Lithospermi radix in mouse peritoneal macrophages

Lithospermi radix (LR, root of Lithospermum erythrorhizon Siebold. et Zuccarinii) has been used to treat various conditions, such as septic shock, eczema and burns. In this study, the effect of LR on lipopolysaccharide (LPS) and recombinant interferon-gamma (rIFN-gamma)-induced production of nitric oxide (NO) and tumor necrosis factor (TNF)-alpha were examined using mouse peritoneal macrophages. At 0.01-1 mg/ml, LR inhibited the LPS/rIFN-gamma-induced expression of inducible nitric oxide synthase (iNOS) and TNF-alpha release. To clarify the mechanism involved, the effect of LR on the activation of nuclear factor (NF)-kappaB was examined. The LPS/rIFN-gamma-induced activation of NF-kappaB was almost completely blocked by LR at 1mg/ml without cytotoxicity. These findings demonstrate that the inhibition of the LPS/rIFN-gamma-induced production of NO and TNF-alpha by LR involves the inhibition of NF-kappaB activation.     

Butanolides from Machilus thunbergii and their inhibitory activity on nitric oxide synthesis in activated macrophages

In activated macrophages the inducible form of nitric oxide synthase (iNOS) generates high amounts of the toxic mediator, nitric oxide (NO), that contributes to the circulatory failure associated with septic shock. Three butanolides were isolated from Machilus thunbergii as active principles which inhibit the production of NO in lipopolysaccharide-activated RAW 264.7 cells, and their structures were identified as litsenolide A2 (1), B1 (2) and B2 (3). They showed dose-dependent inhibition of NO syntheses and the IC(50)s were 3.36, 3.70 and 6.19 micro m, respectively. These new inhibitors of iNOS may have potential in the treatment of endotoxaemia and inflammation accompanied by the overproduction of NO.     

The production of nitric oxide and TNF-alpha in peritoneal macrophages is inhibited by Dichroa febrifuga Lour

Nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) have been suggested to play an important role in endotoxin-mediated shock and inflammation. In this study, we investigated the effect of aqueous extract of Dichroa febrifuga Lour. (Saxifragaceae) roots, a traditional antimalarial drug, on the production of NO and TNF-alpha. The aqueous extract of D. febrifuga roots (AEDF) inhibited the secretion of NO and TNF-alpha in lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma)-stimulated mouse peritoneal macrophages, without affecting cell viability. The protein level of inducible nitric oxide synthase (iNOS) in peritoneal macrophages was also decreased by AEDF. In addition, the serum level of NO was reduced by i.p. administration of AEDF. These results suggest that AEDF suppresses the endotoxin-induced inflammatory responses through inhibiting the production of NO and TNF-alpha, and could be used as an anti-inflammatory drug.     

白术内酯I大鼠在体肠吸收动力学研究

目的:研究白术内酯I在大鼠各肠段的吸收动力学特征.方法:采用大鼠在体肠循环模型和高效液相色谱法研究白术内酯I在大鼠各肠段的吸收特性.结果:白术内酯I在大鼠十二指肠、空肠、回肠和结肠的吸收速度常数没有显著性差异,加入P-糖蛋白抑制剂维拉帕米和地高辛后,都能显著提高白术内酯I的吸收速度常数.结论:白术内酯I属于高渗透性药物,其在大鼠小肠的转运机制为被动扩散,无特殊的吸收窗,P-糖蛋白介导了白术内酯I的小肠吸收.     

Nimbidin suppresses functions of macrophages and neutrophils: relevance to its antiinflammatory mechanisms

Nimbidin is a mixture of tetranortriterpenes and is the major active principle of the seed oil of Azadirachta indica A. Juss (Meliaceae) possessing potent antiinflammatory and antiarthritic activities. The present study revealed that nimbidin significantly inhibited some of the functions of macrophages and neutrophils relevant to the inflammatory response following both in vivo and in vitro exposure. Oral administration of 5-25 mg/kg nimbidin to rats for 3 consecutive days significantly inhibited the migration of macrophages to their peritoneal cavities in response to inflammatory stimuli and also inhibited phagocytosis and phorbol-12-myristate-13-acetate (PMA) stimulated respiratory burst in these cells. In vitro exposure of rat peritoneal macrophages to nimbidin also inhibited phagocytosis and PMA stimulated respiratory burst in these cells. Nimbidin also inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS) stimulated macrophages following in vitro exposure, whereas interleukin 1 (IL-1) was only weakly inhibited. Probing the mechanism of NO inhibition revealed that nimbidin ameliorated the induction of inducible NO synthase (iNOS) without any inhibition in its catalytic activity. In addition, nimbidin also attenuated degranulation in neutrophils assessed in terms of release of beta-glucuronidase, myeloperoxidase and lysozyme. The results suggest that nimbidin suppresses the functions of macrophages and neutrophils relevant to inflammation. Thus nimbidin can be valuable in treating inflammation/inflammatory diseases.     

亮菌多糖IPS-B2对小鼠腹腔巨噬细胞活性及其相关基因转录的影响

目的：观察亮菌多糖IPS-B2对巨噬细胞分泌细胞因子、一氧化氮(NO)生成以及巨噬细胞中白介素-1β(IL-1β)，白介素-6(IL-6)，肿瘤坏死因子-α(TNF-α)和NO合成关键酶诱导型一氧化氮合酶(iNOS)基因转录的影响。方法：采用ELISA和Griess法检测IPS-B2对小鼠腹腔巨噬细胞生成细胞因子IL-1β，IL-6，TNF-α和细胞毒效应分子NO的影响；采用SYBR Green Ⅰ指示的Real-time RT-PCR技术研究IPS-B2对小鼠腹腔巨噬细胞中IL-1β，IL-6，TNF-α和NO合成关键酶iNOS基因在mRNA转录水平上的作用。结果：IPS-B2能显著提高小鼠腹腔巨噬细胞培养上清液中IL-1β，IL-6，TNF-α和NO的含量，增强IL-1β，IL-6，TNF-α和NO合成关键酶iNOS基因的转录水平。结论：IPS-B2可能是通过提高巨噬细胞分泌生物活性物质，并促进此类生物活性物质的基因转录，从而实现其抗肿瘤的重要作用。     

Thai medicinal plants modulate nitric oxide and tumor necrosis factor-alpha in J774.2 mouse macrophages

Centella asiatica (CA) and Rhinacanthus nasutus (RN) are Thai medicinal plants traditionally used to treat a variety of disorders including inflammatory conditions and infections. Nitric oxide (NO) produced from activated macrophages plays a role in both inflammatory and anti-inflammatory processes. This study examined whether CA and RN modulate the production of NO and tumour necrosis factor-alpha (TNF-alpha) by J774.2 mouse macrophages. Expression of the inducible nitric oxide synthase (iNOS) and TNF-alpha genes was also analysed. With CA (water extract) NO production was increased in a dose-dependent manner. An increase also occurred when CA was administered with lipopolysaccharide (LPS), a known macrophage activator. In contrast, an ethanol extract of CA had no effect on NO, and when administered with LPS the extract suppressed production. With RN, neither water nor ethanol extracts alone had an effect on NO, although when the ethanol extract of RN was used in combination with LPS, production was increased. TNF-alpha secretion was correlated with NO production and increases were associated with an elevation in TNF-alpha mRNA. The only effect on iNOS gene expression was an inhibition with the CA ethanol extract in the presence of LPS, consistent with the reduction in NO under these conditions. These studies show that CA and RN extracts can either increase or decrease NO production by macrophages and that these effects are predominantly mediated through an effect on TNF-alpha expression. These data contribute to a better mechanistic understanding of the medicinal properties of these Thai plants.     

Screening of Chinese herbal drug extracts for inhibitory activity on nitric oxide production and identification of an active compound of Zanthoxylum bungeanum

Sixty-eight water and methanol extracts from 34 Chinese herbal drugs, most of which are used for inflammatory diseases, were screened for their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated J774.1 macrophages and in LPS/interferon (IFN)-gamma-stimulated mouse peritoneal exudate macrophages. Among the extracts, methanol extracts of Myristica fragrans, Plantago asiatica, Rubia cordifolia, and Zanthoxylum bungeanum showed significant inhibition in J774.1 macrophages, while in mouse peritoneal exudate macrophages, water extracts of Ru. cordifolia and Scutellaria baicalensis and methanol extracts of Angelica megaphylla, My. fragrans, and Z. bungeanum inhibited the NO production. Among them, inhibition of water extract of Sc. baicalensis was found to be mainly due to direct scavenging of NO radicals, through an examination of its scavenging activity on PAPA NONOate-generated NO radicals, while water extract of Ru. cordifolia and methanol extracts of An. megaphylla, My. fragrans, P. asiatica, and Z. bungeanum showed inhibition on iNOS mRNA expression. At last, an inhibitory compound on iNOS mRNA expression was isolated from a methanol extract of Z. bungeanum and identified as 4-O-beta-D-glucopyranosyldihydroferulic acid by NMR spectral analyses and chemical synthesis.     

Production of nitric oxide in mouse peritoneal macrophages after priming with interferon-gamma by the stem of Sinomenium acutum.

The present study demonstrates that the aqueous extract of Sinomenium acutum stem (SSAE) produces nitric oxide (NO) upon treatment with recombinant interferon gamma (rIFN-gamma) in mouse peritoneal macrophages. Apparently SSAE has no effect on NO production by itself. This production is dependent on L-arginine and can be inhibited by the L-arginine analogue N(G)-monomethyl-L-arginine. The increased production of NO from rIFN-gamma plus SSAE-stimulated cells was decreased by the treatment of protein kinase C inhibitor. Tumor necrosis factor-alpha (TNF-alpha) has been shown to stimulate the oxidative metabolism of L-arginine to produce NO. Mouse peritoneal macrophages secrete high levels of TNF-alpha after incubation with rIFN-gamma plus SSAE. In addition, SSAE-induced NO production is progressively inhibited by anti-murine TNF-alpha neutralizing antibody. These results show that the capacity of SSAE to increase NO production from rIFN-gamma-primed mouse peritoneal macrophages is the result of SSAE-induced TNF-alpha secretion.     

Nitric oxide and tumor necrosis factor-alpha production by Ixeris dentata in mouse peritoneal macrophages

Using mouse peritoneal macrophages, we have examined the mechanism by which Ixeris dentata (IXD) regulates nitric oxide (NO) production. When IXD was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production. However, IXD had no effect on NO production by itself. The increased production of NO from rIFN-gamma plus IXD-stimulated cells was almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kappaB). Furthermore, treatment with IXD alone or rIFN-gamma plus IXD in peritoneal macrophages caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) production. PDTC decreased TNF-alpha production induced by IXD significantly. These findings demonstrate that IXD increases the production of NO and TNF-alpha by rIFN-gamma-primed macrophages and suggest that NF-kappaB plays a critical role in mediating these effects of IXD.     

Polygonum tinctorium extract suppresses nitric oxide production by activated macrophages through inhibiting inducible nitric oxide synthase expression

Despite its beneficial role in host defense mechanisms, excessive nitric oxide (NO) production by activated macrophages has been implicated in several inflammatory diseases. To clarify the mechanisms of anti-inflammatory activities of Polygonum tinctorium, we evaluated whether extracts of P. tinctorium could modulate the production of NO by activated macrophages. An AcOEt extract of P. tinctorium markedly inhibited NO synthesis by interferon-gamma (IFN-gamma)/lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages and the macrophage-like cell line RAW 264.7 in a dose-dependent manner. Inhibition of NO synthesis was achieved by reducing inducible NO synthase (iNOS) expression at protein and mRNA levels. However, the AcOEt extract of P. tinctorium failed to inhibit NO synthesis when iNOS was already expressed following stimulation with IFN-gamma and LPS. The AcOEt extract also exhibited inhibitory activity on iNOS expression in human lung epithelial A549 cells stimulated with a combination of IFN-gamma, TNF-alpha and IL-1 beta without affecting the expression of constitutive isoforms of NOS. Furthermore, in vivo injection of the AcOEt extract of P. tinctorium into LPS-treated mice significantly reduced NO synthesis by peritoneal exudate cells under ex vivo conditions. These results suggest that P. tinctorium extract may be a potential therapeutic modulator of NO synthesis in various pathological conditions.     

Inhibitory activity of plant extracts on nitric oxide synthesis in LPS-activated macrophages

Nitric oxide (NO) produced in large amounts by inducible nitric oxide synthase (iNOS) is known to be responsible for the vasodilation and hypotension observed in septic shock and inflammation. Inhibitors of iNOS, thus, may be useful candidates for the treatment of inflammatory diseases accompanied by overproduction of NO. We prepared alcoholic extracts of woody plants and screened the inhibitory activity of NO production in lipopolysaccharide (LPS)-activated macrophages after the treatment of these extracts. Among 83 kinds of plant extracts, 23 kinds of extracts showed potent inhibitory activity of NO production above 60% at the concentration of 80 micro g/ml. Some of potent extracts showed dose dependent inhibition of NO production of LPS-activated macrophages at the concentration of 80, 40, 20 micro g/ml. Especially, Artemisia iwayomogi, Machilus thunbergii, Populus davidiana and Populus maximowiczii showed the most potent inhibition (above 70%) at the concentration of 40 micro g/ml. Inhibitory activity of NO production was concentrated to nonpolar solvent fractions (ethyl ether and/or ethyl acetate soluble fractions) of Artemisia iwayomogi, Machilus thunbergii and Morus bombycis. These plants are promising candidates for the study of the activity-guided purification of active compounds and would be useful for the treatment of inflammatory diseases and endotoxemia accompanying overproduction of NO.