地黄常见种质的染色体观察

地黄RehmanniaglutinosaLibosch.是一种玄参科多年生草本植物,分布于辽宁、内蒙古、陕西、山西、河北、河南、山东、江苏、安徽、浙江、湖南、湖北、四川等地。其块根入药,年需求量约1.5×107kg,属于最常用中药材。传统认为“怀庆地黄”质量优良。从明朝起,怀地黄的道地地位就已确立,其他地区栽培地黄多引自河南怀庆(现武陟、温县、博爱等县区)。怀地黄的主要特点是块大、油性足、有菊花心,至今已有1000余年的栽培历史[1]。为了探讨怀地黄优良品种的形成原因,近年对地黄种质资源进行了收集和整理,经在相同环境中栽培,发现块根膨大与否是由…     

A fraction of stem bark extract of Entada africana suppresses lipopolysaccharide-induced inflammation in RAW 264.7 cells

Ethnopharmacological relevance

Entada africana is a plant used in African traditional medicine for the treatment of stomachache, fever, liver related diseases, wound healing, cataract and dysentery.

Aims of the study

This study aimed at evaluating the anti-inflammatory activity of fractions of the stem bark extract of the plant using lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages model.

Materials and methods

The crude extract was prepared using the mixture CH₂Cl₂/MeOH (1:1, v/v) and fractionated by flash chromatography using solvents of increasing polarity to obtain five different fractions. The effects of the fractions on the cells viability were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and their inhibitory activity against LPS-induced nitric oxide (NO) production screened by Griess test. The most active fraction was further investigated for its effects on reactive oxygen species (ROS) production using flux cytometry, the expression of inducible nitric oxide synthase (iNOS), pro-and anti-inflammatory cytokines (IL1β, TNFα, IL6, IL10 and IL13) by RT-PCR, and the activity of the enzyme p38 MAPK kinase by enzyme-linked immunosorbent assay (ELISA).

Results

The fractions presented no significant effect on the viability of macrophages at 100 μg/ml after 24 h incubation. The CH₂Cl₂/MeOH 5% (Ea5) fraction was found to be the most potent in inhibiting NO production with a half inhibition concentration (IC₅₀)=18.36 μg/ml, and showed the highest inhibition percentage (89.068%) in comparison with Baicalin (63.34%), an external standard at 50 μg/ml. Ea5, as well as Baicalin significantly (P<0.05) inhibited the expression of TNFα, IL6 and IL1β mRNA, attenuated mRNA expression of inducible NO synthase in a concentration-dependent manner, stimulated the expression of anti-inflammatory cytokines (IL10 and IL13), and showed a 30% inhibition of the activity of p38 MAPK kinase.

Conclusion

The results of the present study indicate that the fraction Ea5 of Entada africana possesses most potent in vitro anti-inflammatory activity and may contain compounds useful as a therapeutic agent in the treatment of inflammatory related diseases cause by over-activation of macrophages.     

小剂量免疫抑制剂与知柏地黄丸联合治疗女性免疫性不孕的临床研究

实验组 6 8例 ,采用隔离法 +强的松阴道用药及口服 ,同时配合口服知柏地黄丸 ;对照 1组采用隔离法即避孕套避孕 ;对照 2组则采用隔离法 +强的松阴道用药。结果 :实验组治愈率达 92 .6 % ,明显高于对照 1组之 4 5 .8%及对照 2组之 76 .5 % ,疗程 (1.8± 0 .9)月 ,较对照 1组 (5 .7± 1.8)月及对照 2组 (4 .8± 1.3)月明显缩短 ,有显著性差异     

Suppression of lipopolysaccharide-induced of inducible nitric oxide synthase and cyclooxygenase-2 by Sanguis Draconis, a dragon's blood resin, in RAW 264.7 cells

Sanguis Draconis (SD) is a kind of dragon's blood resin that is obtained from Daemomorops draco (Palmae). It is used in traditional medicine and has shown anti-inflammatory activity in some diseases. In this study, we examined the effects of Sanguis Dranonis ethanol extract (SDEE) on LPS-induced inflammation using RAW 264.7 cells. Our data indicated that SDEE inhibits LPS-stimulated NO, PGE2, IL-1 beta and TNF-alpha release, and iNOS and COX-2 expression. Furthermore, SDEE suppressed the LPS-induced p65 expression of NF-kappa B, which was associated with the inhibition of I kappa B-alpha degradation. We also found that the expression of HO-1 was significantly increased in RAW 264.7 cells by SDEE. These results suggest among possibilities of anti-inflammation that SDEE inhibits the production of NO and PGE2 by the down-regulation of iNOS and COX-2 gene expression via the suppression of NF-kappaB (p65) activation. SDEE can induce HO-1 over-expression in macrophage cells, which indicates that it may possess antioxidant properties. This result means that SEDD its anti-inflammatory effects in macrophages may be through a novel mechanism that involves the action of HO-1. Thus, SD could provide a potential therapeutic approach for inflammation-associated disorders.     

超临界CO2 萃取法与水蒸气蒸馏法提取桂枝-干姜 药对的化学成分比较

目的:探讨超临界CO2萃取法和水蒸气蒸馏法提取桂枝-干姜药对有效成分的差异,阐释该药配伍前后有效成分的变化情况,以期揭示其药效物质基础。方法:通过超临界CO2萃取法与水蒸气蒸法提取桂枝、干姜、桂枝-干姜药对化学成分,通过GC-MS分析鉴定,比对该药对配伍前后成分变化,总结其变化规律。结果:超临界CO2萃取桂枝、干姜、桂枝-干姜药对萃取物的得率分别为0.69%,3.66%,1.82%,分别从中鉴定出44,57,65个成分,其占萃取物含量分别为84.22%,91.25%,90.13%;水蒸气蒸馏提取桂枝、干姜、桂枝-干姜药对挥发油的得率分别为0.25%,1.75%,1.05%,分别从中鉴定出19,50,57个成分,其占挥发油含量分别为85.35%,93.47%,93.11%。结论:桂枝-干姜配伍有促进成分溶出作用,两者配伍不是成分的简单叠加;超临界CO2萃取法的提取效率明显高于传统水蒸气蒸馏法。     

Acanthopanax senticosus inhibits nitric oxide production in murine macrophages in vitro and in vivo

Excess nitric oxide (NO) production has been implicated in inflammatory diseases. The present study investigated the inhibitory effect of the stem bark extract of Acanthopanax senticosus (A. senticosus) on NO production in murine macrophages in vitro and in vivo. In vitro exposure of RAW264.7 cells to 1, 10, 50, 100, 250, 500 and 1000 microg/mL of A. senticosus extract significantly suppressed NO production induced by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) in a dose-dependent manner. In vitro exposure of mouse resident peritoneal macrophages to 1, 10, 100 and 1000 microg/mL of A. senticosus extract significantly suppressed NO production induced by LPS and IFN-gamma in a dose-dependent manner. In vivo administration of A. senticosus extract (50, 100 and 200 mg/kg) to KM mice dose-dependently inhibited LPS and IFN-gamma induced production of NO in isolated mouse peritoneal macrophages ex vivo. Exposure to A. senticosus extract had no effect on cell viability and systemic toxicity. The results demonstrated that the stem bark extract of A. senticosus extract inhibits NO production in murine macrophages in vitro and in vivo.     

Ethanolic extract and water-soluble polysaccharide from Chaenomeles speciosa fruit modulate lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophage cells

Ethnopharmacological relevance

Chaenomeles speciosa fruits have been widely used in traditional Chinese medicine for treatment of diseases related to inflammatory reaction. This study aims to identify anti-inflammatory and immunomodulatory components of Chaenomeles speciosa fruit and unravel their potential mechanisms.

Materials and methods

Ethanolic extract and its n-hexane, chloroform, ethyl acetate and n-butanol fractions, as well as water-soluble polysaccharide, were prepared from dry fruits of Chaenomeles speciosa. The mouse macrophage-like RAW264.7 cells were induced by lipopolysaccharide (LPS) and used as an inflammatory cell model. Production of nitric oxide in the cells was determined by the Griess assay, and cell viability was tested by the MTT method. Cellular apoptosis was evaluated by fluorescence-activated cell sorting. Relative quantification of inflammation-related genes was analyzed by real-time PCR.

Results

LPS-induced production of nitric oxide in RAW264.7 cells was significantly inhibited by the ethyl acetate fraction (EAF) at 200–800 μg/ml, while Chaenomeles speciosa polysaccharide (CPS) promoted nitric oxide production at 250–750 μg/ml either alone or in an additive fashion with LPS. Both EAF and CPS did not provoke noticeable cytotoxicity and apoptosis at the above effective concentrations. EAF significantly reduced LPS-induced upshift of iNOS mRNA level but showed no significant effect on the induction of IFN-γ and G-CSF, while CPS reduced the gene induction of TNF-α, IFN-γ and G-CSF by LPS.

Conclusions

EAF was able to inhibit nitric oxide production by reducing LPS-induced upshift of iNOS mRNA level. CPS was an activator of nitric oxide production through cytokines such as TNF-α, IFN-γ and G-CSF. These results demonstrate the therapeutic effects of both ethanolic and aqueous extracts of Chaenomeles speciosa fruit, a traditional edible medicine used in health maintenance and disease treatment.     

Stimulation of lymphocyte proliferation and inhibition of nitric oxide production by aqueous Urtica dioica extract

The immunomodulatory and antiinflammatory activities of aqueous Urtica dioica extract were investigated for their effect on the mitogenic response of murine splenocytes and nitric oxide production by murine peritoneal macrophages in vitro. It was found that this extract stimulated the proliferation of T-lymphocytes and suppressed NO production in lipopolysaccharide-stimulated macrophages without affecting cell viability.     

Inhibition of nitric oxide synthesis in mouse macrophage cells by feverfew supercritical extract

Feverfew is the most commonly used medicinal herb against migraine headache. The antimigraine mechanism of feverfew supercritical extract was investigated in vitro using the mouse macrophage cell line (RAW 264.7). Mouse macrophage cells were treated with lipopolysaccharide in the presence and absence of feverfew extracts. Inhibition of lipopolysaccharide-induced nitric oxide and TNF-α synthesis were quantified by ELISA. The mRNA and protein expression of iNOS and eNOS genes were analysed by RT-PCR and western blot analysis, respectively. The feverfew extract inhibited both nitric oxide (NO) and TNF-α production in a dose-dependent manner with complete inhibition of NO occurring at 5 μg/mL of feverfew extract. Both eNOS and iNOS mRNA levels were unchanged with the feverfew treatment. However, eNOS and iNOS proteins were significantly down-regulated by the feverfew extract. Feverfew inhibition of NO is due to the down-regulation of both eNOS and iNOS enzymes at the translational and/or post-translational level.     

The in vitro effect of aqueous extract of Nigella sativa seeds on nitric oxide production

The in vitro effect of aqueous extract of Nigella sativa seeds on nitric oxide (NO) production by murine macrophages was studied. Murine peritoneal macrophages were pre-incubated with the extract and then activated with Escherichia coli lipopolysaccharride. NO production was measured after 24 hours by spectrophotometry. The plant extract caused a dose-dependent decrease in NO production. Dialyzed preparation of the extract did not affect NO production. However, the boiled fraction of the extract resulted in a dose-dependent inhibition of NO apparently comparable to that of the whole extract. These results indicate that the aqueous extract of N. sativa seeds exhibits an inhibitory effect on nitric oxide production by murine macrophages and the active component(s) is/are non-protein in nature. In view of the fact that nitric oxide is a pro-inflammatory mediator, this study validates the traditional use of the Nigella sativa seeds for the treatment of rheumatism.     

Phellinus linteus inhibits inflammatory mediators by suppressing redox-based NF-kappaB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophage

The mushroom Phellinus linteus has been known to exhibit potent biological activity. In contrast to the immuno-potentiating properties of Phellinus linteus, the anti-inflammatory properties of Phellinus linteus have rarely been investigated. Recently, ethanol extract and n-BuOH fractions from Phellinus linteus were deemed most effective in anti-inflammatory activity in RAW 264.7 macrophages. The regulatory mechanisms of Phellinus linteus butanol fractions (PLBF) on the pharmacological and biochemical actions of macrophages involved in inflammation have not been clearly defined yet. In the present study, we tested the role of PLBF on anti-inflammation patterns in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. To investigate the mechanism by which PLBF inhibits NO and PGE2 production as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, we examined the activation of IkappaB and MAPKs in LPS-activated macrophages. PLBF clearly inhibited nuclear translocation of NF-kappaB p65 subunits, which correlated with PLBF's inhibitory effects on IkappaBalpha phosphorylation and degradation. PLBF also suppressed the activation of mitogen-activated protein (MAP) kinases including p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Furthermore, macrophages stimulated with LPS generated ROS via activation of membrane-bound NADPH oxidase, and ROS played an important role in the activation of nuclear factor-kappaB (NF-kappaB) and MAPKs. We demonstrated that PLBF directly blocked intracellular accumulation of reactive oxygen species in RAW 264.7 cells stimulated with LPS much as the NADPH oxidase inhibitors, diphenylene iodonium, and antioxidant pyrrolidine dithiocarbamate did. The suppression of NADPH oxidase also inhibited NO production and iNOS protein expression. Cumulatively, these results suggest that PLBF inhibits the production of NO and PGE2 through the down-regulation of iNOS and COX-2 gene expression via ROS-based NF-kappaB and MAPKs activation. Thus, PLBF may provide a potential therapeutic approach for inflammation-associated disorders.     

Echinacea increases arginase activity and has anti-inflammatory properties in RAW 264.7 macrophage cells,indicative of alternative macrophage activation

Ethnopharmacological relevance

The genus Echinacea is a popular herbal immunomodulator. Recent reports indicate that Echinacea products inhibit nitric oxide (NO) production in activated macrophages.

Aim of the study

In the present study we determined the inhibitory effects of alcohol extracts and individual fractions of alcohol extracts of Echinacea on NO production, and explored the mechanism underlying the pharmacological anti-inflammatory activity.

Materials and methods

Alcohol extracts of three medicinal Echinacea species, Echinacea angustifolia, Echinacea pallida and Echinacea purpurea, were prepared using Soxhlet apparatus and fractionated using HPLC. NO production by LPS activated RAW 264.7 macrophage cells was measured using a Griess reagent and iNOS detected using immunoblotting. In addition, effects on arginase activity were measured in RAW 264.7 cells stimulated with 8-bromo-cAMP +/− LPS.

Results

Alcohol extracts of all three Echinacea species significantly inhibited NO production by lipopolysaccharide (LPS)-activated the RAW 264.7 macrophage cell line; among them Echinacea pallida was the most active. The Echinacea-mediated decrease in NO production was unlikely due to a direct scavenging of NO because the extracts did not directly inhibit NO released from an NO donor, sodium nitroprusside. An immunoblotting assay demonstrated that the extract of Echinacea pallida inhibited inducible nitric oxide synthase (iNOS) protein expression in LPS-treated macrophages. The enzymes iNOS and arginase metabolize a common substrate, l-arginine, but produce distinct biological effects. While iNOS is involved in inflammatory response and host defense, arginase participates actively in anti-inflammatory activation. Arginase activity of RAW 264.7 cells stimulated with 8-bromo-cAMP was significantly increased by alcohol extracts of all three Echinacea species. The polar fraction containing caffeic acid derivatives enhanced arginase activity, while the lipophilic fraction containing alkamides exhibited a potential of inhibiting NO production and iNOS expression.

Conclusions

These results suggest that the anti-inflammatory activity of Echinacea might be due to multiple active metabolites, which work together to switch macrophage activation from classical activation towards alternative activation.     

Suppressive effects of methoxyflavonoids isolated from Kaempferia parviflora on inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells

Ethnopharmacological relevance

The rhizomes of Kaempferia parviflora Wall. ex Baker have been traditionally used in Thailand to treat abscesses, gout, and peptic ulcers.

Aim

Previously, we reported that the chloroform fraction of a Kaempferia parviflora extract had an inhibitory effect on rat paw-edema. In the present study, we isolated the constituents of this fraction and investigated the anti-inflammatory mechanism against nitric oxide (NO) production, tumor necrosis factor-α (TNF-α) and the expression of inducible nitric oxide synthase (iNOS) as well as phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated c-Jun N-terminal kinase (p-JNK). In addition, effects of trimethylapigenin (4) on the enzyme activities of protein kinases possibly leading to iNOS expression were examined to clarify the targets.

Materials and methods

The chloroform fraction was isolated using silica gel column chromatography and HPLC. Isolated compounds were tested against NO and TNF-α using RAW264.7 cells. Cytotoxicity and iNOS, p-ERK and p-JNK expression were also examined.

Results

Three active components, 5,7-dimethoxyflavone (2), trimethylapigenin (4), and tetramethylluteolin (5), markedly inhibited the production of NO in lipopolysaccharide (LPS)-activated RAW264.7 cells. Compounds 2, 4, and 5 moderately inhibited production of TNF-α. Compounds 2, 4, and 5 strongly inhibited expression of iNOS mRNA and iNOS protein in a dose-dependent manner, but did not inhibit p-ERK or p-JNK protein expression. The most active compound, 4, did not inhibit the enzyme activity of inhibitor of κB kinases or mitogen-activated protein kinases, but inhibited that of spleen tyrosine kinase (SYK).

Conclusion

The mechanism responsible for the anti-inflammatory activity of methoxyflavonoids from the chloroform fraction of the rhizomes of Kaempferia parviflora is mainly the inhibition of iNOS expression, and the inhibition of SYK by 4 may be involved in the suppression of LPS-induced signaling in macrophages.     

高良姜超临界萃取物对应激性溃疡大鼠溃疡形成及表皮生长因子和白介素-2水平的影响

目的:探讨高良姜超临界萃取物(CLJ)对应激性胃溃疡模型大鼠溃疡形成及血清表皮生长因子(EGF)和白介素-2(IL-2)的影响.方法:以束缚-水浸应激性胃溃疡大鼠为对象,观察GLJ对大鼠溃疡指数及血清EGF和IL-2水平的影响.结果:GLJ能降低束缚-水浸应激型溃疡大鼠的溃疡指数,并可使该模型大鼠血清IL-2和EGF水平明显回升,接近正常水平.结论:GLJ能降低束缚-水浸应激性胃溃疡的形成,对此模型EGF、IL-2水平有一定的调节作用,可能是其具有黏膜保护作用的机制之一.     

Ability of certain plant extracts traditionally used to treat ciguatera fish poisoning to inhibit nitric oxide production in RAW 264.7 macrophages

Aim of the study

Ciguatera fish poisoning (CFP) is an intertropical ichthyosarcotoxism that manifests in complex assortment of symptoms in humans. Ciguatoxins (CTXs), issued from Gambierdicus spp., are causative agents of this intoxication. We have recently demonstrated that a Pacific CTX (P-CTX-1B) strongly modulated iNOS expression, leading to overproduction of nitric oxide (NO) in RAW 264.7 murine macrophage cells. NO produced in large amounts is involved in a wide range of pathophysiological processes. Many traditional remedies are commonly used in the Pacific against CFP. In this context, bioassay-guided screening was carried out to study NO inhibiting capacity of 28 selected plant extracts.

Materials and methods

We prepared aqueous extracts of plants used in New Caledonia in the treatment of CFP and screened their NO inhibitory activity in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages.

Results

Among 28 plants tested, Euphorbia hirta (Euphorbiaceae), Syzygium malaccense (Myrtaceae), Schinus terebenthifolius (Anacardiaceae), Punica granatum (Punicaceae), Cerbera manghas (Apocynaceae), Vitex trifolia (Labiateae) and Ximenia americana (Olacaceae) showed inhibitory activity, validating their use as traditional remedies in CFP, and the potential for use in the treatment of conditions accompanied by NO overproduction.

Conclusion

These plants are promising candidates for further screening of their active compounds through activity-guided fractionation.     

Inhibitory effects of methanol extract of Cyperus rotundus rhizomes on nitric oxide and superoxide productions by murine macrophage cell line, RAW 264.7 cells

The rhizomes of Cyperus rotundus (C. rotundus) have been used in oriental traditional medicines for the treatment of stomach and bowel disorders, and inflammatory diseases. Nitric oxide (NO) and superoxide (O2-) are important mediators in the pathogenesis of inflammatory diseases. This study was undertaken to address whether the metanol (MeOH) extract of rhizomes of C. rotundus could modulate NO and O2- productions by murine macrophage cell line, RAW 264.7 cells. The MeOH extract of rhizomes of C. rotundus showed the inhibition of NO production in a dose-dependent manner by RAW 264.7 cells stimulated with interferon-gamma plus lipopolysaccharide. The inhibition of NO production by the extract was due to the suppression of iNOS protein, as well as iNOS mRNA expression, determined by Western and Northern blotting analyses, respectively. In addition, the MeOH extract suppressed the production of O2- by phorbol ester-stimulated RAW 264.7 cells in dose- and time-dependent manners. Collectively, these results suggest that the MeOH extract of rhizomes of C. rotundus could be developed as anti-inflammatory candidate for the treatment of inflammatory diseases mediated by overproduction of NO and O2-.     

Increase of cellular recruitment, phagocytosis ability and nitric oxide production induced by hydroalcoholic extract from Chenopodium ambrosioides leaves

The leaves and the oil from the seeds of Chenopodium ambrosioides L. (Chenopodiaceae), a plant known in Brazil as 'mastruz', have been used by native people to treat parasitic diseases. Experimentally it was shown that Chenopodium ambrosioides inhibits the Ehrlich tumor growth, what could be due to an immunomodulatory effect of this product. The aim of this study was to investigate the effect of hydroalcoholic crude extract (HCE) from leaves of Chenopodium ambrosioides on macrophage activity and on lymphoid organs cellularity. C3H/HePas mice received the HCE (5mg/kg) by intraperitoneal via and were sacrificed 2 days later. HCE treatment did not alter the cell number in bone marrow, but it increased the cell number in peritoneal cavity, spleen and lymph node. The spreading and phagocytosis activity, the PMA-induced hydrogen peroxide (H(2)O(2)) release and the nitric oxide (NO) production were also increased when compared to control group. Similar results were obtained with concanavalin A (Con A), used as a positive control, with exception of the NO production that was only detected in HCE-derived macrophages. The in vitro treatment with HCE induced a dose-dependent NO production by resident macrophages, but did not enhance the NO production by HCE-derived macrophage, which however, was enhanced by Con A, suggesting that HCE and Con A induce NO production by different routes. In conclusion, HCE-treatment was able to increase the macrophages activity and also the cellular recruitment to secondary lymphoid organs, what could explain the previously related anti-tumor activity of Chenopodium ambrosioides.