Effects of compounds from Kaempferia parviflora on nitric oxide, prostaglandin E2 and tumor necrosis factor-alpha productions in RAW264.7 macrophage cells

Kaempferia parviflora Wall. ex Baker, is one of the plants in the Zingiberaceae family, locally known in Thai as kra-chai-dam. The rhizome of this plant has been used for treatment of gout, apthous ulcer and abscesses. Since K. parviflora rhizomes have long been used for treatment of inflammation and possessed marked nitric oxide (NO) inhibitory activity (IC(50)=7.8microg/ml), we thus investigated the inhibitory activity of compounds isolated from this plant against lipopolysaccharide (LPS)-induced NO release in RAW264.7 cells. From bioassay-guided fractionation of K. parviflora, seven methoxyflavones were isolated from the hexane fraction and were tested for their anti-inflammatory effects. Among the isolated compounds, compound 5 (5-hydroxy-3,7,3',4'-tetramethoxyflavone) exhibited the highest activity against NO release with an IC(50) value of 16.1microM, followed by 4 (IC(50)=24.5microM) and 3 (IC(50)=30.6microM). Compound 5 was also tested on LPS-induced prostaglandin E(2) (PGE(2)) and tumor necrosis factor-alpha (TNF-alpha) releases from RAW264.7 cells. It was revealed that 5 showed appreciable inhibitory effect on PGE(2) release (IC(50)=16.3microM), but inactive on TNF-alpha (IC(50)>100microM). These findings may support the use in Thai traditional medicine of K. parviflora for treatment of inflammatory-related diseases through the inhibition of NO and PGE(2) releases but partly due to that of TNF-alpha.     

Effect of an extract of Andrographis paniculata leaves on inflammatory and allergic mediators in vitro

Aim of study

Andrographis paniculata has been known to possess widespread traditional application in the treatment of allergy and inflammatory diseases. In the current study, we sought to examine the effects of an extract of Andrographis paniculata leaves on inhibition of lipopolysaccharide (LPS) induced [nitric oxide (NO), prostaglandin E₂ (PGE₂), interleukin-1beta (IL-1 beta), and interleukin-6 (IL-6)] and calcimycin (A23187) induced [leukotriene B₄ (LTB₄), thromboxane B₂ (TXB₂) and histamine] mediators in diverse cell based models.

Materials and methods

Effect of an extract of Andrographis paniculata leaves (AP) was studied on inhibition of LPS induced NO, PGE₂, IL-1 beta and IL-6 in J774A.1 murine macrophages; A23187 induced LTB₄ and TXB₂ in HL-60 promyelocytic leukemic cells and histamine in RBL-2H3 rat basophilic leukemia cells.

Results and conclusion

AP illustrated significant alleviation of pro-inflammatory, inflammatory, and allergic mediators. However, no inhibition was observed against histamine release. This outcome has been summed up to deduce that AP is fairly potent in attenuating the inflammation by inhibiting pro-inflammatory (NO, IL-1 beta and IL-6), inflammatory (PGE₂ and TXB₂) and allergic (LTB₄) mediators.     

Isolation and characterization of an antibacterial biflavonoid from an African chewing stick Garcinia kola Heckel (Clusiaceae)

Ethnopharmacological relevance

The use of African chewing sticks in maintaining oral health is widely practiced in African countries. It has been reported that chewing stick users have a lower rate of dental caries and a better general oral health than non-users. It is generally thought that the beneficial effect of chewing stick is attributed to the mechanical cleansing effect and antimicrobial substances present in the stick. However, the active antimicrobial substances remain uncharacterized.

Aim of the study

To provide a scientific basis for the anti-caries effect of African chewing sticks, the authors purify an active antibacterial compound from Garcinia kola Heckel, a Nigerian chewing stick and examined the antibacterial activity of this compound against the cariogenic bacterium Streptococcus mutans.

Materials and methods

Methanol extract was prepared from Garcinia kola and was further fractionated by solvent extractions. Silica gel chromatography was used to purify the antibacterial compound from the active fraction. The identity of the purified compound was determined by NMR analysis. The antibacterial activity of the purified compound was examined by standard microbiological assays.

Results

The antibacterial activity was found in the ether fraction and the active compound was isolated and determined to be a biflavonoid named GB1. GB1 was active against Streptococcus mutans and other oral bacteria with minimum inhibitory concentration (MIC) values of 32–64 μg/ml. The basis for the antibacterial effect of GB1 was investigated using Streptococcus mutans as the target. At 256 μg/ml, GB1 exhibited some bacteriocidal activity against Streptococcus mutans and induced the aggregation of Streptococcus mutans. GB1 has no apparent effects on protein synthesis and DNA synthesis but inhibited glucose uptake and utilization by Streptococcus mutans suggesting that GB1 exerts its antibacterial effect by inhibiting metabolism. GB1 also inhibited the formation of water-insoluble glucan by the extracellular glucosyltransferases from Streptococcus mutans in a dose-dependent manner. Streptococcus mutans did not develop resistance to GB1 upon subculturing in the presence of sub-MIC level of the biflavonoid.

Conclusion

The antibacterial effect and glucan synthesis-inhibition property of this biflavonoid may account for some of the beneficial effects reported in the chewing stick users.     

Anti-inflammatory mechanism of Kaempferia parviflora in murine macrophage cells (RAW 264.7) and in experimental animals

Ethnopharmacological relevance

The rhizomes of Kaempferia parviflora Wall. ex Baker have been used in Thailand for treatment of gout, apthous ulcer, peptic ulcer and abscesses.

Aim of the study

In our previous study, the crude ethanol extract of Kaempferia parviflora and its compound (5, 5-hydroxy-3,7,3′,4′-tetramethoxyflavone), was reported to show nitric oxide (NO) inhibition in RAW 264.7 cells. The present study is thus investigated the anti-inflammatory mechanism of Kaempferia parviflora extract and compound 5 against inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expressions.

Materials and methods

The extract of Kaempferia parviflora and its compound were tested against NO and prostaglandin E₂ (PGE₂) releases using RAW264.7 cells as well as studied on anti-inflammatory activity in carrageenan-induced rat paw edema and acute toxicity in mice.

Results

The results revealed that the ethanol extract of Kaempferia parviflora markedly inhibited PGE₂ release with an IC₅₀ value of 9.2 μg/ml. This plant extract and compound 5 also suppressed mRNA expression of iNOS in dose-dependent manners, whereas COX-2 mRNA expression was partly affected. According to the in vivo study, chloroform and hexane fractions greater decreased rat paw edema than ethanol, ethyl acetate and water fractions.

Conclusion

The mechanisms for anti-inflammatory activity of Kaempferia parviflora and compound 5 are mainly due to the inhibition of iNOS mRNA expression but partly through that of COX-2 mRNA.     

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.     

Non-toxic melanin production inhibitors from Garcinia livingstonei (Clusiaceae)

Ethnopharmacological relevance

The stem bark of Garcinia livingstonei is used traditionally as a skin lightening agent.

Aim of the study

To isolate and identify compounds responsible for the observed skin lightening activity of Garcinia livingstonei and to evaluate their cytotoxicity.

Materials and methods

Constituents of the stem bark and fruits of Garcinia livingstonei were isolated using chromatographic techniques and structures were determined using 1D and 2D NMR and MS analysis. MeWo cells were used to evaluate the cytotoxicity and impact on melanin levels of extracts and compounds isolated, in vitro.

Results

Twelve known compounds, morelloflavone (1), morelloflavone-7″-sulphate (2), guttiferone A (3), sargaol (4), isojacareubin (5), 6-deoxyisojacareubin (6) and in addition to the common triterpenoids, betulin, betulin aldehyde, lupeol, lupenone, euphol and stigmasterol were isolated in this investigation. Morelloflavone, morelloflavone-7″-sulphate and sargaol, were found to be considerably less cytotoxic and more effective as skin lightening agents than hydroquinone.

Conclusions

A range of compounds was isolated from the stem bark and fruit of Garcinia livingstonei. Although the bark extract contained the cytotoxic guttiferone A, it was found to be less toxic than hydroquinone, and morelloflavone, the 7″-sulphate derivative and sargaol show potential for development as depigmentation/skin lightening agents.     

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.     

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.     

Immunomodulatory properties of Xylaria nigripes in peritoneal macrophage cells of Balb/c mice

Ethnopharmacological relevance

Wu Ling Shen, a folklore name for Xylaria nigripes (XN), is a high value medicinal fungus used in traditional Chinese medicine.

Aim of study

The present study aimed to examine the immunomodulatory properties of aqueous (XN-H) and ethanol (XN-E) XN extracts in lipopolysaccharide (LPS)-induced peritoneal macrophage cells of Balb/c mice.

Materials and methods

After treating the macrophage cells with LPS (1 μg/ml) and different XN extracts, the immunomodulatory properties were determined by the responses of inflammatory mediators, namely nitrite oxide (NO), prostaglandin E2 (PGE₂) and cytokine (IL-1β, IL-6, TNF-α and IFN-γ) production, iNOS, COX-2 and IκB-α expression, and NF-κB activation.

Results

Results showed that treatment of macrophages with 5-30 μg/ml of XN-H or XN-E plus 1 μg/ml LPS exhibited no cytotoxic effect on cell viability. At these concentrations, although both XN-H and XN-E showed a dose-dependent inhibitory effect on NO, PGE₂, IL-1β, IL-6, TNF-α and IFN-γ production in LPS-stimulated macrophages, a greater potency was noted in the XN-H treated group. RT-PCR assay also showed that XN-H possessed a greater inhibition than XN-E on iNOS and COX-2 RNA expression. Furthermore, XN-H also showed a significant stronger suppression than XN-E on the LPS-induced IκB-α phosphorylation and NF-κB activation. XN-E showed a higher total flavonoid and phenol contents but a lower β-glucan content than XN-H.

Conclusion

Taken together, these results conclude that XN-H possesses a stronger anti-inflammatory activity than XN-E, and its mechanism of action could be mediated by inhibiting iNOS and COX-2 expression via the NF-κB signaling pathway, and these activities could be contributed by the β-glucan content.     

Effect of a Garcinia gardneriana (Planchon and Triana) Zappi hydroalcoholic extract on melanogenesis in B16F10 melanoma cells

Ethnopharmacology relevance

Garcinia gardneriana (Planchon and Triana) Zappi (Clusiaceae) is popularly called “bacopari” in southern Brazil. The leaves of this plant are traditionally used to treat skin disorders.

Aim of study

This study evaluated the effects of a hydroalcoholic extract of Garcinia gardneriana leaves (HEGG) on B16F10 murine melanoma cells in order to search for new depigmenting agents.

Materials and methods

The effects of HEGG were assessed in melanin content assays in B16F10 melanoma cells compared with the reference drug kojic acid (500 mM). Melanin content was measured after spontaneous melanogenesis, UVB-induced melanogenesis and melanogenesis induced by α-MSH. At the same time, cell viability assays were conducted. Intracellular and mushroom tyrosinase activity assays were employed to evaluate the effect of HEGG on tyrosinase activity.

Results

HEGG decreased the level of melanin under all three experimental conditions of melanin content evaluation without reducing cell viability. In intracellular tyrosinase assays, the enzyme's activity was reduced about 19% with extract concentrations ranging 0.1–10 µg/mL. In the mushroom tyrosinase activity assay a maximal inhibition of 35% (1000 µg/mL) was observed.

Conclusion

These results suggest that HEGG inhibition relates to its tyrosinase activity. Therefore, the hydroalcoholic extract of Garcinia gardneriana shows great potential for use as a depigmenting agent in hyperpigmentation disorders.     

Constituents isolated from Cordyceps militaris suppress enhanced inflammatory mediator's production and human cancer cell proliferation

Aim of the study

The purpose of this study is to isolate the pure compounds from the extracts of Cordyceps militaris obtained through solid-state cultivation process, and evaluate their anti-inflammatory and anticancer properties.

Materials and methods

Silica gel column chromatographic purification of Cordyceps militaris extracts resulted in the isolation of 10 pure compounds (1-10). The compounds 1-10 were examined for their growth inhibitory properties against nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-12 enhanced production from LPS/IFN-γ-stimulated macrophages. Additionally, the anti-proliferation effects of 1-10 on human cancer cell lines, colon (colon 205), prostate (PC-3), and hepatoma (HepG2) cells were also analyzed.

Results

Compound 8 displayed potent growth inhibition on NO, TNF-α and IL-12 production with an IC₅₀ value of 7.5, 6.3, and 7.6 μg/ml, respectively. A similar inhibitory trend on these inflammatory mediators was observed for 3, 7, 9 and 10 with an IC₅₀ values ranging from 10.8 to 17.2 μg/ml. On the other hand, compounds 3 and 8 were potent anti-proliferative agents with an IC₅₀ value of 35.6 and 32.6 μg/ml toward PC-3 and colon 205 cell lines, respectively. The compounds 1 and 2 showed potent anti-proliferation in PC-3 and colon 205 cells, while only 3 displayed such effect in HepG2 cells.

Conclusion

The present study provides scientific supporting information for the ethnopharmacological use of Cordyceps militaris as an anti-inflammatory and anticancer agent.     

芦荟及其复方提取物对RAW264.7细胞炎症因子的影响

目的:观察芦荟及其复方提取物对小鼠巨噬细胞炎症因子的影响.方法:RAW264.7细胞按2×105/mL稀释,100μL/孔接种入96孔板,药物质量浓度为250,125,62.5 g?L-1下采用噻唑蓝比色法(MTT法)作用4h,检测芦荟及其复方提取物对小鼠巨噬细胞的毒性作用;药物质量浓度为50,25,12.5 g?L-1下采用硝酸还原酶法作用24 h,测定小鼠巨噬细胞一氧化氮(N0)含量,并用酶联免疫吸附法(ELISA法)测定其在单纯药物及脂多糖(LPS)1 mg?L-1诱导情况下小鼠巨噬细胞肿瘤坏死因子-α(TNF-α)、白介素-10(IL-10)的含量.结果:芦荟及其复方提取物具有促进巨噬细胞释放NO的作用,并与药物浓度成正相关(P＜0.01);芦荟有促进TNF-α分泌(P＜0.01)和抑制IL-10分泌(P＜0.05)的作用,其复方在LPS诱导下则对TNF-α和IL-10都有抑制作用(P＜0.01).结论:芦荟在250～7.8 g?L-1下对臣噬细胞无细胞毒性,其复方则有一定毒性;芦荟及其复方对小鼠巨噬细胞炎症因子的释放有一定影响.     

Identification of active compounds from Caesalpinia sappan L. extracts suppressing IL-6 production in RAW 264.7 cells by PLS

Ethnopharmacological relevance

Caesalpinia sappan L. is distributed in Southeast Asia and also used as herbal medicine for the treatment of various diseases such as burning sensations, leprosy, dysentery, osteoarthritis and rheumatoid arthritis (RA). The overproduction of IL-6 plays an important role in the prognosis of RA, but the active compounds from the extracts of Caesalpinia sappan L. suppressing IL-6 production remain unknown.

Aims of the study

Identifying the main active compounds of Caesalpinia sappan L. extracts inhibiting the IL-6 production in LPS-stimulated RAW 264.7 cells by partial least squares (PLS).

Materials and methods

Sixty-four samples with different proportions of compounds were prepared from Caesalpinia sappan L. by supercritical CO₂ fluid extraction (SCFE) and refluxing. Each of 64 samples was applied to RAW 264.7 cells with LPS to evaluate whether IL-6 production by LPS is affected by addition of each sample. The IL-6 production in medium was determined by ELISA and the inhibitory activity of each sample was analyzed. In addition, the fingerprints of these 64 samples were also established by ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC–MS). We used the PLS, a simplified method, to evaluate the results from IL-6 production and fingerprints.

Results

Each of 64 samples markedly suppressed LPS-induced IL-6 production in RAW cells. The fingerprints by UPLC–MS clearly revealed variations among 64 samples produced in different extract conditions. The PLS analysis with IL-6 production and fingerprints by UPLC–MS suggested that the peaks 71, 93, 150, 157, 168 have more influence on the inhibitory activity of Caesalpinia sappan L. extracts. The peaks 71, 93, 150 are likely representing sappanone A, protosappanin E and neoprotosappanin, respectively. The peaks 157 and 168 are still at large.

Conclusion

This is the first report that sappanone A, protosappanin E, neoprotosappanin and two unidentified compounds can be considered as possible active compounds that might inhibit IL-6 production. Further studies are needed to confirm the effectiveness of these five compounds on IL-6 production and possible mechanism.     

Effects of taraxasterol on inflammatory responses in lipopolysaccharide-induced RAW 264.7 macrophages

Aim of the study

Taraxasterol, a pentacyclic-triterpene, was isolated from the Chinese medicinal herb Taraxacum officinale. In the present study, we investigated the in vitro anti-inflammatory activity of taraxasterol in lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages.

Materials and methods

RAW 264.7 cells were pretreated with 2.5, 5, or 12.5 μg/ml of taraxasterol 1 h prior to treatment with 1 μg/ml of LPS. Nitric oxide (NO) level in supernatants from cells was examined by Griess reaction, the concentrations of prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were measured by ELISA. Nuclear factor kappa B (NF-κB) activation was evaluated by immunocytochemical analysis.

Results

We found that taraxasterol inhibited NO, PGE₂, TNF-α, IL-1β and IL-6 production in LPS-induced RAW 264.7 macrophages in a dose-dependent manner. Further studies revealed that taraxasterol prevented the LPS-induced NF-κB translocation from cytoplasm into nuclear.

Conclusions

These results indicate that taraxasterol has anti-inflammatory effect by blocking NF-κB pathway.     

藤黄茎皮中莽吉柿素对人胃癌HGC-27细胞的作用研究

目的 探讨藤黄树茎皮中氧杂环酮类成分莽吉柿素对人胃癌HGC-27细胞的生长抑制作用及潜在的作用机制。方法 采用噻唑蓝（MTT）染色实验检测莽吉柿素对HGC-27细胞的抑制作用。采用克隆形成实验评估莽吉柿素对HGC-27细胞增殖的影响。采用Hoechst染色实验研究莽吉柿素对HGC-27细胞形态变化的影响。采用蛋白免疫印迹法（Western blot）检测莽吉柿素给药后与凋亡过程、Neddylation修饰途径及丝裂原活化蛋白激酶（MAPK）信号通路相关的蛋白表达水平变化。采用裸鼠移植瘤模型进行莽吉柿素裸鼠体内抗肿瘤药效研究。结果 莽吉柿素给药后能够抑制HGC-27细胞的增殖能力和细胞集落形成。与对照组比较,莽吉柿素显著上调HGC-27细胞多聚ADP核糖聚合酶1（PARP1）和Bax蛋白表达（P<0.001）,下调抗凋亡蛋白B细胞淋巴瘤-2（Bcl-2）的表达（P<0.001）,发挥促进胃癌细胞凋亡的作用。莽吉柿素给药后HGC-27细胞中β淀粉样蛋白前体蛋白结合蛋白1（APPBP1）、泛素样修饰激活酶3（UBA3）和泛素结合酶E2M（UBC12）蛋白及MAPK信号通路蛋白c-Jun、c-Jun氨基端激酶（JNK）和p38的磷酸化水平均显著下调（P<0.001）。进一步的体内移植瘤动物实验结果显示,莽吉柿素给药后裸鼠肿瘤体积缩小18.7%,差异具有统计学意义（P<0.05）。与对照组比较,莽吉柿素组裸鼠体内与肿瘤细胞增殖相关的标志蛋白Ki67阳性细胞数明显减少,肿瘤组织中棕色细胞数量明显增加。结论 藤黄树茎皮中氧杂环酮类化学成分莽吉柿素在HGC-27细胞和裸鼠上均显示良好的抗胃癌活性,其作用机制可能与抑制Neddylation修饰途径以及抑制MAPK信号通路相关。     

Hepatic,testicular and spermatozoa antioxidant status in rats chronically treated with Garcinia kolaseed

Ethnopharmacological relevance

Garcinia kola seed is commonly used in African Traditional Medicine as a remedy for liver disorders, hepatitis, bronchitis, throat infections as well as an aphrodisiac and fertility enhancing substance. Owing to the abundance of complex mixture of phenolic compounds in Garcinia kola seed, there is a growing safety concern on its long-term use in folklore medicine. The present study evaluated the hepatic, testicular and spermatozoa antioxidant status in rats chronically treated with Garcinia kolaseed.

Materials and methods

Adult male Wistar rats were randomly assigned to four groups of 10 rats each and were orally administered with Garcinia kola at 0, 250, 500 and 1000 mg/kg for 6consecutive weeks. Clinical observations, serum biochemistry, oxidative stress biomarkers, spermatozoa parameters and histopathological examination of the organs were assessed to monitor treatment-related adverse effects inrats.

Results

Long-term treatment of Garcinia kola had no adverse effect on the spermatozoa characteristics but significantly elevated testosterone concentration when compared to the control group. Improvement of antioxidant systems was accompanied by a significant decrease in malondialdehyde level in the liver, testes and spermatozoa of Garcinia kola-treated rats. Histological observation revealed that chronic administration of Garcinia kola had no effect on the liver and testes at all doses when compared with control.

Conclusion

Garcinia kola seed boosts the antioxidant status and exhibits no adverse effect on the liver, testes and spermatozoa after a long-term oral exposure inrats.     

Aloe-emodin from rhubarb (Rheum rhabarbarum) inhibits lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages

Ethnopharmacological relevance

Rheum rhabarbarum (rhubarb) has long been used for the treatment of inflammation in China and other Asian countries. However, the mechanism underlying the anti-inflammatory activity of this medicinal plant is not fully understood. The present study was designed to investigate the anti-inflammatory effects of anthraquinones, the major constituents in rhubarb, and the molecular mechanism involved in their anti-inflammatory effects.

Materials and methods

RAW264.7 cells were stimulated by lipopolysaccharide (LPS) in the presence or absence of the compounds examined. The proliferation of RAW264.7 cells was assayed by the Alamar-Blue method. The quantity of nitric oxide (NO) was determined by Griess assay. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR. Inducible nitric oxide synthase (iNOS), inhibitor of nuclear factor κBα (IκBα), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), and Akt/phosphoinositide 3-kinase (PI3K) protein expression levels were determined by Western blotting.

Results

Aloe-emodin markedly suppressed the production of NO, interleukin-6 (IL-6), and interleukin-1β (IL-1β) in LPS-stimulated RAW264.7 cells with no apparent cytotoxicity. The mRNA expression levels of iNOS, IL-6, and IL-1β genes were also significantly inhibited by aloe-emodin. Western blot analysis showed that aloe-emodin suppressed LPS-induced iNOS protein expression, IκBα degradation, and the phosphorylation of ERK, p38, JNK, and Akt.

Conclusions

These results demonstrate that aloe-emodin is the bioactive component of rhubarb that confers an anti-inflammatory effect through a likely mechanism involving a decrease in pro-inflammatory cytokine production in LPS-induced RAW264.7 macrophages via inhibition of NF-κB, MAPK, and PI3K pathways.     

Suppression of lipopolysaccharide-induced inflammatory responses in RAW 264.7 murine macrophages by aqueous extract of Clinopodium vulgare L. (Lamiaceae)

Ethnopharmacological relevance

The wild basil Clinopodium vulgare L. is commonly used in Bulgarian folk medicine for treatment of irritated skin, mastitis- and prostatitis-related swelling, as well as for some disorders accompanied with significant degree of inflammation (e.g. gastric ulcers, diabetes, and cancer).

Aim of study

To determine the effect of aqueous extract of Clinopodium vulgare L. on LPS-induced inflammatory responses of murine RAW 264.7 macrophages.

Materials and methods

Cell cytotoxicity was evaluated by MTT assay. Protein expression levels were monitored by Western blot analysis. Production of NO and PGE₂ was measured by the Griess colorimetric method and enzyme immunoassay, respectively. Activation of MMP-9 was visualized by gelatin zymography. Cytokine levels were determined by BioPlex assay. Intracellular ROS and free radical scavenging potential were measured by DCFH-DA and DPPH method, respectively. Xanthine oxidase activity was evaluated spectrophotometrically.

Results

The extract suppresses NF-κB activation by preventing Iκ-B phosphorylation and inhibits the phosphorylation of p38 and SAPK/JNK MAPKs. It down-regulates iNOS expression which manifests as a drastic decrease of NO production, inhibits MMP-9 activation, but does not affect COX-2 protein levels and reduces only slightly the released PGE₂. Secretion of IL-1β and Il-10 is greatly reduced, whereas suppression of TNF-α and GM-CSF production is less dramatic. The extract has strong free radical scavenging properties and exerts inhibitory effect on xanthine oxidase activity, which lowers the levels of intracellular ROS.

Conclusion

The study provides evidence for the anti-inflammatory potential of Clinopodium vulgare L. aqueous extract.     

Anti-Helicobacter pylori compounds from the ethanol extracts of Geranium wilfordii

Ethnopharmocological relevance

Geranium wilfordii Maxim has been extensively used in Chinese Herbal Medicine for treating gastrointestinal disorders, diarrhea and dysentery. In the current study we aimed to investigate the anti-Helicobacter pylori activity of ethanol extracts of Geranium wilfordii Maxim and its main active compounds, corilagin and 1,2,3,6-tetra-O-galloyl-β-d-glucose.

Materials and methods

The plant materials were extracted three times with ethanol and the concentrated filtrate was successively fractioned into chloroform, ethyl acetate, and n-BuOH-soluble portions which were examined in vitro for the anti-Helicobacter. pylori activity. Employing a standard strain and five clinical isolates of Helicobacter pylori, the extract, fractions and compounds of Geranium wilfordii Maxim were assessed in vitro.

Results

The ethanol fraction, ethyl acetate fraction, corilagin, and 1,2,3,6-tetra-O-galloyl-β-d-glucose were found to be strongly inhibitory to Helicobacter. pylori (MICs: 40, 30, 4, and 8 μg/ml respectively).

Conclusions

The results of the present study showed that the ethanol and the ethyl acetate extracts from Geranium wilfordii Maxim displayed as well the most significant inhibition to the growth of Helicobacter. pylori, of which corilagin and 1,2,3,6-tetra-O-galloyl-β-d-glucose have been identified main anti-Helicobacter pylori active constituents.