A herbal formula containing roots of Salvia miltiorrhiza (Danshen) and Pueraria lobata (Gegen) inhibits inflammatory mediators in LPS-stimulated RAW 264.7 macrophages through inhibition of nuclear factor κB (NFκB) pathway

Ethnopharmacological relevance

The herbal formula DG, containing roots of Salvia miltiorrhiza (Danshen) and Pueraria lobata (Gegen), has long history in treating cardiovascular diseases. It has been shown to be able to reduce intima-media thickening in coronary patients in our previous clinical study. Since intima-media thickening is the hallmark of atherosclerotic disease, the etiology of which is inflammation of the arterial wall, the mechanism underlying the effect of DG may be related to its anti-inflammatory activities.

Aim of study

The present study aims to determine the anti-inflammatory activity of DG and elucidate its underlying mechanisms with regards to its molecular basis of action.

Materials and method

The anti-inflammatory effect of DG was studied by using lipopolysaccharide (LPS)-stimulated activation of nuclear factor κB (NFκB) pathway and subsequent production of inflammatory mediators, including nitric oxide (NO), prostaglandin E₂ (PGE₂), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and macrophage chemotactic protein-1 (MCP-1), in mouse RAW 264.7 macrophages.

Results

The present study demonstrated that DG could suppress the production of NO and PGE₂ through the inhibition of iNOS and COX-2 genes. DG could also inhibit the production of IL-1β, IL-6 and MCP-1, but not TNF-α, through the inhibition of respective mRNA expressions. Further investigations showed the inhibitory effect of DG on activation of IKKα/β and degradation of IκBα, thus preventing nuclear translocation of NFκB. All these results suggested the inhibitory effects of DG on the production of inflammatory mediators through the inhibition of the NFκB pathway.

Conclusions

The inhibitory effects of DG on the production of inflammatory mediators by LPS-stimulated RAW 264.7 macrophages, are accomplished by inhibiting the nuclear translocation of NFκB through inactivating IKKα/β and preventing degradation of IκBα.     

Antroquinonol from ethanolic extract of mycelium of Antrodia cinnamomea protects hepatic cells from ethanol-induced oxidative stress through Nrf-2 activation

Aim of the study

In recent years, the medicinal mushroom Antrodia cinnamomea, known as “niu-chang chih” has received much attention with regard to its possible health benefits; especially its hepatoprotective effects against various drugs, toxins, and alcohol induced liver diseases. However, the molecular mechanism underlying this protective effect of Antrodia cinnamomea and its active compound antroquinonol was poorly understood. In the present study we evaluated to understand the hepatoprotective efficacy of antroquinonol and ethanolic extracts of mycelia of Antrodia cinnamomea (EMAC) in vitro and in vivo.

Materials and methods

The protective mechanism of antroquinonol and EMAC against ethanol-induced oxidative stress was investigated in cultured human hepatoma HepG2 cells and ICR mice model, respectively. HepG2 cells were pretreated with antroquinonol (1-20 μM) and oxidative stress was induced by ethanol (100 mM). Meanwhile, male ICR mice were pretreated with EMAC for 10 days and hepatotoxicity was generated by the addition of ethanol (5 g/kg). Hepatic enzymes, cytokines and chemokines were determined using commercially available assay kits. Western blotting and real-time PCR were subjected to analyze HO-1 and Nr-2 expression. EMSA was performed to monitor Nrf-2 ARE binding activity. Possible changes in hepatic lesion were observed using histopathological analysis.

Results

Antroquinonol pretreatment significantly inhibited ethanol-induced AST, ALT, ROS, NO, MDA production and GSH depletion in HepG2 cells. Western blot and RT-PCR analysis showed that antroquinonol enhanced Nrf-2 activation and its downstream antioxidant gene HO-1 via MAPK pathway. This mechanism was then confirmed in vivo in an acute ethanol intoxicated mouse model: serum ALT and AST production, hepatocellular lipid peroxidation and GSH depletion was prevented by EMAC in a dose-dependent manner. EMAC significantly enhanced HO-1 and Nrf-2 activation via MAPKs consistent with in vitro studies. Ethanol-induced hepatic swelling and hydropic degeneration of hepatocytes was significantly inhibited by EMAC in a dose-dependent manner.

Conclusions

These results provide a scientific basis for the hepatoprotective effects of Antrodia cinnamomea. Data also imply that antroquinonol, a potent bioactive compound may be responsible for the hepatoprotective activity of Antrodia cinnamomea. Moreover, the present study highly supported our traditional knowledge that Antrodia cinnamomea as a potential candidate for the treatment of alcoholic liver diseases.     

Anti-inflammatory effects of methanol extract of Antrodia cinnamomea mycelia both in vitro and in vivo

Aims of the study

Antrodia cinnamomea is a folk medicinal mushroom commonly used in Taiwan for the treatment of several types of cancers and inflammatory disorders. This study aimed to explore the folk use of Antrodia cinnamomea on pharmacological grounds to characterize the scientific basis of anti-inflammatory activity.

Materials and methods

The in vitro anti-inflammatory activity of methanol extract of liquid cultured mycelia of Antrodia cinnamomea (MEMAC) was judged by the measurement of the produced levels of pro-inflammatory cytokines and mediators in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and human peripheral blood mononuclear cells (PBMCs). The in vivo anti-inflammatory activity of MEMAC was evaluated using carrageenan-induced hind paw edema in mice, the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver and the levels of malondialdehyde (MDA) and nitrite oxide (NO) in the edema paw. The levels of serum NO and TNF-α were measured. The MEMAC was administered at the concentrations of 100, 200, and 400 mg/kg body weight of mouse.

Results

MEMAC inhibited the production of LPS-induced pro-inflammatory cytokines (TNF-α and IL-6) and mediators (NO and PGE2) in RAW264.7 cells and human PBMCs. Data from Western blotting showed that MEMAC decreased the levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in LPS-stimulated RAW264.7 macrophages. In vivo, MEMAC showed significant (p < 0.05) anti-inflammatory activity by reducing the edema volume in carrageenan-induced paw edema in mice. MEMAC (400 mg/kg) also reduced the carrageenan-induced leukocyte migration (50.92 ± 5.71%). Further, MEMAC increased the activities of CAT, SOD, and GPx in the liver tissue and decreased the levels of serum NO and TNF-α after carrageenan administration.

Conclusions

Our results showed that MEMAC has the anti-inflammatory property both in vitro and in vivo, suggesting that it may be a potential preventive or therapeutic candidate for the treatment of inflammatory disorders.     

Anti-inflammatory activity of ethyl acetate fraction of the seeds of Brucea Javanica

Ethnopharmacological relevance

The seeds of Brucea javanica (L.) Merr. (Yadanzi in Chinese) have been used for the treatment of inflammation, dysentery, malaria, and cancer in Chinese traditional medicine. However, the anti-inflammatory mechanism of Brucea javanica has not been fully elucidated. This study examined the anti-inflammatory activity of ethyl acetate fraction of the seeds of Brucea javanica (EA-BJ) in vitro and in vivo.

Materials and methods

The anti-inflammatory activity of EA-BJ and its ability to modulate the production of NO, PGE2, TNF-α, IL-1β, IL-6 and IL-10 inflammatory mediators in lipopolysaccharide-activated RAW 264.7 macrophage were evaluated. Moreover, the anti-inflammatory activity of EA-BJ was also in vivo assayed by carrageenan induced paw edema in mice.

Results

In vitro assays showed remarkable anti-inflammatory activity of EA-BJ, through the inhibition of production of NO, PGE2, TNF-α, IL-1β and IL-6 inflammatory mediators and induction of production of IL-10 anti-inflammatory cytokine. In vivo assays showed anti-inflammatory activity for decrement of the paw edema in carrageenan induced paw edema test.

Conclusion

The results obtained in vitro and in vivo showed that possible anti-inflammatory effects of EA-BJ may be attributed to inhibition pro-inflammatory mediators production, NO, PGE2, TNF-α, IL-1β and IL-6 and to increase production of IL-10 anti-inflammatory cytokine. The seeds of Brucea javanica may thus prove beneficial in the treatment of inflammatory diseases.     

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.     

Dichroa febrifuga Lour. inhibits the production of IL-1β and IL-6 through blocking NF-κB, MAPK and Akt activation in macrophages

Aim of the study

The roots of Dichroa febrifuga Lour. have been used as a traditional antimalarial drug and also used in the treatment of productive cough and unstable fever caused by infection in China and Korea. In this study, we evaluated the anti-inflammatory effect and underlying molecular mechanism of aqueous extract of Dichroa febrifuga (AEDF) in C57BL/6 mouse peritoneal macrophages.

Materials and methods

The effect of AEDF on proinflammatory cytokine (IL-1β and IL-6) production was analyzed by ELISA and real-time RT-PCR. The effects of AEDF on NF-κB/IκB-α/IKK were measured by reporter assay (in RAW 264.7 cells), EMSA, Western blotting and kinase assay. The effects of AEDF on Akt and MAPKs activity were assayed by Western blotting.

Results

AEDF inhibited the production of IL-1β and IL-6, NF-κB activation, IκB-α degradation, and IKK, Akt, ERK1/2 and JNK activities in LPS-stimulated mouse peritoneal macrophages.

Conclusions

These results suggest that AEDF inhibits proinflammatory cytokine (IL-1β and IL-6) production in LPS-stimulated mouse peritoneal macrophages, and that these effects are mediated by the inhibition of the activity of IKK/IκB/NF-κB and the phosphorylation of Akt, ERK1/2, and JNK. Our results provide a molecular basis for understanding the inhibitory effects of Dichroa febrifuga roots on endotoxin-mediated inflammation.     

The anti-tumor activity of Antrodia salmonea in human promyelocytic leukemia (HL-60) cells is mediated via the induction of G1 cell-cycle arrest and apoptosis in vitro or in vivo

Ethnopharmacological relevance

The medicinal mushroom Antrodia salmonea has been used as a traditional Chinese medicine and has demonstrated antioxidant and anti-inflammatory effects.

Materials and methods

In the present study, we examined the anti-tumor activity of the fermented culture broth of Antrodia salmonea (AS) in vitro and in vivo and revealed its underlying molecular mechanism of action.

Results

Treatment of human promyelocytic leukemia (HL-60) cells with AS (50–150 μg/mL) significantly reduced cell viability and caused G₁ arrest via the inhibition of cell-cycle regulatory proteins, including cyclin D1, CDK4, cyclin E, cyclin A, and phosphorylated retinoblastoma protein (p-Rb). Furthermore, AS treatment induced apoptosis, which was associated with DNA fragmentation, followed by a sequence of events, including intracellular ROS generation; mitochondrial dysfunction; Fas ligand activation; cytochrome c release; caspase-3, -8, -9, and PARP activation; and Bcl-2/Bax dysregulation. The results of the in vitro study suggested that AS-induced apoptosis in HL-60 cells was mediated by both the mitochondrial and death receptor pathways. Furthermore, we found that AS treatment was effective in delaying tumor incidence in HL-60 xenografted nude mice and reducing tumor burden.

Conclusions

To the best of our knowledge, this is the first report confirming the anti-tumor activity of this potentially beneficial mushroom against human promyelocytic leukemia.     

Aqueous extract of unripe Rubus coreanus fruit attenuates atherosclerosis by improving blood lipid profile and inhibiting NF-κB activation via phase II gene expression

Ethnopharmacological relevance

The fruit of Rubus coreanus has been used as a traditional herbal medicine for alleviation of inflammatory and vascular diseases in Asian countries.

Aim of the study

The anti-atherogenic effect of unripe Rubus coreanus fruit extract (URFE) and its underlying mechanism were analyzed in mice fed a high-fat diet (HFD) and in cell culture system.

Materials and methods

Mouse was freely given HFD alone or supplemented with URFE for 14 weeks, followed by analysis of atherosclerotic lesions and serum lipid levels. For in vitro assay, macrophages were pretreated with URFE, followed by stimulation with lipopolysaccharide (LPS). Expression levels of inflammatory genes (TNF-α, IL-1β, and iNOS) and phase II genes (heme oxygenase-1, glutamate cysteine lygase, and peroxiredoxine-1) as well as intracellular reactive oxygen species (ROS) level and NF-κB activation pathway were analyzed in cultured macrophages as well as mouse sera and aortic tissues.

Results

URFE supplementation reduced HFD-induced atherosclerotic lesion formation which was correlated with decreased levels of lipids, lipid peroxides, and inflammatory mediators (TNF-α, IL-1β, and nitric oxide) in sera as well as suppression of inflammatory gene in aortic tissues. In addition, pre-treatment of macrophages with URFE also suppressed LPS-induced NF-κB activation, ROS production, and inflammatory and phase II gene expressions. Inhibition of phase II enzyme and protein activities attenuated the suppressive effects URFE on ROS production, NF-κB activation, and inflammatory gene expression.

Conclusion

These results suggest that URFE attenuates atherosclerosis by improving blood lipid profile and inhibiting NF-κB activation via phase II antioxidant gene expression.     

Experimental study on anti-inflammatory activity of a TCM recipe consisting of the supercritical fluid CO2 extract of Chrysanthemum indicum, Patchouli Oil and Zedoary Turmeric Oil in vivo

Ethnopharmacological relevance

Chrysanthemum indicum (Compositae) Linné, Pogostemon cablin (Blanco) Benth and Curcuma wenyujin (Zingiberaceae) Y. H. Chen et C. Ling are three of the extensively used herbal remedies among traditional Chinese medicines for the purpose of anti-inflammation. A traditional Chinese medicine (TCM) recipe named CPZ consisting extracts of the above three herbs, has shown noteworthy anti-influenza activity, which is closely related to its anti-inflammatory feature.

Aim of this study

To investigated the anti-inflammtory activity of CPZ in vivo for a further exploration of the recipe's anti-inflammatory properties.

Materials and methods

The anti-inflammatory property of CPZ on acute inflammation was evaluated by inflammatory models of dimethylbenzene (DMB)-induced ear vasodilatation and acetic acid-induced capillary permeability enhancement in mice, as well as the carrageenan-induced paw edema rat model, in which inflammation-related cytokine including prostaglandin E₂ (PGE₂), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) in the edematous paw tissue were determined by enzyme linked immunosorbent assay (ELISA). Moreover, effect of CPZ on chronic inflammation was observed through granuloma formation in rats subjected to cotton pellet implantation.

Results

CPZ (340, 170, and 85 mg/kg for mice, p.o.) not only decreased the DMB-induced ear vasodilatation but also attenuated capillary permeability under acetic acid challenge in mice. And the significant inhibition on carrageenan-induced paw edema was observed. Further more, the ELISA results showed that CPZ (170, 85, and 42.5 mg/kg for rats, p.o.) could up-regulate the level of IL-1β in the edema paw tissue of rats significantly while down-regulate that of PGE₂, but no apparent effect on TNF-α or NO was observed in the test. Besides, CPZ had a certain degree of restraining effect on the cotton pellet-induced granuloma formation in rats and the highest dose of 170 mg/kg even showed a significant suppression on it.

Conclusion

The above results indicated that CPZ possessed a potent anti-inflammatory activity, which is indicated to be closely associated with its regulation on IL-1β and PGE₂ thereby mediating the inflammatory response acting at an appropriate level.     

Anti-angiogenic effects and mechanisms of polysaccharides from Antrodia cinnamomea with different molecular weights

Ethnopharmacological relevance

Antrodia cinnamomea is a popular medicinal mushroom in Taiwan that has been widely used for treatment of various cancers and liver diseases.

Aim of the study

This study aimed to investigate the immunomodulatory effect on angiogenesis of polysaccharides from mycelia of Antrodia cinnamomea (PMAC).

Materials and methods

PMAC were extracted in boiling water, precipitated with 95% ethanol, and separated into four different molecular weights (<5, 5–30, 30–100, >100 kDa). Tube formation and chorioallantoic membrane (CAM) assay were used to determine the in vitro and ex vivo anti-angiogenic effects.

Results

Only the PMAC-mononuclear cells (MNCs)-conditioned medium (CM) with MW > 100 kDa significantly and concentration-dependently decreased the secretion of vascular endothelial growth factor in human leukemia cells and inhibited the matrigel tube formation in human umbilical vein endothelial cells. Similarly only the PMAC-MNC-CM with MW > 100 kDa significantly and concentration-dependently increased the levels of interleukin (IL)-12 and interferon-γ (IFN-γ). In addition, the ex vivo CAM assay revealed that only the PMAC with MW > 100 kDa significantly and dose-dependently inhibited neovascularization.

Conclusions

PMAC with MW > 100 kDa are anti-angiogenic in vitro and ex vivo, and the effects are likely through immunomodulation.     

Antioxidant and anti-inflammatory activities of aqueous extract of Centipeda minima

Ethnopharmacological relevance

Centipeda minima (L.) is traditionally used in Chinese folk medicine for the treatments of rhinitis, sinusitis, relieving pain, reducing swelling, and treating cancer for a long history in Taiwan. However, there is no scientific evidence which supports the use in the literature.

Aim of the study

The aim of this study was to evaluate the antioxidant and anti-inflammatory activities of the aqueous extract of Centipeda minima (ACM).

Materials and methods

The following activities were investigated: antioxidant activities [2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), DPPH (1,1-diphenyl-2-picrylhydrazyl)], and anti-inflammatory [lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264.7 macrophages and paw-edema induced by λ-carrageenan (Carr)]. We also investigated the anti-inflammatory mechanism of ACM via studies of the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) and the levels of malondialdehyde (MDA) in the edema paw. Serum NO, tumor necrosis factor α (TNF-α), and interleukin-1β (IL-1β) were also measured in vivo. In HPLC analysis, the fingerprint chromatogram of ACM was established.

Results

ACM showed the highest TEAC and DPPH radical scavenging activities, respectively. ACM also had highest contents of polyphenol and flavonoid contents. We evaluated that ACM and the reference compound of protocatechualdehyde and caffeic acid decreased the LPS-induced NO production in RAW264.7 cells. Administration of ACM showed a concentration dependent inhibition on paw edema development after Carr treatment in mice. The anti-inflammatory effects of ACM could be via NO, TNF-α, and IL-1β suppressions and associated with the increase in the activities of antioxidant enzymes. Western blotting revealed that ACM decreased Carr-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions.

Conclusions

Anti-inflammatory mechanisms of ACM might be correlated to the decrease in the level of Malondialdehyde (MDA), iNOS, and COX-2 via increasing the activities of CAT, SOD, and GPx in the edema paw. Overall, the results showed that ACM demonstrated antioxidant and anti-inflammatory activity, which supports previous claims of the traditional use for inflammation and pain.     

Chrysanthemum indicum Linné extract inhibits the inflammatory response by suppressing NF-κB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophages

Aims of study

Although the flowers of Chrysanthemum indicum Linné (Asteraceae) have long been used in traditional Korean and Chinese medicine to treat inflammatory diseases, the underlying mechanism(s) by which these effects are induced remains to be defined. We investigated the effects of a 70% ethanolic extract of C. indicum (CIE) on the activities of cellular signaling molecules that mediate inflammatory responses.

Materials and methods

Production of NO, PGE₂, TNF-α, and IL-1β by ELISA, mRNA and protein expression of iNOS and COX-2, phosphorylation of MAPKs, and activation of NF-κB by RT-PCR and Western blotting were examined in LPS-induced RAW 264.7 macrophages.

Results

The CIE strongly inhibited NO, PGE₂, TNF-α, and IL-1β production, and also significantly inhibited mRNA and protein expression of iNOS and COX-2 in LPS-induced RAW 264.7 macrophages, in a dose-dependent manner. Furthermore, the CIE clearly suppressed nuclear translocation of NF-κB p65 subunits, which correlated with an inhibitory effect on IκBα phosphorylation. The CIE also attenuated the activation of ERK1/2 and JNK in a dose-dependent manner.

Conclusion

Our results suggest that the anti-inflammatory properties of CIE might result from the inhibition of inflammatory mediators, such as NO, PGE₂, TNF-α, and IL-1β, via suppression of MAPKs and NF-κB-dependent pathways.     

Mechanism of anti-inflammatory activity of umbelliferone 6-carboxylic acid isolated from Angelica decursiva

Aims of the study

We recently reported the potential antioxidant and anti-inflammatory activities of umbelliferone 6-carboxylic acid (UMC) isolated from the whole plants of Angelica decursiva. In this study, we elucidated the anti-inflammatory mechanisms of UMC in vitro and in vivo.

Methods

The inhibitory effects of UMC on the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor-α (TNF-α), the expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the activation of nuclear factor kappa B (NF-κB) were evaluated using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The reactive oxygen species (ROS) generation inhibitory activity of UMC was evaluated using t-butyl hydroperoxide (t-BHP)-induced RAW 264.7 cells. Furthermore, the in vivo anti-inflammatory activity of UMC was evaluated using carrageenan induced mouse paw edema model.

Results

UMC dose-dependently inhibited NO and PGE₂ production by down-regulating iNOS and COX-2 protein expression in LPS-stimulated RAW 264.7 macrophages. UMC also suppressed the production of the proinflammatory cytokine TNF-α in LPS stimulated RAW 264.7 cells in a concentration dependent manner. In addition, UMC dose-dependently prevented LPS-induced nuclear translocation of NF-κB in RAW 264.7 macrophages. Furthermore, UMC exhibited the inhibitory activity against t-BHP-induced ROS generation in RAW 264.7 cells with an IC₅₀ value of 705.1 μg/ml. Moreover, UMC inhibited λ-carrageenan induced mouse paw edema by 70.40 and 60.20% at doses of 50 and 25 mg/kg body weight, respectively.

Conclusion

The combined results of this study indicate that UMC is an important anti-inflammatory constituent of A. decursiva and its anti-inflammatory effect was due to its ability to inhibit the production of inflammatory mediators via inhibition of NF-κB activation pathway.     

Anti-inflammatory activity of a water extract of Acorus calamus L. leaves on keratinocyte HaCaT cells

Ethnopharmacological relevance

Acorus calamus L., sweet flag, is a well-known medicinal plant that grows worldwide wildly along swamps, rivers, and lakes.

Aim of the study

The aim of this study was to evaluate the anti-inflammatory activity of Acorus calamus leaf (ACL) extract and to explore its mechanism of action on human keratinocyte HaCaT cells.

Materials and methods

HaCaT cells treated with polyinosinic:polycytidylic acid (polyI:C) and peptidoglycan (PGN) induced the inflammatory reactions. The anti-inflammatory activities of ACL were investigated using RT-PCR, ELISA assay, immunoblotting, and immunofluorescence staining.

Results

HaCaT cells induced the pro-inflammatory cytokines, interleukin-8 (IL-8) and/or interleukin-6 (IL-6) expressions after treatment with polyI:C or PGN. ACL inhibited the expression of IL-8 and IL-6 RNA and protein levels, and attenuated the activation of NF-κB and IRF3 after polyI:C treatment. ACL also inhibited expression of IL-8 and activation of NF-κB following PGN induction.

Conclusions

These results suggest that ACL inhibits the production of pro-inflammatory cytokines through multiple mechanisms and may be a novel and effective anti-inflammatory agent for the treatment of skin diseases.     

Extracts of Crinum latifolium inhibit the cell viability of mouse lymph oma cell line EL4 and induce activation of anti-tumour activity of macrophages in vitro

Ethnopharmacological relevance

Crinum latifolium L. (CL) leaf extracts have been traditionally used in Vietnam and are now used all over the world for the treatment of prostate cancer. However, the precise cellular mechanisms of the action of CL extracts remain unclear.

Aim of the study

To examine the effects of CL samples on the anti-tumour activity of peritoneal murine macrophages.

Materials and methods

The properties of three extracts (aqueous, flavonoid, alkaloid), one fraction (alkaloid), and one pure compound (6-hydroxycrinamidine) obtained from CL, were studied (i) for redox capacities (DPPH and bleaching beta-carotene assays), (ii) on murine peritoneal macrophages (MTT assay) and on lymphoma EL4-luc2 cells (luciferine assay) for cytotoxicity, (iii) on macrophage polarization (production of ROS and gene expression by PCR), and (iv) on the tumoricidal functions of murine peritoneal macrophages (lymphoma cytotoxicity by co-culture with syngeneic macrophages).

Results

The total flavonoid extract with a high antioxidant activity (IC₅₀=107.36 mg/L, DPPH assay) showed an inhibitory action on cancer cells. Alkaloid extracts inhibited the proliferation of lymphoma cells either by directly acting on tumour cells or by activating of the tumoricidal functions of syngeneic macrophages. The aqueous extract induced mRNA expression of tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin 6 (IL-6) indicating differentiation of macrophages into pro-inflammatory M1 polarized macrophages. The total flavonoid, alkaloid extracts and an alkaloid fraction induced the expression of the formyl peptide receptor (FPR) on the surface of the polarized macrophages that could lead to the activation of macrophages towards the M1 phenotype. Aqueous and flavonoid extracts enhanced NADPH quinine oxido-reductase 1 (NQO1) mRNA expression in polarized macrophages which could play an important role in cancer chemoprevention. All the samples studied were non-toxic to normal living cells and the pure alkaloid tested, 6-hydroxycrinamidine, was not active in any of the models investigated.

Conclusions

Our results indicate that CL extracts and alkaloid fraction (but not pure 6-hydroxycrinamidine) inhibit the proliferation of lymphoma cells in multiple pathways. Our results are in accordance with traditional usage and encourage further studies and in vivo assays.     

Red ginseng extract inhibits the expression of MCP-1 and iNOS in Helicobacter pylori-infected gastric epithelial cells by suppressing the activation of NADPH oxidase and Jak2/Stat3

Ethnopharmcological relevance

Helicobacter pylori induced oxidative stress represents an important mechanism leading to expression of inflammatory mediators. Korean red ginseng is used in traditional medicine to inhibit inflammation. However, the anti-inflammatory mechanism of red ginseng is still under investigation. Thus, we investigated whether Korean red ginseng extract (RGE) inhibits NADPH oxidase, a source of reactive oxygen species (ROS), and the Jak2/Stat3 pathway, which mediates the expression of inflammatory mediators, in Helicobacter pylori-infected gastric epithelial cells.

Materials and methods

A standardized RGE was supplied by the Korea Ginseng Corporation. Human gastric epithelial cells (AGS) were treated with RGE and stimulated with Helicobacter pylori. NADPH oxidase activity, ROS levels, activation of Jak2/Stat3, and induction of MCP-1 and iNOS were determined.

Results

Helicobacter pylori infection resulted in an increase in ROS and activation of NADPH oxidase and Jak2/Stat3, which induced the expression of MCP-1 and iNOS in AGS cells. The induction of MCP-1 and iNOS was inhibited by both the Jak2/Stat3 inhibitor AG490 and RGE in Helicobacter pylori-infected cells. RGE suppressed NADPH oxidase activity by inhibiting translocation of cytosolic subunits p67phox and p47phox to the membrane and reduced ROS levels in Helicobacter pylori-infected cells.

Conclusion

RGE inhibits the expression of MCP-1 and iNOS by suppressing the activation of NADPH oxidase and Jak2/Stat3 in Helicobacter pylori-infected gastric epithelial cells.     

Flower extract of Panax notoginseng attenuates lipopolysaccharide-induced inflammatory response via blocking of NF-κB signaling pathway in murine macrophages

Aim of the study

The root of Panax notoginseng (PN) is commonly used to treat chronic liver disease with its therapeutic abilities to stop haemorrhage in the circulation, while the PN flower (PN-F) is largely unknown in the biological activities on inflammation and mechanisms of its actions. In this study, the pharmacologic effects of PN-F methanol extract on inflammation were investigated to address potential therapeutic or toxic effects in LPS-stimulated mouse macrophage cells, RAW264.7 cells.

Materials and methods

Production of NO, PGE₂ and pro-inflammatory cytokines (TNF-α and IL-1β) in supernatant, the expression of iNOS, COX-2 and cytokines, the phosphorylation of MAPK moleduces (ERK1/2, JNK and p38 MAPK), and the activation of NF-κB in PN-F extract were assayed in LPS-stimulated RAW264.7 cells.

Results

PN-F extract significantly inhibited the productions of NO, PGE₂, TNF-α and IL-1β on the LPS-stimulated RAW264.7 cells. In addition, PN-F extract suppressed the mRNA and protein expressions of iNOS, COX-2, TNF-α and IL-1β in LPS-stimulated RAW264.7 cells. The molecular mechanism of PN-F extract-mediated attenuation in RAW264.7 cells has close a relationship to suppressing the phosphorylation of MAPK molecules such as ERK1/2, JNK and p38 MAPK, and the translocation of NF-κB p65 subunit into nuclear.

Conclusion

These results indicate that PN-F extract inhibits LPS-induced inflammatory response via the blocking of NF-κB signaling pathway in macrophages, and demonstrated that PN-F extract possesses anti-inflammatory properties in vitro.