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.     

Inhibitory effect of eupatilin and jaceosidin isolated from Artemisia princeps on carrageenan-induced inflammation in mice

Ethnopharmacological relevance

Artemisia princeps Pampanini (family Asteraceae) is an herbal medicine widely used as a hepatoprotective, antioxidative, anti-inflammatory, and antibacterial agent in Korea, China, and Japan.

Aim of the study

This study aimed to elucidate the anti-inflammatory effect of the main constituents, eupatilin and jaceosidin, isolated from Artemisia princeps.

Materials and methods

We used carrageenan-induced inflammation in an air pouch on the back of mice and carrageenan-induced hind paw edema in rats to determine the anti-inflammatory effects of eupatilin and jaceosidin. Inflammatory makers, such as expression of pro-inflammatory cytokines and cyclooxygenase (COX)-2, and activation of nuclear factor-kappa B (NF-κB), were measured by enzyme-linked immunosorbent assays and immunoblot analyses.

Results

Eupatilin and jaceosidin blocked carrageenan-induced increase in leukocyte number and protein levels in air pouch exudates. Eupatilin and jaceosidin inhibited COX-2 expression and NF-κB activation, and markedly reduced TNF-α, IL-1β, and prostaglandin E2 (PGE₂) levels. They also inhibited hind paw edema induced by carrageenan. Eupatilin and jaceosidin had similar activity.

Conclusions

These findings suggest that eupatilin and jaceosidin may reduce inflammation by inhibiting NF-κB activation, and that Artemisia princeps inhibits inflammation because of these constituents.     

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.     

Vascular effects of Tanacetum vulgare L. leaf extract: in vitro pharmacological study

Aim of the study

Tanacetum vulgare L. (Asteraceae/Compositae) is principally used in traditional Moroccan medicine as antihypertensive remedy. The aim of the present study was to investigate the in vitro vascular activity of the aqueous extract of Tanacetum vulgare L.

Materials and Methods

The activity of Tanacetum vulgare L. extract was tested on contractile response of Wistar rat aorta to high KCl and noradrenaline and on endothelium-dependent relaxation evoked by acetylcholine.

Results

The addition of Tanacetum extract during the plateau phase of noradrenaline-evoked contraction produced a rapid relaxation that reached a maximum of 30% of the contraction and was suppressed by the NO synthase inhibitor N^G-nitro-l-arginine. At higher extract concentrations this rapid relaxation was followed by a slowly developing, N^G-nitro-l-arginine-resistant, relaxing effect. Tanacetum extract also depressed KCl-evoked contraction by 30% at maximum. This effect was abolished in the presence of N^G-nitro-l-arginine. The endothelium-dependent relaxation induced by acetylcholine was depressed in the presence of Tanacetum extract in the bathing solution.

Conclusion

:This study indicates that the aqueous extract of Tanacetum possesses NO-mediated and NO-independent vasorelaxing properties in vitro.     

The production of nitric oxide and prostaglandin E2 in peritoneal macrophages is inhibited by Andrographis paniculata, Angelica sinensis and Morus alba ethyl acetate fractions

Aim of the study

Traditional Chinese medicine herbs (TCMHs) are used in medicines as well as in daily dietary supplements in Asia. In this study, we employed pNF-κB-Luc or pIFN-γ-Luc and BALB/c mice peritoneal macrophages or splenocytes to investigate both the immune and inflammatory effects of six selected plant species.

Materials and Methods

Specifically, we used ethyl acetate fractions of Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao (Fabaceae) (AM), Andrographis paniculata (Burm. f.) Nees (Acanthaceae) (AP), Angelica sinensis (Oliv.) Diels (Apiaceae) (AS), Eucommia ulmodes Oliv. (Eucommiaceae) leaves (EU leaves), Isatis indigotica Fort. (Brassicaceae) (II) and Morus alba L. (Moraceae) (MA).

Results

We found that ethyl acetate fractions of AP, AS and MA significantly decreased NF-κB luciferase activity and also the secretion of NO and PGE₂ in LPS/IFN-γ stimulated mouse peritoneal macrophages (p < 0.05). In contrast, they did not affect IFN-γ luciferase activity or IFN-γ production in concanavalin A (Con A)-activated mouse splenocytes. Our results indicated that the anti-inflammatory properties of these plant extracts might be resulted from the inhibition of pro-inflammatory mediators (e.g., NO and PGE₂), at least in part via suppression of a signaling pathway such as NF-κB.

Conclusions

Collectively, we have found that three potent bioactive TCMH species exerted significant NF-κB inhibitory activity and acted in a cell type dependent fashion.     

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.     

Suppressive effects of wild bitter gourd (Momordica charantia Linn. var. abbreviata ser.) fruit extracts on inflammatory responses in RAW 264.7 macrophages

Aim of the study

Bitter gourd (Momordica charantia) is used to treat various diseases including inflammation. A wild species of bitter gourd, Momordica charantia Linn. var. abbreviata ser. (WBG), is considered to be more potent in disease prevention than is bitter gourd; however, little is known about the biological and physiological characteristics of WBG.

Materials and methods

The present study investigated the anti-inflammatory effect of WBG on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.

Results

Among the hot water, 95% ethanol, and ethyl acetate extracts of WBG, the ethanol extract showed the greatest reduction of LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production and inducible nitric oxide synthase (iNOS) and pro-interleukin-1β expression. LPS-induced cyclooxygenase-2 expression was not affected by WBG extracts. Compared with WBG, extracts from bitter gourd showed a lesser inhibition of LPS-induced events. Electrophoretic mobility shift assay further showed that both the hot water and the ethanol extracts of WBG inhibited NF-κB activation. Although information is lacking on the bioactive components of WBG, the phenolic compound contents of each extract significantly paralleled its anti-inflammatory ability (r = 0.74, 0.88 and 0.65 for NO, PGE₂ and iNOS expression, respectively, P < 0.05).

Conclusions

These results suggest that WBG is beneficial for reducing LPS-induced inflammatory responses by modulating NF-κB activation.     

Mouririelliptica: Validation of gastroprotective,healing and anti-Helicobacter pylori effects

Ethnopharmacological relevance

Mouriri elliptica Martius (Melastomataceae) is species reputed in folk medicine to heal gastric ulcer and gastritis.

Aim of the study

Methanolic extract (ME) and ethyl acetate fraction (EAF) from leaves of Mouriri elliptica were evaluated for their gastroprotective, healing, immunological, toxicological and anti-Helicobacter pylori activities.

Material and methods

The gastroprotective action of ME and EAF was evaluated in rodent experimental models and to elucidate mechanisms of action, the antisecretory action, involvements of NO, SH, PGE₂, anti-Helicobacter pylori action of ME was evaluated. We also used immunohistochemical (PCNA and COX-2) and immunomodulatory (murine peritoneal macrophages) assays to evaluate Mouriri elliptica effects.

Results

ME present gastroprotective action without antisecretory effect. Otherwise, ME showed anti-Helicobacter pylori action (MIC = 0.025 μg/mL) and was able to inhibit NO production by macrophages. This species also accelerate the healing of ulcerated gastric mucosa by stimulating proliferation factors (PCNA), COX-2 and maintained basal PGE₂ level independent action of NSAID in gastric mucosa. The phytochemical investigation showed that this species possesses phenolic acid derivatives, acylglycoflavonoids and condensed tannins which probably influenced their pharmacological action.

Conclusion

All these results suggest the efficacy and safety of Mouriri elliptica in combating and healing gastric ulcer.     

Anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium on LPS-stimulated Raw264.7 cells

Aim of the study

Lilium lancifolium is commonly used to treat bronchitis, pneumonia, etc. In this study, we investigated the anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium (LL extracts) in LPS-stimulated Raw264.7 cells.

Material and methods

Levels of NO, PGE₂ and pro-inflammatory cytokines (IL-6 and TNF-α) in the supernatant fraction were determined using sandwich ELISA. Expression of COX-2 and iNOS, phosphorylation of MAPK subgroups (ERK and JNK), and NF-κB activation in extracts were detected via Western blot and immunocytochemistry assays.

Results

The LL extract significantly inhibited NO, PGE₂, IL-6 and TNF-α production in LPS-stimulated cells, and suppressed iNOS and COX-2 expression. A mechanism-based study showed that phosphorylation of ERK1/2 and JNK and translocation of the NF-κB p65 subunit into nuclei were inhibited by the LL extract. Furthermore, interleukin-4 and interleukin-13 production in Con A-induced splenocytes was suppressed.

Conclusion

These results indicate that anti-inflammatory effects of methanol extracts from Lilium lancifolium are due to downregulation of iNOS and COX-2 via suppression of NF-κB activation and nuclear translocation as well as blocking of ERK and JNK signaling in LPS-stimulated Raw264.7 cells.     

Chemopreventive action of Lygodium flexuosum extract in human hepatoma PLC/PRF/5 and Hep 3B cells

Ethnopharmacological relevance

Lygodium flexuosum (Lygodiaceae), a medicinal fern used in Indian traditional medicine against liver disorders.

Aim of the study

The rationale of the study was to examine whether the n-hexane extract from plant Lygodium flexuosum affects apoptosis on human hepatoma PLC/PRF/5 and Hep 3B cells.

Materials and methods

Chemopreventive activity of the Lygodium flexuosum extract was determined by MTT assay, annexin-V FITC binding to phosphatidyl serine and cleavage of PARP. Subdiploid condition of cells treated with Lygodium flexuosum was analyzed by flow cytometry. Further, used transiently transfected NF-κB reporter in PLC/PRF/5 cells to evaluate the inhibitive effect of Lygodium flexuosum extract.

Results

Lygodium flexuosum extract inhibited the cell viability and induced apoptosis in hepatoma cells in a concentration dependent manner as evidenced by apoptotic changes such as flipping of phosphatidyl serine, cleavage of PARP. Cell cycle analysis showed the subG1 apoptotic population in cells treated with higher concentrations of the extract. When activated with exogenous TNF-α in transfected hepatoma cells it was observed that NF-κB dependent gene expression was inhibited by treatment with Lygodium flexuosum extract in PLC/PRF/5 cells dose-dependently.

Conclusions

This investigation suggests that the Lygodium flexuosum extract has antiproliferative and apoptotic activity in both cancer cells and has inhibitive role in TNF-α induced NF-κB activation in PLC/PRF/5 cells confirms the potential of the extract as a chemopreventive agent.     

Anti-inflammatory effects of YeongyoSeungma-tang

Ethnopharmacological relevance

YeongyoSeungma-tang which includes Fructus Forsythia, has been used in oriental traditional medicine for treatment of early smallpox and atopic dermatitis.

Aim of the study

YeongyoSeungma-tang was carried out to investigate for anti-inflammatory effects.

Materials and methods

YeongyoSeungma-tang was tested for anti-inflammatory effects against lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor alpha (TNF-α) releases as well as nuclear factor kappa B (NF-κB) expression using RAW264.7 macrophage cells.

Results

YeongyoSeungma-tang significantly inhibited generation of NO (42% and 59% inhibition at doses of 5 μg/mL and 10 μg/mL, respectively), PGE₂ (46% and 80% inhibition at doses of 5 μg/mL and 10 μg/mL, respectively) and TNF-α (6% and 23% inhibition at doses of 5 μg/mL and 10 μg/mL, respectively) on LPS-stimulated RAW 264.7 cells in a concentration-dependent manner. Consistently in these observations, the expression of inducible NO synthase (iNOS) enzyme was also inhibited by YeongyoSeungma-tang. However, YeongyoSeungma-tang did not show any influence on the expression of cyclooxygenase-2. The cream containing 0.075% YeongyoSeungma-tang showed good skin moisturizing effect without any irritation.

Conclusion

The present study may support the fact that YeongyoSeungma-tang can have the good possibility as an anti-inflammatory agent for troubled skins.     

Depressant effects of Clinopodium mexicanum Benth. Govaerts (Lamiaceae) on the central nervous system

Ethnopharmacological relevance

The decoction of leaves of Clinopodium mexicanum Benth. Goaverts (Lamiaceae), commonly known as “Toronjil de Monte”, is used in the Mexican traditional medicine to induce sleep, as well as sedative and analgesic remedy.

Aim of the study

To evaluate the putative depressant effects of an aqueous extract of the medicinal plant Clinopodium mexicanum on the central nervous system (CNS).

Materials and methods

The effects of the extract (AECM) on mice were tested in several animal paradigms, including sodium pentobarbital-induced sleep, open field tests, and hole-board tests. The effects of AECM on pentylenetetrazole- and picrotoxin-induced convulsions in mice and on the antithermonociceptive response in the hot-plate paradigm were also tested. Additionally, the active extract (AECM) was analyzed with HPLC–ESI-MS techniques.

Results

Mice acutely treated with AECM at 100, 200, 500 and 1000 mg/kg doses prolonged the sleeping time induced by sodium pentobarbital (42 mg/kg). This extract, at 100 and 200 mg/kg doses, showed a sedative effect in the hole-board paradigm and decreased spontaneous activity in mice. AECM at 10, 100 and 200 mg/kg prolonged the onset of seizures induced by pentylenetetrazole (90 mg/kg) and antagonized tonic convulsions induced by picrotoxin (10 mg/kg). Additionally, AECM inhibited the response to a thermonociceptive stimulus. The intraperitoneal AECM treatment produced mortality with an LD₅₀ = 2154 mg/kg. Chemical analysis showed that the flavanone glycosides neoponcirin, poncirin, and isonaringenin are the main compounds of the active extract.

Conclusions

This study demonstrates that an acutely administered single dose of an aqueous extract of Clinopodium mexicanum can exert depressant effects on the CNS. These findings are in agreement with the traditional use of Clinopodium mexicanum to induce sleep as well as sedative and analgesic remedy. The chemical analysis of AECM revealed the presence of the flavanone glycosides neoponcirin, poncirin, and isonaringin.     

Protective effects of butanol fraction from Betula platyphyla var. japonica on cartilage alterations in a rabbit collagenase-induced osteoarthritis

Aim of this study

Many cartilage protective agents have been developed from natural products, and they have resulted in the development of treatments for osteoarthritis. In this study, we determined the osteoarthritic efficacy and mechanism of butanol fraction from the bark of Betula platyphylla var. japonica (BFBP) in collagenase-induced rabbit model of osteoarthritis (CIA).

Materials and methods

The right knees of rabbits were injected intra-articularly with collagenase, and rabbits were orally administrated with distilled water (vehicle), BFBP (50, 100 and 200 mg/kg) or celecoxib (100 mg/kg) once a day for 28 days after the initiation of the CIA.

Results

Oral administration of BFBP dose-dependently suppressed the stiffness and global histologic score. Proteoglycan intensity was considerably increased in a dose-dependent manner. As well, the mRNA expression of MMP-1, and MMP-3 was decreased. On the contrary, the level of TIMP-1 in the synovial fluids was significantly increased in the BFBP treated group. The pathologic inflammatory molecules such as PGE₂ and COX-2 were inhibited by BFBP, but COX-1 expression not affected.

Conclusion

We suggest that BFBP has shown the protective effect on cartilage alternations through balance of MMPs/TIMP-1 and regulates inflammatory-related molecules in vivo model of osteoarthritis, and great candidate for development of osteoarthritis treatment.     

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.     

Potential anticancer properties of the water extract of Inontus obliquus by induction of apoptosis in melanoma B16-F10 cells

Ethnopharmacological relevance

Inonotus obliquus (Chaga mushroom), one of the widely known medicinal mushrooms, has been used to treat various cancers in Russia and most of Baltic countries for many centuries.

Aim of the study

To examine the anti-proliferative effects of Inonotus obliquus extract on melanoma B16-F10 cells. Furthermore, to assess the anti-tumor effect of Inonotus obliquus extract in vivo in Balb/c mice.

Materials and methods

The water extract of Inonotus obliquus was studied for anti-proliferative effects on the growth and morphology of B16-F10 melanoma cells and for anti-tumor effect using in vivo in Balb/c mice.

Results

Inonotus obliquus extract not only inhibited the growth of B16-F10 cells by causing cell cycle arrest at G₀/G₁ phase and apoptosis, but also induced cell differentiation. These effects were associated with the down-regulation of pRb, p53 and p27 expression levels, and further showed that Inonotus obliquus extract resulted in a G₀/G₁ cell cycle arrest with reduction of cyclin E/D1 and Cdk 2/4 expression levels. Furthermore, the anti-tumor effect of Inonotus obliquus extract was assessed in vivo in Balb/c mice. Intraperitoneal administration of Inonotus obliquus extract significantly inhibited the growth of tumor mass in B16-F10 cells implanted mice, resulting in a 3-fold (relative to the positive control, ^*p < 0.05) inhibit at dose of 20 mg/kg/day for 10 days.

Conclusion

This study showed that the water extract of Inonotus obliquus mushroom exhibited a potential anticancer activity against B16-F10 melanoma cells in vitro and in vivo through the inhibition of proliferation and induction of differentiation and apoptosis of cancer cells.