Drosera rotundifolia and Drosera tokaiensis suppress the activation of HMC-1 human mast cells

Ethnopharmacological relevance

Several Northern Hemisphere Drosera species have been used in the therapy of respiratory tract infections as the traditional medicine Droserae Herba.

Aim of the study

To determine the anti-inflammatory effects of Drosera species and to investigate a substitute material for Droserae Herba, we examined the effect of extracts of Drosera rotundifolia, Drosera tokaiensis and Drosera spatulata on activated T cell membrane (aTc-m)-induced inflammatory gene expression in HMC-1 human mast cells.

Materials and methods

Drosera rotundifolia, Drosera spatulata and Drosera tokaiensis were collected in Japan. Herbs were extracted with 80% EtOH, and subsequently applied to OASIS HLB column. HMC-1 cells were treated with each Drosera column-adsorbed fraction for 15 min, and subsequently added to aTc-m and incubated for 16 h. Inflammatory gene and protein expressions were determined by DNA microarray, RT-PCR and Western blotting.

Results

Drosera rotundifolia and Drosera tokaiensis fractions, but not the Drosera spatulata fraction, suppressed inflammatory gene expression induced by aTc-m in HMC-1 cells.

Conclusions

Drosera rotundifolia and Drosera tokaiensis suppressed activation of HMC-1 cells induced by aTc-m. Since the Drosera tokaiensis fraction was more effective than the traditionally used Drosera rotundifolia, Drosera tokaiensis is a likely substitute as a source of Droserae Herba.     

Antiallergic effects of Scutellaria baicalensis on inflammation in vivo and in vitro

Ethnopharmacological relevance

Scutellaria baicalensis (SB) is one of the most widely used medicinal herbs for the treatment of inflammation. In this study, we investigated the antiallergic effect of SB in vivo and in vitro.

Materials and methods

Sprague–Dawley (SD) rats received intradermal injections of anti-DNP IgE at each of three dorsal skin sites. Forty-eight hours later, each rat received an injection of DNP-HSA in saline containing 4% Evans blue through the dorsal vein of the penis. One hour before injection, SB extract was administered orally. The dorsal skin of the rats was removed and the pigment area measured. In addition, rat peritoneal mast cells (RPMCs) were cultured and purified to investigate histamine release. In vitro, human mast cells (HMC-1) were pretreated with SB extract for 30 min before stimulation with phorbol 12-myristate 13-acetate (PMA) plus A23187. The effects on pro-inflammatory cytokine expression and mitogen activated protein (MAP) kinase expression were investigated using TNF-α and IL-8 assays, and Western blotting analysis of HMC-1 cells.

Results and conclusions

SB treatment inhibited the passive cutaneous anaphylaxis reaction compared to the control group, and histamine release decreased significantly following treatment of RPMCs with SB. In HMC-1 cells, SB restored IL-8 and TNF-α expression and inhibited MAP kinase expression in compound 48/80-induced HMC-1 cells. These data suggest that SB may prove to be a useful anti-inflammatory agent through its downregulation of the expression of various inflammatory mediators.     

Inula japonica extract inhibits mast cell-mediated allergic reaction and mast cell activation

Ethnopharmacological relevance

The flowers of Inula japonica (Inulae Flos) have long been used in traditional medicine for the treatment of bronchitis, digestive disorders, and inflammation. However, the mechanisms underlying its anti-inflammatory effects remain yet to be elucidated. The objectives of this study were 1) to assess the anti-allergic activity of the ethanol extract of flowers of Inula japonica extract (IFE) in vivo, 2) to investigate the mechanism of its action on mast cells in vitro, and 3) to identify its major phytochemical compositions.

Materials and methods

The anti-allergic activity of IFE was evaluated using mouse bone marrow-derived mast cells (BMMCs) in vitro and a passive cutaneous anaphylaxis (PCA) animal model in vivo. The effects of IFE on mast cell activation were evaluated in terms of degranulation, eicosanoid generation, Ca²⁺ influx, and immunoblotting of various signaling molecules.

Results

IFE inhibited degranulation and the generation of eicosanoids (PGD₂ and LTC₄) in stem cell factor (SCF)-stimulated BMMCs. Biochemical analysis of the SCF-mediated signaling pathways demonstrated that IFE inhibited the activation of multiple downstream signaling processes including mobilization of intracellular Ca²⁺ and phosphorylation of the mitogen-activated protein kinases (MAPKs), PLCγ1, and cPLA₂ pathways. When administered orally, IFE attenuated the mast cell-mediated PCA reaction in IgE-sensitized mice. Its major phytochemical composition included three sesquiterpenes, 1-O-acetylbritannilactone, britanin and tomentosin.

Conclusions

This study suggests that IFE modulates eicosanoids generation and degranulation through the suppression of SCF-mediated signaling pathways that would be beneficial for the prevention of allergic inflammatory diseases. Anti-allergic activity of IFE may be in part attributed particularly to the presence of britanin and tomentosin as major components evidenced by a HPLC analysis.     

Cheongyeolsaseuptang inhibits production of TNF-alpha, IL-6 and IL-8 as well as NF-kappa B activation in human mast cells

Traditional Korean medicine, Cheongyeolsaseuptang (CYSST) has been widely applied as a treatment of rheumatoid arthritis (RA) in Korea. However, its effect in experimental models remains unknown. Recent reports suggest that in patients with RA, synovial mast cells increase in number and show signs of activation and production of cytokines. In this study, we investigated the effect of CYSST on production of cytokines by activated human mast cell line, HMC-1. When CYSST (1mg/ml) was added, the production of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-8 was significantly inhibited about 37, 33.6, and 48%, respectively on phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187-stimulated HMC-1 cells. In addition, CYSST inhibited PMA plus A23187-induced activation of nuclear factor-kappaB. These findings may help understanding the mechanism of action of this medicine leading to control activated mast cells on inflammatory condition like RA.     

Suppressive effects of Houttuynia cordata Thunb (Saururaceae) extract on Th2 immune response

ETHNOPHARMACOLOGICAL RELEVANCE: Houttuynia cordata Thunb (Saururaceae), known as 'E-Sung-Cho' in Korea, has been traditionally used for the treatment of herpes simplex, chronic sinusitis, and allergy. AIM OF THE STUDY: To investigate the inhibitory activity of Houttuynia cordata Thunb fractions (HcFs) on the T helper 2 (Th2) immune response, we evaluated the alternation of the release of Th2-type cytokines and chemokines such as interleukin (IL)-4 and IL-5, and thymus and activation-regulated chemokine (TARC/CCL17). MATERIALS AND METHODS: Ethanol fraction was obtained from dried and powdered whole plants of Houttuynia cordata Thunb using ethanol. The residue was diluted with water and was then successively partitioned with n-hexane, EtOAc and BuOH. HcFs include ethanol, n-hexane, EtOAc, BuOH and water fractions. RT-PCR and ELISA were performed to measure mRNA and protein expression of cytokines. RESULTS: HcFs inhibited the expression of IL-4 and IL-5 in response to phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (CaI) in Jurkat T cells and the human mast cell line, HMC-1. IL-4- and tumor necrosis factor-alpha (TNF-alpha)-induced TARC production was blocked by HcFs in skin fibroblast CCD-986sk cells, particularly by the ethanol extract of Hc. Stimulants included in PMA, phytohemagglutinin (PHA) and CaI, increased the mRNA level of CC chemokine receptor 4 (CCR4), a receptor of TARC, in Jurkat T cells, and the ethanol extract of HcF weakly blocked the increased mRNA level. However, the stimulants and ethanol extract had no effect on the CCR4 protein level. The ethanol extract inhibited TARC-induced migration, as well as basal migration of Jurkat T cells. CONCLUSIONS: This study may show the usefulness of HcFs in the ethnopharmacological treatment of Th2-mediated or allergic inflammation, through the down-regulation of the production of Th2 cytokines and TARC, as well as cell migration.     

Induction of apoptotic cell death by Pharbitis nil extract in HER2-overexpressing MCF-7 cells

Aim of the study

We performed this study to investigate the anti-cancer activity of Pharbitis nil (PN) ethanol extract which has been used for herbal medicinal treatment against diseases in East Asia.

Materials and methods

We analyzed the effects of PN extract on proliferation of breast cancer cell lines, MCF-7 control vector (vec) and MCF-7 human epidermal growth factor receptor 2 (HER2) cells engineered to overexpress oncogenic HER2 via retroviral infection. We performed the proliferation assay to measure the growth rate of the cells. FACS analysis was used to analyze the cell cycle. Western blot analysis was used to investigate the effect of PN on the level and activation of intracellular molecules.

Results

We found that PN extract inhibited the proliferation of both MCF-7 vec and MCF-7 HER2 cells. This growth inhibition was accompanied with the increase of sub G0/G1 apoptotic fractions. When we check the efficiency of PN on the level of intracellular signaling molecules, we found that PN extract induced the inhibition of phosphorylation of HER2 and its downstream effectors, Akt and extracellular signal-regulated kinases (ERK). Active forms of both Akt and ERK were gradually decreased in PN-treated MCF-7 vec and MCF-7 HER2 cells suggesting that the growth suppressive activity of PN is related to signaling pathway. The level of cyclin D also diminished in PN-treated both cells suggesting that PN may inhibit the growth of MCF-7 vec and MCF-7 HER2 cells by perturbing cell cycle progression. It should be noted that PN decreased the growth rate of both MCF-7 vec and MCF-7 HER2 cells without changing the level and activation of p53.

Conclusion

PN extract suppressed the proliferation rate of HER-2 overexpressing MCF-7 breast cancer cells inducing apoptotic cell death in vitro. Our data demonstrates that PN extracts contain useful anti-tumor activity especially against HER2 overexpressing breast cancer.     

Mast cell stabilizing effects of Sphaeranthus indicus

Aim of the study

We investigated the effects of Sphaeranthus indicus on mast cell stabilizing activity to provide scientific basis for the clinical use of S. indicus.

Materials and methods

The protective effect of different extracts of whole plant of S. indicus against compound 48/80 and sheep serum induced mast cell degranulation was evaluated.

Results

Ethanol extract of S. indicus at the doses of 150 mg/kg and 300 mg/kg and ethyl acetate extract at the dose of 100 mg/kg, 150 mg/kg and 300 mg/kg showed slightly better protection of mast cell degranulation (77–86%) than the standard drug ketotifen (75%) in the sheep serum model. These extracts also showed better mast cell stabilizing activity (77–88%) than the standard drug (69%) when peritoneal mast cells are treated with compound 48/80.

Conclusion

These results suggest that S. indicus has potent mast cell stabilizing effects thereby inhibiting mediator release from mast cells.     

Regulatory effect of atopic allergic reaction by Carpopeltis affinis

Carpopeltis affinis Okamura (CA, Halymeniaceae) has long been used as therapeutics for various allergic diseases in Korea. The precise effects of CA in experimental models, however, have remained unknown. We studied the effects of a methanol extract of CA on atopic allergic reaction. Histamine content was measured by the o-phthalaldehyde spectrofluorometric procedure. Cytokines were measured by a modified enzyme-linked immunosorbent assay. Cytotoxicity was determined by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. CA significantly inhibited the histamine release and beta-hexosaminidase release from rat peritoneal mast cells. CA also inhibited interleukin-8 and tumor necrosis factor-alpha secretion from the phorbol 12-myristate 13-acetate and A23187-induced HMC-1 cells (human mast cell line). 48 h exposure to CA (1.0, 10, and 100 microg/ml) had little effect on HMC-1 cell viability. Our results suggest that CA has an inhibitory effect on mast cell-dependent allergic reaction and thus may be useful in the treatment of atopic dermatitis.     

Evaluation of the genotoxicity of extracts of Houttuynia cordata Thunb

The present study was conducted to evaluate the activity of methanol extracts from Houttuynia cordata Thunb. (HC) in a reverse mutation assay in Salmonella typhimurium, and a chromosome aberration assay in the Chinese hamster ovary (CHO) cell line and to evaluate its effect on the occurrence of polychromatic erythrocytes in mice. In the reverse mutation assay using Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2urvA(-), methanol extracts of HC (5, 2.5, 1.25, 0.62, or 0.312 mg/plate) did not induce reverse mutations in the presence or absence of an S9 metabolic activation mixture. In the chromosome aberration test using CHO cells, methanol extracts (1.25, 2.5 or 5 μg/ml) caused a few incidences of structural and numerical aberrations, in both of absence or presence of an S9 metabolic activation mixture, but in comparison with the positive control group, these incidences were not significantly increased. In the mouse micronucleus test, no significant increases in the occurrence of micronucleated polychromatic erythrocytes were observed in male ICR mice that were orally administered methanol extracts of HC at doses of 2.0, 1.0, or 0.5 g/kg. From these results, we concluded that the methanol extracts of HC did not induce harmful effects on genes in bacteria, a mammalian cell system or in mouse bone marrow cells. Thus, HC's use for health promotion and/or a sick remedy for humans may be safe.     

Antileukemic activity of Bidens pilosa L. var. minor (Blume) Sherff and Houttuynia cordata Thunb.

To evaluate the anti-leukemic activity of Bidens pilosa L. var. minor (Blume) Sherff and Houttuynia cordata Thunb., cytotoxicity tests with an XTT-based colorimetric assay were used. Five leukemic cell lines, namely L1210, U937, K562, Raji and P3HR1, were cultured with hot water extracts of B. pilosa var. minor or H. cordata. Hot water extracts of B. pilosa var. minor inhibited these five leukemic cells with IC50s between 145 microg/ml and 586 microg/ml. The effect was greatest on four cell lines, namely L1210, P3MR1, Raji and K562, with IC50s below 200 microg/ml and a selective index of more than 5. Hot water extract of H. cordata inhibited these five leukemic cells with IC50s between 478 microg/ml and 662 microg/ml. The selective index was between 1.5 and 2.1. B. pilosa var. minor was more effective than H. cordata in inhibiting most of the leukemic cells in our study. We suggest that B. pilosa L. var. minor (Blume) Sherff may prove to be a useful medicinal plant for treating leukemia.     

Anti-inflammatory activity of herbal medicines: inhibition of nitric oxide production and tumor necrosis factor-alpha secretion in an activated macrophage-like cell line

Houttuynia cordata Thunb. (HC), Glycyrrhiza uralensis Fischer (GU), Forsythia suspense (Thunb.) Vahl (FS), and Lonicera japonica Thunb. (LJ) are Chinese herbs known to possess anti-inflammatory properties. The effects of aqueous extracts of these herbs on the production of the pro-inflammatory mediators, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) were examined in an activated macrophage-like cell line, RAW 264.7 cells. Aqueous extracts from FS at 0.0625-2.0 mg/ml inhibited in vitro production of NO and secretion of TNF-alpha in a dose-dependent manner. FS at 1.0-2.0 mg/ml and 0.125-2.0 mg/ml significantly inhibited NO production and TNF-alpha, respectively. An extract of LJ demonstrated potent inhibition of both NO production and TNF-alpha secretion in a dose-dependent manner. An aqueous extract from HC inhibited NO production in a dose-dependent manner, but minimally (approximately 30%) inhibited TNF-a secretion at 0.0625 and 0.125 mg/ml. In contrast, an aqueous extract of GU had a minimal effect on both the production of NO and the secretion of TNF-alpha. Viability of cells at all concentrations studied was unaffected as determined by MTT cytotoxicity assay and trypan blue dye exclusion. These results suggest that aqueous extracts from FS, LJ and HC have anti-inflammatory actions as measured by inhibition of NO production and/or TNF-alpha secretion.     

Antihistaminic and mast cell stabilizing activity of Striga orobanchioides

The ethanolic and aqueous extracts of the whole plant of S. orobanchioides were evaluated for antihistaminic and mast cell stabilizing activities. Both extracts inhibited histamine-induced contractions of the guinea-pig ileum at the concentration range of 2.5-25 microg/ml in a dose-related manner. At 25 microg/ml, both extracts inhibited the response of histamine (0.5 microg/ml) almost completely. The effect of these two extracts on the degranulation rate of sensitized peritoneal cells of albino rats when challenged with antigen (horse serum) was studied. Triple vaccine was used as adjuvant. Ketotifen and prednisolone were used for comparison. The ethanolic extract at 100 and 200 mg/kg body weight was found to significantly inhibit degranulation of mast cells to an extent of 52.14+/-3.24 and 67.96+/-3.70%, respectively. At the same doses, the aqueous extract showed 42.09+/-2.91 and 60.67+/-3.50% reduction in degranulation of mast cells, respectively. Hence, both extracts markedly protected the rats against antigen-induced challenge of mast cells.     

Physalins from Witheringia solanacea as modulators of the NF-kappaB cascade

Crude extracts of Witheringia solanacea leaves showed inhibition of NF-kappaB activation at 100 microg/mL induced by phorbol 12-myristate-13-acetate (PMA) in HeLa cells stably transfected with a luciferase reporter gene controlled by the IL-6 promoter. Three physalins were isolated from an active fraction, namely, physalins B (1), F (2), and D (3). Of these compounds, 1 and 2 demonstrated inhibitory activities on PMA-induced NF-kappaB activation at 16 and 8 microM and induced apoptosis after 24 h in a cell-cycle analysis using a human T cell leukemia Jurkat cell line. Compound 2 also inhibited TNF-alpha-induced NF-kappaB activation at 5 microM through the canonical pathway, but was inactive in the Tet-On-Luc assay, indicating specificity of action, although it interfered with Tet-On-Luc at higher concentrations. It is suggested that the presence of a double bond and an epoxy ring between carbons 5 and 6 in compounds 1 and 2, respectively (which are not present in compound 3), are related to their anti-inflammatory activity.     

Growth inhibition and induction of apoptosis in human oral squamous cell carcinoma Tca-8113 cell lines by Shikonin was partly through the inactivation of NF-kappaB pathway

Shikonin, a naphthoquinone pigment isolated from the Chinese herbal therapeutic, Zicao, has been shown to exhibit antioxidant and anticancer effects. In this study, its ability to induce apoptosis in cultured Tca-8113 oral cancer cells was studied. Treatment of the Tca-8113 cells with a variety of concentrations of Shikonin (10-40 microm) resulted in dose- and time-dependent sequences of events marked by apoptosis, as shown by the loss of cell viability, chromatin condensation, internucleosomal DNA fragmentation and sub-G1 phase accumulation. Furthermore, apoptosis in the Tca-8113 cells was accompanied by the activation of protease caspase-8, -9, -3 and low expression of Bcl-2 protein. Interestingly, inactivation of the NF-kappaB pathway was found in shikonin-induced apoptosis in Tca-8113 cells. These results raise the possibility that the anti-tumor effects of Shikonin in Tca-8113 cells are at least partly through the inactivation of the NF-kappaB pathway and subsequent activation of protease caspase family. Pharmacological inhibition of the NF-kappaB activity by Shikonin might be a powerful treatment option for OSCC in which activation of NF-kappaB plays a critical role in tumor growth and progression.