Anti-inflammatory effect of Inonotus obliquus, Polygala senega L., and Viburnum trilobum in a cell screening assay

Aim of the study

The purpose of the study was to assess the anti-inflammatory effects of the mushroom Inonotus obliquus (Chaga), Polygala senega (Senega) and Viburnum trilobum (Cranberry) bark extract fractions from locally produced materials in lipopolysaccharide (LPS) induced murine macrophage RAW 164.7 cells.

Materials and methods

Four fractions from each of the three extracts were obtained: (80% ethanol extracted; Fa), (water-soluble polysaccharide fraction; Fb), (Polyphenolic fraction; Fc) and (ETOAc/H₂O extracted fraction; Fd). These extract fractions were tested in the cell screening system at 50,100 and 500 μg/ml for their ability to inhibit LPS induced inflammatory cytokines IL-1β, TNFα and IL-6. Supernatants from LPS alone treated cells were used as control. The cytokines in the cell culture supernatants following treatments with extract fractions were quantified by ELISA method, using 96 well ELISA plates.

Results

All fractions of the extracts significantly inhibited (p < 0.05) the levels of IL-1β, IL-6 and TNFα except the polyphenolic Fc fraction of Senega which showed an increased production of IL-6. Furthermore, each fraction showed a dose-dependant anti-inflammatory effect. Nitric oxide production was not affected by cranberry and senega, while Chaga significantly reduced NO production in murine macrophage cell assay.

Conclusions

These results demonstrate that the extracts obtained from the root of Polygala senega L., bark of Viburnum trilobum, and the mushroom Inonotus obliquus possess anti-inflammatory properties when tested in a RAW 264.7 macrophage cell system.     

Anti-tumor activity of Annona squamosa seeds extract containing annonaceous acetogenin compounds

Ethnopharmacological relevance

Seeds of Annona squamosa L. have been used in the south of China as a folk remedy to treat “malignant sores” (cancer).

Aim of the study

To investigate the chemical constituents and the anti-tumor activity of the standardized A. squamosa seeds extract in vitro and in vivo.

Materials and methods

Annonaceous acetogenin profiles of the standardized extract were determined by using Fourier transform infrared (FT-IR) and high performance liquid chromatography (HPLC) techniques. The anti-tumor activity of the extract was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity in vitro and H₂₂ hepatoma cells transplantation tumor model in vivo.

Results

The FT-IR spectroscopy showed the presence of annonaceous acetogenin compounds in the extract. Two major annonaceous acetogenins: 12, 15-cis-squamostatin-A and bullatacin were identified and quantified by HPLC. The seed extract showed significant anti-tumor activity against four human tumor cell lines, especially for MCF-7 (IC₅₀. 0.25 μg/ml) and Hep G2 (IC₅₀. 0.36 μg/ml) cells in vitro. The extract inhibited the growth of H₂₂ tumor cells in mice with a maximum inhibitory rate of 69.55% by oral administration.

Conclusion

A. squamosa seed extract showed significant anti-tumor activities against human hepatoma cells in vitro and in vivo, indicating a potential for developing the extract as a novel anti-liver cancer drug.     

Anticancer potential of aqueous extract of alocasia macrorrhiza against hepatic cancer in vitro and in vivo

Ethnopharmacological relevance

Alocasia macrorrhiza has been used as a folk medicine for cancer treatment in the Southwest of China.

Aim of the study

The purpose of this study is to confirm the anticancer activity of aqueous extract of alocasia macrorrhiza against hepatic cancer and to elucidate its mechanism of action.

Materials and methods

Human normal liver cells and hepatocellular carcinoma cells were tested in vitro for cytotoxicity, colony formation inhibition, EdU incorporation, AO/EB staining apoptotic cells, apoptotic DNA fragmentation, and cell cycle distribution in response to alocasia macrorrhiza extract. The mRNA and protein expressions of PPARγ, Cyclin D1, Rb, P21, Bax, Bcl-2 and caspase-3 were detected through RT-PCR and Western blotting; the tumor growth inhibition in vivo was tested by oral administration of the extract.

Results

Alocasia macrorrhiza aqueous extract exhibited proliferation inhibition and apoptosis effects on human hepatocellular carcinoma cells in vitro, inhibited hepatoma growth in vivo.

Conclusion

Alocasia macrorrhiza extract has potential cytotoxic and apoptotic effect on human hepatocellular carcinoma cells and inhibits hepatoma growth in vivo, its mechanism of action might be associated with the inhibition of DNA synthesis, cell cycle (G₀/G₁) arrest, apoptosis induction through up-regulation the mRNA and protein expressions of PPARγ, Rb, Bax and capase-3genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes.     

Anti-tumor activity and relative mechanism of ethanolic extract of Marsdenia tenacissima (Asclepiadaceae) against human hematologic neoplasm in vitro and in vivo

Ethnopharmacological relevance

Marsdenia tenacissima, a traditional Chinese medicinal herb, endemic to Yunnan Province is widely used to treat cough, asthma, expectorant, esophageal cancer, gastric cancer, lung cancer, and hepatocellular carcinoma. The aim of this study was to evaluate in vitro and in vivo anti-hematologic neoplasm potential of the ethanolic extract of this herb (crude ethanolic extract of Marsdenia tenacissima, CME) and by using different assays to elucidate its possible mechanism of action.

Materials and methods

The cytotoxicity of CME on tumor cells and peripheral blood mononuclear cells (PBMCs) was evaluated using MTT and apoptosis assays. We also tested the effect of CME on colony formation inhibition and cell cycle distribution of tumor cells. The protein expressions of Cyclin D1, Bax, Bcl-2, caspase-3 and caspase-9 were detected through Western blotting. In vivo anti-tumor effect was evaluated by measuring tumor volume changes, measuring tumor weight, evaluation of tumor microvessel density (MVD) and TUNEL staining by using immunohistochemistry staining in tumor models of nude mice.

Results

Marsdenia tenacissima ethanolic extract exhibited effects of proliferation inhibition and induction of apoptosis on human hematologic neoplasm tumor cells in vitro, as well as hematologic neoplasm growth in vivo.

Conclusion

This study clearly indicated that the ethanolic extract of Marsdenia tenacissima displayed strong anti-tumor effects against hematologic neoplasm cells and could induce tumor cells apoptosis in vitro and in vivo, and also had a significant anti-angiogenic effect in vivo against tumor cell apoptosis. Its multi-mechanism of action might be associated with the cell cycle (G0/G1) arrest, induction of apoptosis through up-regulation protein expressions of Bax, caspase-9 and caspase-3 genes and down-regulation of the expressions of Cyclin D1 and Bcl-2 genes, a decrease in tumor microvessel density and an increase of TUNEL-positive cells in vivo. These findings provided the molecular theoretical basis of clinical application.     

Ganoderma tsugae extracts inhibit colorectal cancer cell growth via G(2)/M cell cycle arrest

Ethnopharmacological relevance

Ganoderma, known as Lingzhi or Reishi, has been traditionally administered throughout Asia for centuries as a cancer treatment and for other medicinal purposes.

Aim of the study

To investigate the inhibitory activity and explore the molecular mechanisms of anti-tumor effect on colorectal cancer cells in vitro and in vivo as well as to test the side effects of Ganoderma tsugae.

Materials and methods

Methanol fraction was obtained from dried fruiting bodies of Ganoderma. TLC and HPLC were performed to differentiate and confirm the identification of different species as well as to quantify the bioactive molecules in methanol extracts of Ganoderma species. MTT and Trypan blue exclusion assay as well as tumorigenesis study were used to assess the anti-tumor effect in vitro and in vivo. Using flow cytometry and Western Blots, we examined further the molecular mechanisms of anti-tumor effect. Finally, biochemical and hematological profiles and pathological examinations were used to evaluate the safety.

Results

The Ganoderma tsugae extracts inhibit colorectal cancer cell proliferation caused by accumulating cells in G₂/M phase, and it may be through downregulation of cyclin A and B1 and upregulation of p21 and p27. Tumorigenesis study in nude mice revealed the extracts caused tumor shrinkage. Additionally, safety assay showed Ganoderma tsugae extracts caused no significant side effects in an animal model.

Conclusions

This study provides molecular evidence that Ganoderma tsugae extracts exert anti-tumor effects both in vitro and in vivo on colorectal adenocarcinoma cells by inducing G₂/M cell cycle arrest. More importantly, no significant physiological changes resulting from treatment with Ganoderma tsugae extracts were observed in the animal model. Therefore, these data provide new insights into the possible therapeutic use of Ganoderma tsugae for treating colorectal cancer.     

Identification of Escherichia coli enterotoxin inhibitors from traditional medicinal herbs by in silico, in vitro, and in vivo analyses

Ethnopharmacological relevance

Glycyrrhiza uralensis has been used for the treatment of gastrointestinal disorders, such as diarrhea, in several ancient cultures. Glycyrrhizin is the principal component of liquorice and lots of pharmacological effects have been demonstrated.

Aim of the study

Heat-labile enterotoxin (LT), the virulence factor of enterotoxigenic Escherichia coli, induces diarrhea by initially binding to the G_M1 on the surfaces of intestinal epithelial cells and consequently leading to the massive loss of fluid and ions from cells. Therefore, we evaluated the inhibitory effects of traditional medicinal herbs (TMH) on the B subunit of LT (LTB) and G_M1 interaction.

Materials and methods

The inhibitory effects of TMH on LTB-G_M1 interaction were evaluated by G_M1-enzyme-linked immunosorbent assay (ELISA). The likely active phytochemicals of these TMH were then predicted by in silico model (docking) and analyzed by in vitro (G_M1-ELISA) and in vivo (patent mouse gut assay) models.

Results

We found that various TMH, which have been ethnomedically used for the treatment of diarrhea, inhibited the LTB-G_M1 interaction. Docking data showed that triterpenoids were the most active phytochemicals and the oleanane-type triterpenoids presented better LTB-binding abilities than other types of triterpenoids. Moreover, by in vitro and in vivo models, we demonstrated that glycyrrhizin was the most effective oleanane-type triterpenoid that significantly suppressed both the LTB-binding ability (IC₅₀ = 3.26 ± 0.17 mM) and the LT-induced fluid accumulation in mice.

Conclusions

We found an LT inhibitor, glycyrrhizin, from TMH by in silico, in vitro, and in vivo analyses.     

Neuroprotective effects of Fructus Chebulae extracts on experimental models of cerebral ischemia

Objective

To investigate the neuroprotective effects of Fructus Chebulae extract using both in vivo and in vitro models of cerebral ischemia.

Methods

As an in vitro model, oxygen glucose deprivation followed by reoxygenation (OGD-R) and hydrogen peroxide (H₂O₂) induced cellular damage in rat pheochromocytoma (PC12) cells was used to investigate the neuroprotective effects of extract of Fructus Chebulae. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to calculate cell survival. For in vivo, occlusion of left middle cerebral artery on rats was carried out as a focal cerebral ischemic model.

Results

Fructus Chebulae extract increases the PC12 cell survival against OGD-R and H₂O₂ by 68% and 91.4% respectively. Fructus Chebulae also decreases the cerebral infarct volume by 39% and extent of hemisphere swelling from 17% in control group to 10% in Fructus Chebulae treated group.

Conclusion

Fructus Chebulae, as a traditional medicine, can rescue the neuronal cell death against ischemia related damage. The possible mechanism for the neuroprotection might be the inhibition of oxidative damages occurring after acute phase of cerebral ischemia.     

Orally administered aqueous extract of Inonotus obliquus ameliorates acute inflammation in dextran sulfate sodium (DSS)-induced colitis in mice

Ethnopharmacological relevance

Chaga mushroom (Inonotus obliquus) has been used in folk medicine to treat several disorders through its various biological functions. I. obliquus is claimed to produce general immune-potentiating and strengthening, antiinflammatory, and antitumor properties, but its effects on intestinal inflammation (ulcerative colitis) are clearly not understood.

Aim of the study

To determine the effects and mode of action of an aqueous extract of I. obliquus (IOAE) on experimental colitis in mice induced by dextran sulfate sodium (DSS).

Materials and methods

Female 5-week-C57BL/6 mice were randomized into groups differing in treatment conditions (prevention and treatment) and doses of IOAE (50 and 100 mg/kg body weight). Mice were exposed to DSS (2%) in their drinking water over 7 day to induce acute intestinal inflammation. In colon tissues, we evaluated histological changes by hematoxylin and eosin staining, levels of iNOS by immuno-histochemical staining, and neutrophil influx by myeloperoxidase assay. mRNA expression of pro-inflammatory mediators TNF-α, IL-1β, IL-6, and IFN-γ was determined by RT-PCR.

Results

Histological examinations indicated that IOAE suppressed edema, mucosal damage, and the loss of crypts induced by DSS. IOAE markedly attenuated DSS-induced iNOS levels and myeloperoxidase accumulation in colon tissues, demonstrating its suppressive effect on infiltration of immune cells. In addition, IOAE significantly inhibited mRNA expression of pro-inflammatory cytokines induced by DSS in colon tissues.

Conclusion

Our results suggest anti-inflammatory effect of IOAE at colorectal sites due to down-regulation of the expression of inflammatory mediators. Suppression of TNF-α and iNOS together with IL-1β by IOAE denotes that it might be a useful supplement in the setting of inflammatory bowel disease.     

Antiproliferative and apoptosis inducing activity of Markhamia tomentosa leaf extract on HeLa cells

Ethnopharmacological relevance

Markhamia tomentosa (Benth) K. Schum ex. Engl. (Bignoniaceae), a tree widely dispersed in West Tropical Africa, is used traditionally to treat various diseases as it possesses antimicrobial, antioxidant, analgesic, anticancer and anti-inflammatory activities.

Materials and methods

This study evaluates the cytotoxic effect and underlying mechanisms of the ethanolic extract of Markhamia tomentosa on HeLa and MCF-7 cancer cell lines and non-cancerous Vero cell line. Brine shrimp lethality test was used for preliminary screening. Cytotoxicity was determined using the MTT assay and IC₅₀ was calculated. Effect of Markhamia tomentosa on the cell cycle was monitored by flow cytometry and the apoptosis-induction capability confirmed by exposure of phosphatidylserine to the outer leaflet of the plasma membrane. Loss of mitochondrial membrane potential was analysed by flow cytometry using JC-1.

Results

Markhamia tomentosa was toxic to brine shrimps with LD₅₀ of 31.62 µg/ml. Cell viability and growth of HeLa cells was inhibited by the extract with an IC₅₀ of 189.1±1.76 µg/ml at 24 h post treatment. However, no cytotoxic effect was observed in MCF-7 and Vero cell lines. The extract induced cell cycle arrest in HeLa cells in the G₀/G₁ phase resulting in cell death after 24 h exposure. Induction of apoptosis in HeLa cells was substantiated by Annexin V-FITC/PI double staining showing phosphatidylserine translocation and depolarisation of the mitochondrial membrane potential by flow cytometry of JC-1 stained cells.

Conclusion

The ethanolic extract of Markhamia tomentosa induces G₀/G₁ in HeLa cells followed by induction of the intrinsic pathway of apoptosis.     

Acorus gramineus inhibits microglia mediated neuroinflammation and prevents neurotoxicity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease

Ethnopharmacological relevance

Acorus gramineus Solander (Acoraceae, AG), is a widely distributed plant in Asian countries. Rhizome part of this plant has long been used as a traditional medicine for treating various symptoms including central nervous system (CNS) disorders.

Aim of study

The anti-neuroinflammatory effect of AG aqueous extract was investigated using in vitro cellular and in vivo Parkinson's disease (PD) mouse model.

Materials and methods

Lipopolysaccharide (LPS) is used to stimulate BV-2 microglial cells in vitro and the changes in neuroinflammatory expressional levels were measured using ELISA, Western blotting, RT-PCR and immunofluorescence techniques. In in vivo experiments, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mouse model of PD was developed followed by immunohistochemical analysis of specific brain tissues.

Results

LPS-stimulation to BV-2 cells increased the production of nitric oxide (NO) and proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β. Pretreatment with AG extract inhibited the increased levels of NO and pro-inflammatory cytokines in LPS-stimulated BV-2 cells. Mechanistic study revealed that AG acts via the regulation of nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPKs) and TRIF-dependent signaling pathways. Further, AG protected MPTP-induced neuronal cell death and inhibited neuroinflammation in vivo.

Conclusion

Our results indicated that AG extract exerted anti-neuroinflammatory effects against activated microglia mediated insults through multiple signaling pathways and prevented in vivo neuronal cell death in mouse model of PD substantiating the traditional claims for its use in CNS disorders.     

Hyperpigmentant activity of leaves and flowers extracts of Pyrostegia venusta on murine B16F10 melanoma

Ethnopharmacological relevance

Pyrostegia venusta is a native Brazilian plant which has a variety of uses in traditional folk medicine including the treatment of vitiligo. However, its effectiveness on melanogenesis is not yet elucidated.

Aim of the study

This study aimed to investigate the melanogenic activity of hydroalcoholic extracts from the leaves and flowers of P. venusta on murine B16F10 melanoma cells.

Materials and methods

Different concentrations of the hydroalcoholic extracts of flowers and leaves of P. venusta were evaluated in trials of spontaneous melanin content (4 days), and cell viability by the MTT assay in murine B16F10 cells, and in the mushroom tyrosinase activity in vitro.

Results

Both extracts, leaves (0.1; 0.3; 1 and 3 μg/mL) and flowers (0.03 and 0.1 μg/mL) increased the melanin content in a concentration dependent manner after 4 days of incubation on melanoma cells. Leaves extract promoted enhancement of melanogenesis with maximum effect of 33.3 ± 3% (3 μg/mL), and the flower extract increased in 23.4 ± 3% (0.1 μg/mL). The cell viability test using MTT showed that in the same tested concentrations of both extracts no cell death was detected. Actually, either extract was not able to cause any change in the tyrosinase activity. HPLC analysis of P. venusta extracts found 0.09% and 1.08% of allantoin on leaves and flowers extracts, respectively.

Conclusions

The leaves and flowers extracts of P. venusta stimulates B16F10 melanogenesis at very low concentrations. These findings support the folk medicinal use of P. venusta on the treatment of hypopigmentation diseases, such as vitiligo.     

Anti-tumor and pro-apoptotic activity of ethanolic extract and its various fractions from Polytrichum commune L.ex Hedw in L1210 cells

Ethnopharmacological relevance

Polytrichum commune L.ex Hedw is a traditional Chinese herb for treatment of fever, hemostatic, uterine prolapse and especially for lymphocytic leukemia, but the antitumor effect and its potential mechanism remains unclear.

Aims of the study

The present study was to investigate the possible anti-proliferative activity of ethanolic extract and the organic fractions from P. commune on murine leukemia L1210 cells.

Materials and methods

The content of ethanolic extract and its fractions was performed on HPLC analysis with gradient elution. L1210 cells were treated with different concentrations of ethanolic extract and its fractions at different time intervals. Cell viability was evaluated using MTT assay. Apoptotic cell death was monitored by nuclear condensation and confirmed by exposure of phosphatidylserine to outer leaflet of plasma membrane. Loss of mitochondrial membrane potential was analyzed by flow cytometry using rhodamine 123 staining.

Results

The obtained results showed that the cell viability of L1210 cells was reduced by ethanolic extract of P. commune in a concentration-dependent manner, and the IC₅₀ value was about 77.22 μg/ml at 24 h post treatment. The ethylacetate fraction displayed higher anti-tumor effect than that of chloroform and butanol fractions with 32.29 μg/ml (IC₅₀ value, 48 h). Microscopy studies revealed that ethanolic extract and ethylacetate fraction treated cells showed morphological characteristics of apoptosis such as chromatin condensation and DNA aggregation. Further, Annexin V-PE/ 7-AAD double staining showing the out leaflet of phosphatidylserine and the decline of mitochondrial membrane potential by flow cytometry confirmed that the extracts do, in fact, induce apoptosis in L1210 cells.

Conclusion

This is the first report on anti-tumor and pro-apoptotic effect of P. commune in cultured leukemia cells, which provides scientific basis for its usefulness as traditional medicine. Further studies are needed to confirm the precise mechanism not only the crude extract but as well the monomeric chemical substances of Polytrichum commune L.ex Hedw.     

Aqueous extract of Taxus Chinensis (Pilger) Rehd inhibits lung carcinoma A549 cells through the epidermal growth factor receptor/mitogen-activated protein kinase pathway in vitro and in vivo

Objective

To explore the anticancer mechanism of aqueous extract of Taxus Chinensis (Pilger) Rehd (AETC).

Methods

The serum pharmacological method was used to avoid interference from administration of the crude medicinal herbs. Eight purebred New Zealand rabbits were used for preparation of serum containing various concentrations of AETC. Forty-eight Balb/c-nu mice were used for in vivo experiments. The effects of serum containing AETC on the proliferation of A549 cells and expression levels of the epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) pathway-related proteins in vitro were investigated. Additionally, the effects on the growth of A549 xenografts in nude mice, and expression levels of the EGFR/MAPK pathway-related proteins in the xenografts, were investigated.

Results

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the serum containing AETC significantly decreased the viability of A549 cells in a dose-dependent manner. Western blot showed that the serum containing various concentrations of AETC strongly reduced the levels of phospho-Jun N-terminal kinase (p-JNK) and phospho-extracellular signal-regulated kinasel/2 (ERK1/2) while it increased the level of p-p38. However, no significant effects on the expression levels of JNK, ERK1/2, and p38 MAPK were found. In addition, an anticancer effect from AETC was observed in vivo in the Balb/c-nu mice bearing A549 xenografts.

Conclusion

AETC has significant effects on the growth of A549 xenografts and on the activity of the EGFR/MAPK pathway. Therefore, AETC may be beneficial in lung carcinoma treatment.     

In vitro and in vivo antioxidant activity of the ethanolic extract from Meconopsis quintuplinervia

Aims of the study

Meconopsis quintuplinervia, a medicinal herb endemic to the Tibetan region, is used to treat hepatitis. The aim of this study is to evaluate the antioxidant potential of the ethanolic extract of this herb using different assays.

Materials and methods

The antioxidant capacity of Meconopsis quintuplinervia was investigated using various established in vitro systems. An in vivo study of carbon tetrachloride (CCl₄)-induced antioxidant activity in mice was also conducted by examining the levels of malondialdehyde (MDA) and the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH).

Results

The extract showed strong in vitro antioxidant ability. In the in vivo study, CCl₄-induced oxidative stress caused significant decreases in the SOD, CAT, and GSH levels and a significant increase in the MDA level, most of which were significantly reversed (except for SOD in the liver.) by treatment with the extract and standard Vitamin E.

Conclusion

This study clearly indicates that the ethanolic extract of Meconopsis quintuplinervia is a valuable source of natural antioxidants. These findings provide scientific support for the traditional use of this herb as a Tibetan medicine for liver diseases.     

Genotoxic and antigenotoxic properties of Baccharis trimera in mice

Ethnopharmacological relevance

Baccharis trimera (Less.) (Asteraceae) is a native plant from Brazil. Also known as “carqueja”, it is popularly used to treat liver diseases, diabetes, as well as digestive disorders, mainly by women with lower socioeconomic status.

Aim of the study

The aim of the present study was to investigate the in vivo genotoxic/antigenotoxic and mutagenic potential of this plant, using the comet and the micronucleus assays.

Material and methods

Female adult mice were treated with 500, 1000 or 2000 mg/kg of B. trimera aqueous extract (Bt-AE) by gavage for three consecutive days.

Results

Independently of the dose, no genotoxic effect was observed in blood and liver samples analyzed by the comet assay. Conversely, B. trimera showed an antigenotoxic effect in blood from treated mice, thus protecting cells against oxidative DNA damage induced by the ex vivo treatment with hydrogen peroxide. In addition, Bt-AE showed in vitro antioxidant activity, assessed by DPPH and xanthine oxidase assays, suggesting that the observed antigenotoxic effects might be related to its antioxidant properties.

Conclusions

However, the extract increased the frequency of micronucleus in bone marrow of treated mice, indicating a chromosomal mutagenic activity. Thus, medicines prepared from this plant should be used with caution, although the results also suggest antigenotoxic effects for B. trimera aqueous extract.     

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.     

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.     

Genotoxic and mutagenic properties of Bauhinia platypetala extract,a traditional Brazilian medicinal plant

Ethnopharmacological relevance

Bauhinia platypetala Burch. is a traditionally used Brazilian medicinal plant, although no evidence in the literature substantiates the safety of its use.

Aim of the study

The aim of this study was to investigate the safety of the ethanolic extract and the ethereal fraction of B. platypetala leaves.

Materials and methods

The identification of chemical compounds from the B. platypetala ethanolic extract and its ethereal fraction was performed by GC/MS and ESI-MS/MS. The plant’s toxicological, cytotoxic, mutagenic and genotoxic properties were determined in Saccharomyces cerevisiae strains and V79 cell culture by survival assays and comet assay.

Results

The major compound identified in the B. platypetala ethanolic extract is palmitic acid, kaempferitirin and quercitrin, while the B. platypetala ethereal fraction was found to be rich in phytol, gamma-sitosterol and vitamin E. Moreover, the results indicated that the B. platypetala ethanolic extract has an anti-oxidative effect against H₂O₂ in yeast. In addition, the B. platypetala ethanolic extract did not induce mutagenic effects on the S. cerevisiae N123 strain, but the ethereal fraction of B. platypetala at higher concentrations (250–500 μg/mL) induced cytotoxicity and mutagenicity. A slight cytotoxic effect was observed in mammalian V79 cells; however, both the B. platypetala ethanolic extract and its ethereal fraction were able to induce DNA strand breaks in V79 cells, as detected by the alkaline comet assay.

Conclusion

The B. platypetala ethanolic extract has antioxidant action and showed absence of mutagenic effects in yeast S. cerevisiae. On the other hand B. platypetala ethereal fraction is mutagenic and does not show antioxidant activity in yeast. In mammalian cells B. platypetala ethanolic extract and it's ethereal fraction induce cyotoxic and genotoxic action.     

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.     

Anti-inflammatory and anti-allergic effects of Agrimonia pilosa Ledeb extract on murine cell lines and OVA-induced airway inflammation

Ethnopharmacological evidence

Agrimonia pilosa Ledeb (Rosaceae, AP) has long been used as a traditional medicine in Korea and other Asian countries to treat various diseases.

Aim of the study

In the present study, the anti-inflammatory and anti-allergic effects of AP extract in in vitro cell lines and in vivo mouse model of inflammation and the molecular mechanisms involved were reported.

Materials and methods

Using Raw 264.7 murine macrophages the effects of methanol extract of AP in lipopolysaccharide (LPS)-induced production of inflammatory mediators were measured. Further IgE-DNP-induced interleukin (IL)-4 production and degranulation in RBL-2H3 rat basophilic cell lines was also estimated. To investigate the anti-asthmatic effect of AP in vivo, airway inflammation in ovalbumin (OVA)-induced mouse model was used.

Results

AP attenuated the production of inflammatory mediators such as NO, PGE₂ and pro-inflammatory cytokines in LPS-induced Raw 264.7 cells. Further, AP inhibited IL-4 production and degranulation in IgE-DNP-induced RBL-2H3 cells. Furthermore, AP attenuated the infiltration of immune cells into lung, cytokines production in broncho-alveolar lavage fluid (BALF) and airway-hyperresponsiveness (AHR) on OVA-induced mouse model of inflammation.

Conclusion

Our results showed that AP attenuated the activation of macrophages, basophils, and inhibited the OVA-induced airway inflammation. The molecular mechanisms leading to AP's potent anti-inflammatory and anti-allergic effects might be through regulation of TRIF-dependent and Syk-PLCγ/AKT signaling pathways, suggesting that AP may provide a valuable therapeutic strategy in treating various inflammatory diseases including asthma.