Kaempferitrin induces immunostimulatory effects in vitro

Ethnopharmacological relevance

Justicia spicigera is a plant used as immunostimulatory in Mexican traditional medicine. Recently, we showed that Justicia spicigera extracts exerted immunostimulatory effects and the major component of this extract was kaempferitrin (KM). This work shows a correlation between the medical traditional use of Justicia spicigera and kaempferitrin, its active compound.

Materials and methods

The in vitro immunostimulatory effects of KM were evaluated on the proliferation of murine splenocytes and macrophages, and human peripheral blood mononuclear cells (PBMC). The effects of KM on NO production, lysosomal enzyme activity and neutral red uptake were assayed in murine macrophages RAW 264.7. The effects of KM on the NK cell activity were also assayed.

Results

KM at 25 μM, the highest concentration tested, increased the proliferation of murine macrophages (23%) and splenocytes (17%), and human PBMC (24%) in the absence of lipopolysaccharides (LPS), compared to untreated cells. KM also stimulated the pinocytosis (25%) and lysosomal enzyme activity (57%) in murine macrophages with a similar potency than LPS 1 μg/ml. In addition, KM induced the NK cell activity (11%).

Conclusion

KM exerts immunostimulatory effects on immune responses mediated by splenocytes, macrophages, PBMC and NK cells.     

Antitumor and immunomodulatory effects of Justicia spicigera Schltdl (Acanthaceae)

Ethnopharmacological relevance

Medicinal plants are an important source of antitumor compounds. This study evaluated the acute toxicity in vitro and in vivo, as well as the cytotoxic, antitumor and immunomodulatory effects of ethanolic extracts of Justicia spicigera leaves (JSE).

Materials and methods

The in vitro and in vivo toxicity of JSE was evaluated with comet assay in peripheral blood mononuclear cells (PBMC) and acute toxicity in mice, according to the Lorke procedure, respectively. The apoptotic effect of JSE on human cancer cells and human noncancerous cells was evaluated using flow cytometry with annexin-Alexa 488/propidium iodide. Also, different doses of JSE were injected intraperitoneally daily into athymic mice bearing tumors of HeLa cells during 18 days. The growth and weight of tumors were measured. The in vitro immunomodulatory effects of JSE were evaluated estimating the effects of JSE on the phagocytosis of the yeast Saccharomyces cerevisiae, NO production and H₂O₂ release in macrophages, as well as the proliferation of splenocytes and NK activity.

Results

The comet assay showed that only JSE tested at 200 and 1000 μg/ml induced a significantly DNA damage in PBMC, compared to untreated cells, whereas the LD₅₀ was >5000 mg/kg by intraperitoneal route (i.p.) and by oral route. JSE showed pro-apoptotic (Annexin/PI) effects by 35% against HeLa cells, but lack toxic effects against human normal cells.JSE administrated at 10, 50 and 100 mg/kg i.p. inhibited the tumor growth by 28%, 41% and 53%, respectively, in mice bearing HeLa tumor. JSE stimulated, in a concentration dependent manner, the phagocytosis of Saccharomyces cerevisiae yeasts, the NO production and H₂O₂ release by human differentiated macrophages. In addition, JSE stimulated the proliferation of murine splenocytes and induced the NK cell activity.

Conclusion

Justicia spicigera shows low toxic effects in vitro and in vivo, exerts apoptotic effects on HeLa cells, has antitumor effects in mice bearing HeLa tumor and induces immunomodulatory activities in vitro.     

Antidiabetic effects of Justicia spicigera Schltdl (Acanthaceae)

Ethnopharmacological importance

Justicia spicigera is a plant species used for the Teenak (Huesteca Potosina) and Mayan (Yucatan peninsula) indigenous for the empirical treatment of diabetes, infections and as stimulant.

Aim of the study

To evaluate the cytotoxicity, antioxidant and antidiabetic properties of J. spicigera.

Materials and methods

The effects of ethanolic extracts of J. spicigera (JSE) on the glucose uptake in insulin-sensitive and insulin-resistant murine 3T3-F442A and human subcutaneous adipocytes was evaluated. The antioxidant activities of the extract of JSE was determined by ABTS and DPPH methods. Additionally, it was evaluated the antidiabetic properties of JSE on T2DM model.

Results

JSE stimulated 2-NBDG uptake by insulin-sensitive and insulin-resistant human and murine adipocytes in a concentration-dependent manner with higher potency than rosiglitazone 1 mM. JSE showed antioxidant effects in vitro and induced glucose lowering effects in normoglycemic and STZ-induced diabetic rats.

Conclusion

The antidiabetic effects of administration of J. spicigera are related to the stimulation of glucose uptake in both insulin-sensitive and insulin-resistant murine and human adipocytes and this evidence justify its empirical use in Traditional Medicine. In addition, J. spicigera exerts glucose lowering effects in normoglycemic and STZ-induced diabetic rats.     

Antitumor effect of Croton lechleri Mull. Arg. (Euphorbiaceae)

Ethnopharmagological relevance

Medicinal plants are an important source of antitumor compounds. This study evaluated the acute toxicity in mice, as well as the cytotoxic and antitumoral effects of methanolic extracts of Croton lechleri leaves (CLE).

Materials and methods

The cytotoxicity of CLE on human cancer cell lines and human non-cancerous cells was evaluated using the MTT and apoptosis assays. Apoptosis induced by CLE on human cancer cell lines was determined using flow cytometry with annexin-Alexa 488/propidium iodide. The acute toxicity in mice was performed according to the Lorke procedure. Different doses of CLE were injected intraperitoneally daily into athymic mice bearing tumor during 18 days. The growth and weight of tumors was measured.

Results

CLE showed low IC₅₀ values on HeLa (17 μg/ml) cells but lack toxic effects against human normal cells. Induction of cell death in HeLa cells by CLE was confirmed by an increase of apoptosis (Annexin/PI) by 30% compared to untreated cells. The LD₅₀ was 356 mg/kg by intraperitoneal route (i.p.) and 500 mg/kg by oral route. CLE administrated at 1, 10 and 50 mg/kg i.p. inhibited the tumor growth by 38%, 48% and 59%, respectively, in mice bearing HeLa tumor.

Conclusion

Croton lechleri shows moderate toxic effects in vivo, exerts cytotoxic effects on HeLa cells and has antitumor effects in mice bearing HeLa tumor.     

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.     

Rhizoma Paridis ethanol extract selectively inhibits the proliferation of HUVECs comparing to Lovo cells and shows anti-angiogenesis effects in a mouse model

Ethnopharmacological relevance

Rhizoma Paridis, a traditional Chinese Medicine, was identified to be cytotoxic to cancer cells. The present study was designed to investigate the potential anti-angiogenic and antitumor effect of the ethanol extract of Rhizoma Paridis (RPE) in vitro and in vivo.

Materials and methods

The cytotoxic effect of RPE against human colon cancer Lovo cells and human umbilical vein endothelial cells (HUVECs) was examined using MTT assay. We also tested the effect of RPE on tube formation, migration, apoptosis and cell cycle of HUVECs. Moreover, Lovo subcutaneous xenograft was applied to study the antitumor and anti-angiogenesis effect of RPE in vivo.

Results

RPE exerted a higher inhibition effect on the proliferation of HUVECs than Lovo cells. The tube formation and cell migration were also significantly inhibited in the presence of RPE in a concentration-dependent manner though the significant inhibition effects were observed at the cytotoxic dose. RPE induced cell apoptosis and G0–G1 cell cycle arrest of HUVECs. In vivo, significant tumor growth inhibition was observed in human colon cancer xenografts established by Lovo cells, accompanying by a marked decrease in MVD.

Conclusions

Our current study exhibited that RPE has a selective cytotoxity against HUVECs comparing to Lovo cells and also demonstrated significant anti-angiogenic effect in vivo.     

Cytotoxic and apoptotic activity of Cheilanthes farinosa (Forsk.) Kaulf. against human hepatoma,Hep3B cells

Aim of the study

Cheilanthes farinosa (Forsk.) Kaulf., family: Adianthaceae, is a fern of immense medicinal properties used in ethno-medicine. The Gaddis tribe of Himachal Pradesh, India, has been using this fern to treat liver damage. Aim of the current study was to determine the apoptosis inducing and cytotoxic activity, if any, of this fern towards hepatic cancer cells.

Materials and methods

Water extract of the plant was used in the study. MTT assay was performed in hepatocellular carcinoma cell line, Hep3B as well as murine macrophage cell line, RAW264.7 to analyze the cytotoxic activity of the plant. Further, the apoptosis inducing action of water extract of the plant was evaluated using comet assay, DNA fragmentation analysis, DAPI staining of chromatin and Annexin V-FITC staining.

Results

This plant was found to produce considerable cytotoxicity in hepatoma cell line, Hep3B without inducing substantial damage to non-cancerous cell line RAW264.7. In addition, this plant was found to induce apoptosis in Hep3B cells. This was substantiated by comet assay, DNA fragmentation analysis, DAPI staining of chromatin and Annexin V-FITC staining for detecting early stage of apoptosis.

Conclusions

This investigation shows that the water extract of Cheilanthes farinosa has antiproliferative and apoptotic activity in human liver cancer cells and is not deleterious towards non-cancerous macrophage cell line.     

Mirabijalone E: A novel rotenoid from Mirabilis himalaica inhibited A549 cell growth in vitro and in vivo

Ethnopharmacological relevance

The roots of Mirabilis himalaica have been used in Tibetan folk medicine for treatment of uterine cancer, nephritis edematous, renal calculus and arthrodynia. In our previous work, the ethanol extract of roots had shown potent cytotoxicity against human cancer cells. However, no information is available on the antitumor effect of Mirabilis himalaica. The aim of the present study was to investigate the active constituents guided by bioassay and evaluate the related antitumor efficacy in vitro and in vivo.

Materials and methods

The active subextract (ethyl acetate) was subjected to successive chemical separation using a combination of silica gel, LH-20 chromatography and semi-preparative HPLC. The structures were determined by spectroscopic analysis techniques such as nuclear magnetic resonance (NMR) and mass spectrometry. Three human cancer cell lines, A549, HepG2 and HeLa were used for in vitro cytotoxicity evaluation of all isolated compounds by MTT-assay. Then, the potent and novel compound mirabijalone E was employed to the mechanism study againstA549 cells. BrdU immunofluorescence, soft agar assay and cell cycle analysis were employed to detect the cell proliferation effects. Annexin V-FITC/PI staining assay was used for examining apoptotic effects. Expression levels of apoptosis-related proteins were determined by western blot assay. in vivo tumorigenic assay was used to evaluate the xenograft tumor growth treated with mirabijalone E.

Results

One new rotenoid compound, mirabijalone E, together with eight known rotenoids was isolated from Mirabilis himalaica. Mirabijalone E, 9-O-methyl-inone B, boeravinone C and boeravinone H exhibited cytotoxicity against A 549 and HeLa cells. Further study on mirabijalone E was carried out in vitro and in vivo. Mirabijalone E inhibited A549 cells growth in a time and dose-dependent manner, which arrested cell cycle in S phase. Mechanistically, mirabijalone E treatment resulted in the increase of Bax expression level, the decrease of Bcl-2 level and the activation of caspase-3, which suggested the activation of apoptosis cascades. Consequently, the xenograft treated with mirabijalone E showed markedly suppressed tumor growth.

Conclusions

The result suggested that mirabijalone E, together with active compounds, 9-O-methyl-4-hydroxyboeravinone B, boeravinone C and boeravinone H could be a promising candidate for cancer therapy.     

Swietenia macrophylla King induces mitochondrial-mediated apoptosis through p53 upregulation in HCT116 colorectal carcinoma cells

Ethnopharmacological relevance

Swietenia macrophylla King is a traditional herb used to treat various diseases including hypertension, diabetes and cancer. Previous study demonstrated its anti-tumor effect but the potential mechanisms have not been clearly defined. The current study was to further investigate the underlying mechanism of ethyl acetate fraction of Swietenia macrophylla (SMEAF)-induced anti-proliferative effect and apoptosis in HCT116 colorectal carcinoma cell.

Materials and methods

Cell viability was evaluated in HCT116 cells by trypan blue exclusion assay. Apoptotic cell death was detected by Hoechst 33342/propidium iodide (PI) staining and intracellular reactive oxygen species (ROS) was analyzed by flow cytometry. The apoptotic gene and protein expression were determined by Real-time quantitative PCR (q-PCR) and immunofluorescence staining using flow cytometry, respectively.

Results

SMEAF significantly inhibited HCT116 cell viability and induced apoptosis in a dose-dependent manner. SMEAF-induced apoptosis was triggered by the activation of p53 and intracellular reactive oxygen species (ROS) production. Moreover, the significant increase in p53 was accompanied by a decrease murine double minute 2 (MDM2) expression. SMEAF significantly increased the expression of the Bax protein resulting in a markedly elevated Bax/Bcl-2 ratio which may have triggered the mitochondrial apoptotic pathway, resulting in caspase-3/7 and caspase-9 activation.

Conclusion

These results suggested that SMEAF exerts its antitumor activity in HCT116 cells by activating proapoptotic signaling pathway through intracellular ROS formation triggering the mitochondrial-mediated pathway via p53 activation.     

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.     

In vitro cytotoxic activity of nine plants used in Mayan traditional medicine

Ethnopharmacological relevance

Plants have been used in folk medicine by Mayan ancient people from the Yucatan Peninsula, Mexico, to treat some diseases considered as cancer diseases such as chronic wounds or tumors.

Aim of the study

We collected a selection of nine plants in order to investigate their cytotoxic activity against cancer cell lines.

Materials and methods

Methanolic extracts were tested for their cytotoxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on four cancer cell lines; nasopharynx carcinoma (KB), laryngeal carcinoma (Hep-2), cervix adenocarcinoma (HeLa), and cervix squamous carcinoma cells (SiHa) and one normal cell line; canine kidney (MDCK).

Results

All species exhibited some degree of cytotoxic activity. The root bark extract of Hamelia patens exhibited the highest cytotoxic activity on HeLa cells with a CC₅₀ of 13 μg/mL and selectivity index of 13.3, higher than docetaxel. Gossypium schottii and Dioon spinulosum showed similar good cytotoxic activity and selectivity index on HeLa and Hep-2 cells, respectively.

Conclusions

Hamelia patens, Dioon spinulosum and Gossypium schottii demonstrated promising cytotoxic activity and have been selected for future bio-guided fractionation and isolation of active cytotoxic compounds.     

Cordyceps pruinosa extracts induce apoptosis of HeLa cells by a caspase dependent pathway

Aim of the study

Cordyceps is a parasitic fungus and has long been used as a traditional Chinese medicine to treat illnesses, promote longevity, increase athletic power, and relieve exhaustion and cancer. In this study, we reveal the mechanisms underlying apoptosis induced by Cordyceps pruinosa butanol fraction (CPBF) in the human cervical adenocarcinoma cell line, HeLa.

Materials and methods

Proliferation and apoptosis of cells were examined by MTT assay, DNA fragmentation, phosphatidyl serine distribution assay, Western blot analysis, and immunocytochemistry. To determine the association between CPBF related apoptosis and ROS, electron spin resonance (ESR) trapping experiments were used.

Results

CPBF inhibited proliferation and induced apoptosis in HeLa cells in a dose-dependent manner using a MTT assay, DNA fragmentation, and a phosphatidyl serine distribution assay. Western blot analysis showed that apoptosis in HeLa cells was caspase-3- and -9-dependent. Proteolytic cleavage of PARP and the release of cytochrome c from the mitochondria into the cytosol were significantly increased and the Bcl-2/Bax protein ratio was decreased. Apoptosis induced by CPBF was not prevented by various antioxidants.

Conclusions

These results indicate that apoptotic effects of CPBF on HeLa cells are mediated by mitochondria-dependent death-signaling pathway independent of reactive oxygen species, suggesting that CPBF might be effective as an anti-proliferative agent for cancer.     

In vitro anti-breast cancer activity of ethanolic extract of Wrightia tomentosa: Role of pro-apoptotic effects of oleanolic acid and urosolic acid

Ethnopharmacological relevance

Wrightia tomentosa Roem. & Schult. (Apocynaceae) is known in the traditional medicine for anti-cancer activity along with other broad indications like snake and scorpion bites, renal complications, menstrual disorders etc. However, the anti-cancer activity of this plant or its constituents has never been studied systematically in any cancer types so far.

Aim of the study

To evaluate the anti-cancer activities of the ethanolic extract of W. tomentosa and identified constituent active molecule(s) against breast cancer.

Material and methods

Powdered leaves of W. tomentosa were extracted with ethanol. The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa were tested for its anti-proliferative and pro-apoptotic effects in breast cancer cells MCF-7 and MDA-MB-231.

Results

The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa inhibited the proliferation of human breast cancer cell lines, MCF-7 and MDA-MB-231. The fraction F-4 obtained from hexane fraction inhibited proliferation of MCF-7 and MDA-MB-231 cells in concentration and time dependent manner with IC₅₀ of 50 μg/ml and 30 μg/ml for 24 h, 28 μg/ml and 22 μg/ml for 48 h and 25 μg/ml and 20 μg/ml for 72 h respectively. The fraction F-4 induced G1 cell cycle arrest, reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and subsequent apoptosis. Apoptosis is indicated in terms of increased Bax/Bcl-2 ratio, enhanced Annexin-V positivity, caspase 8 activation and DNA fragmentation. The active molecule isolated from fraction F-4, oleanolic acid and urosolic acid inhibited cell proliferation of MCF-7 and MDA-MB-231 cells at IC₅₀ value of 7.5 μM and 7.0 μM respectively, whereas there is devoid of significant cell inhibiting activity in non-cancer originated cells, HEK-293. In both MCF-7 and MDA-MB-231, oleanolic acid and urosolic acid induced cell cycle arrest and apoptosis as indicated by significant increase in Annexin-V positive apoptotic cell counts.

Conclusion

Our results suggest that W. tomentosa extracts has significant anti-cancer activity against breast cancer cells due to induction of apoptosis pathway. Olenolic and urosolic acid are important constituent molecules in the extract responsible for anti-cancer activity of W. tomentosa.     

Cyto-/genotoxic effects of the ethanol extract of Chan Su,a traditional Chinese medicine,in human cancer cell lines

Ethnopharmacological relevance

Chan Su, an ethanolic extract from skin and parotid venom glands of the Bufo bufo gargarizans Cantor, is widely used as a traditional Chinese medicine for cancer therapy. Although the anti-cancer properties of Chan Su have been investigated, no information exists regarding whether Chan Su has genotoxic effects in cancer cells. The aim of the present study was to examine the cyto-/genotoxic effect of Chan Su in human breast carcinoma (MCF-7 cells), human lung carcinoma (A-549 cells), human T cell leukemia (Jurkat T cells), and normal human lymphocytes.

Materials and methods

Effects on the viability of MCF-7, A-549, Jurkat T cells, and normal lymphocytes were evaluated by Trypan blue exclusion assays. The DNA content in the sub-G1 region was detected by propidium iodide (PI) staining and flow cytometry. The genotoxicity of Chan Su was assessed by single-cell gel electrophoresis (comet assay) and the cytokinesis-block micronucleus assay (CBMN assay).

Results

Chan Su significantly inhibited the viability of MCF-7, A-549, and Jurkat T cells dose dependently, but had no effect on normal human lymphocytes. Apoptotic death of the cancer cells was evident after treatment. Chan Su also induced genotoxicity in a dose-dependent manner, as indicated by the comet and cytokinesis-block micronucleus assays.

Conclusions

These findings suggest that Chan Su can induce apoptotic death of, and exert genotoxic effects on, MCF-7, A-549, and Jurkat T cells.     

Hempseed oil induces reactive oxygen species- and C/EBP homologous protein-mediated apoptosis in MH7A human rheumatoid arthritis fibroblast-like synovial cells

Ethnopharmacological relevance

The medicinal efficacy of hempseed (Cannabis sativa L.), which is rich in polyunsaturated fatty acids, in atopic dermatitis, inflammation, and rheumatoid arthritis (RA) has been suggested for centuries. Hempseed has been used as a treatment for these diseases in Korean and Chinese folk medicine. The aim of the study is to investigate the effects of hempseed oil (HO) on MH7A human RA fibroblast-like synovial cells.

Materials and methods

MH7A cells were used to study the anti-rheumatoid effects of hempseed (Cannabis sativa L., cv. Cheungsam/Cannabaceae) oil by investigating cell viability, apoptosis, lipid accumulation, oxidative stress, and endoplasmic reticulum (ER) stress-induced apoptosis.

Results

HO treatment reduced the survival rate of MH7A cells and promoted apoptotic cell death in a time- and dose-dependent manner. Both lipid accumulation and the level of intracellular reactive oxygen species (ROS) increased in HO-treated MH7A cells. Co-treatment with the antioxidant Tiron effectively abrogated the cytotoxic effects of HO; the ROS level was reduced, cell viability was recovered, and apoptotic cell death was significantly diminished. Moreover, HO-treated cells exhibited increased expression of the major ER stress markers, glucose-regulated protein 78 and C/EBP homologous protein (CHOP). The siRNA-mediated knockdown of CHOP prevented HO-induced apoptosis.

Conclusions

Our results suggest that HO treatment induced lipid accumulation, ROS production, CHOP expression, and apoptosis in MH7A cells, and that CHOP functions as an anti-rheumatoid factor downstream of HO in MH7A cells.     

Chemopreventive and anti-cancer properties of the aqueous extract of flowers of Butea monosperma

Ethnopharmacological relevance

Butea monosperma (Lam.) (Fabaceae) popularly known as ‘flame of the forest’ has been widely used in the traditional Indian medical system of ‘Ayurveda’ for the treatment of a variety of ailments including liver disorders.

Aim of the study

To evaluate the antioxidative, anti-inflammatory, hepatoprotective and anti-cancer activities of the aqueous extract of Butea monosperma flowers.

Materials and methods

Dried flowers of Butea monosperma were extracted with water. The extract was tested for its anti-proliferative, pro-apoptotic and anti-carcinogenic effects in hepatoma cell lines. The chemopreventive and anti-angiogenic effects of the extract were evaluated by its daily oral administration in a HBV-related X15-myc mouse model of hepatocellular carcinoma (HCC).

Results

Treatment with the aqueous extract inhibited cell proliferation and accumulation of cells in G1 phase. This was accompanied by a marked reduction in the levels of activated Erk1/2 and SAPK/JNK and induction of apoptotic cell death. Oral administration of the extract in transgenic mice conferred hepatoprotection as is evident from normal serum ALT levels and improved liver histopathology and lowered serum VEGF level.

Conclusions

The ability of aqueous extract of Butea monosperma flowers to impose growth arrest and trigger pro-apoptotic death in cell culture strongly correlated with its strong chemopreventive effect in vivo when given orally.     

Identification of antihyperlipidemic constituents from the roots of Rubia yunnanensis Diels

Ethnopharmacological relevance

The roots of Rubia yunnanensis Diels (Rubiaceae) have been used as an alternative for Rubia cordifolia for the treatment of various diseases including cardiovascular disease and metabolic disease for a long history in traditional Chinese medicine. To evaluate antihyperlipidemic activity of the roots of Rubia. yunnanensis Diels and to identify active compounds from the active fraction.

Material and methods

Antihyperlipidemic effects of extract and compounds of the roots of Rubia yunnanensis were studied in HepG2 cells and in vivo model in olive oil-loaded mice. The isolation of compounds was conducted using various chromatographic methods and the structures of the isolated compounds were elucidated by NMR and MS techniques.

Results

Methanol extract and ethyl acetate fraction of Rubia yunnanensis potently decreased the triglycerides accumulation in HepG2 cells and the methanol extract of Rubia yunnanensis (125, 250 and 500 mg/kg) significantly inhibited the TG elevation in a dose dependent manner in olive oil-loaded mice. Through various chromatographic methods, twenty-three compounds (1–23) were isolated in which arborinane-type triterpenoids and a free anthraquinone potently inhibited TG levels in HepG2 cells.

Conclusion

Arborinane-type triterpenoids, especial rubiarbonone C (16) and an anthraquinone, 2-methyl-1, 3, 6-trihydroxy-9, 10-anthraquinone (MTHA) (22) were identified as the main active compounds responsible for antihyperlipidemic activity. Based on these findings, we proposed that the extract of Rubia yunnanensis and its active components, arborinane-type triterpenoids and the free anthraquinone might be beneficial in the treatment and prevention of hyperlipidemic disease.     

The antitumoral effect of the American mistletoe Phoradendron serotinum (Raf.) M.C. Johnst. (Viscaceae) is associated with the release of immunity-related cytokines

Ethnopharmacological relevance

Phoradendron serotinum is commonly used in Mexican traditional medicine for the empirical treatment of cancer. However, there are no studies regarding the antitumoral or immunomodulatory activities of Phoradendron serotinum.

Materials and methods

The in vivo toxicity of ethanolic extracts of Phoradendron serotinum (PSE) was evaluated in mice according to the Lorke procedure. The in vitro immunomodulatory effects of PSE were evaluated estimating the effects of PSE on the pinocytosis, NO production and lysosomal enzyme activity in murine macrophages RAW 264.7. The effects of PSE on the proliferation of murine splenocytes and NK cell activity were also assayed. The cytotoxic effects on TC-1 (lung murine cancer cells) were evaluated using the MTT assay, whereas the apoptotic effect of PSE on TC-1 cells was evaluated using TUNEL assay. Also, different doses of PSE were injected intraperitoneally daily into C57BL/6 mice bearing tumors of TC-1 cells during 25 days. The growth and weight of tumors was measured. In addition, the levels of IL-2, IL-6, IL-12, IL-23 and IFN-γ in murine serum and supernatants of K562 cell—murine splenocyte cocultures were measured.

Results

PSE stimulated the proliferation, pinocytosis and lysosomal enzyme activity in murine macrophages with a similar potency than lypopolisaccharides 1 μg/ml. In addition, PSE stimulated the proliferation of murine splenocytes and induced the NK cell activity. PSE showed cytotoxic (IC₅₀ = 1.9 μg/ml) and apoptotic effects against TC-1 cells. The LD₅₀ was 125 mg/kg by intraperitoneal route (i.p.) and 375 mg/kg by oral route. PSE administrated at 1, 5 and 10 mg/kg i.p. inhibited the tumor growth by 18%, 40% and 69%, respectively, in mice bearing TC-1 tumor. PSE increased the in vitro and in vivo release of IL-2, IL-6 and IFN-γ but lacked effect on IL-12 and IL-23 release.

Conclusions

Phoradendron serotinum shows moderate toxic effects in vivo, exerts cytotoxic and apoptotic effects on TC-1 cells. Phoradendron serotinum also has antitumor effects in mice bearing TC-1 tumor and induces immunomodulatory activities in vivo. The results suggest that antitumoral effects of PSE are related with the production of immunity-related cytokines.     

Baicalin can scavenge peroxynitrite and ameliorate endogenous peroxynitrite-mediated neurotoxicity in cerebral ischemia-reperfusion injury

Ethnopharmacological relevance

Baicalin is one of the principal flavonoids isolated from the dried root of Scutellaria baicalensis Georgi that has long been used to treat ischemic stroke. However, its neuroprotective mechanisms against cerebral ischemia injury are poorly understood.

Aim of the study

To explore the neuroprotective mechanisms of baicalin against cerebral ischemia reperfusion injury.

Material and methods

In chemical systems, we conducted electron paramagnetic resonance (EPR) spin trapping experiments to evaluate the scavenging effects of baicalin on superoxide and nitric oxide, and mass spectrometry (MS) studies on the reaction of baicalin and peroxynitrite. In cellular experiments, we investigated the effects of baicalin against extraneous and endogenous peroxynitrite mediated neurotoxicity in SH-SY5Y cells treated with peroxynitrite donor, synthesized peroxynitrite and exposed to oxygen glucose deprivation and reoxygenation (OGD/RO) in vitro. Moreover, we studied the neuroprotective effects of baicalin by using a rat model of middle cerebral artery occlusion in vivo. FeTMPyP, a peroxynitrite decomposition catalyst, was used as positive control. Cell viability and apoptotic cell death was accessed by MTT assay and TUNEL assay respectively; 3-nitrotyrosine formation and infarction volume were detected by immunostaining experiments and TTC staining respectively.

Results

Baicalin revealed strong antioxidant ability by directly scavenging superoxide and reacting with peroxynitrite. Baicalin protected the neuronal cells from extraneous and endogenous peroxynitrite-induced neurotoxicity. In ischemia-reperfused brains, baicalin inhibited the formation of 3-nitrotyrosine, reduced infarct size and attenuated apoptotic cell death, whose effects were similar to FeTMPyP.

Conclusions

Baicalin can directly scavenge peroxynitrite and the peroxynitrite-scavenging ability contributes to its neuroprotective mechanisms against cerebral ischemia reperfusion injury.     

An in vitro study on antiproliferative and antiestrogenic effects of Boerhaavia diffusa L. extracts

Ethnopharmacological relevance

Boerhaavia diffusa L. (Nyctinaceae) is a plant of tropical region used in Indian traditional medicine for the treatment of human ailments including abdominal tumor, jaundice, dyspepsia, menstrual disorders, etc. This plant also has antilymphoproliferative, antimetastatic and immunomodulatory effects.

Aim of the study

This study aimed to assess the antiproliferative and antiestrogenic properties of methanol extract of Boerhaavia diffusa (BME) in MCF-7 breast cancer cell lines.

Materials and methods

The effective concentration range of BME on cell viability was analyzed using MTT assay. Hydroxylapatite assay (HAP) was carried out to confirm the competitive binding of BME to the estrogen receptor (ER). The effect of BME on the expression of a selected estrogen responsive gene pS2 was analyzed by RT-PCR. The ability of BME to alter the cell cycle phases and distributions were studied using FACS analysis.

Results

Treatment with varying concentrations of BME (20–320 μg/mL) resulted in moderate to very strong growth inhibition in MCF-7 cell lines. BME competed with [³H]-estradiol for binding to ER with IC₅₀ value of 320 ± 25 μg/mL. RT-PCR analysis revealed that BME reduced the mRNA expression of pS2 indicating the antiestrogenic action of BME. BME treatment for 48 h resulted in a remarkable increase in the number of MCF-7 cells in the G0-G1 fraction from 69.1% to 75.8%, with a reciprocal decrease of cells in all other phases indicating cell cycle arrest at G0-G1 phase.

Conclusions

The results demonstrate that Boerhaavia diffusa possess antiproliferative and antiestrogenic properties and suggest that it may have therapeutic potential in estrogen dependent breast cancers.