The flavonoid-rich fraction of Coreopsis tinctoria promotes glucose tolerance regain through pancreatic function recovery in streptozotocin-induced glucose-intolerant rats

Aim of the study

Infusions of Coreopsis tinctoria Nutt. flowering tops have been used traditionally in Portugal to control hyperglycaemia and a previous study revealed that daily administration of the infusion during a 3-week period promoted the recovery of glucose tolerance by a mechanism different from inhibition of glucose absorption and direct promotion of insulin secretion.We know report the study of the ethyl acetate fraction of Coreopsis tinctoria flowers infusion aiming to confirm flavonoids as bioactive metabolites. To give one step forward into the antihyperglycaemic mechanism of action of this traditionally used plant we also studied the activity of Coreopsis tinctoria flavonoids on the pancreatic function of glucose-intolerant rats. A standard antioxidant, Trolox, was also studied for comparative purposes as the antioxidant mechanism has been frequently purposed as one of the mechanisms mediating antihyperglycaemic effects of flavonoid-rich extracts.

Material and methods

Thirteen compounds, mainly of flavanone and chalcone flavonoidal type, have been identified in this fraction by HPLC-DAD-ESI-MS/MS, and the major one (marein) quantified by HPLC-UV. The fraction (125 mg containing 20 mg of marein/kg b.w.) and Trolox (50 mg/kg b.w.) were administered daily by oral gavage to normal and STZ (40 mg/kg b.w.)-induced glucose-intolerant Wistar rats for 3 weeks. Blood glucose levels were measured weekly by Oral Glucose Tolerance Test. Pancreatic function was evaluated by plasma lipase of treated and non-treated glucose-tolerant and- intolerant rats after the 3-week treatment period.

Results

After 2 weeks oral treatment with Coreopsis tinctoria AcOEt fraction the animals were no longer glucose-intolerant, an effect maintained over the remaining experimental period. Additionally, plasma lipase values of glucose-intolerant animals treated with the AcOEt fraction (13.5 ± 0.84 U/L) showed a clear reduction when compared with the glucose-intolerant group (34.60 ± 1.76 U/L; P < 0.001) and normoglycaemic control (8.35 ± 0.69 U/L) demonstrating recovery of pancreatic function. On the other hand, treatment with standard antioxidant Trolox had no effect on glucose homeostasis of glucose-intolerant rats. The oral treatment with Coreopsis tinctoria fraction caused no hepatotoxicity, as determined by blood alanine and aspartate transaminases, and had also no effect on glucose homeostasis and pancreatic function of normal rats.

Conclusions

AcOEt fraction, containing the same amount of marein as the infusion, promoted glucose tolerance regain in the rats more quickly, which means that the bioactivity is probably due to the several flavonoids present in Coreopsis tinctoria extracts and not to marein alone. The results also strongly suggest that these compounds act by promoting pancreatic cell function recovery from STZ-induced injury, possibly through a mechanism of action other than merely antioxidant mediated.     

新疆金鸡菊提取物对2型糖尿病小鼠氨基酸代谢谱的影响

目的:研究新疆金鸡菊(Xinjiang Coreopsis tinctoria)乙酸乙酯提取物对2型糖尿病小鼠血清氨基酸代谢谱的影响。方法:高脂高糖喂养联合腹腔注射链脲佐菌素(streptozotocin,STZ)建立2型糖尿病模型,造模成功后随机分组,灌胃给予新疆金鸡菊提取物8 g·kg-1·d-1,阳性药二甲双胍0.16 g·kg-1·d-1,每周测空腹血糖(FBG),给药4周后处死,收集小鼠血清,检测甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)水平。采用柱前衍生高效液相色谱法(HPLC)检测小鼠血清中氨基酸代谢谱的变化。结果:结合SIMCA-P软件的偏最小二乘法判别(PLS-DA)对氨基酸数据进行分析,结果显示新疆金鸡菊乙酸乙酯提物给药后对血清氨基酸代谢谱的改变较模型组有很大区别,有4种氨基酸对组间贡献较大(牛磺酸、天冬氨酸、苏氨酸、蛋氨酸)。小鼠成模后体内氨基酸发生了明显变化。与空白组、模型组、阳性组相比,新疆金鸡菊乙酸乙酯给药组小鼠血清氨基酸代谢谱发生了显著变化。结论:新疆金鸡菊乙酸乙酯提取物显著影响小鼠血清中与糖尿病相关的氨基酸代谢标志物,为进一步阐释新疆金鸡菊抗糖尿病药理作用的研究提供了依据。     

Studies of biological properties of Uncaria tomentosa extracts on human blood mononuclear cells

Ethnopharmacological relevance

Uncaria tomentosa (Willd.) DC is a lignified climbing plant from South and Central America, which (under the name of “vilcacora” or “cat's claw”) has become highly popular in many countries due to its proven immunostimmulatory and anti-inflammatory activities and also with respect to its anticancer and antioxidative effects. There are insufficient data on the mechanism of U. tomentosa action on normal blood mononuclear cells.

Aim of the study

The aim of the study was to analyze the impact of ethanol and aqueous extracts from bark and leaves of Uncaria tomentosa on the structure and function of human mononuclear cells and to find out whether the kind of extractant used modulates biological activity of the extracts studied.

Materials and methods

Plant material consisted of four different extracts: (1) ethanol extract from leaves, (2) aqueous extract from leaves, (3) ethanol extract from bark and (4) aqueous extract from bark. The effect of these extracts on protein damage as well as on free-radical formation in human peripheral blood mononuclear cells was analyzed. Moreover, changes in viability, size, and granularity as well as apoptotic alterations in human blood mononuclear cells exposed to U. tomentosa extracts were investigated.

Results

The oxidative changes were observed in mononuclear blood cells exposed to both ethanol and aqueous extracts obtained from bark and leaves. Moreover, in the cells studied the extracts from U. tomentosa induced apoptosis and a decrease in viability of mononuclear blood cells, with the exception of aqueous extract from leaves. Additionally, no statistically significant changes in the cell size were observed both for aqueous extracts from leaves and bark. Changes in the blood mononuclear cell granularity were observed at 250 μg/mL for all extracts examined. The strongest changes were observed for the ethanol extract of the bark, which increased cell granularity at 50 μg/mL and changed cell size at 100 μg/mL.

Conclusion

The conducted research showed differences in biological activity between aqueous and ethanol extracts. It was observed that ethanol extracts exhibited stronger negative effects on mononuclear blood cells. The kind of extractant used had a significant influence of the chemical composition of the tested extracts. The ethanol extract from bark containing a high amount of polyphenols and alkaloids revealed the highest pro-apoptotic effect.     

Induction of apoptosis in human hepatocarcinoma SMMC-7721 cells in vitro by flavonoids from Astragalus complanatus

Aim of the study

Flavonoids extracted from the seeds of Astragalus complanatus R.Br. reduce the proliferation of many cancer cells. The present study was carried out to evaluate the effects of these flavonoids from Astragalus complanatus (FAC) on human hepatocarcinoma cell viability and apoptosis and to investigate its mechanisms of action in SMMC-7721 cells.

Materials and methods

Cell viability was measured using the MTT assay. To detect apoptotic cells, SMMC-7721 cells treated with FAC were stained with Hoechst 33258 and subjected to agarose gel electrophoresis. Quantitative detection of apoptotic cells was performed by flow cytometry. The effects of FAC on apoptosis and cell cycle regulatory genes and proteins in SMMC-7721 cells were examined using an S series apoptosis and cell cycle gene array and Western blot analysis.

Results

The growth of SMMC-7721 and HepG2 cells was inhibited by treatment with FAC. Cell death induced by FAC was characterized by nuclear condensation and DNA fragmentation. Moreover, the cell cycle was arrested in the G0/G1 and S phases in FAC-treated SMMC-7721 cells. A sub-G1 peak with reduced DNA content was also formed. The activity of caspase-3 was significantly increased following FAC treatment. Microarray data indicated that the expression levels of 76 genes were changed in SMMC-7721 cells treated with FAC: 35 genes were up-regulated and 41 were down-regulated. Western blot analysis showed that caspase-3, caspase-8, Bax, P21, and P27 protein levels in SMMC-7721 cells were increased after 48 h of FAC treatment, while cyclinB1, cyclinD1, CDK1, and CDK4 protein levels were decreased.

Conclusions

These results suggest that FAC may play an important role in tumor growth suppression by inducing apoptosis in human hepatocarcinoma cells via mitochondria-dependent and death receptor-dependent apoptotic pathways.     

Acanthopanax senticosus extracts have a protective effect on Drosophila gut immunity

Ethnopharmacological relevance

Aanthopanax senticosus (A. senticosus) Harms is a classical adaptogenic agent used in China. It has been applied as an analeptic aid to improve weakened physical status. However, little is known about the effects of A. senticosus on inflammatory disease processes.

Materials and methods

Flies fed with standard cornmeal–yeast medium were used as controls, and the treatment groups contained 10% of A. senticosus aqueous extracts (root or fruit) in standard medium. Survival rate was performed by feeding a vial containing five layers of filter paper hydrated with 5% sucrose solution contaminated with pathogenic or toxic compounds. Imaging of the guts was viewed under the microscope. Death cells were detected by 7-AAD staining.

Results

The A. senticosus extract improved the survival rate, attenuated the death of intestinal epithelial cells, promoted the expression of antimicrobial peptide genes, and decreased the formation of melanotic masses. Moreover, our results indicated that the protective effect of fruit is much higher than that of root extracts.

Conclusions

A. senticosus extracts have a protective effect on Drosophila gut immunity and stress response, and may contribute to the prevention of inflammatory diseases induced by pathogenic and toxic compounds.     

In vitro effects of Sutherlandia frutescens water extracts on cell numbers,morphology, cell cycle progression and cell death in a tumorigenic and a non-tumorigenic epithelial breast cell line

Sutherlandia frutescens is a South African herb traditionally used for internal cancers, diabetes, a variety of inflammatory conditions and recently to improve the overall health in cancer and HIV/AIDS patients. The in vitro effects of S. frutescens extracts were evaluated on cell numbers, morphology, cell cycle progression and cell death. Dose-dependent studies (2–10 mg/ml) revealed a decrease in malignant cell numbers when compared to their controls. S. frutescens extracts (10 mg/ml) decreased cell growth in a statistically significantly manner to 26% and 49% (P < 0.001) in human breast adenocarcinoma (MCF-7) and human non-tumorigenic epithelial mammary gland cells (MCF-12A) respectively after 72 h of exposure. Cell density was significantly compromised and hypercondensed chromatin, cytoplasmic shrinking, membrane blebbing and apoptotic bodies were more pronounced in the MCF-7 cell line. Both S. frutescens-treated cell lines exhibited and increased tendency for acridine orange staining, suggesting increased lysosomal and/or autophagy activity. Flow cytometry showed an increase in the sub G₁ apoptotic fraction and an S phase arrest in both the 5 mg/ml and 10 mg/ml S. frutescens-treated cells. S. frutescens induced an increase in apoptosis in both cell lines as detected by Annexin V and propidium iodide flow cytometric measurement. At 10 mg/ml, late stages of apoptosis were more prominent in MCF-7 S. frutescens-treated cells when compared to the MCF-12A cells. Transmission electron microscopy revealed hallmarks of increased vacuolarization and hypercondensed chromatin, suggesting autophagic and apoptotic processes. The preliminary study demonstrates that S. frutescens water extracts exert a differential action mechanism in non-tumorigenic MCF-12A cells when compared to tumorigenic MCF-7 cells, warranting future studies on this multi-purpose medicinal plant in southern Africa.     

Comparative in vitro bioactivities of tea extracts from six species of Ardisia and their effect on growth inhibition of HepG2 cells

Aim of the study

Ardisia species, notably A. compressa, are used in some regions of the world as food or in traditional medicine for prevention and treatment of certain health conditions including liver disease. We investigated the chemical composition and relative anticancer potential of six Ardisia species [A. japonica (AJ), A. escallonioides (AES), A. mamillata (AM), A. compressa (AC), A. crenata (ACR), and A. elliptica (AE)].

Materials and methods

Antioxidant capacity, DNA human topoisomerase II catalytic inhibition, and cytotoxicity on human liver cancer cells (HepG2) were determined in vitro in tea extracts of the 6 Ardisia species evaluated. Selected pure phenolic compounds present in Ardisia species were also evaluated.

Results

AC showed the highest topoisomerase II catalytic inhibition (IC₅₀ = 12 μg/ml) and cytotoxicity (IC₅₀ = 117 μg/ml) against HepG2 cells, followed by ACR and AJ. Total polyphenols ranged from 21 to 72 mg equivalents of gallic acid (GA)/g solid extract (SE). LC–MS analysis revealed the presence of GA, quercetin derivatives, ardisenone, ardisiaquinone, ardisianone, bergenin, norbergenin, and embelin. However, neither total polyphenol concentration nor antioxidant capacity correlated with anticancer capacity. Significant HepG2 cytotoxicity was also achieved by bergenin (IC₅₀ = 18 μM) and embelin (IC₅₀ = 120 μM). AC, bergenin, embelin, and quercetin showed a tendency to accumulate cells in the G1 phase and reduced G2/M leading to apoptosis.

Conclusions

Although the mechanism is not entirely clear, AC, ACR, and AJ are the Ardisia species with the greatest anticancer potential against liver cancer cells in vitro and deserve further investigation.     

The apoptosis inducing effects of Sutherlandia spp. extracts on an oesophageal cancer cell line

Aim of study

Oesophageal cancer is the ninth most common cancer in the world and the second most common cancer among South African men. It also has one of the lowest possibilities of cure, with the 5-year survival rate estimated to be only 10% overall. Sutherlandia frutescens, or the “cancer bush”, is a medicinal plant indigenous to southern Africa that is believed to have anti-cancer and anti-proliferative properties. The aim of this study was to investigate the potential apoptosis-inducing effects of two S. frutescens extracts and one Sutherlandia tomentosa extract on the SNO oesophageal cancer cell line.

Materials and methods

Cell viability and morphology of SNO cells were evaluated following exposure to the extracts. Apoptotic markers including cytochrome c translocation and phosphatidylserine externalisation were quantified by flow cytometry. The activity of caspases 3 and 7 was evaluated with spectrofluorometry. Apoptosis was evaluated in the presence of the pan-caspase inhibitor, Z-VAD-fmk. The effect of the extracts was compared to non-cancerous peripheral blood mononuclear cells (PBMCs).

Results

Time- and dose-response studies were conducted to establish treatment conditions of 2.5 and 5 mg/ml of crude plant extracts. Microscopy studies revealed that S. frutescens- and S. tomentosa-treated SNO cells had morphological features characteristic of apoptosis. Annexin V/propidium iodide flow cytometry confirmed that the extracts do, in fact, induce apoptosis in the SNO cells. Caspase inhibition studies seem to indicate that extracts A (S. frutescens (L.) R. Br. subsp. microphylla from Colesberg), B (S. frutescens (L.) R. Br. subsp. microphylla from Platvlei) and C (S. tomentosa Eckl. & Zeyh from Stil Bay) are able to induce caspase-dependent as well as -independent cell death. The S. frutescens and S. tomentosa extracts were found to be more cytotoxic to cancerous SNO cells when compared to the PBMCs.

Conclusions

S. frutescens and S. tomentosa extracts show promise as apoptosis-inducing anti-cancer agents.     

Cytotoxic effects of Echinacea root hexanic extracts on human cancer cell lines

Echinacea is one of the most widely used alternative medicine in the world. Intake of Echinacea preparations is common among patients with advanced malignancies enrolled onto phase I chemotherapy trials; however, to our knowledge, no data are available regarding the possible direct effect of Echinacea species on human cancer cells. The purpose of the present study was to investigate potential in vitro cytotoxic and pro-apoptotic properties of hexanic root extract of the three medicinal Echinacea (Asteraceae) species (Echinacea pallida (Nutt.) Nutt., Echinacea angustifolia DC. var. angustifolia, Echinacea purpurea (L.) Moench.) on the human pancreatic cancer MIA PaCa-2 and colon cancer COLO320 cell lines. We demonstrated, for the first time, that all the three species reduced cell viability in a concentration- and time-dependent manner; Echinacea pallida was the most active species with IC(50)s of 46.41+/-0.87 and 10.55+/-0.70 microg/ml in MIA PaCa-2 and COLO320 cells, respectively. Echinacea pallida extract was able to induce apoptosis by increasing significantly caspase 3/7 activity and promoting nuclear DNA fragmentation. These results represent the starting point to establish viable scientific evidence on the possible role of Echinacea species in medical oncology.     

昆仑雪菊黄酮类提取物抗脂多糖诱导的小胶质细胞活化作用及网络药理学机制分析

目的:研究昆仑雪菊干预小胶质细胞功能的作用机制。方法:采用CCK-8法检测昆仑雪菊总黄酮对BV2细胞的增殖抑制活性,Griess法和ELISA检测细胞炎症因子一氧化氮(NO)、白细胞介素-6(IL-6)的分泌。借助自建昆仑雪菊成分-靶点数据集对昆仑雪菊中9个主要黄酮成分和相关作用靶点进行筛选。通过GeneCards、GENE、STRING数据库及R语言收集并筛选小胶质细胞功能靶点,筛选昆仑雪菊改善小胶质细胞功能的靶点,构建蛋白质-蛋白质相互作用(PPI)网络,并用Cytoscape软件的NetworkAnalyzer功能筛选核心靶点。利用基因集富集分析(GSEA)软件对分子特征数据库MSigDB中的相关靶点进行Hallmark基因分析。使用Bioconductor数据库进行基因本体(GO)功能注释和京都基因与基因组百科全书(KEGG)通路富集分析。结果:昆仑雪菊黄酮类提取物在4μg·mL–1时可以显著抑制小胶质细胞激活分泌NO和IL-6。昆仑雪菊黄酮类成分可能作用于蛋白激酶B(Akt)、肿瘤坏死因子(TNF)、IL-6、TP53、酪氨酸激酶C(SRC)、表皮生长因子受体(EGFR)、JUN、信号转导和转录激活因子3(STAT3)、丝裂原活化蛋白激酶3(MAPK3)、CTNNB1等靶点干预小胶质细胞的功能。涉及的通路包括磷脂酰肌醇3-激酶(PI3K)-Akt信号通路、MAPK信号通路、TNF信号通路、信号转导子和转录激活子(JAK)-STAT信号通路等。结论:昆仑雪菊黄酮类成分可能通过参与调控PI3K-Akt信号通路、MAPK信号通路、TNF信号通路、JAK-STAT信号通路等的相关靶点,实现调节胶质细胞活化的作用。     

Protective effect of hexane extracts of Uncaria sinensis against photothrombotic ischemic injury in mice

Ethnopharmacological relevance

Uncaria sinensis (US) has been used in traditional Korean medicine to treat vascular disease and to relieve various neurological symptoms.

Aim of the study

Scientific evidence related to the effectiveness or action mechanism of US on cerebrovascular disease has not been examined experimentally. Here, we investigated the cerebrovascular protective effect of US extracts on photothrombotic ischemic injury in mice.

Materials and methods

US hexane extracts (HEUS), ethyl acetate extracts (EAEUS) and methanol extracts (MEUS) were administered intraperitoneally 30 min before ischemic insults. Focal cerebral ischemia was induced in C57BL/6J mice and endothelial nitric oxide synthase knockout (eNOS KO) mice by photothrombotic cortical occlusion. We evaluated the infarct volume, neurological score and the activation of Akt and eNOS in ischemic brain.

Results

HEUS more significantly reduced infarct volume and edema than did EAEUS and MEUS following photothrombotic cortical occlusion. HEUS produced decreased infarct volume and edema size, and improved neurological function in a concentration-dependent manner (10, 50, and 100 mg/kg). However, HEUS did not reduce brain infarction in eNOS KO mice, suggesting that the protective effect of HEUS is primarily endothelium-dependent. Furthermore, HEUS (10-300 μg/ml) produced a concentration-dependent relaxation in mouse aorta and rat basilar artery, which was not seen in eNOS KO mouse aorta, suggesting that HEUS cause vasodilation via an eNOS-dependent mechanism. This correlated with increased phosphorylation of Akt and eNOS in the brains of HEUS-treated mice.

Conclusion

HEUS prevent cerebral ischemic damage by regulating Akt/eNOS signaling. US, herbal medicine, may be the basis of a novel strategy for the therapy of stroke.     

Apoptotic effect of Oldenlandia diffusa on the leukaemic cell line HL60 and human lymphocytes

Oldenlandia diffusa is traditionally prescribed in the treatment of a number of cancers and studies suggest that it exerts a cytotoxic action specific to cancer cells. To further investigate this suggested action, the effect(s) of Oldenlandia diffusa on leukaemic cells (HL60) and stimulated and unstimulated human blood lymphocytes (PBLs) was investigated. For the HL60s, cell growth, apoptotic induction, alterations in cell cycle characteristics and genotoxicity were investigated. For the PBLs, apoptotic induction and alterations in cell cycle characteristics were investigated. Preliminary chemical analysis to identify the cytotoxic constituents of Oldenlandia diffusa was also carried out. Results showed that Oldenlandia diffusa significantly inhibited the growth of the HL60s and induced apoptosis in a cell cycle-independent fashion, possibly through the induction of genotoxic damage. Oldenlandia diffusa did not induce apoptosis in the PBLs however progression through the cell cycle was not evident (in stimulated PBLs) suggesting some degree of cytotoxicity. Preliminary chemical analysis indicated that a number of compounds appear to be responsible for the cytotoxic action of Oldenlandia diffusa. These results indicate that HL60s are more sensitive to Oldenlandia diffusa than stimulated PBLs and thus support a cytotoxic action for Oldenlandia diffusa that has some degree of specificity.     

苦菊提取物对HepG2细胞的抗氧化作用及其机制研究

目的：考察苦菊提取物（CEE）对H₂O₂致HepG2细胞氧化应激损伤的保护作用,并探讨苦菊提取物在HepG2细胞中抗氧化作用机制。方法：通过MTT法和DCFH-DA荧光染色,检测H₂O₂处理HepG2细胞的活性和胞内ROS水平;在抗氧化反应元件（ARE）报告基因转染稳定的HepG2细胞内检测ARE-Luciferase活性;并通过荧光定量RT-PCR测定HepG2细胞内含有ARE序列相关基因的mRNA表达水平。结果：H₂O₂处理HepG2细胞后,细胞存活率下降,胞内ROS水平上升,而加入不同浓度的CEE则可明显改善上述结果。不同浓度的CEE处理转染ARE报告基因的HepG2细胞,可使细胞内的ARE活性呈浓度依赖性增强。此外,CEE可使HepG2细胞中含ARE序列的调控基因GCLC,GCLM和HMOX-1的mRNA表达水平上调,并呈浓度依赖性。结论：CEE通过降低ROS水平,抑制H₂O₂诱导的HepG2细胞损伤,而CEE对HepG2细胞的抗氧化保护作用机制可能与其激活HepG2细胞内Nrf2-ARE通路相关的抗氧化防御系统密切相关。     

Typhonium flagelliforme induces apoptosis in CEMss cells via activation of caspase-9, PARP cleavage and cytochrome c release: Its activation coupled with G0/G1 phase cell cycle arrest

Ethnopharmacological relevance

The plant Typhonium flagelliforme (TF), commonly known as ‘rodent tuber’ in Malaysia, is often used as traditional remedy for cancer, including leukemia.

Aim of the study

We had previously identified morphologically that the linoleic acid rich fraction (DCM/F7) from the tubers of this plant induces selective anti-proliferative effects and apoptosis in CEMss cells. In this present study, we subjected the same DCM/F7 fraction to cell based activity analyses in order to determine the possible mechanism of cell death in leukemic CEMss cells in vitro.

Materials and methods

Extraction of Typhonium flagelliforme tuber has done and fractionation has been done by vacuum liquid column chromatography. The anti-proliferative activity was assayed using MTT and the apoptosis detection was done by Annexin V and DNA laddering assay. Colorimetric caspase assay and immunoblot analysis were employed to detect the expression of protein associated with cell death. Cell cycle analysis was done using flow cytometry.

Results

We found that the cancer inhibitory effect of the DCM/F7 fraction in CEMss cells was 3 ± 0.08 μg/ml (IC₅₀). An early apoptotic induction in CEMss cells was observed by Annexin V assay, which showed a clear dose-dependent DNA fragmentation being observed in gel electrophoresis at 10 and 20 μg/ml. The DCM/F7 fraction at 3 μg/ml significantly arrested CEMss cells at G0/G1 phase (p < 0.05). A constant but increasing pattern-related Sub-G0/G1 index was observed between 12 and 72 h treatment. In relation to this, we further investigated the biochemical events leading to cell death and found that the DCM/F7 fraction increased the cellular levels of caspase-3 and -9 on treated cells. Our results indicated that cytochrome c from mitochondria into the cytosol increased gradually as the DCM/F7 concentration increases, which later lead to the subsequent cleavage of PARP in to 85 kDa fragments. On the contrary, Bcl-2 protein was found to decrease concomitantly during treatment.

Conclusions

Collectively, results presented in this study demonstrated that the DCM/F7 fraction inhibited the proliferation of leukemia cells, leading to the programmed cell death, which was confirmed to be through the mitochondrial pathway.     

In vitro and in vivo trypanocidal effect of lipophilic extracts of medicinal plants from Mali and Burkina Faso

AIM OF THE STUDY: To determine the in vitro and in vivo antitrypanosomal activity of extracts of traditionally used plants. MATERIALS AND METHODS: 47 dichloromethane extracts were tested in vitro in the Long-term Viability Assay (LtVA) on Trypanosoma brucei brucei. The most active ones were also tested in vivo using a standardised mouse test. RESULTS: 13 extracts (28%) were active in vitro with MIC-values相似文献   

Cytotoxic effects of Echinacea purpurea flower extracts and cichoric acid on human colon cancer cells through induction of apoptosis

Ethnopharmacological relevance

Echinacea is a top-selling herbal supplement that acts as immunostimulant. It has been used to treat common cold, coughs, bronchitis and upper respiratory infections. It is also a popular product used in anticancer therapy. The cytotoxic effects of Echinacea on cancer cells are still not clear. The aims of this study were to provide a preliminary validation of the effects of 50% aqueous ethanol extract of Echinacea purpurea flowers and its major compound, cichoric acid, on human colon cancer cells Caco-2 and HCT-116.

Materials and methods

The cytotoxic effects of Echinace flower extracts and cichoric acid on cell viability, telomerase activity, DNA fragmentation, β-catenin, caspase-9, and cleavage of poly-ADP-ribose polymerase (PARP) of human colon cancer cell were examined.

Results

The results showed a significant inhibition of proliferation in E. purpurea flower extract and cichoric acid in a dose- and time-dependent manner. Cichoric acid treatment decreased telomerase activity in HCT-116 cells. Moreover, cichoric acid effectively induced apoptosis in colon cancer cells, which were characterized by DNA fragmentation, activation of caspase-9, cleavage of PARP and downregulation of β-catenin.

Conclusions

Our data indicate that cichoric acid has a strong growth-inhibitory effect against colon cancer cells, presumably resulting from the reduced telomerase activity and the induction of apoptosis. The exact mechanism of action should still be determined in future studies. Overall, the effects of 50% aqueous ethanol extract of E. purpurea flowers and cichoric acid may have provided in vitro evidence for the use as chemotherapeutic agents.     

Influence of Sutherlandia frutescens extracts on cell numbers, morphology and gene expression in MCF-7 cells

Sutherlandia frutescens is a well-known South African herbal remedy traditionally used for stomach problems, internal cancers, diabetes, various inflammatory conditions and recently to improve the overall health in cancer and HIV/AIDS patients. The influence of crude Sutherlandia frutescens extracts (prepared with 70% ethanol) was investigated on cell numbers, morphology, and gene expression profiles in a MCF-7 human breast adenocarcinoma cell line. Time-dependent (24, 34, 48 and 72 h) and dose-dependent (0.5-2.5 mg/ml) studies were conducted utilizing spectrophotometrical analysis with crystal violet as DNA stain. A statistically significant decrease to 50% of malignant cell numbers was observed after 24 h of exposure to 1.5 mg/ml Sutherlandia frutescens extract when compared to vehicle-treated controls. Morphological characteristics of apoptosis including cytoplasmic shrinking, membrane blebbing and apoptotic bodies were observed after 24h of exposure. A preliminary global gene expression profile was obtained by means of microarray analysis and revealed valuable information about the molecular mechanisms and signal transduction associated with 70% ethanolic Sutherlandia frutescens extracts.     

Evaluation of the anti-inflammatory and anti-proliferation tumoral cells activities of Antrodia camphorata, Cordyceps sinensis, and Cinnamomum osmophloeum bark extracts

The extracts of chloroform (1) and methanol (2) from Antrodia camphorata (AC), and chloroform (3) and n-butanol (4) fractions of methanol extract from Cordyceps sinensis (CS), and hexane (5), ethyl acetate (6), and methanol (7) from Cinnamomum osmophloeum bark (CO) were evaluated for their anti-inflammatory as well as tumor-cell growth inhibitory activities in vitro. All the tested extracts dose dependently inhibited the enhanced production of inflammatory mediators such as nitric oxide (NO) through reducing inducible NO synthase expression, and cytokines (tumor necrosis factor (TNF)-alpha and interleukin (IL)-12 in LPS/IFN-gamma activated murine peritoneal macrophages. In addition, extracts 1 from AC, and 5 and 6 from CO significantly arrest the mitogen-stimulated spleen cells in G0/G1 stage. On the other hand, all these extracts were also evaluated for their tumor-cell proliferation activities in different type of cancer cell lines such as Jurkat, HepG2, PC 3, Colon 205, and MCF 7 as well as normal PBMCs. Compared to untreated controls, the extracts 1, 2, and 4-7 were most active and inhibited Jurkat cells with IC50 value of 22, 40, 18, 4, 5, and 45 microg/ml, respectively. In addition, the extracts 5, 6, and 7 from CO showed potent growth inhibition of HepG2 and PC 3 with IC50 values of 35, 80, 55 microg/ml; and 42, 125, and 50 microg/ml, respectively. Similarly, the extracts 1 and 5 inhibited the growth of Colon 205 and MCF 7 cells with IC50 values of 65, 33; and 95 and 30 microg/ml, respectively. Interestingly, none of the tested extract has shown cytotoxicity towards normal PBMCs up to the concentration range studies (0-150 microg/ml). Taken together, these data suggest that the anti-inflammatory and anti-cancer properties of AC, CS, and CO might result from the growth inhibition of NO, TNF-alpha and IL-12, and tumor cells proliferation, respectively.     

Differential signaling involved in Sutherlandia frutescens-induced cell death in MCF-7 and MCF-12A cells

Ethnopharmacological relevance

The scientific study of natural products traditionally used in anticancer preparations has yielded several therapeutically relevant compounds. One of these traditional preparations with potentially beneficial properties is aqueous extracts of Sutherlandia frutescens, a shrub indigenous to the Western Cape region of South Africa. The aims of this study were to evaluate in vitro efficacy of these preparations on the MCF-7 breast adenocarcinoma and MCF-12A non-tumorigenic cell lines in terms of cell proliferation, cell morphology and possible induction of cell death.

Materials and methods

Crystal violet staining was used to evaluate cell proliferation, light-and fluorescence microscopy were used to investigate both intracellular and extracellular morphological features of apoptosis and autophagy (e.g. membrane blebbing, condensed chromatin and intracellular lysosomes), while flow cytometry quantified cell cycle changes and induction of apoptosis through analysis of the flip-flop translocation of phosphatidylserine.

Results

Crystal violet staining showed a time- and dose specific response to aqueous Sutherlandia frutescens extracts, revealing exposure to 1 mg/ml aqueous extract for 48 h to be ideal for comparing the differential effects of Sutherlandia frutescens in the MCF-7 and MCF-12A cell lines. Microscopy showed distinct morphological changes with hallmarks of apoptosis being observed in both cell lines. Flow cytometry revealed a decrease in actively cycling cells in both cell lines, and a 4.36% increase in phosphatidylserine translocation in the MCF-7 cell line, indicative of apoptosis induction, while fluorescence microscopy showed evidence of the induction of autophagy.

Conclusions

Analyses revealed the carcinogenic MCF-7 cell line to be more susceptible to the cytostatic and cytotoxic effects of aqueous extracts of Sutherlandia frutescens when compared to the non-tumorigenic MCF-12A cell line, thus warranting further research into the exact cellular mechanisms involved and the possible synergistic activities of Sutherlandia frutescens ingredients.     

Spatholobus suberectus inhibits cancer cell growth by inducing apoptosis and arresting cell cycle at G2/M checkpoint

Aim of the study

Although herbs have long been alternatively applied for cancer treatment in China, its treatment effects and their potential mechanisms have not been sufficiently investigated. The chinese herb Spatholobus suberectus (SS) is commonly prescribed to cancer patients. In this study, the anti-cancer effect of SS and its molecular mechanisms have been investigated.

Materials and methods

The effect of SS on cell proliferation was studied by cell growth assay and flow cytometry on breast cancer cell lines MCF-7 and colon cancer cell line HT-29. The role of SS in apoptosis was studied by flow cytometry, DNA fragmentation assay and mitochondrial membrane potential assay. Expression of proteins associated with cell cycle and apoptosis was determined by Western blot analysis. The in vivo effect of SS was tested in nude mouse cancer xenografts.

Results

Cell growth assay showed that SS effectively inhibits tumor cell growth in a dose-dependent manner. Flow cytometry analysis showed that SS could arrest the cell cycle at G2/M checkpoint, which is associated with DNA damage and activation of phosphor-Chk1/Chk2. The pro-apoptotic effect of SS was demonstrated by Annexin V-PI staining and mitochondrial membrane potential assay. In vivo experiments show that the efficiency of SS alone group was superior to docetaxel or to docetaxel and SS combined. No obvious body weight loss or blood toxicity was observed in SS tested animals.

Conclusions

Our data demonstrates that SS is a potential herb for cancer treatment by inhibiting tumor growth via induction of apoptosis and arrest of the cell cycle at G2/M phase.