Origanum Sipyleum Methanol Extract in Combination with Ponatinib Shows Synergistic anti-Leukemic Activities on Chronic Myeloid Leukemia Cells.

Origanum sipyleum is used in folk medicine due to its anti-inflammatory, antimicrobial, and antioxidant properties. Ponatinib, an effective tyrosine kinase inhibitor in the treatment of chronic myeloid leukemia (CML), has severe side effects. Thus, we aimed to determine a novel herbal combination therapy that might not only increase the anti-leukemic efficacy but also reduce the dose of ponatinib in targeting CML cells. Origanum sipyleum was extracted with methanol (OSM), and secondary metabolites were determined by phytochemical screening tests. The cytotoxic effects of OSM on K562 cells were measured by WST-1 assay. Median-effect equation was used to analyze the combination of ponatinib and OSM (p-OSM). Apoptosis, proliferation, and cell-cycle were investigated by flow-cytometry. Cell-cycle-related gene expressions were evaluated by qRT-PCR. OSM that contains terpenoids, flavonoids, tannins, and anthracenes exhibited cytotoxic effects on K562 cells. The median-effect of p-OSM was found as synergistic; OSM reduced the ponatinib dose ∼5-fold. p-OSM elevated the apoptotic and anti-proliferative activity of ponatinib. Consistently, p-OSM blocked cell-cycle progression in G0/G1, S phases accompanied by regulations in TGFB2, ATR, PP2A, p18, CCND1, CCND2, and CCNA1 expressions. OSM enhanced the anti-leukemic activity of ponatinib synergistically via inducing apoptosis, suppressing proliferation, and cell-cycle. As a result, OSM might offer a potential strategy for treating patients with CML.

[Investigation of the Anti-Leishmanial Effects of Prangos ferulacea and Ferula orientalis Extracts Collected from Sirnak Province Against Leishmania tropica Isolated in Turkey]. / Sirnak Ili ve Çevresinden Toplanan Prangos ferulacea ve Ferula orientalis Ekstrelerinin Türkiye'den Izole Edilmis Leishmania tropica'ya Karsi Anti-Leishmanial Etkilerinin Arastirilmasi.

Leishmaniasis is a vector-borne disease that is caused by the protozoa of Leishmania genus. Leishmaniasis is endemic in tropical, subtropical, and large areas of the Mediterranean basin, and covers a total of 98 countries worldwide. It is estimated, according to the World Health Organization (WHO) data, that approximately 350 million people are at risk in these areas, and approximately 12 million people are infected. Increased drug resistance has been documented lately, in the treatment of leishmaniasis which causes almost 1.2 million new cases annually. Thus, interest in plant-derived active substances has increased in recent years, and new anti-leishmanial agents are investigated with in vitro studies. The aim of the present study was to investigate the anti-leishmanial effects of Prangos ferulacea and Ferula orientalis plant extracts collected from the rural areas of Sirnak province against Leishmania tropica. The water, chloroform, and ethanol extracts of the roots, stems, and fruits of P.ferulaceae and F.orientalis plants were obtained, and the cytotoxic activity tests of the extracts were performed. L.tropica isolate obtained from the Parasite Bank in Manisa Celal Bayar University in Turkey (MHOM/TR/2012/CBCL-LT) was grown on NNN and RPMI 1640 broth medium. The cytotoxicity of each extract on the L.tropica isolate was evaluated with the XTT test. Amphotericin B (AmpB) was used as the positive control, and the IC50 values were determined. The lowest IC50 values of the plant extracts were found to be as follows: P.ferulaceae root chloroform extract 36 µg/ml and fruit chloroform extract 20 µg/ml, F.orientalis root ethanol extract 2.5 µg/ml, and fruit ethanol extract 48 µg/ml, stem chloroform extract 24 µg/ml, and fruit chloroform extract 3.1 µg/ml. It was also determined in our study that only P.ferulaceae root ethanol extract showed cytotoxic activity on the WI-38 fetal lung fibroblast cell line at 65.19 µg/ml at 72 hours. This is the first study that assessed the anti-leishmanial activities of P.ferulaceae and F.orientalis plants that grow in high altitude areas of our country. It was determined that P.ferulaceae root ethanol extract and fruit chloroform extract had the lowest IC50 values among the 18 plant extracts that we examined for their anti-leishmanial activities. The outcomes of this study will be useful in further studies for the determination of active compounds in P.ferulaceae and F.orientalis plant extracts.

Propolis Extract Regulates microRNA Expression in Glioblastoma and Brain Cancer Stem Cells.

BACKGROUND: Grade IV gliomas are classified as glioblastoma (GBM), which is the most malignant brain cancer type. Various genetic and epigenetic mechanisms play a role in the initiation and progression of GBM. MicroRNAs (miRNAs) are small, non-coding RNA molecules that belong to the main epigenetic regulatory RNA class that plays different roles in either physiological or pathological conditions, including GBM pathogenesis regulating expression levels of the target genes. Brain Cancer Stem Cells (BCSCs) are responsible for poor prognosis, including therapy resistance and relapse. Epigenetic regulation mediated by miRNAs is also a critical component of BCSC selfrenewal and differentiation properties. Propolis is a resinous substance collected by honey bees from various plant sources. The flavonoid content of propolis varies depending on the collection region and the extraction method. Although there are studies that include the effects of different originated-propolis on the miRNA expression levels of the glioblastoma cells, the impact on the BCSCs has not been studied yet. OBJECTIVE: This study aims to evaluate the effects of propolis obtained from Aydin, a city in western Turkey, on miRNA expression levels of BCSCs and GBM cells. METHODS: Aydin propolis was dissolved in 60% ethanol, and after evaporation, distilled water was added to prepare the propolis stock solution. The flavonoids content of the Aydin propolis was determined by MS Q-TOF analysis. Commercially obtained U87MG and BCSCs were used as in-vitro brain cancer models. Cytotoxic and apoptotic effects of Aydin propolis were determined via WST-1 assay and Annexin V test, respectively. The miRNA expression profile was investigated using the real-time qRT-PCR method. The fold changes were calculated by the2-ΔΔCt method. The miRNA-mRNA-pathway interactions, including significantly altered miRNAs, were determined using different bioinformatics tools and databases. RESULTS: Quercetin 3-methyl ether was the main component of the Aydin propolis. Aydin propolis did not show significant cytotoxic and apoptotic effects on both GBM and BCSCs up to 2mg/ml concentration. Aydin propolis treatment decreased the expression of nine miRNAs in the U87MG and five miRNAs in the BCSCs. Moreover, ten miRNAs have upregulated from 2.22 to 10.56 folds in propolis treated GBM cells compared to the control group significantly (p<0.05). In the study, the potential roles of two new miRNAs, whose regulations in glioma were not previously defined, were identified. One of them was miR-30d-5p, a novel potential oncomiR in GBM, which was 2.46 folds downregulated in Aydin propolis treated GBM cells. The other one is miR-335-5p, which is a potential tumor suppressor miR in GBM, that was 5.66 folds upregulated in Aydin propolis treated GBM cells. FOXO pathway, its upstream and downstream regulators, and critically neuronal developmental regulators, NOTCH and WNT pathways, were determined as the most deregulated pathways in Aydin propolis treated cells. CONCLUSION: The determination of the anti-cancer effect of Aydin propolis on the miRNA expression of GBM, especially on cancer stem cells, may contribute to the elucidation of brain cancer genetics by supporting further analyses.

In vitro anti-Trichomonas vaginalis activity of Haplophyllum myrtifolium.

INTRODUCTION: In the classic treatment of Trichomonas vaginalis infection, although metronidazole has been used since the 1960s, there has been an increase in MTZ-resistant T. vaginalis strains and failure in the treatment of trichomoniasis causes serious concerns. Therefore, the present study aimed to investigate the in vitro antitrichomonal activities of extracts (ethanol and total alkaloid) and pure compounds (chrysosplenetin, dictamnine, gamma-Fagarine, skimmianine) of H. myrtifolium against T. vaginalis. METHODOLOGY: H. myrtifolium was collected from the town of Honaz in Denizli, located in the Aegean region of Turkey, and preparation of extracts and isolation and structure elucidation of pure compounds were performed. Later, different concentrations of extracts and pure compounds were incubated with T. vaginalis trophozoites isolated from Turkey, which are known to be sensitive to metronidazole. RESULTS: It was found that ethanol extract caused a more effective lysis on T. vaginalis trophozoites compared with total alkaloid extract (P < 0.05). No compounds except for furoquinoline alkaloid skimmianine prepared above 37.5 µg/mL were found to have any inhibitory effect on T. vaginalis trophozoites. CONCLUSION: The ethanol extract of H. myrtifolium and skimmianine can be considered as potential candidates for antitrichomonal drug development.

Effect of Extracts of the Endemic Plants Centaurea lydia and Phlomis nissolii on Toxoplasma gondii.

OBJECTIVE: This study aimed to investigate the potential antitoxoplasma activities of extracts of the endemic plants Centaurea lydia and Phlomis nissolii in a fibroblast cell culture infected with T. gondii trophozoites. METHODS: WI-38 cell lines treated with plant extracts (55 µg/mL each) and an untreated control were infected with 5×105 T. gondii trophozoites, and the number of parasites in the medium was determined on days 7, 14, and 24. RESULTS: No cytotoxic effects of C. lydia and P. nissolii extracts were detected at concentrations of 0.86-55 µg/mL in the WI-38 cell line, and the absence of the cytotoxicity of these extracts on the fibroblast cell line was considered as a positive effect. C. lydia extract at 55 µg/mL had marked activity against T. gondii trophozoites. A 47.5-fold increase was observed in the number of trophozoites in the control group, while a 84-fold decrease was found in the C. lydia extract group. However, a 36-fold increase was detected in the P. nissolii extract group, indicating no antitoxoplasma activity. CONCLUSION: The extract of C. lydia, an endemic plant, was found to be a good drug candidate for treating toxoplasmosis. The in vitro activity of the extract of this endemic plant should be further investigated in animal models in vivo.

Evaluation of the miRNA profiling and effectiveness of the propolis on B-cell acute lymphoblastic leukemia cell line.

Acute lymphoblastic leukemia (ALL) is one of the most frequent causes of death from cancer. Since the discovery of chemotherapeutic agents, ALL has become a model for improvement of survival. In parallel to this, serious side effects were observed and new natural therapeutic options has been discussed. One of these substances is called propolis which is a resinous substance gathered by honeybees. In the molecular era, miRNAs have been shown to play crucial roles in the development of many clinical conditions. The aim of this study is to evaluate the effect of Aydin propolis on 81 human miRNA activity in CCRF-SB leukemia cell line. Apoptotic effects of propolis on cell lines were also evaluated and apoptosis were found to be induced 1.5 fold in B-cell leukemia cells. The expression of 63 miRNAs (46 miRNAs were downregulated, 19 miRNAs were upregulated) in propolis treated leukemia cells have changed significantly (p<0.05). In conclusion propolis has changed expression of miRNAs which have epigenetic effects on leukemic cells. It is thought that it can be a promising agent for ALL treatment for future studies.

Olive Leaf Extract Improves the Atherogenic Lipid Profile in Rats Fed a High Cholesterol Diet.

Coronary heart disease because of atherosclerosis is still the most common cause of mortality. Elevated levels of low-density lipoprotein and total cholesterol are major risk factors for atherosclerotic cardiovascular disease. The aim of this study was to evaluate the effects of the olive leaf extract on serum lipid profile, early changes of atherosclerosis and endothelium-dependent relaxations in cholesterol-fed rats. For this purpose, rats were fed by 2% cholesterol-enriched or standard chow for 8 weeks. Some rats in each group were also fed orally by olive leaf extract at doses of 50 or 100 mg/kg/day. Atorvastatin at dose of 20 mg/kg of body weight daily was also given as positive control. After 8 weeks, lipid profiles of rat serums were analyzed. Antioxidant enzyme activities (superoxide dismutase and glutathione peroxidase) and degree of lipid peroxidation (malondialdehyde levels) were also measured in the hearts isolated from rats. In addition, expression of adhesion molecules and endothelium-dependent relaxations of isolated thoracic aortas of rats were evaluated. Total cholesterol and LDL-cholesterol levels were found to be increased in cholesterol-fed rats, and both doses of olive leaf extract and atorvastatin significantly decreased those levels. In conclusion, because the olive leaf extract attenuates the increased cholesterol levels, it may have beneficial effects on atherosclerosis.

Effects of Ganoderma lucidum (Higher Basidiomycetes) Extracts on the miRNA Profile and Telomerase Activity of the MCF-7 Breast Cancer Cell Line.

Ganoderma lucidum is a medicinal higher Basidiomycetes mushroom that exerts anticancer effects through several different mechanisms. This study investigated the effects of G. lucidum on the telomerase activity and microRNA (miRNA) profiles of MCF-7 cells. According to the cytotoxicity results, the G. lucidum ether extract exhibits the highest cytotoxic potency; therefore it was chosen for the subsequent telomerase activity assay and miRNA profiling. The telomerase activity observed in the cells treated with a half-maximal inhibitory concentration of G. lucidum ether extract (100 µg/mL in dimethyl sulfoxide) was 32.2% lower than that of the control cells treated with 1% dimethyl sulfoxide. Among 1066 miRNAs, the most downregulated miRNA was hsa-miR-27a* (4.469-fold), and the most upregulated miRNA was hsa-miR-1285 (10.462-fold). A database search revealed the predicted miRNAs that target the catalytic subunit of the telomerase enzyme telomerase reverse transcriptase, and only miR-3687 (upregulated 2.153-fold) and miR-1207-5p (upregulated 2.895-fold) were changed by at least 2-fold. The miRNA profile changes demonstrated in this study provide a data set regarding their effects on the pathways that regulate telomerase activity in MCF-7 breast cancer cells treated with G. lucidum. These data should aid the development of novel cancer treatment strategies.

Antimicrobial and antioxidant activities with acute toxicity, cytotoxicity and mutagenicity of Cystoseira compressa (Esper) Gerloff & Nizamuddin from the coast of Urla (Izmir, Turkey).

The aim of the study was to evaluate the biological activities with toxic properties of the methanol, hexane, and chloroform extracts of Cystoseira compressa (Esper) Gerloff & Nizamuddin from the Coast of Urla in the Aegean Sea. The extracts of C. compressa were tested for their antimicrobial and antioxidant activities in this study. Cytotoxic and mutagenic potentials of the extracts were also evaluated using cell culture and mutagenicity assays. Hexane extract was found to have higher total flavonoid and phenolic contents than the other extracts and exerted higher antioxidant activity than other extracts. All extracts exhibited moderate antimicrobial activity against tested microorganisms (minimum inhibitory concentration ranges are 32-256 µg/mL). The results indicated that the extracts had no significant cytotoxic activity against human hepatocellular carcinoma Hep 3B cell line in all treated concentrations (5-50 µg/mL) and did not show mutagenicity in the Ames test. Lethality was not observed among mice treated with oral doses of the extracts. In conclusion, results of investigations indicate that brown alga C. compressa is a natural source of antioxidant. It has moderate antimicrobial activities with no toxicity.

Assessment of in vivo antimalarial activities of some selected medicinal plants from Turkey.

Resistant infections lead to increased necessity of searching novel drugs and drug combinations. The purpose of this paper was to investigate antimalarial properties of some selected medicinal plants that have been traditionally used in Turkey for antipyretic and analgesic purposes. Lavandula stoecheas subsp. cariensis, Phlomis nissolii, Phlomis bourgaei, Phlomis leucophracta, Centaurea hierapolitana, Centaurea polyclada, Centaurea lydia, Scrophularia cryptophila, Scrophularia depauperata, Scrophularia floribunda, Rubia davisiana, and Alkanna tinctoria subsp. subleiocarpa were investigated for their in vivo antimalarial activities in mice infected with Plasmodium yoelii. Two hundred fifty to 500 mg/kg doses of plant extracts were given to mice as a single daily dose for 4 days. P. nissolii water extract, C. lydia chloroform extract, S. cryptophila ethanol extract, and C. polyclada methanol extract showed antimalarial activity with reducing parasitaemia. The chemotherapeutic effects of plant extracts ranged between 13.5% and 66.91%. The chemosuppressions exerted by combined plant extracts of P. nissolii, S. cryptophila, and C. lydia with C. polyclada methanol extract were detected as 51.25%, 57.33%, and 58.33%, respectively. Investigation of cytotoxic activities against brine shrimps revealed that methanol extract of C. polycada, chloroform extract of C. lydia, and ethanol extract of S. cryptophila showed cytotoxic activities, while water extract of P. nissolii was not active against brine shrimps.

Antioxidant, anti-inflammatory activities and acute toxicity of the polyherbal formulation: Romix®.

CONTEXT: Polyherbal formulations containing different plants are used for the treatment of various diseases. Romix® powder is a polyherbal formulation consisting of 14 traditionally used herbs and is used as a food supplement. There is no information about pharmaceutical activities of Romix®. OBJECTIVE: This study determined the total phenolic and total flavonoid content, and investigated the antioxidant and anti-inflammatory activities and acute toxicity of Romix®. MATERIAL AND METHODS: The total phenolics in the extracts were determined colorimetrically by using the Folin-Ciocalteu reagent. The total flavonoid content of the extracts was evaluated by a spectrophotometric method. The quercetin content of the extract was analyzed using the high-performance liquid chromatography (HPLC) method. Antioxidant activity of the extracts was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assays. The anti-inflammatory activity was evaluated by the carrageenan-induced paw edema test in the rat. RESULTS: The flavonoid and phenolics contents of Romix® were 50.58 and 265.83 mg/g in ethanol extract and 18.60 and 222.50 mg/g in water extract, respectively. Total quercetin content of Romix® was determined as 2.857 mg/g. Antioxidant activity results showed that ethanol extract in 1 mg/mL concentration (4.49775 µg/mL) had moderate antioxidant activity than water extract in the same concentration (4.28191 µg/mL). Intraperitoneal administration of 25 mg/kg Romix® extract exhibited anti-inflammatory activity and inhibited paw swelling at 1, 2, 3, 4, 5 and 6 h in rats with no acute toxicity. CONCLUSION: These findings suggest that Romix® due to its antioxidant and anti-inflammatory activities may be useful in the prevention or treatment of aging-related and inflammatory diseases.