Induction of apoptosis and autophagy by dichloromethane extract from Patrinia scabiosaefolia Fisch on acute myeloid leukemia cells.

Patrinia scabiosaefolia Fisch (PS), a perennial herb belonging to the genus Pinus in the family Pinnacle Sauce, has been previously known for its analgesic, anti-inflammatory, antibacterial, and antitumor properties. However, the specific mechanism behind its antileukemic effect remains unknown. This study focused on the cytotoxicity and potential modes of action of the dichloromethane extract from PS (DEPS) in acute myeloid leukemia (AML) cells. Our results demonstrated that DEPS reduced cell viability, arrested the cell cycle in the G2/M phase, disrupted the mitochondrial membrane potential, increased reactive oxygen species (ROS) production, and upregulated the expression of Bax/Bcl-2 and Cleaved caspase-3. However, the impact of DEPS on cell viability and the expression of apoptosis-associated proteins was reversed upon pretreatment with the caspase-3 inhibitor (Z-DEVD-FMK) in HL-60 cells, which demonstrated that DEPS could induce apoptosis through the mitochondria-associated apoptotic pathway. Interestingly, DEPS also influenced autophagy by upregulating the expression of LC3II/I, P62, and Beclin-1 proteins, and the autophagy inhibition chloroquine(CQ) could attenuate the apoptotic effects of DEPS in HL-60 cells. Furthermore, SMART 2.0 analysis predicted that the main components present in DEPS were likely terpenoids. In conclusion, DEPS possibly exerts antileukemic effects by downregulating the PI3K/AKT and ERK pathways, thereby promoting intracellular ROS production, activating the mitochondrial apoptotic pathway, and affecting autophagy, providing valuable insights for the potential future application of PS in the treatment of AML.

Anti-cancer Effect and Active Phytochemicals of Houttuynia cordata Thunb. against Human Breast Cancer Cells.

BACKGROUND: Houttuynia cordata Thunb (HCT) is a medicinal herb used in Southeast Asia. Aim of this work: This study aimed at investigating the cytotoxicity of this plant extract and fractions towards human breast cancer MDA-MB-231 and MCF-7 cells. HCT's phytoactive compounds are determined. MATERIALS AND METHODS: Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mode of cell death was measured by staining with annexin V-FITC and propidium iodide (PI) employing flow cytometry technique. The oxidative stress was measured by using 2',7'-dihydrodichlorofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE+) fluorescent probes and using a fluorescence microplate reader. HCT phytochemicals were characterized by high performance liquid chromatography (HPLC). RESULTS: The proliferation of MDA-MB-231 and MCF-7 cells was dramatically decreased by the crude extract and individual fraction of HCT. Ethyl acetate was the solvent fraction with the highest toxicity against MCF-7 cells, followed by dichloromethane, crude, and hexane fractions, respectively, whereas in MDA-MB231 cells, dichloromethane, crude, hexane, and ethyl acetate fractions each had the strongest impact, respectively. The methanol fraction had no effect on either cell line up to 200 µg/ml. The extract and fractions were less harmful to the NIH3T3 normal murine fibroblast cell line. The mode of both cell death was apoptosis evidenced by the increase of cell population stained with annexin V-FITC and PI. The fluorescence probes of both DCFH-DA and DHE in MDA-MB-231 cell line were enhanced. Phenolic acids included chlorogenic acid (CA), gallic acid (GA), transcoumaric acid (TCA), vanillic acid (VA), and syringic acid (SA), as well as flavonoids like quercetin and rutin, were identified as the active phytochemicals in the crude and fractions by using HPLC method. CONCLUSION: MDA-MB-231cells underwent apoptosis via oxidative stress when induced with HCT hexane fraction. Phenolic acids and flavonoids were identified in HCT's extract and fractions.

In Vitro effects of petroleum ether, dichloromethane, methanolic and aqueous leaf extracts of Eucalyptus grandis on selected multidrug-resistant bacteria.

BACKGROUND: The emergence and spread of antimicrobial resistance is of grave concern, requiring the search for newer and more effective antimicrobials to combat infections caused by resistant microbes. This study assessed the antimicrobial effects of Eucalyptus grandis crude extracts against selected multidrug resistant bacteria. METHODOLOGY: Four different crude leaf extracts of E. grandis were prepared using petroleum ether, dichloromethane, methanol, and water, with the aid of the Soxhlet extraction method. These were screened against methicillin-resistant Staphylococcus aureus (MRSA), multidrug resistant Pseudomonas aeruginosa, and multidrug resistant Escherichia coli, using the agar well diffusion method. Phytochemical screening was carried out to evaluate the bioactive phytochemical constituents responsible for the antimicrobial effect. RESULTS: Each of the extracts, except for the one prepared from water, had antimicrobial activity against the screened bacteria. The non-polar petroleum ether extract had the highest antimicrobial activity (19.33-24.33 mm), including bactericidal effects, compared to the medium polar dichloromethane and polar methanol extracts, which recorded zone diameter ranges of 14.33-16.67 mm and 16.33-17.67 mm, respectively. The Gram-negative bacteria (E. coli and P. aeruginosa) were the least susceptible in comparison with the Gram-positive bacterium (MRSA), probably owing to differences in their cell wall structures. Furthermore, phytochemical screening indicated the presence of alkaloids, tannins, saponins, terpenoids, and flavonoids. CONCLUSION: The findings suggest that E. grandis could be potentially useful in the treatment of infections caused by multidrug resistant bacteria.

[Effect of Dichloromethane Extraction Phase of Patrinia Scabiosaefolia Fisch. Stem on Proliferation and Differentiation of K562 Cells].

OBJECTIVE: To explore the effect of dichloromethane extraction phase of ethanol extract from stem of Patrinia scabiosaefolia Fisch.(DPSS) on proliferation and differentiation of K562 cells and its related mechanism. METHODS: MTT assay was used to detect the effects of DPSS at 0, 25, 50, 100 and 200 µg/ml on the proliferation of K562 cells at 24, 48 and 72 hours. Flow cytometry was used to analyze the changes of cell cycle and apoptosis at 24 and 48 hours. Wright-Giemsa staining was used to observe the morphological changes of K562 cells. The cell surface antigens CD33 and CD11b were detected by flow cytometry. RESULTS: The proliferation of K562 cells treated with different concentrations of DPSS was inhibited in a time-dose dependent manner (r=-0.96). Cell cycle analysis showed that with the increase of DPSS concentration, cells in G2/M phase increased (r=0.88), and cells were blocked in G2/M phase. Flow cytometry results showed that with the apoptosis rate of K562 cells was the highest when treated with 200 µg/ml DPSS for 48 h. Morphological observation showed that the K562 cell body increased, the amount of cytoplasm increased, the ratio of nucleus to cytoplasm decreased, and the nuclear chromatin was rough after DPSS treatment. Cell differentiation antigen, CD33 and CD11b, were positively expressed after treated with DPSS. CONCLUSION: DPSS can induce apoptosis through cell cycle arrest, inhibit the proliferation of K562 cells, and induce K562 cells to differentiate into monocytes, which has a potential anti-leukemia effect.

Development and in vivo evaluation of lipid-based nanocarriers containing Jatropha isabellei dry extract from the dichloromethane fraction intended for oral treatment of arthritic diseases

In this study, a dichloromethane fraction dry extract from the underground parts of Jatropha isabellei (DFJi) was used to prepare lipid nanocarriers (LNCJi) aimed at providing the oral delivery of terpenic compounds in the treatment of arthritis. The lipid nanocarriers were prepared by the spontaneous emulsification method. The lipid nanocarriers displayed sizes ranging from 180 to 200 nm and zeta potential values of around -18 mV. A high value of entrapment efficiency (> 90%) was obtained for jatrophone, which was used as the chemical marker of DFJi. LNCJi stored at 4°C were demonstrated to be stable through measurements of transmitted light after analytical centrifugation of the samples. In vitro drug release studies conducted in biorelevant dissolution media demonstrated that jatrophone release was faster from LNCJi than from free DFJi. When tested in an acute arthritis model, the LNCJi exhibited antinociceptive properties after oral administration of a 50 mg/kg dose, unlike the free DFJi, although no reduction in articular diameter was observed. These results suggest that an increase in the oral absorption of DFJi constituents may have occurred through the carrying of this fraction in LNCJi, thus improving the antinociceptive activity of this compound

Falcarindiol and dichloromethane fraction are bioactive components in Oplopanax elatus: Colorectal cancer chemoprevention via induction of apoptosis and G2/M cell cycle arrest mediated by cyclin A upregulation.

Oplopanax elatus (Nakai) Nakai has a long history of use as an ethnomedicine by the people living in eastern Asia. However, its bioactive constituents and cancer chemopreventive mechanisms are largely unknown. The aim of this study was to prepare O. elatus extracts, fractions, and single compounds and to investigate the herb's antiproliferative effects on colon cancer cells and the involved mechanisms of action. Two polyyne compounds were isolated from O. elatus, falcarindiol and oplopandiol. Based on our HPLC analysis, falcarindiol and oplopandiol are major constituents in the dichloromethane (CH2Cl2) fraction. For the HCT-116 cell line, the dichloromethane fraction showed significant effects. Furthermore, the IC50 for falcarindiol and oplopandiol was 1.7 µM and 15.5 µM, respectively. In the mechanistic study, after treatment with 5 µg/ml for 48 h, dichloromethane fraction induced cancer cell apoptosis by 36.5% (p < 0.01% vs. control of 3.9%). Under the same treatment condition, dichloromethane fraction caused cell cycle arrest at the G2/M phase by 32.6% (p < 0.01% vs. control of 23.4%), supported by upregulation of key cell cycle regulator cyclin A to 21.6% (p < 0.01% vs. control of 8.6%). Similar trends were observed by using cell line HT-29. Data from this study filled the gap between phytochemical components and the cancer chemoprevention of O. elatus. The dichloromethane fraction is a bioactive fraction, and falcarindiol is identified as an active constituent. The mechanisms involved in cancer chemoprevention by O. elatus were apoptosis induction and G2/M cell cycle arrest mediated by a key cell cycle regulator cyclin A.

Decaffeination and Neuraminidase Inhibitory Activity of Arabica Green Coffee (Coffea arabica) Beans: Chlorogenic Acid as a Potential Bioactive Compound.

Coffee has been studied for its health benefits, including prevention of several chronic diseases, such as type 2 diabetes mellitus, cancer, Parkinson's, and liver diseases. Chlorogenic acid (CGA), an important component in coffee beans, was shown to possess antiviral activity against viruses. However, the presence of caffeine in coffee beans may also cause insomnia and stomach irritation, and increase heart rate and respiration rate. These unwanted effects may be reduced by decaffeination of green bean Arabica coffee (GBAC) by treatment with dichloromethane, followed by solid-phase extraction using methanol. In this study, the caffeine and chlorogenic acid (CGA) level in the coffee bean from three different areas in West Java, before and after decaffeination, was determined and validated using HPLC. The results showed that the levels of caffeine were reduced significantly, with an order as follows: Tasikmalaya (2.28% to 0.097% (97 ppm), Pangalengan (1.57% to 0.049% (495 ppm), and Garut (1.45% to 0.00002% (0.2 ppm). The CGA levels in the GBAC were also reduced as follows: Tasikmalaya (0.54% to 0.001% (118 ppm), Pangalengan (0.97% to 0.0047% (388 ppm)), and Garut (0.81% to 0.029% (282 ppm). The decaffeinated samples were then subjected to the H5N1 neuraminidase (NA) binding assay to determine its bioactivity as an anti-influenza agent. The results show that samples from Tasikmalaya, Pangalengan, and Garut possess NA inhibitory activity with IC50 of 69.70, 75.23, and 55.74 µg/mL, respectively. The low level of caffeine with a higher level of CGA correlates with their higher levels of NA inhibitory, as shown in the Garut samples. Therefore, the level of caffeine and CGA influenced the level of NA inhibitory activity. This is supported by the validation of CGA-NA binding interaction via molecular docking and pharmacophore modeling; hence, CGA could potentially serve as a bioactive compound for neuraminidase activity in GBAC.

Aquilariae Lignum Methylene Chloride Fraction Attenuates IL-1ß-Driven Neuroinflammation in BV2 Microglial Cells.

Microglial hyperactivation and neuroinflammation are known to induce neuronal death, which is one of the main causes of neurodegenerative disorders. We previously found that Aquilariae Lignum extract attenuated both neuronal excitotoxicity and neuroinflammation in vivo and in vitro. For further analysis, we extracted the methylene chloride fraction of Aquilariae Lignum to determine the bioactive compounds. In this study, we investigated the anti-neuroinflammatory effects and underlying mechanisms of the Aquilariae Lignum fraction (ALF) using lipopolysaccharide (LPS)-stimulated BV2 microglial cells. BV2 cells were pretreated with ALF (0.5, 1, and 2.5 µg/mL) before treatment with LPS (1 µg/mL). Pretreatment with ALF significantly attenuated the LPS-induced overproductions of nitric oxide (NO), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and interleukin (IL)-1ß. These anti-inflammatory effects were supported by ALF-mediated modulation of the nuclear factor-kappa B (NF-κB) pathway. Furthermore, ALF exerted strong anti-inflammasome effects, as shown by IL-1ß-specific inhibitory activity, but not activity against tumor necrosis factor (TNF)-α, along with inhibition of caspase-1 activity and NACHT, LRR, and PYD domain-containing protein 3 (NLRP3)-related molecules. These results indicate the potent anti-neuroinflammatory activity of ALF and that its underlying mechanism may involve the regulation of NLRP3 inflammasome-derived neuroinflammation in microglial cells.

NOx-, IL-1ß-, TNF-α-, and IL-6-Inhibiting Effects and Trypanocidal Activity of Banana (Musa acuminata) Bracts and Flowers: UPLC-HRESI-MS Detection of Phenylpropanoid Sucrose Esters.

Banana inflorescences are a byproduct of banana cultivation consumed in various regions of Brazil as a non-conventional food. This byproduct represents an alternative food supply that can contribute to the resolution of nutritional problems and hunger. This product is also used in Asia as a traditional remedy for the treatment of various illnesses such as bronchitis and dysentery. However, there is a lack of chemical and pharmacological data to support its consumption as a functional food. Therefore, this work aimed to study the anti-inflammatory action of Musa acuminata blossom by quantifying the cytokine levels (NOx, IL-1ß, TNF-α, and IL-6) in peritoneal neutrophils, and to study its antiparasitic activities using the intracellular forms of T. cruzi, L. amazonensis, and L. infantum. This work also aimed to establish the chemical profile of the inflorescence using UPLC-ESI-MS analysis. Flowers and the crude bract extracts were partitioned in dichloromethane and n-butanol to afford four fractions (FDCM, FNBU, BDCM, and BNBU). FDCM showed moderate trypanocidal activity and promising anti-inflammatory properties by inhibiting IL-1ß, TNF-α, and IL-6. BDCM significantly inhibited the secretion of TNF-α, while BNBU was active against IL-6 and NOx. LCMS data of these fractions revealed an unprecedented presence of arylpropanoid sucroses alongside flavonoids, triterpenes, benzofurans, stilbenes, and iridoids. The obtained results revealed that banana inflorescences could be used as an anti-inflammatory food ingredient to control inflammatory diseases.

Satureja hortensis induces cell death and inhibited cell cycle progression in K562 myelogenous and Jurkat T cell leukemia cell lines.

Several plants of Satureja genus have shown anti-tumor activity. We investigated the antileukemia effects of different fractions of Satureja hortensis (Summer savory). The growth inhibitory effect of S. hortensis fractions on K562 and Jurkat leukemia cells were determined by MTT assay. The most effective fractions were analyzed by flow cytometry and colorimetric assay for apoptosis induction and cell cycle changes. Various fractions from S. hortensis showed growth inhibitory effects on leukemia cells, among them two hexane and dichloromethane fractions with IC50 values of 32.1-47.8 µg/ml (K562) and 44.3-45.7 µg/ml (Jurkat) were the most effective. According to annexin V staining, both of these fractions significantly induced apoptosis at 50µg/ml in K562 (hexane; 73.06 ± 5.11% and dichloromethane; 96.14 ± 2.33%) and Jurkat cells (hexane; 78.85 ± 11.9% and dichloromethane; 94.05 ± 2.47%) 48 h after treatment. They increased cell accumulation in sub-G1 phase (>50%, p < .001) and decreased number of cells in G0-G1, S and G2M phases. The fractions significantly increased the caspase-3 activity in both cell lines (≈2.5-3.5 fold of untreated cells). Hexane and dichloromethane fractions of S. hortensis had the capacity to induce death and change the cell cycle distribution in leukemia cells; therefore they might be good candidates for more studies in regard to their possible therapeutic usefulness in leukemia.

N-terminome and proteogenomic analysis of the Methylobacterium extorquens DM4 reference strain for dichloromethane utilization.

Methylobacterium strains can use one-carbon compounds, such as methanol, for methylotrophic growth. In addition to methanol, a few strains also utilize dichloromethane, a major industrial chlorinated solvent pollutant. With a fully assembled and annotated genome, M. extorquens DM4 is the reference bacterium for aerobic dichloromethane degradation. The doublet N-terminal oriented proteomics (dN-TOP) strategy was applied to further improve its genome annotation and a differential proteomics approach was performed to compare M. extorquens DM4 grown either with methanol or dichloromethane as the sole source of carbon and energy. These approaches led to experimental confirmation of 259 hypothetical proteins, correction of 78 erroneous predicted start codons, discovery of 39 new proteins and annotation of 66 signal peptides, including essential enzymes involved in methylotrophic growth. SIGNIFICANCE: Dichloromethane (methylene chloride, CH2Cl2, DCM) is one of the most widely used industrial halogenated solvents and a potential carcinogen. Microbial rehabilitation of worldwide-contaminated sites involves DCM breakdown by bacteria that are able to grow using this pollutant as their sole carbon and energy source. The most-studied methylotrophic DCM degrader is Methylobacterium extorquens strain DM4. Proteomic studies of the Methylobacterium genus have been performed previously, but genome-wide investigation of N-termini of expressed proteins has not yet been performed. Differential quantitative proteomic analysis also opens new research perspectives to better monitor and understand bacterial growth with DCM.

In vitro and in vivo anti-cancer activity of dichloromethane fraction of Triticum aestivum sprouts.

Triticum aestivum sprouts (TA) contain significant amounts of chlorophyll, minerals, enzymes, and other functional entities. Furthermore, TA extracts have been shown to possess anti-obesity, anti-diabetic and hepatoprotective effects and are believed to help blood flow, digestion, and general detoxification of the body. In this study, the mechanism underlying the anti-cancer effects of a dichloromethane fraction of TA (TDF) was investigated in vitro and in vivo. In vitro study was done by examining cancer cells growth, morphological changes, cell cycles, expressions of death receptors and apoptosis-linked proteins in wide range of human cancer cell lines. To investigate the effect of TDF in vivo, C57BL/6 mice were injected with B16 melanoma cells and orally administered TDF. TDF markedly inhibited cancer cell growth and induced cellular morphological alterations, cell cycle arrest and apoptosis, and enhanced the expressions of death receptors (DR)-4, 5, and 6 in cell lines. In addition, TDF regulated the expressions mitochondrial apoptosis-linked proteins and induced caspase-dependent cell death. It also significantly enhanced phosphorylation of ERK1/2 and JNK, but not p38, whereas inhibited the activation of NF-κB in cancer cells. In our mouse model, TDF significantly suppressed B16 melanoma growth, to an extent similar to cisplatin (reference control) and augmented immunomodulatory cytokines. In brief, this study presents the mechanism responsible for the anti-cancer effects of TDF in vitro and in vivo.

Influence of GSH S-transferase on the mutagenicity induced by dichloromethane and 1,2-dichloropropane.

It has been suggested that dichloromethane (DCM) and 1,2-dichloropropane (DCP) are responsible for occupational cholangiocarcinoma. Dihaloalkanes are metabolically activated by GSH S-transferase theta1 (GSTT1) to yield products such as episulfonium ions. However, whether the GSTT1-mediated step of these dihaloalkanes is related to occupational cholangiocarcinoma is not known. In the present study, we investigated the influence of GSTT1 activation on the mutagenicity of DCM and 1,2-DCP using GSTT1-expressing Salmonella typhimurium TA100 (TA100-GST). Since the mutagenicity of DCM was significantly increased in TA100-GST compared with mock control (TA100-pCTC), GSTT1 is thought to be involved in the mutagenicity of DCM. Mutation spectrum analysis on the hisG gene revealed that C:G to A:T transversions were the predominant form observed in DCM-treated TA100-pCTC. However, C:G to T:A transitions were dramatically increased in TA100-GST. We also analysed the DCM-DNA adduct, N2-GSH-Me-dG, and formation of N2-GSH-Me-dG was increased in TA100-GST compared with TA100-pCTC. On the other hand, 1,2-DCP did not increase the numbers of revertants in TA100-GSTT1. In mutation spectrum analysis, C:G to T:A transitions was predominant in both TA100-pCTC and TA100-GSTT1. These findings suggest that GSTT1 has little involvement in DCP mutagenicity, and other mechanisms might be more important for bioactivation and consequent genotoxicity. Clarification of the mechanisms underlying the development of DCM- and/or 1,2-DCP-related human cholangiocarcinoma may help establish risk assessment and prevention strategies against occupational cancer.

Analysis of chemical constituents, antimicrobial and anticancer activities of dichloromethane extracts of Sordariomycetes sp. endophytic fungi isolated from Strobilanthes crispus.

Plants are primary source of natural product drugs. However, with every new bioactive molecule reported from a plant source, there follows reports of endangered status or even extinction of a medicinally important plant due to over-harvesting. Hence, the attention turned towards fungi namely the endophytes, which reside within medicinally important plants and thus may have acquired their medicinal properties. Strobilanthes crispus is a traditional medicinal plant which has been used traditionally to treat kidney stones, diabetes, hypertension and cancer as well as having antimicrobial activities. In our efforts to bioprospect for anticancer and antimicrobial metabolites, two fungal endophytes most closely related to the Sordariomycetes sp. showed promising results. Sample (PDA)BL3 showed highest significant antimicrobial activity against 6 bacteria at 200 µg/disc whereas sample (PDA)BL5 has highest significant anticancer activity against all 5 cancer cell lines at concentrations ranging from 30 to 300 µg/ml. As for the gas chromatography coupled with mass spectrometry (GC-MS) results, a total of 20 volatile metabolites identified from sample (PDA)BL3 and 21 volatile metabolites identified from sample (PDA)BL5 having more than 1% abundance. Both GC-MS analysis showed that compound Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) has the highest abundance at 15.10% abundance for sample (PDA)BL3 and 19.00% abundance for sample (PDA)BL5 respectively. In conclusion, these results have shown bio-prospecting potential of endophytic fungi having antimicrobial and anticancer activities as well as its potential secondary metabolites of interest. Therefore, this work has further indicated the medicinal and industrial potential of endophytic fungi.

CYTOTOXIC, α-CHYMOTRYPSIN AND UREASE INHIBITION ACTIVITIES OF THE PLANT Heliotropium dasycarpum L.

BACKGROUND: The aim of this study was to investigate Cytotoxic, α-Chymotrypsin and Urease inhibition activities of the plant Heliotropium dasycarpum. MATERIALS & METHODS: Dichloromethane and methanol extracts of the plant were evaluated for cytotoxic, α-Chymotrypsin and Urease inhibition by using in vivo Brine Shrimp lethality bioassay and in vitro enzymatic inhibition assays respectively. RESULTS: The methanol extract of the plant exhibited significant cytotoxic activity. Out of 30 brine shrimp larvae, 2 (6%), 26 (86%) and 28 (93%) larvae were survived at concentration of 1000µg/ml, 100µg/ml and 10µg/ml respectively with LD50; 215.837. Similarly 21 (70%), 25 (83%), 29 (96%) larvae were survived of dichloromethane plant extract with LD50; 6170.64. The methanol and dichloromethane extract exhibited 10.50±0.18% and 41.51±0.15% α-chymotrypsin enzyme inhibition respectively with IC50 values of greater than 500 µmol. The methanol extract showed 24.39±0.21% Urease enzyme inhibition with IC50 values of greater than 400 µmol While dichloromethane extract has 11.46±0.09% enzyme inhibition with IC50 values of greater than 500 µmol. CONCLUSION: The results clearly indicated that Heliotropium dasycarpum has cytotoxic potential and enzyme inhibition properties. Further study is needed to screen out antitumor and anti-ulcerative agents.

Dichloromethane fractions of Scrophularia oxysepala extract induce apoptosis in MCF-7 human breast cancer cells.

Breast cancer is a prevalent malignancy among women, especially in developing countries. A large number of anticancer agents with herbal origins have been reported. Hence, herbals may play an essential role in prevention and treatment of cancers. We investigated cytotoxic effects of dichloromethane fractions of Scrophularia oxysepala extract on the MCF-7 breast cancer cell line. The cytotoxic activity of Scrophularia oxysepala fractions on the MCF-7 cells was assessed using Trypan Blue dye exclusion and MTT (3-(4, 5-dimetylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) assays. In addition, apoptosis induction was determined using TUNEL (terminal deoxy transferase (TdT)-mediated dUTP nick- end labeling) assay and DNA fragmentation analysis. Quantitative Real-Time PCR was also used for analyzing the changes in Caspase-3, Caspase-9, and Bcl-2 genes' expression. Results revealed an effective inhibition of growth and viability in MCF-7 cells treated with dichloromethane fractions. Cell death assay and DNA fragmentation analysis using the TUNEL test confirmed the induction of apoptosis in the MCF-7 cell line. Further, the fractions have resulted in an increased expression of Caspase-3 and Caspase-9 mRNA, which highlights the possibility of apoptosis in the treatments. The expression study of Caspase-9 mRNA confirmed that, the fractions have triggered apoptosis via intrinsic mitochondrial pathway. In summary, fractions of Scrophularia oxysepala extract were found to be promising in growth inhibition and induction of apoptosis in MCF-7 breast cancer cells.

Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae)

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.

In vitro and in vivo antileishmania activity of sesquiterpene lactone-rich dichloromethane fraction obtained from Tanacetum parthenium (L.) Schultz-Bip.

The discovery of new treatments for neglected diseases, including leishmaniasis, is a substantial challenge for scientific research. Plant extracts have shown potential in the selective treatment of tropical diseases. The present study evaluated the in vitro and in vivo antileishmania effects of a sesquiterpene lactone-rich dichloromethane fraction (DF) obtained from the aerial parts of Tanacetum parthenium (L.) Schultz-Bip. In vitro studies of the DF indicated an IC50 of 2.40±0.76 µg mL(-1) against the promastigote form and 1.76±0.25 µg mL(-1) against the axenic amastigote form of Leishmania amazonensis. In vivo intramuscular treatment with DF decreased the growth and size of footpad lesions in mice. The DF also significantly decreased the parasite population compared with animals that were treated with the reference drug. Plasma malondialdehyde levels were increased slightly by the DF, attributable to its parthenolide-rich composition that causes cellular apoptosis, compared with the control group, demonstrating treatment efficacy without toxicity or genotoxicity. Because the isolation and purification of plant compounds are costly and time-consuming and generate low yields, extract fractions, such as the DF studied herein, represent a promising alternative for the treatment of leishmaniasis.

Novel antimicrobial activity of a dichloromethane extract obtained from red seaweed Ceramium rubrum (Hudson) (Rhodophyta: Florideophyceae) against Yersinia ruckeri and Saprolegnia parasitica, agents that cause diseases in salmonids

Background Enteric red mouth disease and Saprolegniasis, which are caused by the bacteria Yersinia ruckeri and the oomycete Saprolegnia parasitica, respectively, are important illnesses that affect salmonid farming. Sanitary problems in farms are addressed by the prevention of disease outbreaks or by the treatment of diseases with chemicals. Environmental and governmental restrictions, toxicity and high treatment costs limit the use of drugs. Marine organisms, such as algae, sponges and corals, have developed an antimicrobial defense strategy based on the production of bioactive metabolites. Among these organisms, seaweeds offer a particularly rich source of potential new drugs. Hence, many pharmacologically active substances have been isolated from seaweeds. In the Ceramium genus, Ceramium rubrum has been emphasized by several authors for its antimicrobial properties. Based on this background, the present study focused on the antimicrobial activity of a lipophilic extract of C. rubrum on Y. ruckeri and S. parasitica. Results The alga, collected from the Pacific coast of Chile, underwent an ethanol extraction, and the concentrated extract was partitioned between water and dichloromethane. From the dichloromethane extract, fatty acids, fatty acid esters, one hydrocarbon and phytol were identified by Gas Chromatography-Mass Spectrometry (GC/MS) analysis. The antimicrobial study showed that the whole extract was more active than the individual components, which suggests a strong synergistic effect among the components. Conclusions These results may constitute a basis for promising future applied research that could investigate the use of C. rubrum seaweed as a source of antimicrobial compounds against fish pathogens.

Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae).

We evaluated the in vitro anti-Mycobacterium tuberculosis activity and the cytotoxicity of dichloromethane extract and pure compounds from the leaves of Calophyllum brasiliense. Purification of the dichloromethane extract yielded the pure compounds (-) mammea A/BB (1), (-) mammea B/BB (2) and amentoflavone (3). The compound structures were elucidated on the basis of spectroscopic and spectrometric data. The contents of bioactive compounds in the extracts were quantified using high performance liquid chromatography coupled to an ultraviolet detector. The anti-M. tuberculosis activity of the extracts and the pure compounds was evaluated using a resazurin microtitre assay plate. The cytotoxicity assay was performed in J774G.8 macrophages using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colourimetric method. The quantification of the dichloromethane extract showed (1) and (2) at concentrations of 31.86 ± 2.6 and 8.24 ± 1.1 µg/mg of extract, respectively. The dichloromethane and aqueous extracts showed anti-M. tuberculosis H37Rv activity of 62.5 and 125 µg/mL, respectively. Coumarins (1) and (2) showed minimal inhibitory concentration ranges of 31.2 and 62.5 µg/mL against M. tuberculosis H37Rv and clinical isolates. Compound (3) showed no activity against M. tuberculosis H37Rv. The selectivity index ranged from 0.59-1.06. We report the activity of the extracts and coumarins from the leaves of C. brasiliense against M. tuberculosis.