Suppression of VEGF-induced angiogenesis and tumor growth by Eugenia jambolana,Musa paradisiaca,and Coccinia indica extracts

Context: Abnormal angiogenesis and evasion of apoptosis are hallmarks of cancer. Accordingly, anti-angiogenic and pro-apoptotic therapies are effective strategies for cancer treatment. Medicinal plants, namely, Eugenia jambolana Lam. (Myrtaceae), Musa paradisiaca L. (Musaceae), and Coccinia indica Wight &; Arn. (Cucurbitaceae), have not been greatly investigated for their anticancer potential.

Objective: We investigated the anti-angiogenic and pro-apoptotic efficacy of ethyl acetate (EA) and n-butanol (NB) extracts of E. jambolana (seeds), EA extracts of M. paradisiaca (roots) and C. indica (leaves) with respect to mammary neoplasia.

Materials and methods: Effect of extracts (2–200?μg/mL) on cytotoxicity and MCF-7, MDA-MB-231 and endothelial cell (EC) proliferation and in vitro angiogenesis were evaluated by MTT, ³[H]thymidine uptake and EC tube formation assays, respectively. In vivo tumour proliferation, VEGF secretion and angiogenesis were assessed using the Ehrlich ascites tumour (EAT) model followed by rat corneal micro-pocket and chicken chorioallantoic membrane (CAM) assays. Apoptosis induction was assessed by morphological and cell cycle analysis.

Results: EA extracts of E. jambolana and M. paradisiaca exhibited the highest cytotoxicity (IC₅₀ 25 and 60?μg/mL), inhibited cell proliferation (up to 81%), and tube formation (83% and 76%). In vivo treatment reduced body weight (50%); cell number (16.5- and 14.7-fold), secreted VEGF (～90%), neoangiogenesis in rat cornea (2.5- and 1.5-fold) and CAM (3- and 1.6-fold) besides EAT cells accumulation in sub-G1 phase (20% and 18.38%), respectively.

Discussion and conclusion: Considering the potent anti-angiogenic and pro-apoptotic properties, lead molecules from EA extracts of E. jambolana and M. paradisiaca can be developed into anticancer drugs.     

The chorioallantoic membrane: A novel approach to extrapolate data from a well-established method

The chorioallantoic membrane (CAM) of the chicken embryo is a highly vascularized extra-embryonic structure that has been widely used as an in vivo model for the evaluation of angiogenesis. This study was designed to optimize data extrapolation from the most exploited experimental protocol to improve its efficiency and the reliability of the obtainable results. In our study, we followed the most common procedure for CAM assay, employing retinoic acid and vascular endothelial growth factor as standards. CAMs were photographed at t₀, t₂₄, and t₄₈; then, the main parameters of the predefined vascular network/area were evaluated. Subsequently, their variations in each CAM were calculated comparing them within the same CAM over the course of the whole treatment (t₂₄ and t₄₈), also comparing the treated CAMs respect to the untreated ones. Thus, we provide a novel approach aimed at extrapolating data from CAM assay that allows to (i) have a greater reliability and richness of data; (ii) better estimate the potential pro- and anti-angiogenic activity of new candidate drugs; (iii) save both eggs and time for the experiments.     

Cytotoxic effect of Amphotericin B in a myofibroblast cell line

In this study we investigate whether Amphotericin B (AmB), a widely used antifungal agent, could decrease the proliferation of a myofibroblast cell line – GRX, a model of activated hepatic stellate cells (HSC). Three different hepatic cell lines (GRX, Hep G2 and ARL-6) were treated with two concentrations of AmB (1.25 μg/mL or 2.50 μg/mL). Cytotoxicity was assessed by MTT assay. The effects of AmB on GRX migration was evaluated by Wound-healing Assay. Cell cycle arrest was investigated by flow cytometry. Apoptosis and autophagy were analyzed by Caspase 3 and LC3 immunostaining, respectively. Treatment with AmB 1.25 or 2.50 μg/mL showed a decrease in viability of GRX cells. This decrease was not observed for Hep G2 or ARL-6 in any of the two AmB concentrations tested. GRX cells treated with 1.25 μg/mL AmB were unable to close the wound after 96 h. Cell cycle analysis showed an increase in sub-G1 population and a decrease in G2/M population in AmB-treated cells. In addition, AmB-treated GRX cells showed increased expression of LC-3 and Caspase-3 by immunohistochemistry, suggesting an increase in both autophagy and apoptosis. Here we show that AmB is cytotoxic for GRX cells, a model of activated HSC, but not for hepatic lineages HepG2 and ARL6.     

In vitro inhibition of angiogenesis by heat and low pH stable hydroalcoholic extract of Peganum harmala seeds via inhibition of cell proliferation and suppression of VEGF secretion

Abstract

Context: Progression of cancer cells is completely dependent on its angiogenesis. Inhibition of tumor angiogenesis has shed new light on cancer treatment. As a result, anti-angiogenesis therapy represents one of the most significant advances in clinical oncology. Peganum harmala L. (Zygophyllaceae) is a native plant from the eastern Iranian region, which is used as a traditional folk medicine. Although some biological properties of this plant are determined, its effect on angiogenesis is still unclear.

Objective: We investigated the anti-angiogenic effects of heat and low pH stable hydroalcoholic extract of P. harmala seeds on endothelial cells (ECs) proliferation and VEGF secretion.

Materials and methods: Dried Peganum seeds were purchased from Kermanshah Traditional Bazar in 2011. Hydroalcoholic extract of dried seeds (0, 10, 20, 40, 60, 80, 100, 120, and 150?μg/ml) was used for in vitro evaluation of its cytotoxicity, anti-proliferative, and anti-angiogenic effects on ECs. In vitro effect of the extract on VEGF secretion was assayed using ELISA.

Results: Treatment with hydroalcoholic extract at seven different concentrations resulted in significant decrease of ECs proliferation and angiogenesis with an ID₅₀ of ～85?μg/ml. VEGF secretion was (inhibited) decreased by the extracts at concentrations higher than 10?μg/ml.

Discussion and conclusion: Herbal plant extracts still attract attention owing to their fewer side effects comparing to synthetic drug agents. Current study indicated that hydroalcoholic extract of P. harmala seeds contains a potent anti-angiogenic component, which exerts its inhibitory effect mainly through down-regulation of essential mediators such as VEGF.     

Anti-angiogenic activities associated with exposure of environmental smoke solutions from 2-stroke auto-rickshaw

Angiogenesis, the formation of new blood vessels, is vital for embryonic development and disruption of this process can be a powerful mechanism of abortion. Over the last few decades there has been increasing global concern regarding the public health impact attributed to environmental smoke pollution. However, no study has yet examined the relation between exhaust from 2-stroke auto-rickshaws and angiogenesis. The current experiment was carried out to elucidate the possible detrimental effects of 2-stroke auto-rickshaw smoke solutions (2SARSS) on physiological angiogenesis, using a well-defined chicken chorioallantoic membrane (CAM) assay. Gross computer based 3D image probing and histopathologic modalities were utilized to quantify different detrimental effects of 2SARSS on the fundamental processes of angiogenesis. Macroscopic investigations of 2SARSS treated CAMs revealed severe disruption in the orientation and normal branching pattern of the blood vessels with profound disorganization. Application of 2SARSS caused substantial decrease in the total vascular area of CAM (p<0.001) diameters of the primary, secondary (p<0.01) and tertiary blood vessels (p<0.001) as well as capillary plexuses formation (p<0.001). Evaluation of different 3D parameters of 2SARSS treated CAMs unveiled diminished surface roughness, angular distribution, and height of the Abbott curves. Moreover, histological evaluations of 2SARSS treated CAMs also revealed disruption of the normal architecture of the blood vessel with marked thinning of ectodermal layer and mesodermal extracellular matrix. The anti-angiogenic effects of 2SARSS clearly demonstrate its toxicity to those travelling and/or living in the vicinity of these vehicles and these populations may suffer from several angiogenesis related pathologies.     

Piqueria trinervia as a source of metabolites against Giardia intestinalis

Context: Piqueria trinervia Cav. (Asteraceae) is a plant species with a long history in traditional medicine to cure diarrhoea and other digestive disorders.

Objective: The study investigates the antigiardial activity of piquerol, trinervinol, red oil and two fractions (F1 and F2) from P. trinervia.

Materials and methods: P. trinervia was collected in the Ajusco in Mexico City. Aerial parts were ground and mixed with water to obtain the extract, which was treated with dichloromethane to isolate piquerol and trinervinol (P &; T). Remnants were the red oil, fractions 1 and 2 (RO, F1 &; F2). Trophozoites of Giardia intestinalis were treated with P, T, RO, F1 and F2 at different concentrations (0.78–200?μg/mL) for 48?h. Antigiardial activity was measured using the methylene blue reduction, and the cytotoxicity assayed on human fibroblasts and Vero cells by reduction of tetrazolium salts.

Results: Trinervinol and piquerol showed antigiardial activity with an IC₅₀?=?2.03 and 2.42?μg/mL, and IC₉₀?=?13.03 and 8.74?μg/mL, respectively. The concentrations of trinervinol (CC₅₀?=?590?μg/mL) and piquerol (CC₅₀?=?501?μg/mL) were not cytotoxic to human fibroblasts.

Conclusions: Compounds from P. trinervia showed antigiardial activity; to enhance this activity, piquerol and trinervinol can be chemically modified.     

The in vivo evaluation of anti-angiogenic effects of Hypericum essential oils using the chorioallantoic membrane assay

Context. Hypericum species including Hypericum confertum Choisy, H. hircinum L., H. hyssopifolium Chaix. subsp. elongatum (Ledeb.) Woron var. microcalycinum (Boiss. &; Heldr.) Boiss. and H. perforatum L. (Clusiaceae) are used as medicinal plants in Turkey.

Objective: The anti-angiogenic evaluation of Hypericum essential oils using the chick embryo chorioallantoic membrane (CAM) assay are performed with this study for the first time.

Materials and methods: The anti-angiogenic activity of Hypericum essential oils (0.5–5.0?mg/ml) was evaluated in vivo using the CAM assay, compared to standard anti-angiogenic substances at the same concentrations, in trice replicated independent assays. GC and GC-MS analyses were carried out simultaneously to identify the chemical compositions of the Hypericum essential oils.

Results: The CAM treated with H. perforatum essential oil showed anti-angiogenic effect (score 0.6?±?0.3) at 50?µg/pellet concentration, whereas other tested Hypericum essential oils showed no effect compared to the standards (e.g. suramin score 0.5?±?0.2). Furthermore, the tested oils showed neither membrane toxicity nor irritation at the tested concentrations. The major compound of the essential oil of H. confertum was identified as germacrene D (30.2%). The major compound of the essential oils of the H. hircinum. H. hyssopifolium subsp. elongatum var. microcalycinum and H. perforatum was identified as α-pinene (88.3, 57.8, 33.3%), respectively.

Discussion and conclusion. Hypericum species and in particular H. perforatum essential oil may have important effect toward wound healing and various inflammations. The data obtained in this experiment suggest further investigations on various cancers due to its anti-angiogenic effects observed.     

Unlocking the in vitro anti-inflammatory and antidiabetic potential of Polygonum maritimum

Context: Several Polygonum species (Polygonaceae) are used in traditional medicine in Asia, Europe and Africa to treat inflammation and diabetes.

Objective: Evaluate the in vitro antioxidant, anti-inflammatory and antidiabetic potential of methanol and dichloromethane extracts of leaves and roots of the halophyte Polygonum maritimum L.

Material and methods: Antioxidant activity was determined (up to 1?mg/mL) as radical-scavenging activity (RSA) of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), copper (CCA) and iron (ICA) chelating activities and iron reducing power (FRAP). NO production was measured in lipopolysaccharide (LPS)-stimulated macrophages for 24?h at concentrations up to 100?μg/mL and antidiabetic potential was assessed by α-amylase and α-glucosidase inhibition (up to 10?mg/mL) assays. The phytochemical composition of the extracts was determined by gas chromatography-mass spectrometry (GC-MS).

Results: The methanol leaf extract had the highest activity against DPPH? (IC₅₀ =?26?μg/mL) and ABTS⁺? (IC₅₀ =?140?μg/mL), FRAP (IC₅₀ =?48?μg/mL) and CCA (IC₅₀ =?770?μg/mL). Only the dichloromethane leaf extract (LDCM) showed anti-inflammatory activity (IC₅₀ =?48?μg/mL). The methanol root (IC₅₀ =?19?μg/mL) and leaf (IC₅₀ =?29?μg/mL) extracts strongly inhibited baker’s yeast α-glucosidase, but LDCM had higher rat’s α-glucosidase inhibition (IC₅₀ =?2527?μg/mL) than acarbose (IC₅₀ =?4638?μg/mL). GC-MS analysis identified β-sitosterol, stigmasterol, 1-octacosanol and linolenic acid as possible molecules responsible for the observed bioactivities.

Conclusions: Our findings suggest P. maritimum as a source of high-value health promoting commodities for alleviating symptoms associated with oxidative and inflammatory diseases, including diabetes.     

Assessment of the mutagenic,recombinogenic, and carcinogenic potential of amphotericin B in somatic cells of Drosophila melanogaster

Amphotericin B (AmB) is an antifungal antibiotic extracted from Streptomyces nodosus. Its fungicidal activity depends primarily on its binding to the sterol group that is present in fungal membranes. In view of the toxicity of this drug, the purpose of this study was to evaluate its mutagenic, carcinogenic, and recombinogenic activity, based on the wing somatic mutation and recombination test (SMART) and the epithelial tumor detection test (wts) applied to Drosophila melanogaster. Larvae were chronically treated with different concentrations of AmB (0.01, 0.02, and 0.04?mg/mL). The results revealed that AmB is a promutagen exhibiting increase in the number of spots on individuals from high bioactivation (HB) cross with a high level of cytochrome P450. The results also indicate that the main genotoxic event induced by AmB is recombinogenicity. Homologous recombination can act as a determinant at different stages of carcinogenesis. For verification of carcinogenic potential of this compound, larvae from the wts/mwh and wts/ORR, flr³ were treated with the same three AmB concentrations used in the SMART assay. The results did not provide evidence that AmB has carcinogenic potential in wts/mwh individuals. However, individuals from wts/ORR, flr³ developed tumors at the highest concentration tested.     

Plasma and tissue disposition of florfenicol in Escherichia coli lipopolysaccharide-induced endotoxaemic sheep

1.?The purpose of this study was to understand the effects of the acute inflammatory response (AIR) induced by Escherichia coli lipopolysaccharide (LPS) on florfenicol (FFC) and FFC-amine (FFC-a) plasma and tissue concentrations.

2.?Ten Suffolk Down sheep, 60.5?±?4.7?kg, were distributed into two experimental groups: group 1 (LPS) treated with three intravenous doses of 1?μg/kg bw of LPS at 24, 16, and 0.75?h (45?min) before FFC treatment; group 2 (Control) was treated with saline solution (SS) in parallel to group 1. An IM dose of 20?mg FFC/kg was administered at 0.75?h after the last injection of LPS or SS. Blood and tissue samples were taken after FFC administration.

3.?The plasma AUC_0–4?h values of FFC were higher (p?=?0.0313) in sheep treated with LPS (21.8?±?2.0?μg·min/mL) compared with the control group (12.8?±?2.3?μg·min/mL). Lipopolysaccharide injections increased FFC concentrations in kidneys, spleen, and brain. Low levels of plasma FFC-a were observed in control sheep (C_max?=?0.14?±?0.01?μg/mL) with a metabolite ratio (MR) of 4.0?±?0.87%. While in the LPS group, C_max increased slightly (0.25?±?0.01?μg/mL), and MR decreased to 2.8?±?0.17%.

4.?The changes observed in the plasma and tissue concentrations of FFC were attributed to the pathophysiological effects of LPS on renal hemodynamics that modified tissue distribution and reduced elimination of the drug.     

Cytotoxicity and anti-Leishmania amazonensis activity of Citrus sinensis leaf extracts

Context: Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease characterized by lesional polymorphism and the commitment of skin surface. Previous reports demonstrated that the Citrus genus possess antimicrobial activity.

Objective: This study evaluated the anti-L. amazonensis activity of Citrus sinensis (L.) Osbeck (Rutaceae) extracts.

Materials and methods: Citrus sinensis dried leaves were subjected to maceration with hexane (CH), ethyl acetate (CEA), dichloromethane/ethanol (CD/Et – 1:1) or ethanol/water (CEt/W – 7:3). Leishmania amazonensis promastigotes were treated with C. sinensis extracts (1–525?μg/mL) for 120?h at 27?°C. Ultrastructure alterations of treated parasites were evaluated by transmission electron microscopy. Cytotoxicity of the extracts was assessed on RAW 264.7 and J774.G8 macrophages after 48-h treatment at 37?°C using the tetrazolium assay. In addition, Leishmania-infected macrophages were treated with CH and CD/Et (10–80?μg/mL).

Results: CH, CD/Et and CEA displayed antileishmanial activity with 50% inhibitory activity (IC₅₀) of 25.91?±?4.87, 54.23?±?3.78 and 62.74?±?5.04?μg/mL, respectively. Parasites treated with CD/Et (131.2?μg/mL) presented severe alterations including mitochondrial swelling, lipid body formation and intense cytoplasmic vacuolization. CH and CD/Et demonstrated cytotoxic effects similar to that of amphotericin B in the anti-amastigote assays (SI of 2.16, 1.98 and 1.35, respectively). Triterpene amyrins were the main substances in CH and CD/Et extracts. In addition, 80?μg/mL of CD/Et reduced the number of intracellular amastigotes and the percentage of infected macrophages in 63% and 36%, respectively.

Conclusion: The results presented here highlight C. sinensis as a promising source of antileishmanial agents.     

Antifungal and immunomodulatory activity of a novel cochleate for amphotericin B delivery against Sporothrix schenckii

Introduction: Sporotrichosis is an emergent subcutaneous mycoses caused by species of the Sporothrix schenckii complex. Amphotericin B (AmB) remains the main antifungal drug for the treatment of systemic infections, but its use is limited by toxicity reasons. AFCo3 is a novel cochleate containing detoxified LPS, which exhibits drug delivery and immunomodulating properties. Here, AFCo3 was used as the vehicle for AmB to evaluate the immunomodulatory and antifungal efficacy against S. schenckii in vitro and in vivo. Methods and results: The minimum inhibitory concentrations of AFCo3-AmB and AmB were 0.25 and 1 μg/mL respectively. The minimum fungicidal concentration was 0.5 μg/mL for AFCo3-AmB and 2 μg/mL for AmB. AFCo3-AmB was less cytotoxic than AmB for peritoneal macrophages, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and reduced the AmB-induced hemolysis in murine erythrocytes. AFCo3-AmB improved the intracellular killing of phagocytized yeast and it enhanced the in vitro production of IL-1β, TNF-α and NO in peritoneal macrophages. Moreover, AFCo3-AmB was more effective than AmB in reducing spleen and liver fungal burden after repeated (five days) intraperitoneal administration of 5 mg/kg of AmB, in a Balb/c model of systemic infection, associated to a significant induction of Th1/Th17 response. Finally, blood chemistry revealed that AFCo3-AmB did not cause changes suggestive of nephrotoxicity, such as increases in total proteins, albumin, creatinine and blood urea nitrogen that were caused by free AmB. Conclusions: AFCo3-AmB exhibited a significant immunomodulator action, reduced toxicity and improved antifungal action against S. schenckii, suggesting a potential use as AmB delivery for systemic sporotrichosis treatment.     

Bioactive potential of endophytic Myrothecium sp. isolate M1-CA-102, associated with Calophyllum apetalum

Context: Endophytes colonizing medicinal plants are diverse, constituting a rich bioresource for novel natural products.

Objective: Myrothecium sp. isolate M1-CA-102 was the most promising among the 16 Myrothecium isolates screened. The bioactive potential of the crude extract from the Calophyllum apetalum Willd. endophytic Myrothecium sp. (Alb. &; Schwein.) Ditmar (Incertae sedis) isolate M1-CA-102 and its thin layer chromatography (TLC) fractions were screened based on antioxidant, anti-inflammatory, antimicrobial activities, and cytotoxicity.

Materials and methods: The antioxidant activity was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical scavenging capacities. Further, 15-lipoxygenase (15-LOX) and human cyclooxygenase-2 (COX-2) inhibition were assessed at different concentrations (25, 50, and 100?μg/mL for the crude extract, 5, 25, and 50?μg/mL for the TLC fractions). DNA-nicking assay as an indicator of the capacity of extracts to scavenge hydroxyl radical was recorded at a concentration of 50?μg/mL. Cell cytotoxicity was recorded by colorimetric 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Antibacterial (Bacillus subtilis) and anti-Candida (Candida albicans) assays were performed by the microdilution method.

Results: The DPPH and ABTS IC₅₀ values of M1-CA-102 extract were 10 and 6?μg/mL compared with 6.1 and 7.03?μg/mL for the positive control quercetin. The cytotoxicity IC₅₀ value of M1-CA-102 extract was 37?μg/mL, while the M-I TLC fraction was 21?μg/mL. The M1-CA-102 extract gave an IC₅₀ value of 58 and 8?μg/mL for 15-LOX and COX-2, respectively. The MIC values for antimicrobial activity for M1-CA-102 extract ranged from 35 to 54?μg/mL, while for the TLC fractions, it ranged from 91 to 515?μg/mL.

Conclusion: The results indicate that Myrothecium M1-CA-102 isolated from C. apetalum is a potential source of natural metabolites of pharmaceutical importance.     

Wound healing activity of Pluchea indica leaf extract in oral mucosal cell line and oral spray formulation containing nanoparticles of the extract

Context: Pluchea indica (L.) Less (Asteraceae) is an herb used as a traditional medicine for wound healing. The chemical compounds found in Pluchea indica leaves are phenolic acids, flavonoids, anthocyanins and carotenoids.

Objective: This study investigates the effect of Pluchea indica leaf ethanol extract and its nanoparticles (NPs) on cytotoxicity, cell survival and migration of human oral squamous carcinoma cell line.

Materials and methods: Cell viability was measured using MTT assay to assess the effect of Pluchea indica leaf extract and NPs (1–500?μg/mL) on cytotoxicity and cell survival. The effect of Pluchea indica leaf extract and NPs on cell migration was determined by scratch assay. The % relative migration was calculated after 24, 48 and 72?h of treatment.

Results: The sizes of Pluchea indica leaf extract NPs were in a range of nanometers. NPs possessed negative charge with the polydispersity index (PDI) smaller than 0.3. After the treatment for 24, 48 and 72?h, Pluchea indica leaf extract had IC₅₀ value of 443.2, 350.9 and 580.5?μg/mL, respectively, whereas the IC₅₀ value of NPs after the treatment for 24, 48 and 72?h were 177.4, 149.2 and 185.1?μg/mL, respectively. The % relative migration of cells was significantly increased when the cells were treated with 62.5 and 125?μg/mL of the extract and 62.5?μg/mL of NPs.

Discussion and Conclusions: NPs increased cytotoxicity of the Pluchea indica leaf extract, increased the migration of cells at low concentration and increased colloidal stability of the extract in an oral spray formulation.     

Leishmanicidal and cytotoxic activities of Nigella sativa and its active principle,thymoquinone

Abstract

Context: Leishmaniasis is a complex disease with a broad spectrum of clinical presentations.

Objective: We evaluated the anti-leishmanial effects of Nigella sativa L. (Ranunculaceae) against Leishmania tropica and Leishmania infantum with an in vitro model.

Materials and methods: Antileishmanial effects of essential oil and methanolic extract of N. sativa (0–200?µg/mL) and thymoquinone (0–25?µg/mL) on promastigotes of both species and their cytotoxicity activities against murine macrophages were evaluated using the MTT assay at 24, 48, and 72?h. Moreover, their leishmanicidal effects against amastigotes were investigated in a macrophage model, for 48 and 72?h.

Results: The findings showed that essential oil (L. tropica IC₅₀ 9.3?μg/mL and L. infantum IC₅₀ 11.7?μg/mL) and methanolic extract (L. tropica IC₅₀ 14.8?μg/mL and L. infantum IC₅₀ 15.7?μg/mL) of N. sativa, particularly thymoquinone (L. tropica IC₅₀ 1.16?μg/mL and L. infantum IC₅₀ 1.47?μg/mL), had potent antileishmanial activity on promastigotes of both species after 72?h. In addition, essential oil (L. tropica IC₅₀ 21.4?μg/mL and L. infantum IC₅₀ 26.3?μg/mL), methanolic extract (L. tropica IC₅₀ 30.8?μg/mL and L. infantum IC₅₀ 34.6?μg/mL), and thymoquinone (L. tropica IC₅₀ 2.1?μg/mL and L. infantum IC₅₀ 2.6?μg/mL) mediated a significant decrease in the growth rate of amastigote forms of both species. Thymoquinone (CC₅₀ 38.8?μg/mL) exhibited higher cytotoxic effects against murine macrophages than the other extracts.

Conclusion: N. sativa, especially its active principle, thymoquinone, showed a potent leishmanicidal activity against L. tropica and L.infantum with an in vitro model.     

Evaluation of genotoxic effects of 3-methyl-5-(4-carboxycyclohexylmethyl)-tetrahydro-2H-1,3,5-thiadiazine-2-thione on human peripheral lymphocytes

Context: Tranexamic acid is commonly used for curing abnormal bleeding in a variety of diseases. In a previous study, 12 different tetrahydro-2H-1,3,5-thiadiazine derivatives were synthesized from the amine group of tranexamic acid. Their antifibrinolytic and antimicrobial activities were compared with tranexamic acid. 3-Methyl-5-(4-carboxycyclohexylmethyl)-tetrahydro-2H-1,3,5-thiadiazine-2-thione (3-MTTT) was the most remarkable one, which may be used as a drug.

Objectives: In vitro genotoxicity of 3-MTTT was investigated using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronucleus (MN) and comet assays.

Materials and methods: Various concentrations 0.78, 1.56, 3.13, 6.25, 12.50 and 25.00?μg/mL of 3-MTTT were applied to lymphocytes obtained from two donors for periods of 24 and 48?h. A negative (distilled water), a solvent (2:1 PBS:10% NaOH for cultured lymphocyte, and PBS for isolated lymphocytes) and a positive control (MMC for cultured lymphocytes and H₂O₂ for isolated lymphocytes) were also maintained.

Results: While this compound did not increase the frequency of abnormal cells and CA/cell ratio compared to negative control (except 48?h, 25?μg/mL), it significantly increased the frequency of SCEs at the four highest concentrations at both treatment periods (except 6.25?μg/mL, 48?h). It significantly decreased the MI in all the concentrations at 24?h (except 0.78?μg/mL) and in the highest three concentrations at 48?h. This compound did not significantly increase the frequency of MN and DNA damage compared to negative control. This compound did not affect the replication and nuclear division index.

Discussion and conclusion: Our results demonstrated that this compound does not represent a significant risk at the genetic level in in vitro human lymphocytes.     

Glutathione sulfotransferase inhibition activity of a self-fermented beverage,Kanji

Context: Kanji, a liquid preparation of roots of Daucus carota L. ssp. sativus (Hoffm.) Arcang. var. vavilovii Mazk. (Apiaceae), may inhibit glutathione sulfotransferase (GST) activity due to ferulic acid content.

Objectives: GST inhibition activity and characterization of Kanji and methanol extract of D. carota roots, and oral absorption pattern of ferulic acid from Kanji in rats.

Materials and methods: GST inhibition activity of Kanji and methanol extract of D. carota roots in concentration range 0.001–100.00?mg/mL was determined using Sprague Dawley rat liver cytosolic fraction. Methanol extract upon column chromatography gave ferulic acid, which was used to characterize Kanji and determine its oral absorption pattern in Wistar rats.

Results: The GST inhibition activity of Kanji (100.00?μg/mL), methanol extract of D. carota roots (100.00?μg/mL) and tannic acid (10.00?μg/mL, positive control) was found to be 0.162?±?0.016, 0.106?±?0.013 and 0.073?±?0.004?μM/min/mg, respectively. Different Kanji samples and methanol extract contained ferulic acid (0.222–0.316?mg/g) and 0.77?mg/g, respectively. Ferulic acid did not appear in plasma after oral administration of Kanji.

Discussion: Kanji having solid contents 80.0?μg/mL, equivalent to 0.0025?μg/mL ferulic acid, does not inhibit the activity of GST. The oral administration of Kanji, in human equivalent dose (528?mg/kg, 16.67?μg ferulic acid), to rats indicated poor absorption of ferulic acid.

Conclusion: Kanji having solid contents 14–36?mg/mL does not inhibit GST activity, hence may not interfere with drugs that are the substrates of GST, if taken concomitantly.     

Antimicrobial activity of pomegranate fruit constituents against drug-resistant Mycobacterium tuberculosis and β-lactamase producing Klebsiella pneumoniae

Abstract

Context: The global surge in multi-drug resistant bacteria and the imminence of tuberculosis pandemic necessitate alternative therapeutic approaches to augment the existing medications. Pomegranate, the fruit of Punica granatum Linn. (Punicaceae), widely recognized for potency against a broad spectrum of bacterial pathogens, deserves further investigation in this respect.

Objective: This study determines the therapeutic potential of pomegranate juice, extracts of non-edible peel prepared with methanol/water, and its four polyphenolic constituents, namely caffeic acid, ellagic acid, epigallocatechin-3-gallate (EGCG) and quercetin, against drug-resistant clinical isolates.

Materials and methods: Phenotypic characterisation of Mycobacterium tuberculosis, extended-spectrum β-lactamase (ESBL) and KPC-type carbapenemase producing Klebsiella pneumoniae was performed by biochemical and molecular methods. Resistance profiles of M. tuberculosis and K. pneumoniae were determined using LJ proportion and Kirby–Bauer methods, respectively. Pomegranate fruit extracts, and the compounds, were evaluated at a dose range of 1024–0.5?µg/mL, and 512–0.25?µg/mL, respectively, to determine minimum inhibitory (MIC) and bactericidal concentrations (MBC) against the drug-resistant isolates by the broth micro-dilution method.

Results: The peel extracts exhibited greater antimycobacterial activity (MIC 64–1024?μg/mL) than the potable juice (MIC 256?-?>?1024?μg/mL). EGCG and quercetin exhibited higher antitubercular (MIC 32–256?μg/mL) and antibacterial (MIC 64–56?μg/mL) potencies than caffeic acid and ellagic acid (MIC 64–512?μg/mL).

Discussion and conclusion: The pomegranate fruit peel and pure constituents were active against a broad panel of M. tuberculosis and β-lactamase producing K. pneumoniae isolates. EGCG and quercetin need further investigation for prospective application against respiratory infections.     

Methanol extract of the aerial parts of barley (Hordeum vulgare) suppresses lipopolysaccharide-induced inflammatory responses in vitro and in vivo

Abstract

Context: Recently, there has been renewed interest in barley (Hordeum vulgare L. Poaceae) as a functional food and for its medicinal properties.

Objective: This study examines the anti-inflammatory potential of the active fractions of barley and the mechanisms involved.

Materials and methods: The macrophages were exposed to 100?μg/mL of each of the barley extracts in the presence of 1?μg/mL lipopolysaccharide (LPS) and after 24 or 48?h of incubation, cells or culture supernatants were analyzed by various assays. The anti-inflammatory potential of barley fractions was also investigated using the LPS-injected septic mouse model. The active constituents in the fractions were identified using gas chromatography-mass spectrometry (GC-MS).

Results: The active fractions, named F₄, F₇, F₉ and F₁₂, inhibited almost completely the LPS-induced production of nitric oxide (NO) and inducible NO synthase. Pre-treatment with these fractions at 100?μg/mL diminished the tumor necrosis factor-α (TNF-α) levels to 19.8, 3.5, 1.2 and 1.7?ng/mL, respectively, compared to LPS treatment alone (41.5?ng/mL). These fractions at 100?μg/mL also suppressed apparently the secretion of interleukin (IL)-6 and IL-1β and the DNA-binding activity of nuclear factor-κB in LPS-stimulated cells. Mice injected intraperitoneally with LPS (30?mg/kg BW) showed 20% survival at 48?h after injection, whereas oral administration of the fractions improved the survival rates to 80%. GC-MS analysis revealed the presence of the derivatives of benzoic and cinnamic acids and fatty acids in the fractions.

Discussion and conclusion: The aerial parts of barley are useful as functional food to prevent acute inflammatory responses.     

A novel model of image acquisition and processing for holistic quantification of angiogenesis disrupted by application of mainstream and sidestream cigarette smoke solutions

Angiogenesis is a vital process in the growth of new blood vessels from pre-existing vasculature. Among several approaches being used for studies related to angiogenesis, chicken chorioallantoic membrane assay (CAM) is an excellent model system. However, its utility has been limited due to difficulty in quantifying putative angiogenic and anti-angiogenic response to an experimental compound in an objective and quantifiable manner. Herein, we report a novel approach of image acquisition and processing for better evaluation of neovascularization. The effects of mainstream cigarette smoke solutions (MSCSS) and sidestream cigarette smoke solutions (SSCSS) from different commercially available cigarettes on angiogenesis were quantified, using CAM assay. Different gross and nanometer scale topographies of CAMs were quantified, which are vital for 3D image scrutiny and can precisely enumerate angiogenesis. Pattern formation of blood vessels, diameter, area and 3D surface roughness of CAMs were substantially disrupted by application of cigarette-smoke extracts. An important point revealed in our study that SSCSS appeared to be significantly more toxic than MSCSS with respect to their effects on angiogenesis. This new imaging technique combined with other modalities, will provide a robust platform to optimize trial design and more patent studies in angiogenesis.