In vitro inhibitory effects of thymol and carvacrol on dendritic cell activation and function

Context: Thyme has been used in traditional medicine for medicinal purposes since ancient times.

Objective: The objective of this study was to investigate the effects of thymol and carvacrol as two major constituents of thyme on dendritic cells (DCs) maturation and T cell activation.

Materials and methods: Splenic DCs were treated with non-cytotoxic concentrations of the components and then analyzed for MHC II, CD86, and CD40 expression by flow cytometry. The effects of compounds on mitogenic, as well as allogenic T cell responses in mixed lymphocyte culture (MLR) and the release of cytokines were investigated.

Results: At 0.1?µg/ml, reduced mean fluorescent intensity (MFI) of CD86 for thymol (80.3?±?0.2% of untreated control) and CD40 for carvacrol (79.5?±?0.14%) was observed (p?<?0.001). Decreased mitogenic T cell proliferation by thymol [proliferation index (PI) from 0.93?±?0.11 at 1?µg/ml to 0.42?±?0.16 at 100?µg/ml (p?<?0.01)] and carvacrol [PI from 1.08?±?0.3 at 1?µg/ml to 0.28?±?0.1 at 100?µg/ml (p?<?0.001)] was seen. Ten micrograms/ml thymol (PI, 0.85?±?0.04) and carvacrol (PI, 0.89?±?0.03) inhibited allogenic T cell response (p?<?0.05). Decreased IFN-γ level in MLR supernatant from 1441?±?27.7?pg/ml in untreated cells to 944?±?32.1 at 10?µg/ml of thymol and of carvacrol (886?±?31.7?pg/ml) (p?<?0.01) was found. IL-4 levels were decreased in the presence of both compounds (p?<?0.01).

Conclusion: These data showed the suppressive effects of thymol and carvacrol on DCs maturation and function, as well as T cell responses.     

In vitro antioxidant and anti-cholinesterase activities of Rhizophora mucronata

Context: Rhizophora mucronata Lam. (Rhizophoraceae), commonly known as Asiatic mangrove, has been used traditionally among Asian countries as folk medicine.

Objective: This study investigates the cholinesterase inhibitory potential and antioxidant activities of R. mucronata.

Materials and method: Rhizophora mucronata leaves were successively extracted using solvents of varying polarity and a dosage of 100–500?µg/ml were used for each assay. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were assessed according to the method of Ellman. In vitro antioxidant activity was assessed using free radical scavenging, reducing power, and metal-chelating activity (duration – 3 months). Total phenolic and flavonoid content were quantified spectrophotometrically. Compound characterization was done using column chromatography, NMR, FTIR, and LC-MS analysis.

Results: Methanolic leaf extract (500?µg/ml) exhibited the highest inhibitory activity against AChE (92.73?±?0.54%) and BuChE (98.98?±?0.17%), with an IC₅₀ value of 59.31?±?0.35 and 51.72?±?0.33?µg/ml, respectively. Among the different solvent extracts, methanolic extract exhibited the highest antioxidant activity with an IC₅₀ value of 47.39?±?0.43, 401.45?±?18.52, 80.23?±?0.70, and 316.47?±?3.56?µg/ml for DPPH, hydroxyl, nitric oxide radical, and hydrogen peroxide, respectively. Total polyphenolic and flavonoid contents in methanolic extract were observed to be 598.13?±?1.85?µg of gallic acid equivalent and 48.85?±?0.70?μg of rutin equivalent/mg of extract. Compound characterization illustrated (+)-catechin as the bioactive compound responsible for cholinesterase inhibitory and antioxidant activities.

Conclusion: The presence of rich source of flavonoids, in particular catechin, might be responsible for its cholinesterase inhibitory and antioxidant activities.     

Phytochemical investigation and radical scavenging activities of Melia azedarach and its DNA protective effect in cultured lymphocytes

Abstract

Context: Melia azedarach Linn (Meliaceae) is an Ayurvedic medicinal plant which is native to India. It is traditionally used for the treatment of leprosy, inflammation, scrofula, anthelmintic, antilithic, diuretic, deobstruent and cardiac disorders.

Objective: To evaluate the phytochemical constituents and antioxidant activities of the ethanol leaf extract of Melia azedarach (MA) and its protective effect against H₂O₂-induced cellular damage in cultured lymphocytes.

Materials and methods: The dose-dependent study of MA (20, 40, 60, 80, 100?µg/ml) was used to study in vitro radical scavenging assays. The effective dose of MA (60?µg/ml) was further used to study the H₂O₂-induced DNA damage (comet assay and DNA fragmentation assay) in cultured lymphocytes.

Results: The ethanol extract of MA (20, 40, 60, 80, 100?µg/ml) exhibited a significant dose-dependent inhibition of in vitro radical scavenging assays and their corresponding IC₅₀ values as follows: hydroxyl radical (26.50?±?0.26?µg/ml), superoxide anion (30.00?±?0.32?µg/ml), nitric oxide radical (48.00?±?0.48?µg/ml), DPPH radical (30.55?±?0.32?µg/ml) and reducing power (22.00?±?0.22?µg/ml). The increase in the severity of DNA damage and TBARS was increased significantly (p?<?0.05) at 500?µM H₂O₂-treated cultured lymphocytes and RBC cellular membranes. The phytochemical screening studies identified 13 chemical constituents present in the leaf extract of MA.

Discussion and conclusion: The results of this study demonstrate that MA offers protection against H₂O₂-induced cellular damage and it can be developed as an effective antioxidant during oxidative stress.     

Antioxidant,antimicrobial and wound healing properties of Struthanthus vulgaris

Context: Struthanthus vulgaris (Vell.) Mart. (Loranthaceae) has been widely used in traditional medicine in Brazil to bathe wounds.

Objective: The objective of this study is to investigate the in vitro wound healing effects, together with the antioxidant and antimicrobial activities of S. vulgaris leaf and branch extracts.

Material and methods: Ethanol leaf and branch extracts of S. vulgaris were investigated at 1–100?µg/ml concentrations in the scratch assay after 14?h. Antioxidant activity was investigated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay, and the antibacterial activity was tested at concentrations up to 1000?µg/ml against Gram-positive and Gram-negative bacteria by the microdilution test after 24?h. The total phenolic and flavonoid contents were determined by colorimetric methods.

Results: Struthanthus vulgaris leaf and branch extracts at 100?µg/ml concentration stimulated migration and proliferation of fibroblasts and enhanced cell numbers by 56.2% and 18.6%, respectively. Antioxidant activity exhibited IC₅₀ values of 24.3 and 18.9?µg/ml for the leaf and branch extracts, respectively. The ethanol leaf extract showed antimicrobial activity against the Gram-positive Staphylococcus mutans and Staphylococcus aureus bacteria, exhibiting minimum inhibitory concentration values of 125 and 500?µg/ml, respectively. An appreciable total phenolic content in the leaves (813.6?±?2.7?mg/g) and branches (462.8?±?9.6?mg/g), and relatively low concentration of flavonoids in the leaves (13.3?±?4.3?mg/g) and branches (1.9?±?0.2?mg/g), was detected.

Discussion and conclusion: The antioxidant and antibacterial activities, together with the strong ability to stimulate proliferation and migration of fibroblasts, provide some support for the traditional use of S. vulgaris.     

Immunomodulatory effects of Santolina chamaecyparissus leaf extracts on human neutrophil functions

Context: Santolina chamaecyparissus L. (Asteraceae) is an aromatic plant wide spread in the Mediterranean region. It is used in folk medicine for its anti-inflammatory properties.

Objective: The effects of S. chamaecyparissus aqueous extract (SCAE) and polyphenolic extract (SCPE) on human polymorphonuclear neutrophil (PMN) degranulation, chemotaxis, phagocytosis, and microbicidal capacity were examined in vitro.

Materials and methods: Aqueous and polyphenolic extracts were prepared from S. chamaecyparissus leaves. The elastase release was used as a marker for measuring PMN degranulation, while chemotaxis was performed using a 48-microwell chemotaxis chamber. The phagocytosis and the microbicidal capacity were evaluated using fresh cultures of Candida albicans.

Results: The treatment of neutrophils with different concentrations (10–200?µg/ml) of SCAE and SCPE caused a significant (p?<?0.001) and dose-dependent inhibitory effect on elastase release in fMLP/Cytochalasin B (CB)-stimulated neutrophils. Indeed, 100?µg/ml of SCAE exerted an inhibitory effect of 51.97?±?6.2%, whereas SCPE at the same concentration abolished completely PMN degranulation. Moreover, both extracts inhibited markedly (p?<?0.01) fMLP-induced chemotactic migration. At 200?µg/ml, SCAE and SCPE exerted an inhibitory effect of 54.61?±?7.3% and 57.71?±?7.44%, respectively. In addition, a decline in both phagocytosis and microbicidal capacity against Candida albicans was observed when PMNs were exposed to 100 and 200?µg/ml of SCAE or SCPE.

Conclusion: The exerted effects on neutrophil functions support the anti-inflammatory activity and show new mechanisms of action and effectiveness of S. chamaecyparissus leaf extracts. This plant may be considered as an interesting source of anti-inflammatory and immunomodulatory agents.     

Chemical profile and biological activities of Veronica thymoides subsp. pseudocinerea

Abstract

Context: In Turkey, Veronica species (Plantaginaceae) have been used as a diuretic and for wound healing in traditional medicine.

Objective: To examine the fatty acid and essential oil profiles, the antioxidant, anticholinesterase, antimicrobial, and DNA damage effects of Veronica thymoides P.H. Davis subsp. pseudocinerea M.A. Fischer as a potential source of natural active compounds.

Materials and methods: GC/MS was used to analyze essential oil and fatty acid obtained from whole plant. The antioxidant activity was evaluated by the β-carotene-linoleic acid test system, DPPH-free and ABTS cation radicals scavenging, and cupric reducing antioxidant capacity assays. The anticholinesterase and antimicrobial activities were determined by Ellman and broth macrodillution methods, respectively. The effect of the methanol extract on DNA cleavage was investigated.

Results: Hexatriacontene (21.0%) was found to be the main constituent in essential oil, and linoleic acid (25.2%) and palmitic acid (20.6%) in fatty acid. Methanol extract demonstrated the best IC₅₀ values in lipid peroxidation (49.81?±?0.31?µg/ml) and DPPH-free radical scavenging activity (15.32?±?0.17?µg/ml). Methanol and water extracts possessed strong ABTS cation radical scavenging activity with IC₅₀ values 9.15?±?0.28 and 8.90?±?0.14?µg/ml, respectively. The acetone extract exhibited moderate butyrylcholinesterase inhibitory activity. The highest antimicrobial activity was determined in methanol extract against Escherichia coli with 31.25?µg/ml MIC value. Inhibition of methanol extract on plasmid DNA cleavage by OH radicals was found to be 93.32% at 500?µg/ml.

Conclusion: The methanol extract having strong antioxidant and DNA damage effects could be investigated phytochemically to find natural active compounds.     

Mineral pitch stimulates humoral,cellular and innate immune responses in mice

Abstract

Context: Mineral pitch (MP), a traditional medicine, is proposed to boost immunity in conditions that suppress Th1 cytokines such as AIDS/HIV, tuberculosis, leishmaniasis and cancer.

Objective: This study investigates the immunoregulatory mechanisms of MP in innate, humoral and cell-mediated immunity.

Materials and methods: Mice given MP (100, 200, 300 or 400?mg/kg, orally) for 10 consecutive days were immunized intravenously with goat RBC or ovalbumin, and investigated for plaque-forming cells (PFC), hemagglutination titer, hypersensitivity response, lymphocyte proliferation and macrophage function.

Results: MP increased PFC (330.2 versus 182.2/10⁶ splenocytes) in mice immunized with goat RBC and elicited ovalbumin-specific IgG titer at 400?mg/kg. Increase in Th1 immunity was correlated with the increased level of IFN-γ (724 versus 470?pg/ml) and decreased IL-4 (96 versus 178?pg/ml). CD4⁺/CD3⁺ ratio and delayed-type hypersensitivity response also increased to, respectively, 20.62?±?0.59 (versus 16.47?±?0.72) and 1.59?±?0.12 (versus 0.87?±?0.10?mm) in MP-treated mice. MP increased lymphocyte proliferation (11.14?±?0.60 versus 5.81?±?0.40 SI) and macrophage phagocyte response (0.24?±?0.02 versus 0.15?±?0.009), expressed as absorbance at 570?nm, but decreased nitrite production (17.4?±?1.10 versus 24.3?±?1.30?µM/10⁶ cells). We also observed an increased bone marrow cellularity (24.5?±?1.10 versus 17.10?±?0.70 cells/femur) and WBC count (12?667?±?377 versus 9178?±?213 cells/mm³) following MP treatment. There was no sign of toxicity at 400?mg/kg, 1/12th of reported LD₅₀.

Conclusion: MP elicits a dose-dependent Th1 immune response.     

Buccal films of prednisolone with enhanced bioavailability

Abstract

The conventional formulation of prednisolone is considered to be low in efficacy, primarily on account of their failure in providing and maintaining effective therapeutic drug levels. This study aims to focus on development of a mucoadhesive buccal delivery system with a twofold objective of offering a rapid as well as a prolonged delivery of prednisolone coupled with enhanced therapeutic efficacy. Buccoadhesive films of prednisolone were prepared by solvent-casting method using hydroxyl propyl methyl cellulose (K100), Carbopol 940 and/or Eudragit® NE 40?D. Placebo films possessing the most desirable physicomechanical properties were selected for drug loading. The effect of polymer and its content on film properties, i.e. mucoadhesive strength, swelling and hydration, in vitro drug release was studied. Based on these studies, film F7D was selected for ex vivo permeation across porcine cheek mucosa. The steady state flux of prednisolone across the buccal mucosa was found to be 105.33?±?32.07?µg/cm²/h. A comparative pharmacokinetic study of prepared film (F7D) and oral suspension of prednisolone was conducted. In vivo data of buccal film show greater bioavailability (AUC_0–α: 24.26?±?4.06?µg.h/ml versus 10.65?±?2.15?µg.h/ml) and higher C_max (2.70?±?0.38?µg/ml versus 2.29?±?0.32?µg/ml) value when compared to oral suspension. The data observed from this study highlight the feasibility of the buccal route as a viable option for delivery of prednisolone.     

Effect of hydroalcoholic extract of Aegle marmelos fruit on radical scavenging activity and exercise-endurance capacity in mice

Context: Aegle marmelos L. Corr (Rutaceae) is an important Indian Ayurvedic medicinal plant used for the treatment of various ailments. However, little information is available on the anti-fatigue properties of its fruit.

Objective: Evaluation of the physical endurance and exercise-induced oxidative stress modulating properties of A. marmelos fruit in mice.

Material and methods: Radical scavenging activity of the fruit hydroalcoholic extract was evaluated using in vitro systems. The extract was further evaluated for its endurance-enhancing properties at three oral doses (100, 200 and 400?mg/kg?b.wt) in BALB/c mice for 21?d using a swimming test.

Results and discussion: The extract exhibited significant scavenging activity against DPPH (IC₅₀, 351?±?37?µg/ml) and ABTS radicals (IC₅₀, 228?±?25?µg/ml), respectively, with the polyphenol content of 95?µg/mg extract. It also inhibited AAPH radical-induced oxidation of biomolecules such as BSA protein (63%), plasmid DNA (81%) and lipids (80.5%). Administration of extract resulted in an increase in the duration of swimming time to exhaustion by 23.4 and 47.5% for medium and higher doses, respectively. The extract significantly normalized the fatigue-related biochemical parameters and also down-regulated the swim stress-induced over-expression of heat shock protein-70 and up-regulated the skeletal muscle metabolic regulators (GLUT-4 and AMPK1-α) by 2- and 3-fold, respectively, at the higher dose in muscle tissues.

Conclusion: Our study demonstrates the anti-fatigue properties of A. marmelos fruit, most probably manifested by delaying the accumulation of serum lactic acid, increasing the fat utilization and up-regulating the skeletal muscle metabolic regulators.     

Bioassay guided fractionation and identification of active anti-inflammatory constituent from Delonix elata flowers using RAW 264.7 cells

Abstract

Context: Delonix elata (L.) Gamble (Fabaceae) has been used in the Indian traditional medicine system to treat rheumatism and inflammation.

Aim: To assess the anti-inflammatory effect of Delonix elata flowers and to isolate the active principle.

Materials and methods: The prompt anti-inflammatory constituent was isolated from Delonix elata flower extracts using bioassay guided fractionation in liposaccharide (LPS) stimulated RAW 264.7 macrophage cell line. The anti-inflammatory activity of extracts/fractions/sub-fractions/compounds (10, 25, and 50?µg/ml) was evaluated by estimating the levels of nitric oxide (NO), TNF-α, and IL-1β after 24?h of LPS induction (1?μg/ml). The isolated active compound was subjected to NMR, IR, and UV analyses for structure determination.

Results: In an attempt to search for anti-inflammatory constituents, the active pure principle was isolated and crystallized as a white compound from Delonix elata flowers methanol extract. This active compound (50?µg/ml) decreased the release of inflammatory mediators levels such as NO (0.263?±?0.03?µM), TNFα (160.20?±?17.57?pg/ml), and IL-1β (285.79?±?15.16?pg/ml) significantly (p?<?0.05); when compared to the levels of NO (0.774?±?0.08?µM), TNFα (501.71?±?25.14?pg/ml), and IL-1β (712.68?±?52.25?pg/ml) from LPS-stimulated macrophage cells. The active compound was confirmed as hesperidin with NMR, IR, and UV spectroscopy data. This is the first report of this compound from Delonix elata flowers.

Conclusion: The findings of the study support the traditional use of Delonix elata flowers to treat inflammation.     

A novel sesquiterpene acid and an alkaloid from leaves of the Eastern Nigeria mistletoe,Loranthus micranthus with potent immunostimulatory activity on C57BL6 mice splenocytes and CD69 molecule

Context: The Eastern Nigeria mistletoe, Loranthus micranthus Linn. (Loranthaceae), is used in the treatment of several diseases including immune-modifying diseases and thus there is a need to identify the immunoactive constituents.

Objective: This research isolated and characterized the immunoactive constituents in the Eastern Nigeria mistletoe.

Materials and methods: Bioassay-guided fractionation was employed in the isolation and purification of the constituents. The characterized compounds were screened for immunostimulatory activities on isolated C57BL/6 mice splenocytes and early activation marker, CD69 at concentrations of 10, 25, and 100 µg/mL using flow cytometry techniques and compared to lipopolysaccharide (LPS; 10 µg/mL) and concanavalin A (ConA; 2 µg/mL) as standards.

Results: Two compounds, a novel sesquiterpene, 2, 3-dimethoxy-benzo [a, b] cyclopentenyl-3′,3′,5′-trimethyl pyran-4-carboxylic acid (1), and a known alkaloid, lupinine (2) were isolated and characterized. The compounds (25 µg/mL) showed statistically significantly (p?<?0.05) stimulatory activity on the splenocytes with values of 56.34?±?0.26% and 69.84?±?0.19%, respectively, compared to 7.58?±?0.42% recorded for the unstimulated control. Similarly, the CD69 expression assay showed immunostimulation with statistically significant values (p?<?0.05) of 2.31?±?0.07% and 2.71?±?0.03%, respectively, compared to 1.69?±?0.05% recorded for the nonstimulated control.

Discussion: These data suggest that the isolated compounds possess immunomodifying abilities. In addition, the activation of the CD69 molecule is possibly one of its mechanisms of action.

Conclusion: These compounds may be responsible in part, for the immunostimulatory activities already established for the Eastern Nigeria mistletoes.     

Antioxidant activity and cytotoxicity of Cyrtosperma johnstonii extracts on drug sensitive and resistant leukemia and small cell lung carcinoma cells

Context: The number of patients with cancer is increasing. New therapeutic agents to overcome drug-resistant tumors are urgently needed. Cyrtosperma johnstonii N.E. Br. (Araceae) is used for treatment of cancer in Thai traditional medicine. This study aimed to evaluate antioxidant activity and cytotoxicity of C. johnstonii extracts on human cancer cells.

Materials and methods: Dried powder of C. johnstonii rhizomes was extracted with several solvents. The 0.1?mg/ml extract solution was tested for antioxidant activity by 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) assays. Color formation from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to determine cell viability. Standardization of the extract was performed by high-performance liquid chromatography (HPLC) with photodiode array detector at 254 and 360?nm. Cell cycle arrest was evaluated by flow cytometry after 5?min, 12?h and 24?h treated with 20 µg/ml of the acetone extract.

Results: The acetone extract exhibited the highest phenolic content and antioxidant activity (TEAC and EC values = 19.2?±?0.14 and 19.2?±?0.31?mM/mg, respectively). The IC₅₀ values for leukemia ranged from 11?±?1 to 29?±?3 µg/ml and from 5?±?2 to 6?±?0 µg/ml for small cell lung carcinoma cells. Cell cycle arrest occurred at the G2/M phase followed by apoptosis. HPLC analysis revealed that rutin is the major constituents of the extract.

Discussion and conclusion: The acetone extract of C. johnstoni is a promising source of natural antioxidants and anticancer. The extract inhibits cancer cells effectively with less effect on normal cells.     

Phytochemical standardization and biological activities of certain desert plants growing in Saudi Arabia

The phytochemical screening, antimicrobial and antitumor activities of Calendula tripterocarpa, Centarea sinaica, Centaurea pseudosinaica, Koelpinia linearis, Plectranthus arabicus, Plectranthus asirensis and Tripleurospermum auriculatum determined. The best antibacterial activity; 41.8?±?0.23?mm, 39.7?±?0.25?mm, 35.8?±?0.58?mm, 34.7?±?0.51?mm and 32.7?±?0.25?mm was obtained by Plectranthus arabicus against Klebsiella pneumonia, Tripleurospermum auriculatum against Bacillus subtilis, Centaurea pseudosinaica against Bacillus subtilis, Centaurea pseudosinaica against Stroptococcus pyogenes and Plectranthus arabicus against Staphylococcus epidermidis, respectively. While the highest antifungal activity; 35.9?±?1.15?mm, 34.6?±?0.34, 30.6?±?0.26?mm and 29.9?±?0.63?mm was obtained by Tripleurospermum auriculatum against Geotricum candidum, Candida albicans, C. tropicalis and Aspergillus fumigatus, respectively. The antitumor activity (IC₅₀) obtained by Centarea sinaica; 3.1?±?6.9?µg/ml, 14.3?±?3.1?µg/ml and 22.7?±?4.1?µg/ml was better than activity of vinblastine sulphate; 5.9?±?0.4?µg/ml, 59.7?±?2.1?µg/ml and 30.3?±?1.4?µg/ml against breast carcinoma (MCF-7), cervical carcinoma (Hela) and colorectal carcinoma (CACO), respectively. Plectranthus arabicus alcoholic extract showed higher antitumor activity; 15.3?±?5.3?µg/ml, 28.6?±?3.6?µg/ml and 24.3?±?4.1?µg/ml than vinblastine; 21.2?±?0.9?µg/ml, 59.7?±?2.1?µg/ml and 30.3?±?1.4?µg/ml against prostate carcinoma (Pc3), cervical carcinoma (Hela) and colorectal carcinoma (CACO), respectively. Also, the antitumor activity of Plectranthus asirensis against cervical carcinoma (Hela) (37.1?±?2.6?µg/ml) was potent than vinblastine sulphate (59.7?±?2.1?µg/ml). The obtained results of LD₅₀ and sub-chronic toxicity revealed that the plants have no toxicity.     

Essential oil of Psidium cattleianum leaves: Antioxidant and antifungal activity

Abstract

Context: Psidium cattleianum Sabine (Myrtacea) is rich in vitamin C and phenolic compounds, including epicatechin and gallic acid as the main components.

Objective: To evaluate the antifungal and antioxidant capacity in vitro of the essential oil of araçá (EOA). The acute toxicity of the EOA also was evaluated in mice.

Materials and methods: The leaves of the P. cattleianum were extracted by steam distillation. The antioxidant capacity was evaluated by in vitro tests [1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), ferric ion reducing antioxidant power (FRAP), linoleic acid oxidation, thiobarbituric acid reactive species (TBARS)], and ex vivo analysis [TBARS, δ-aminulevunilate dehydratase (δ-Ala-D) and catalase activity, non-protein thiols (NPSH), and ascorbic acid levels]. The toxicity was studied in mice by a single oral administration of EOA; and the antifungal activity was performed with five strains of fungi.

Results: The EOA exhibited antioxidant activity in the FRAP assay and reduced lipid peroxidation in the cortex (I_max?=?32.90?±?2.62%), hippocampus (IC₅₀?=?48.00?±?3.00?µg/ml and I_max?=?32.90?±?2.62%), and cerebellum (I_max?=?45.40?±?14.04%) of mice. Acute administration of the EOA by the oral route did not cause toxicological effects in mice (LD₅₀?>?500?µg/ml). The EOA also showed antifungal activity through of the determination minimum inhibitory concentration (MIC) values ranging from 41.67?±?18.04 to 166.70?±?72.17?µg/ml for tested strains.

Conclusion: The results of present study indicate that EOA possess antioxidant properties, antifungal and not cause toxicity at tested doses.     

Antidiabetic components of Cassia alata leaves: Identification through α-glucosidase inhibition studies

Context: Cassia alata Linn. [syn. Senna alata (L.) Roxb.] (Caesalpiniaceae) is used for treating various disease conditions including diabetes but its mechanism(s) of action and active principles remain to be elucidated.

Objective: The antidiabetic principles were identified using an in vitro α-glucosidase inhibition study.

Materials and methods: The methanol extract of leaves of C. alata, which showed potent α-glucosidase inhibitory activity (IC₅₀, 63.75?±?12.81 µg/ml), was fractionated. Active fractions were taken for further analysis by a variety of techniques including HPLC and Combiflash chromatography. The identity of the isolated compounds was established by spectroscopic analysis while their potential antidiabetic activity was assessed by in vitro enzyme inhibition studies.

Results: The α-glucosidase inhibitory effect of the crude extract was far better than the standard clinically used drug, acarbose (IC₅₀, 107.31?±?12.31 µg/ml). A subsequent fractionation of the crude extract was made using solvents of ascending polarity (petroleum ether, chloroform, ethyl acetate, n-butanol and water). The ethyl acetate (IC₅₀, 2.95?±?0.47 µg/ml) and n-butanol (IC₅₀, 25.80?±?2.01 µg/ml) fractions which contained predominantly kaempferol (56.7?±?7.7 µM) and kaempferol 3-O-gentiobioside (50.0?±?8.5 µM), respectively, displayed the highest carbohydrate enzyme inhibitory effect.

Discussion: One of the possible antidiabetic mechanisms of action of C. alata is by inhibiting carbohydrate digestion. This is the first report on α-glucosidase activity of kaempferol 3-O-gentiobioside.

Conclusion: Considering the activity profile of the crude extract and isolated bioactive compounds, further in vivo and clinical studies on C. alata extracts and compounds are well merited.     

Effect of Musca domestic maggot polypeptide extract on HUVEC dysfunction induced by early-activated macrophages

Context: Musca domestica Linn. maggot is a traditional Chinese medicine. In our previous studies, Musca domestica maggot protein-enriched fraction and polypeptide extract (molecular weight <30?kD) were found to reverse endothelial cell dysfunction in atherosclerotic lesions.

Objective: This study determines whether and how M. domestica maggot polypeptide extract affects the dysfunction of human umbilical vein endothelial cells (HUVEC) induced by macrophages (M?).

Materials and methods: HUVEC and early-activated THP-1?M? (incubated with LPS of 1?μg/ml for 2?h) were co-cultured in a Transwell system. The effects of Musca domestica maggot polypeptide extract (with increasing concentrations, i.e., 1.0, 2.5, 5.0, 10.0, 20.0, and 40.0?µg/ml) on the proliferation and migration HUVEC and their secretion of vascular endothelial growth factor (VEGF) were determined by flow cytometry, modified Boyden chamber assay, and enzyme-linked immunosorbent assay (ELISA) after incubation for 24?h.

Results: Musca domestica maggot polypeptide extract decreased the proliferation of HUVEC in a concentration-dependent manner, with a 50% effective concentration (EC₅₀) of 22.16?±?1.48?µg/ml, and effectively inhibited HUVEC migration (EC₅₀?=?35.15?±?2.03?µg/ml) and VEGF secretion (EC₅₀?=?28.64?±?1.29?µg/ml).

Discussion and conclusion: Musca domestica maggot polypeptide extract can inhibit the dysfunction of HUVEC induced by early-activated THP-1?M?.     

Potent enhancement of transdermal absorption and stability of human tyrosinase plasmid (pAH7/Tyr) by Tat peptide and an entrapment in elastic cationic niosomes

Abstract

Enhancement of transdermal absorption through rat skin and stability of the human tyrosinase plasmid (P) using Tat (T) and an entrapment in elastic cationic niosomes (E) were described. E (Tween61:cholesterol:DDAB at 1:1:0.5 molar ratio) were prepared by the freeze-dried empty liposomes (FDELs) method using 25% ethanol. TP was prepared by a simple mixing method. TPE was prepared by loading T and P in E at the T:P:E ratio of 0.5:1:160 w/w/w. For gel formulations, P, TP, PE and TPE were incorporated into Carbopol 980 gel (30?µg of plasmid per 1?g of gel). For the transdermal absorption studies, the highest cumulative amounts and fluxes of the plasmid in viable epidermis and dermis (VED) were observed from the TPE of 0.31?±?0.04?µg/cm² and 1.86?±?0.24?µg/cm²/h (TPE solution); and 4.29?±?0.40?µg/cm² and 25.73?±?2.40?µg/cm²/h (TPE gel), respectively. Only plasmid from the PE and TPE could be found in the receiving solution with the cumulative amounts and fluxes at 6?h of 0.07?±?0.01?µg/cm² and 0.40?±?0.08?µg/cm²/h (PE solution); 0.10?±?0.01?µg/cm² and 0.60?±?0.06?µg/cm²/h (TPE solution); 0.88?±?0.03?µg/cm² and 5.30?±?0.15?µg/cm²/h (PE gel); and 1.02?±?0.05?µg/cm² and 6.13?±?0.28?µg/cm²/h (TPE gel), respectively. In stability studies, the plasmid still remained at 4?±?2?°C and 25?±?2?°C of about 48.00–65.20% and 27.40–51.10% (solution); and 12.34–38.31% and 8.63–36.10% (gel), respectively, whereas at 45?±?2?°C, almost all the plasmid was degraded. These studies indicated the high potential application of Tat and an entrapment in elastic cationic niosomes for the development of transdermal gene delivery system.     

Ethyl acetate extract from Glycosmis parva leaf induces apoptosis and cell-cycle arrest by decreasing expression of COX-2 and altering BCL-2 family gene expression in human colorectal cancer HT-29 cells

Abstract

Context: Glycosmis parva Craib (Rutaceae) is reported to have cytotoxic and anti-inflammatory activities by decreasing COX-2 expression.

Objective: To investigate the effect of G. parva on human colorectal cancer cells expressing COX-2, HT-29 cells.

Materials and methods: HT-29 cells were treated with ethyl acetate extract from the leaves of G. parva (GPE 6.25–100?µg/ml) for 24–72?h. Cell viability was evaluated by the resuzurin reduction assay. An apoptotic study was performed using annexinV/FITC-PI staining. The cell-cycle pattern was investigated by PI staining. The expression of BCL-2 family genes was analyzed by quantitative RT-PCR and expression of cyclins and COX-2 were done by RT-PCR.

Results: GPE at 6.25–100?µg/ml reduced HT-29 cell viability with IC₅₀ values of 69.49, 55.89, and 48.94?µg/ml at 24, 48, and 72?h, respectively. HT-29 apoptosis was induced by 18.23% at 100?µg/ml. Cells in S phase decreased by 5.22% and 13.28% at 50 and 100?µg/ml, respectively, causing G0/G1 (10.6% at 50?µg/ml) and G2/M (15.67% at 100?µg/ml) accumulation. GPE at 50?µg/ml downregulated cyclin A (11.46%), cyclin E (17.98%), BCL-2 (0.32-fold), and COX-2 (29.06%) expression with an increased BAK expression (1.79-fold).

Discussion and conclusion: GPE reduced HT-29 cell viability, inhibited cell proliferation, induced apoptosis, and arrested the cell cycle. Underlying mechanisms may involve decreases in COX-2, cyclin A, and cyclin E expression in addition to changes in BCL-2 family gene expression. Fundamental knowledge of GPE anticancer effects found in this study could lead to future use of this compound for colorectal cancer treatment.     

Antimicrobial,antioxidant and anticancer activities of Laurencia catarinensis,Laurencia majuscula and Padina pavonica extracts

The antimicrobial, antioxidant, and anticancer activities of ethanolic extract of Laurencia catarinensis, L. majuscula and Padina pavonica were determined. The highest antibacterial activity; 23.40?±?0.58?mm (00.98?µg/ml) and 22.60?±?2.10?mm (03.90?µg/ml) were obtained against Klebsiella pneumonia by Laurencia catarinensis and Padina pavonica, respectively. However, Padina pavonica showed excellent antibacterial activity against Bacillus subtilis (21.7?±?1.5?mm; 1.95?µg/ml), Staphylococcus aureus (21.7?±?0.58?mm; 1.95?µg/ml), Streptococcus pyogenes (20.7?±?1.2?mm; 1.95?µg/ml) and Acinetobacter baumannii (20.1?±?1.2?mm; 3.9?µg/ml). Moreover, the highest antifungal activity; 24.7?±?2.0?mm (0.98?µg/ml), 23.7?±?1.5?mm (0.98?µg/ml), 23.6?±?1.5?mm (0.98?µg/ml) was obtained by Padina pavonica against Candida tropicalis, C. albicans and Aspergillus fumigatus, respectively. The algal extracts showed DPPH radical scavenging activity in a concentration–dependent manner with maximum scavenging activity (77.6%, IC₅₀?=?5.59?µg/ml and 77.07%, IC₅₀?=?14.3?µg/ml) was provided by Padina pavonica and Laurenica majuscula, respectively. The in vitro antitumor activity revealed that the IC₅₀ values of Padina pavonica were 58.9, 115.0, 54.5, 59.0, 101.0, 101.0, and 97.6?µg/ml; Laurencia catarinensis were 55.2, 96.8, 104.0, 78.7, 117.0, 217.0, 169.0?µg/ml; and Laurencia. majuscula were 115.0, 221.0, 225.0, 200.0, 338.0, 242.0, and 189.0?µg/ml; respectively against A-549 (Lung carcinoma), Caco-2 (Intestinal carcinoma), HCT-116 (Colon carcinoma), Hela (Cervical carcinoma), HEp-2 (Larynx carcinoma), HepG-2 (Hepatocellular carcinoma), and MCF-7 (Breast carcinoma) cell lines.     

Antidiabetic effects of Mukia maderaspatana and its phenolics: An in vitro study on gluconeogenesis and glucose uptake in rat tissues

Context: Traditional medicine is used by over 60% of the world’s population for health care. Mukia maderaspatana (L.) M. Roem. (Cucurbitaceae) (Mukia) is extensively used in folklore medicine as an antidiabetic plant. It is rich in phenolics that contribute to its medicinal properties.

Objective: Mukia extract and phenolics such as quercetin and phloroglucinol are investigated for their in vitro antidiabetic activity.

Materials and methods: Quercetin, phloroglucinol, and methanol extract of the dried whole plant (0.25 and 0.5?mg/ml) were studied for the inhibition of gluconeogenesis in rat liver slices and glucose uptake in isolated rat hemi-diaphragm (50 and 100?µg/ml). Phenolics of Mukia were analyzed by HPLC.

Results and discussion: Glucose (1.2?mg/g/h) was synthesized from pyruvate and the synthesis was completely inhibited by insulin (1?U/ml). Quercetin at 0.25 and 0.5?mg/ml caused 65% and 89% inhibition (0.42?mg/g/h and 0.13?mg/g/h glucose). Addition of insulin did not increase inhibition. Phloroglucinol inhibited 100% glucose production with or without insulin. Mukia (0.25?mg/ml) inhibited gluconeogenesis (0.65?mg/g/h) by 45%, and with insulin, inhibition increased to 50% (0.59?mg/g/h). At 0.5?mg/ml, glucose production was stimulated by1.2-fold, but with insulin it was inhibited by 89% (0.13?mg/g/h glucose). Mukia had no effect on glucose uptake, but potentiated the action of insulin mediated glucose uptake (152.82?±?13.30?mg/dl/g/30?min) compared with insulin control (112.41?±?9.14?mg/dl/g/30?min) (p?0.05). HPLC analysis revealed the presence of phenolics.

Conclusion: Results provide scientific rationale for the use of Mukia in folk medicine as an antidiabetic nutraceutical.