Evaluation of the antioxidant activity and antitumor activity of marine invertebrates extracts

Dissertação de Mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologias, Universidade do Algarve, 2014

Anti-vibrio potentials of acetone and aqueous leaf extracts of Ocimum gratissimum (Linn)

Purpose: To evaluate the anti-vibrio potentials of acetone and aqueous leaf extracts of Ocimum gratissimum and determine its relevance in the treatment of vibrios infection. Methods: The agar-well diffusion method was used for screening the extracts for their anti-vibrio activity. Broth micro-dilution assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts. Time-kill assay was used to assess bactericidal and/or bacteriostatic activity. Results: The acetone extract showed activity against 47.5 % (19/40) of the test bacteria, while the aqueous extract had activity against 30 % (12/40). MIC and MBC values range for the acetone extract were 0.625 – 5.0 mg/mL and 2.5 – 10 mg/mL respectively. The range of MIC exhibited by the antibiotic (gentamicin) against the vibrios is 0.002 mg/mL and >0.256 mg/mL. Significant reduction in the bacterial density was at 2 × MIC after a 4 h interaction period, while bacterial density after 6 and 8 h interactions with extract was highly bactericidal. Growth inhibition and efficacy of the crude acetone extract were observed to be both concentration- and time-dependent. Conclusion: The bacteriostatic and bactericidal activities observed for Ocimum gratissimum leaf suggest that the plant is a potential source of bioactive components that may be effective in the treatment of vibrios infections.

Cytotoxic, anti-inflammatory and antioxidant activities of four different extracts of Galega officinalis L (Goat’s rue)

Purpose: To evaluate the cytotoxic, anti-inflammatory and antioxidant activities of four different solvent extracts obtained from the aerial parts of Galega officinalis L Methods: The hexane, DCM, methanol and water extracts of G. officinalis were successively obtained by soxhlet extraction method. The cytotoxic activity of the extracts was assessed against human lung carcinoma (A-549), human colorectal adenocarcinoma (HT-29), human brain glioblastoma (U-87), and colon adenocarcinoma (DLD-1) by Resazurine test. The antioxidant activity of extracts were determined by Folin-Ciocalteau, oxygen radical absorbing capacity (ORAC), and 2’.7’-dichlorofluorescin-diacetate (DCFH-DA) cell-based assay while their anti-inflammatory activity was determined by nitric oxide (NO) assay. Results: DCM extract showed strong cytotoxic activity against lung adenocarcinoma and brain glioblastoma cell lines, with IC50 (concentration inhibiting 50 % of cell growth) values of 11 ± 0.4 and 16 ± 3 μg/mL, respectively. The hexane extract showed moderate anticancer activity against the same cell lines (59 ± 13 and 63 ± 16 μg/mL, respectively). DCM extract also showed significant anti-inflammatory activity, inhibiting NO release by 86.7 % at 40 μg/mL in lipopolysaccharide (LPS) - stimulated murine RAW 264.7 macrophages. Of all test extracts, the methanol extract of G. officinalis showed the highest antioxidant activity with 2.33 ± 0.09 μmol Trolox/mg , 7.10 ± 0.9 g tannic acid equivalent (TAE), and IC50 of 44 ± 4 μg/mL. Conclusion: The findings of this study suggest that DCM extract may possess anticancer effect against lung adenocarcinoma and brain glioblastoma, as well as serve as an anti-inflammatory agent.

Phytochemical screening and evaluation of cytotoxicity of stem bark extracts of Genus dolichocarpa and Duguetia chrysocarpa (Annonaceae)

Purpose: To investigate the phytochemistry and cytotoxic activity of stem bark extracts from Genus dolichocarpa and Duguetia chrysocarpa - two species of the Annonaceae family. Methods: The crude ethanol bark extracts (EtOH) of the plants were obtained by maceration. The crude extracts were suspended in a mixture of methanol (MeOH) and water (H2O) (proportion 3:7 v/v) and partitioned with hexane, chloroform (CHCl3) and ethyl acetate (AcOEt) in ascending order of polarity to obtain the respective fractions. The extracts were evaluated on thin layer chromatography (TLC) plates of silica gel to highlight the main groups of secondary metabolites. Cytotoxicity was tested against human tumor cell lines - OVCAR-8 (ovarian), SF-295 (brain) and HCT-116 (colon) - using 3- (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Results: The screening results demonstrated that all the extracts were positive for the presence of flavonoids and tannins. The presence of alkaloids also was detected in some extracts. The hexane extract of A. dolichocarpa showed the strongest cytotoxicity against HCT-116 with cell growth inhibition of 89.02 %. Conclusion: The findings demonstrate for the first time the cytotoxic activity of the extracts of A. dolichocarpa and D. chrysocarpa, thus providing some evidence that plants of the Annonaceae family are a source of active secondary metabolites with cytotoxic activity.

Evaluación de la eficacia antibacteriana in vitro de un colutorio de aceite esencial de Origanum vulgare L. (Oregano) sobre Sterptococcos mutans

con el objetivo de evaluar la eficacia antibacteriana in vitro de dos formulaciones de un colutorio de aceite esencial de orégano sobre Streptococcus mutans (S. mutans) el principal agente causal de la placa bacteriana dental, para determinar dicha actividad, primero se realizó la determinación de la concentración inhibitoria mínima (CIM) del aceite esencial de orégano (AEO) sobre S. mutans mediante el método de microdilución en caldo y se obtuvo una CIM de 0.0025 %, esa concentración o una mayor podría ser útil en una forma farmacéutica con propósito antibacteriano. se realizó la determinación de la aptitud de los métodos de recuento microbiano que permitió indicar la metodología adecuada para evaluar la biocarga microbiana en los colutorios. Esta prueba indicó que el polisorbato 80 al 1% es un agente neutralizante idóneo para recuperar el S. mutans si se encuentra presente en los colutorios de AEO al 0.05 % y 0.10 %, y la mezcla de tiosulfato de sodio al 0.3 %, lecitina al 0.5% y polisorbato 80 al 1 % es la indicada para neutralizar las propiedades antimicrobianas del colutorio de clorhexidina al 0.12 % ylograr una recuperación adecuada del microorganismo en estudio cuando estuviera presente. Finalmente, se realizó la determinación de la eficacia antibacteriana de los colutorios de AEO, el vehículo de estos y un colutorio comercial de clorhexidina sobre S. mutans, los resultados reflejaron que el porcentaje máximo alcanzado de reducción para los colutorios de AEO al 0.05% y 0.10 % fue de 48.280 % a los 15 minutos y de 78.535 % en 1 minuto respectivamente, dichas reducciones fueron mayores que la que presento el vehículo, la cual fue de 29.292 % a los 15 minutos. El colutorio de clorhexidina al 0.12 % fue el que produjo la máxima reducción del microorganismo de estudio, la cual fue de99.996% en 6 minutos. Todos los ensayos se realizaron en el laboratorio del Centro de Investigación y Desarrollo en Salud, durante el periodo de julio a septiembre de 2015. Se concluyó que, ningún colutorio alcanzó el 99.999 % de reducción especificado por la AOAC para germicidas y sanitizantes, se observaron reducciones que sugieren buenas perspectivas de desarrollo para este tipo de formulaciones que incluyen activos de origen natural, por lo tanto se recomienda la necesidad de realizar más estudios sobre la composición y diferentes actividades del aceite esencial de orégano que pueden aprovecharse en futuras formulaciones.

Characterization of volatile compounds of Albertisia papuana Becc root extracts and cytotoxic activity in breast cancer cell line T47D

Purpose: To evaluate the cytotoxic activity of chloroform and water root extracts of Albertisia papuana Becc. on T47D cell line and identify the volatile compounds of the extracts. Methods: The plant roots were extracted with chloroform and water using maceration and boiling methods, respectively. The cytotoxicity of the extracts on T47D were determined using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Doxorubicin was used as reference drug in the cytotoxicity test while Probit analysis was used to calculate the Median Growth Inhibitory Concentration IC50 of the extracts. The volatile compounds in the chloroform and water root extracts were analyzed using Gas Chromatography-Mass Spectrophotometry GC-MS. Results: The IC50 of the chloroform and water extracts were 28.0 ± 6.0 and 88.0 ± 5.5 μg/mL, respectively whereas that of doxorubicin was 8.5 ± 0.1 μg/mL. GC-MS results showed that there were 46 compounds in the chloroform extract, out of which the five major components are ethyl linoleate (49.68 %), bicyclo (3.3.1) non-2-ene (29.29 %), ethyl palmitate (5.06 %), palmitic acid (3.67 %) and ethyl heptadecanoate (1.57 %).The water extract consisted of three compounds, butanoic acid (15.58 %); methyl cycloheptane (3.45 %), and methyl 2-O-methylpentofuranoside (80.96 %). Conclusion: The chloroform root extract of A. papuana Becc. had a fairly potent anticancer activity against breast cancer cells and may be further developed as an anticancer agent. Its major components were fatty acids and fatty acid esters.

Leishmanicidal and cytotoxic activity of extracts and saponins from Ilex laurina (Aquifoliaceae)

Purpose: To evaluate the leishmanicidal and cytotoxic activity of alcohol and non-alcohol extracts and saponins from Ilex laurina . Methods: Extracts were obtained by percolation with solvents of different polarities: hexane, dichloromethane, ethyl acetate and ethanol. The ethyl acetate extract was subjected to silica gel column chromatography eluting with a step gradient of dichloromethane-methanol. All products were evaluated in vitro for leishmanicidal activity against amastigotes of leishmania panamensis and cytotoxicity on U- 937 cells. Results: Two saponins were isolated from the ethyl acetate extract. The ethyl acetate extract showed high leishmanicidal activity against intracellular amastigotes of L. panamensis (EC50, 7.5 ± 1.5 μg/mL) and low activity against axenic amastigotes (EC50, 52.8 ±1.6 μg/mL); this extract showed also high cytotoxicity (LC50, 57.7 ± 12.1 μg/mL). Saponin 2 exhibited high activity against intracellular amastigotes (EC50, 5.9 ± 0.5 μg/mL) but also showed high cytotoxicity on U-937 cells (EC50, 25.7 ± 6.1 μg/mL). This compound showed similar leishmanicidal activity and cytotoxicity to meglumine antimoniate and amphotericin B, respectively, drugs currently used for the treatment of leishmaniasis. Conclusions: Based on these results, Ilex laurina is a potential source of compounds that can lead to the development of new therapeutic alternatives against leishmaniasis.

In vivo analgesic activity and safety assessment of Vitis vinifera L and Punica granatum L fruits extracts

Purpose: To investigate the analgesic properties of fruit extracts of Vitis vinifera (grape) and Punica granatum (pomegranate) in Albino mal mice. Methods: The analgesic activity of fruit extracts of V. vinifera and P. granatum were examined in vivo using thermal stimulus assays (i.e., tail immersion and hot plate) and acetic acid-induced writhing test using acetylsalicylic acid (0.1 g/kg, per os) as standard. The extracts were administered orally in doses of 1.0, 2.0 and 3.0 g/kg. Results: In acetic acid writhes test, both fruit extract pretreatments (1.0, 2.0 and 3.0 g/kg, per os) significantly decreased the number of writhes (p < 0.0001) in a dose-dependent manner compared to control. The Index of Pain Inhibition (IPI) values following V. vinifera extract treatments were 36.52 % (1.0 g/kg), 66.67 % (2.0 g/kg) and 89.71 % (3.0 g/kg) which were significantly different from those for P. granatum extracts (45.39 %, 1.0 g/kg), 70.93 %, 2.0 g/kg) and 86.88 %, 3.0 g/kg) at equivalent doses of 2.0 and 3.0 g/kg of the extracts The fruit extracts of both species increased the reaction latency time. In tail-immersion assay, only the fruit extract of P. granatum significantly increased the response to heat stimulus at doses of 2.0 g/kg (p < 0.05). Conclusion: The hydroalcohol fruit extracts of P. granatum and V. vinifera have potential analgesic effects. Further studies are needed to determine the active component responsible for this effect.

Antifungal activity of Stryphnodendron adstringens (Mart.) extracts.

2009

Multiplexed detection: fast comprehensive sample analysis of tobacco leaf extracts using HPLC with AFT columns

Detector-based comprehensive screening analysis of complex samples of natural origin using High Performance Liquid Chromatography (HPLC) can be a complicated and time-consuming task. There are a number of ways multidetection characterization can be achieved; however, there are limitations associated with each technique. Active Flow Technology (AFT) in Parallel Segmented Flow (PSF) mode allows for multiplexed detection HPLC analysis within a single injection, whereas maintaining chromatographic performance and allowing the use of multiple destructive detectors to achieve a comprehensive yet efficient screening of a complex sample. In this study, a comprehensive characterization analysis of tobacco leaf extract was carried out through multiplexed detection using a PSF column for the detection of biomolecules by UV-Vis detection, DPPH• for reactive-oxygen species (ROS) detection, and mass spectrometry, the latter two detection methods being sample destructive.

Enhancement of the total phenolic compounds and antioxidant activity of aqueous Citrus limon L. pomace extracts using microwave pre-treatment on the dry powder

The effect of microwave pre-treatment on the levels of total phenolic compounds, flavonoids, proanthocyanidins and individual major compounds as well as the total antioxidant activity of the dried lemon pomace was investigated. The results showed that microwave pre-treatment significantly affected all the examined parameters. The total phenolic content, total flavonoids, proanthocyanidins, as well as the total antioxidant activity significantly increased as the microwave radiation time and power increased (e.g., 2.5 folds for phenolics, 1.4 folds for flavonoids and 5.5 folds for proanthocyanidins), however irradiation more than 480 W for 5 min resulted in the decrease of these parameters. These findings indicate that microwave irradiation time and power may enhance higher levels of the phenolic compounds as well as the antioxidant capacity of the dried lemon pomace powder. However, higher and longer irradiation may lead to a degradation of phenolic compounds and lower the antioxidant capacity of the dried lemon pomace.

Antimicrobial activity of Brazilian propolis extracts for Staphylococcus aureus of sheep and goats mastitis origin

Twenty-four S. aureus isolates were analysed. From those, 22 were isolated from milk of goats and sheep with clinical and subclinical mastitis, from the region of Vale do São Francisco in the Brazilian Sertão and S. aureus ATCC 25923 plus a MRSA strain were added. Alcoholic extracts were produced from several batches of green, red and brown propolis consisting of 300 g of raw propolis in 700 mL of 70 % ethanol. Four genes related to antimicrobial resistance were assessed: blaZ that determines the resistance to β-lactam antibiotics, and genes icaA, icaD and bap that influence the production of biofilm. For the tests of susceptibility to different types of propolis the microdilution method was used, in triplicate, and dilutions between 0.003672 and 15% were tested, 70 % ethanol consisted of a negative control. The gene blaZ was found in 15 isolates; icaA gene was present in 3 isolates, icaD gene in 2 and bap gene was detected in 6 isolates. All the propolis tested exhibited antimicrobial activity, ranging from 44 to 100 % of susceptible isolates depending on different propolis batches. According to the results of this experiment the green and red propolis appear to have better antimicrobial activity than the brown variety.

Antibiofilm activity of Brasilian propolis extracts against Staphylococcus aureus producers isolated from goat and sheep mastitic milk

The aim of this study was to investigate the action of inhibiting S. aureus biofilm formation, and the ability to eliminate formed biofilm, by alcoholic extracts of green, red and brown propolis from Brazil. Ten isolates of S. aureus have been tested, 8 field isolates, 1 MRSA and 1 ATCC 25923, by microplate quantitative method. For the evaluation of inhibitory action, the isolates were inoculated, in triplicate, in TSB 1% glucose in the presence of green (1), red (2) and brown (4) propolis extracts. Biofilm formation was evaluated by optical reading, compared to a negative control consisting of a mixture of TSB and extract. For biofilm elimination assay, extracts were added to plates with 24h cultures of the same isolates. Assays were repeated three times on three different days. Eight out of the 10 isolates produced less biofilm in the presence of the green propolis extracts, so the inhibitory effect is 80%. Brown propolis extracts inhibited the formation of biofilm in 10% to 70% of the isolates and the red extracts in 30% to 80%. Regarding the biofilm elimination activity, green propolis extract was positive for 9 out of the 10 isolates (90%), the brown propolis extracts were positive for 0% to 100% isolates and red extracts for 0% to 10% isolates.

In vitro effects of Pilocarpus microphyllus extracts and pilocarpine hydrochloride on Rhipicephalus (Boophilus) microplus.

The aim of this study was to assess the activity of aqueous (AE) and ethanolic extracts (EE) and pilocarpine hydrochloride, which were extracted and isolated from Pilocarpus microphyllus (Jaborandi), respectively, on Rhipicephalus (Boophilus) microplus. High performance liquid chromatography (HPLC) was performed to quantify these compounds. Larval packet and adult immersion tests were conducted with different concentrations. Five AE and EE concentrations, ranging from 6.2 to 100.0 mg mL?1, and six concentrations of pilocarpine hydrochloride, ranging from 0.7 to 24.0 mg mL?1, were tested. The lethal concentration (LC50) of each extract for larvae and engorged females was calculated through Probit analysis. The concentration of pilocarpine hydrochloride obtained from the EE and the AE was 1.3 and 0.3% (m/m), respectively. Pilocarpine hydrochloride presented the highest acaricidal activity on larvae (LC50 2.6 mg mL?1) and engorged females (LC50 11.8 mg mL?1) of R.(B.) microplus, followed by the EE which presented LC50 of 56.4 and 15.9 mg mL?1, for larvae and engorged females, respectively. Such results indicate that pilocarpine hydrochloride has acaricidal activity, and may be the primary compound responsible for this activity by P. microphyllus EE.

Parametric building development during early design stage

Objectives The objectives of this project were two-fold: • Assess the ease with which current architectural CAD systems supported the use ofparametric descriptions in defining building shape, engineering system performance and cost at the early stages of building design; • Assess the feasibility of implementing a software decision support system that allowed designers to trade-off the characteristics and configuration of various engineering systems to move towards a “global optimum” rather than considering each system in isolation and expecting humans to weigh up all of the costs and benefits. The first stage of the project consisted of using four different CAD systems to define building shells (envelopes) with different usages. These models were then exported into a shared database using the IFC information exchange specifications. The second stage involved the implementation of small computer programs that were able to estimate relevant system parameters based on performance requirements and the constraints imposed by the other systems. These are presented in a unified user interface that extracts the appropriate building shape parameters from the shared database Note that the term parametric in this context refers to the relationships among and between all elements of the building model - not just geometric associations - which will enable the desired coordination.