Betulinic acid and its spray dried microparticle formulation: In vitro PDT effect against ovarian carcinoma cell line and in vivo plasma and tumor disposition.

The race against ovarian cancer continue to motivate the research worldwide. It is known that many antitumor drugs have limited penetration into solid tumor tissues due to its microenvironment, thus contributing to their low efficacy. Therapeutic modalities have been exploited to elicit antitumor effects based on microenvironment of tumor, including Photodynamic therapy (PDT). Prospection of natural small molecules and nanotechnology are important tools in the development of new ways of obtaining photoactive compounds that are biocompatible. The Betulinic acid (BA) has shown potential biological effect as bioactive drug, but it has low water solubility. Thus, in the present study, owing to the poor solubility of the BA, its free form (BAF) was compared to a spray dried microparticle betulinic acid/HP-ß-CD formulation (BAC) aiming to assess the BAF and BAC efficacy as a photosensitizer in PDT for application in ovarian cancer. BAF and BAC were submitted to assays in the presence of LED (λ = 420 nm) under different conditions (2.75 J/cm2, 5.5 J/cm2, and 11 J/cm2) and in absence of irradiation, after 5 min or 4 h of contact with ovarian carcinoma cells (A2780) or fibroblast murine cells (3T3). Furthermore, HPLC-MS/MS and MALDI-MSI methods were developed and validated in plasma and tumor of mice proving suitable for in vivo studies. The results found a greater photoinduced cytotoxic effect for the BAC at low concentration for A2780 when irradiated with LED with similar results for fluorescence microscopy. The results motivate us to continue the studies with the BA as a potential antitumor bioactive compound.

Biopharmaceutical and antifungal properties of ellagic acid-cyclodextrin using an in vitro model of invasive candidiasis.

Aim: To investigate biopharmaceutical and antifungal properties of pure and complexed ellagic acid. Materials & methods: Caco-2 cells cultured in a Transwell® inserts were infected with Candida albicans to develop an in vitro model. Ellagic acid was complexed with cyclodextrins. Microbial compositions, ellagic acid concentration as function of time and characterization studies of complexes were evaluated. Results: Ellagic acid presented ability to reduce C. albicans invasion, although this was not statistically significant. Its poor water solubility and absorption probably limited this ability. Water solubility was increased after complexation with hydroxypropyl-ß-CD; however, ellagic acid/hydroxypropyl-ß-CD did not improve the antifungal activity. Conclusion: Although ellagic acid presented a promising antifungal activity, its biopharmaceutical properties limit such activity and should be improved.

Cytotoxicity and bacterial membrane destabilization induced by Annona squamosa L. extracts.

This study aimed to further investigate the cytotoxicity against tumor cell lines and several bacterial strains of Annona squamosa and its mode of action. Methanol extracts of A. squamosa leaves (ASL) and seeds (ASS) were used. ASL showed significant antibacterial activity against S. aureus, K. pneumoniae and E. faecalis with MIC values of 78, 78 and 39 µg/mL respectively. Moreover, ASL exhibited significant biofilm disruption, rapid time dependent kinetics of bacterial killing, increased membrane permeability and significantly reduced the cell numbers and viability. Regarding the cytotoxicity against tumor cell lines, ASS was more active against Jurkat and MCF-7 cells, with CI50 1.1 and 2.1 µg/mL, respectively. ASL showed promising activity against Jurkat and HL60, with CI50 4.2 and 6.4 µg/mL, respectively. Both extracts showed lower activity against VERO cells and reduced the clonogenic survival at higher concentrations (IC90) to MCF-7 and HCT-116 lineages. The alkaloids anonaine, asimilobine, corypalmine, liriodenine nornuciferine and reticuline were identified in extracts by UPLC-ESI-MS/MS analysis. This study reinforced that A. squamosa presents a remarkable phytomedicinal potential and revealed that its antimicrobial mechanism of action is related to bacterial membrane destabilization.

Cytotoxicity and bacterial membrane destabilization induced by Annona squamosa L. extracts

ABSTRACT This study aimed to further investigate the cytotoxicity against tumor cell lines and several bacterial strains of Annona squamosa and its mode of action. Methanol extracts of A. squamosa leaves (ASL) and seeds (ASS) were used. ASL showed significant antibacterial activity against S. aureus, K. pneumoniae and E. faecalis with MIC values of 78, 78 and 39 µg/mL respectively. Moreover, ASL exhibited significant biofilm disruption, rapid time dependent kinetics of bacterial killing, increased membrane permeability and significantly reduced the cell numbers and viability. Regarding the cytotoxicity against tumor cell lines, ASS was more active against Jurkat and MCF-7 cells, with CI50 1.1 and 2.1 µg/mL, respectively. ASL showed promising activity against Jurkat and HL60, with CI50 4.2 and 6.4 µg/mL, respectively. Both extracts showed lower activity against VERO cells and reduced the clonogenic survival at higher concentrations (IC90) to MCF-7 and HCT-116 lineages. The alkaloids anonaine, asimilobine, corypalmine, liriodenine nornuciferine and reticuline were identified in extracts by UPLC-ESI-MS/MS analysis. This study reinforced that A. squamosa presents a remarkable phytomedicinal potential and revealed that its antimicrobial mechanism of action is related to bacterial membrane destabilization.

Polycarpol in Unonopsis, Bocageopsis and Onychopetalum Amazonian species: chemosystematical implications and antimicrobial evaluation

Polycarpol, a recurrent lanostane-type triterpene in Annonaceae family, was confirmed by thin layer chromatography and mass spectrometry analysis in the aerial parts (twigs and trunk barks) of Unonopsis duckei R.E. Fr., U. floribunda Diels, U. rufescens (Baill.) R.E. Fr., U. stipitata Diels, Onychopetalum amazonicum R.E. Fr. and Bocageopsis pleiosperma Maas. Its chemotaxonomic significance was discussed for these three genera, as well for the Annonaceae family. In addition, the antimicrobial activity against several strains of microorganisms was evaluated for the first time for this compound, being observed significant antibacterial activity against Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (ATCC 1228) and Escherichia coli (ATCC 10538 and ATCC 10799) with minimal inhibitory concentration values between 25 and 50 μg ml−1.

Mauritic acid: a new dammarane triterpene from the roots of Mauritia flexuosa L.f. (Arecaceae).

A new dammarane triterpene named mauritic acid (1) was isolated from the roots of Mauritia flexuosa L.f. The complete structural assignment of this new compound was elucidated from spectroscopic methods. Moreover, this compound was evaluated for its cytotoxicity against human cancer cell lines (OVCAR-8, PCM3, NCIH358M and different leukaemia cell strains). The mauritic acid presented significant cytotoxicity against OVCAR-8, PCM3 and NCIH358M cell lines with IC50 3.02, 2.39 and 6.19 µM, respectively. The triterpenes 1 and 2 were also tested for their antimicrobial activity against 15 strains of microorganisms, including fungi and bacteria, with the best minimal inhibitory concentration values ranging from 50.8 to 203.5 µM.

Bioactivity-guided isolation of cytotoxic sesquiterpene lactones of Gochnatia polymorpha ssp. floccosa.

Phytochemical study of Gochnatia polymorpha (Less) Cabr. ssp. floccosa Cabr. trunk bark, guided by antiproliferative assays on 10 human cancer cell lines and the VERO cell line, yielded six known compounds identified as the triterpene bauerenyl acetate, the guaianolide 11α,13-dihydrozaluzanin C and the dimeric guaianolides 10-desoxygochnatiolide A, gochnatiolide A, 8-hydroxi-10-desoxygochnatiolide A and 8-hydroxigochnatiolide A. Extracts, fractions of extracts and isolated compounds were tested in vitro against a panel of human cancer cell lines, including U251 (glioma, CNS), UACC-62 (melanoma), MCF-7 (breast), NCI-ADR/RES (drug-resistant ovarian), 786.0 (kidney), NCI-H460 (lung, no small cells), PC-3 (prostate), OVCAR-3 (ovarian), HT-29 (colon), K562 (leukemia) and against the VERO no cancer cell line. Bauerenyl acetate was inactive, while 11α,13-dihydrozaluzanin C showed weak activity against UACC62 and the VERO cell line. The most active compounds were 10-desoxygochnatiolide A and gochnatiolide A, which inhibited the growth of kidney, melanoma, ovarian-resistant and glioma cell lines with values of TGI (total growth inhibition) varying 0.21-1.09 µg/mL. This is the first report about cytotoxic activity of dimeric lactones against cell lines.

Triterpenes from the flowers of Gochnatia polymorpha subsp. floccosa

Phytochemical study of the flowers of Gochnatia polymorpha subsp. floccosa, Asteraceae, yielded eleven known triterpenes identified as lupeol, lupeyl acetate, lupeyl palmitate, taraxasterol, taraxasteryl acetate, pseudotaraxasterol, pseudotaraxasterol acetate, α-amyrin, α-amyryl palmitate, β-amyrin and β-amyryl palmitate, along with sitosterol, stigmasterol, palmitic and stearic acids. These compounds are been reported for the first time in the species. The compounds were identified by analysis of NMR spectra (¹H, 13C and DEPT), GC-MS and comparison with literature data. Previous work have reported the isolation of triterpenes, diterpenes, sesquiterpenes, flavonoids, coumarins and phenolic compounds from aerial parts and roots from G. polymorpha.

Extracts from Alternanthera maritima as natural photosensitizers in photodynamic antimicrobial chemotherapy (PACT).

This study investigated the effect of photodynamic antimicrobial chemotherapy (PACT) using extracts from Alternanthera maritima on the viability of Candida dubliniensis. Human infections constitute a great health problem. Several antifungal drugs are currently available, but their uses are limited by a number of factors, such as low potency, poor solubility, microbial resistance, and drug toxicity. Therefore, the search for new and more effective antimicrobial agents and the development of alternative therapies, such as PACT, are necessary. Crude hexane and ethanol extracts of A. maritima were produced. The prepared extracts presented absorption at 650-700 nm. For bioassays, 50 microL of culture medium, 50 microL of extract (25 mg/mL) or control, and 5 microL of a suspension of the microorganism to be tested (C. dubliniensis ATCC 778157 or ATCC 777, 10(7)CFU/mL) were placed in a sterile 96-well microtiter plate (well cross section=0.38 cm(2)). The contents of each well were irradiated with a 685-nm diode laser with an output power of 35 mW, which was distributed through the well cross section yielding an energy dosage of 28 J/cm(2). In each assay (n=6), one plate was subjected to irradiation, and one was not. For each active sample, the number of colony-forming units per milliliter (CFU/mL) was obtained, and data were analyzed by the Tukey test. The chemical compositions of the extracts were determined by chromatographic and spectroscopic techniques. The results suggest inhibition of the growth of C. dubliniensis when irradiated with a diode laser in the presence of hexane and ethanol extracts from A. maritima as photosensitizers. Laser irradiation alone or crude extracts at 25mg/mL did not significantly reduce the number of CFU/mL. Steroids, triterpenes, and flavonoids were identified in the analyzed extracts. In conclusion, the photoactivation of crude hexane and ethanol extracts of A. maritima by red laser radiation at 685 nm promoted an antimicrobial effect, showing that these natural products can be used as photosensitizers in PACT.

Evaluation of the antimicrobial activity of crude extracts and isolated constituents from Chresta scapigera

Crude extracts and eight isolated compounds from Chresta scapigera were evaluated for antibacterial and antifungal activities by the agar-well diffusion method. Twenty strains, including Gram-positive and Gram-negative bacteria and yeasts were used in the bioassay. Hexane extracts presented the best results while ethanol extracts did not indicate inhibition of the microbial growth. Amongst the evaluated compounds b-amyrin acetate, tiliroside and luteolin showed the strongest antimicrobial effect.

Os extratos brutos e oito constituintes isolados de Chresta scapigera foram avaliados para as atividades antibacteriana e antifúngica, utilizando o método de difusão em ágar. Vinte cepas indicadoras, incluindo bactérias (Gram-positivas e Gram-negativas) e leveduras, foram utilizadas no bioensaio. Os melhores resultados foram obtidos para os extratos hexânicos, enquanto os extratos etanólicos não inibiram o crescimento microbiano. Acetato de b-amirina, tilirosídeo e luteolina foram os mais eficazes dentre os constituintes avaliados.

Avaliação da atividade antimicrobiana e citotóxica de extratos de Gochnatia polymorpha ssp floccosa / Evaluation of antimicrobial and cytotoxic activitiy of extracts from Gochnatia polymorpha (Less) ssp floccosa

Neste estudo procedeu-se a avaliação da atividade antimicrobiana e citotóxica de extratos de Gochnatia polymorpha ssp floccosa, espécie empregada na medicina popular contra doenças respiratórias. Folhas, cascas do tronco e ramos foram extraídos com hexano, diclorometano e etanol, sucessivamente, sendo obtidos os respectivos extratos brutos. A atividade antimicrobiana foi determinada pelo método de difusão em ágar utilizando-se bactérias Gram-positivas, Gram-negativas e fungos. A avaliação da citotoxicidade foi realizada empregando-se o ensaio de letalidade contra Artemia salina. Nenhum dos extratos mostrou atividade citotóxica. Os extratos das folhas apresentaram uma fraca atividade antimicrobiana frente a algumas cepas de Staphylococcus aureus e Streptococcus mutans, enquanto o extrato em diclorometano dos ramos e o extrato em etanol das cascas foram ativos contra Micrococcus luteus, Staphylococcus aureus e S. epidermidis. A maior atividade antimicrobiana foi observada para o extrato em diclorometano das cascas, que inibiu o crescimento de Micrococcus luteus, Staphylococcus aureus, S. epidermidis, Streptococcus mutans, Enterococcus faecalis e Candida albicans. Esta atividade parece estar relacionada à presença de diterpenos no extrato. Nenhum dos extratos estudados (a 5,0 mg/mL) mostrou atividade frente a cepas de bactérias Gram-negativas. Esses resultados demonstram o potencial dessa planta como fonte de compostos antibacterianos e justificam, parcialmente, seu uso popular.

The antimicrobial and cytotoxic activities of Gochnatia polymorpha ssp floccosa, a medicinal plant used against respiratory diseases, were evaluated. Successive petroleum ether, dichloromethane and ethanol extracts of dried leaves, trunk bark and stems were used in the brine shrimp lethality bioassay and tested against 22 strains of microorganisms (Gram-positive and Gram-negative bacteria and fungi) by the well-diffusion agar method. None of the extracts showed cytotoxicity on the brine shrimp bioassay. The extracts of leaves showed a mild activity against some strains of Staphylococcus aureus and Streptococcus mutans. The ethanol extracts of trunk bark and dichloromethane extract of stems also showed activity against Micrococcus luteus, Staphylococcus aureus and S. epidermidis. The highest activity was found in the dichloromethane extract of trunk bark, which showed significant antibacterial and antifungal activities against Micrococcus luteus, Staphylococcus aureus, S. epidermidis, Streptococcus mutans, Enterococcus faecalis and Candida albicans. Diterpenes are present in this extract and could be responsible for the activity. All the screened extracts were shown to be inactive (at 5.0 mg/mL) against strains of Gram-negative bacteria tested. These results indicate the potential of G. polymorpha ssp floccosa as a source of antibacterial compounds and partly support the use of this plant in folk medicine.

Characterization of hypoglycemiant plants by total reflection X-ray fluorescence spectrometry.

In this work, synchrotron radiation total reflection X-ray fluorescence spectrometry (SRTXRF) was used to determine trace elements in eight hypoglycemiant plants (Trigonella foenum graecum, Panax ginseng, Pfaffia paniculata, Myrcia speciosa, Zea mays, Harpagophytum procumbens, Syzygium jambolona, and Bauhinia forficate). The elements P, K, Ca, Ti, Mn, Fe, Cu, Zn, Rb, and Sr were detected in all medicinal plants investigated, whereas Si, S, Sc, V, Cr, Co, Ni, Se, Nb, Mo, Sn, Sb, Ba, Hg, and Pb were detected only in some of the samples. The concentration of elements in hypoglycemiant plants varied from 0.15 microg/g of Co to 3.0 x 10(4) microg/g of K and the mean of experimental limit of detection for these elements were 0.14 and 3.6 microg/g, respectively.

Comparative study of antibacterial and antifugal activity of callus culture and adult plants extracts from Alternanthera maritima (Amaranthaceae)

Neste estudo procedeu-se a avaliação da atividade antibacteriana e antifúngica dos extratos brutos de Alternanthera maritima (Amaranthaceae) planta in natura de duas coletas distintas e obtidos por cultura de células buscando-se averiguar a manutenção da atividade antimicrobiana dos extratos obtidos da planta in vivo e in vitro. A ação antibacteriana e antifúngica foi determinada pelo método de difusão em ágar (técnica do poço) utilizando-se trinta cepas de microrganismos indicadores (bactérias Gram-positivas e Gram-negativas, leveduras e dermatófitos). Todos os extratos obtidos com solventes orgânicos avaliados apresentaram-se bioativos com halos de inibição de 6 a 20 mm. Os extratos da planta in natura das duas coletas (Restinga de Marica (RJ), verão de 1995 e 1998) inibiram o desenvolvimento de diferentes microrganismos (bactérias, leveduras e dermatófitos). Os extratos obtidos da cultura de calos desenvolvidos em duas condições de cultivo diferentes, também se mantiveram bioativos. Assim, os resultados obtidos encorajam a realização de novos estudos com esta espécie vegetal para se determinar quais as substâncias presentes nos extratos e que contribuem para a atividade biológica, como também para entender seu mecanismo de ação e avaliar sua toxicidade, visando uma possível aplicação farmacêutica.