Chlorophyll treatment combined with photostimulation increases glycolysis and decreases oxidative stress in the liver of type 1 diabetic rats

Photodynamic therapy (PDT) promotes cell death, and it has been successfully employed as a treatment resource for neuropathic complications of diabetes mellitus (T1DM) and hepatocellular carcinoma. The liver is the major organ involved in the regulation of energy homeostasis, and in pathological conditions such as T1DM, changes in liver metabolic pathways result in hyperglycemia, which is associated with multiple organic dysfunctions. In this context, it has been suggested that chlorophyll-a and its derivatives have anti-diabetic actions, such as reducing hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, but these effects have not yet been proven. Thus, the biological action of PDT with chlorophyll-a on hepatic parameters related to energy metabolism and oxidative stress in T1DM Wistar rats was investigated. Evaluation of the acute effects of this pigment was performed by incubation of isolated hepatocytes with chlorophyll-a and the chronic effects were evaluated by oral treatment with chlorophyll-based extract, with post-analysis of the intact liver by in situ perfusion. In both experimental protocols, chlorophyll-a decreased hepatic glucose release and glycogenolysis rate and stimulated the glycolytic pathway in DM/PDT. In addition, there was a reduction in hepatic oxidative stress, noticeable by decreased lipoperoxidation, reactive oxygen species, and carbonylated proteins in livers of chlorophyll-treated T1DM rats. These are indicators of the potential capacity of chlorophyll-a in improving the status of the diabetic liver.

Antimicrobial effects of Piper hispidum extract, fractions and chalcones against Candida albicans and Staphylococcus aureus.

Three chalcones, 2'-hydroxy-4,4',6'-trimethoxychalcone, 2'-hydroxy-4,4',6'-tetramethoxychalcone, and 3,2'-dihydroxy-4,4',6'-trimethoxychalcone, were isolated from the leaves of Piper hispidum in a bioguided fractionation of crude extract. The antimicrobial activity of crude extract of P. hispidum leaves was determined against bacteria Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and yeasts Candida albicans, C. parapsilosis and C. tropicalis. Fractions and chalcones were tested against C. albicans and S. aureus. The checkerboard assay was performed to assess synergic interactions between extract and antifungal drugs, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay was used to evaluate anti-biofilm effects of extract. The extract was active against yeasts, S. aureus and B. subtilis with MIC values between 15.6 and 62.5µg/mL. Synergistic effects of extract associated with fluconazole and nystatin were observed against C. albicans, with fractional inhibitory concentration indices of 0.37 and 0.24, respectively. The extract was also effective against C. albicans and S. aureus biofilm cells at concentrations of 62.5 and 200µg/mL, respectively. Thus, P. hispidum may be a possible source of bioactive substances with antimicrobial properties.

Antidermatophytic activity of hydroalcoholic extracts from Rosmarinus officinalis and Tetradenia riparia.

Rosmarinus officinalis and Tetradenia riparia are used in folk medicine for the treatment of disease, including infectious diseases and skin disorders. The purpose of this study was to investigate the antifungal activity of hydroalcoholic extracts from R. officinalis and T. riparia against strains of Trichophyton rubrum, T. mentagrophytes and Microsporum gypseum. Hydroalcoholic extracts prepared with dried leaves from R. officinalis, Psidium guajava and T. riparia were assayed against dermatophyte species by the microdilution technique and by microscopy. R. officinalis and T. riparia were the most active against dermatophytes, as determined from the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC), and were investigated further. Fluorescence microscopy and scanning electron microscopy were used to investigate inhibition of hyphal growth by the two extracts, and showed a strong inhibition and an irregular growth pattern. Both extracts showed good action against dermatophytes, inhibiting fungal growth and causing alterations in their hyphae. Therefore, R. officinalis and T. riparia are potential sources of new compounds for the development of antifungal drugs.

Further evidence of the trypanocidal action of eupomatenoid-5: confirmation of involvement of reactive oxygen species and mitochondria owing to a reduction in trypanothione reductase activity.

Our group assays natural products that are less toxic and more effective than available nitroheterocycles as promising therapeutic options for patients with Chagas disease. Our previous study reported the trypanocidal activity of eupomatenoid-5, a neolignan isolated from the leaves of Piper regnellii var. pallescens, against the three main parasitic forms of Trypanosoma cruzi. The present study further characterizes the biochemical and morphological alterations induced by this compound to elucidate the mechanisms of action involved in the cell death of T. cruzi. We show that eupomatenoid-5 induced oxidative imbalance in the three parasitic forms, especially trypomastigotes, reflected by a decrease in the activity of trypanothione reductase and increase in the formation of reactive oxygen species (ROS). A reduction of mitochondrial membrane potential was then triggered, further impairing the cell redox system through the production of more ROS and reactive nitrogen species. Altogether, these effects led to oxidative stress, reflected by lipid peroxidation and DNA fragmentation. These alterations are key events in the induction of parasite death through various pathways, including apoptosis, necrosis, and autophagy.

Effect of inactive dry yeast from sugar cane as protein source on rumen fermentation in Saanen goats / Efeito da levedura seca inativa da cana-de-açúcar como fonte de proteína sobre a fermentação ruminal de caprinos Saanen

Five castrated and ruminally cannulated Saanen goats (±48.19kg) were used to evaluate intake, digestibility and rumen fermentation parameters of diets with inactive dry yeast as a soybean meal substitute (0, 25, 50, 75 and 100 percent). Goats were randomly assigned to a 5×5 (five levels of dry yeast x five periods) Latin square design. Diets were composed of corn silage (40 percent), ground corn, soybean meal and/or dry yeast and mineral supplement. The intake and digestibility of DM, OM, CP, NDF and TC were not influenced by the treatments. However, EE intake showed negative linear effect. The TDN content did not change with the inclusion of dry yeast in the diets. The pH, N-NH3 concentration and rumen short-chain fatty acids content did not differ among diets. Rumen content scanning electron microscopy observations did not suggest microbial colonization and degradation changes. Dry yeast from sugar cane can replace soybean meal in diets for Saanen goats without changing the rumen fermentation pattern, intake and digestibility.

Com o objetivo de avaliar a ingestão, a digestibilidade e os parâmetros de fermentação ruminal de rações com de levedura seca em substituição ao farelo de soja (0; 25; 50; 75 e 100 por cento), foram utilizados cinco caprinos Saanen (± 48,19kg) castrados e canulados no rúmen distribuídos em quadrado latino 5 x 5 (cinco níveis x cinco períodos). As rações foram compostas de silagem de milho (40 por cento), milho moído, farelo de soja e/ou levedura seca e suplemento vitamínico-mineral. A ingestão e a digestibilidade da MS, MO, PB, FDN e dos CT não foram alteradas. A ingestão de EE apresentou efeito linear negativo. O NDT não foi alterado com a inclusão da levedura seca. O pH e as concentrações de N-NH3 e de ácidos graxos de cadeia curta no rúmen não apresentaram alterações. As observações por microscopia eletrônica de varredura não indicaram mudanças na colonização microbiana e na degradação da digesta ruminal. A levedura seca da cana-de-açúcar não altera o padrão de fermentação ruminal, a ingestão e a digestibilidade, podendo substituir o farelo de soja em rações de caprinos Saanen.

Effects of (-) mammea A/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial membrane of Leishmania amazonensis.

We have previously demonstrated antileishmanial activity on Leishmania amazonensis of the natural (1-2), synthetic (7) and derivatives of coumarin (-) mammea A/BB (3-6) isolated from the dichloromethane extract of Calophyllum brasiliense leaves. The aim of the present study was to evaluate morphological and ultrastructural alterations in Leishmania amazonensis induced by these compounds. In promastigote forms, all seven compounds produced significant morphological and ultrastructural alterations, as revealed by scanning and transmission electron microscopy. The compound 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3), the most active antileishmanial with LD50 of 0.9 µM), induced cell shrinkage and a rounded appearance of the cells. Parasites incubated in the presence of compound (3) showed ultrastructural changes, such as the appearance of mitochondrial swelling with a reduction in the density of the mitochondrial matrix and the presence of vesicles inside the mitochondrion, indicating damage and significant change in this organelle; abnormal chromatin condensation, alterations in the nuclear envelope, intense atypical cytoplasmic vacuolization, and the appearance of autophagic vacuoles were also observed. In addition, the compound (3) may be acting to depolarize the mitochondrial membrane potential of the cells, leading to death of the parasite.

Beta-carboline-3-carboxamide derivatives as promising antileishmanial agents.

Leishmaniasis has an overwhelming impact on global public health especially in tropical and subtropical countries and the currently available antileishmanial drugs have serious side effects and low efficacy. Natural and synthetic compounds have been tested in the past few years against Leishmania and the beta-carboline class of compounds have shown great results in antiparasitic chemotherapy. In the present study, three 1-substituted beta-carboline-3-carboxamides (3-5) and 1-substituted beta-carboline-3-carboxylic acid (2) were synthesized and screened for in vitro activity against L. amazonensis. Compound 5 (N-benzyl 1-(4-methoxy)phenyl-9H-beta-carboline-3-carboxamide) had the best activity against promastigote and axenic amastigote forms with IC(50) of 2·6 and 1·0 µM, respectively. Its CC(50) on macrophages cell line was higher than 2457·0 µM with an SI ratio of 930·2. Against intracellular amastigote forms, it had a dose-dependent relationship with a 50% growth inhibitory concentration of 1·0 µM. Through morphological and ultrastructure analysis of promastigote forms treated with compound 5, alterations on cell shape and number of flagella and nuclear membrane damage were observed. For this, compound 5 supports the idea for more in vitro and in vivo studies.

Antileishmanial activity of an essential oil from the leaves and flowers of Achillea millefolium.

An essential oil was recently extracted from the leaves and flowers of yarrow (Achillea millefolium) and tested for in-vitro activity against Leishmania amazonensis and murine macrophages (i.e. the J774G8 cell line). The median inhibitory concentration (IC(50)) against L. amazonensis promastigotes was 7.8 µg/ml whereas the survival of amastigotes of this pathogen, within peritoneal murine macrophages, was halved by treatment with the oil at 6.5 µg/ml. The mean value for the median cytotoxic concentration of the oil, measured against adherent (uninfected) J774G8 macrophages, was 72.0 µg/ml (i.e. 9.2 and 11.0 times higher, respectively, than the IC(50) against the promastigotes and intracellular amastigotes). Scanning electron microscopy revealed that the oil caused morphological changes in the treated parasites, including alterations in their shape and size. In transmission electron microscopy, promastigotes treated with the oil (at the IC(50) of 7.8 µg/ml) showed various ultrastructural alterations, including changes in the flagellar membrane, abnormal membrane structures, rupture of the plasma membrane, atypical vacuoles, myelin-like figures, and vesicles that resembled autophagic vacuoles.

Antiviral activity and mode of action of a peptide isolated from Sorghum bicolor.

In this paper, we describe the purification of an antiviral peptide from seeds of Sorghum bicolor L. by a procedure that included gel filtration, ion exchange, and high-performance liquid chromatography (HPLC) in a reversed-phase column. Its molecular weight, determined by chromatographic mobility on the Shim-pack DIOL-150 gel permeation column in HPLC, was found to be 2000Da. The peptide designated 2kD peptide strongly inhibited the replication of herpes simplex virus type 1 (HSV-1), dose-dependently, at 40-90% of the control level, after incubation with 10-50 microM of the peptide, with EC(50) and EC(90) values of 6.25 and 15.25 microM, respectively. The IC(50) value of the 2kD peptide against Vero cells was 250 microM. Pre-incubation of HSV-1 with various concentrations of the 2kD peptide showed dose-dependent cytopathic effects (CPE) reduction patterns at concentrations from 6.25 to 50 microM. The presence of the 2kD peptide before HSV-1 infections showed moderate inhibition of virus-induced CPE as compared to during or after infections, with EC(50) values of 12.5, 6.25, and 6.25 microM, respectively. Similar results were observed when the 2kD peptide was assayed against bovine herpes virus (BHV), an enveloped virus like HSV-1. On the other hand, the 2kD peptide showed weak activity against poliovirus type 1, a non-enveloped virus. Taken together, these results indicate that the 2kD peptide was able not only to inhibit the initiation and the spread of infection, but also had an in vitro prophylactic effect against HSV-1 infection.

Antibacterial activity of indole alkaloids from Aspidosperma ramiflorum

We evaluated the antibacterial activities of the crude methanol extract, fractions (I-V) obtained after acid-base extraction and pure compounds from the stem bark of Aspidosperma ramiflorum. The minimum inhibitory concentration (MIC) was determined by the microdilution technique in Mueller-Hinton broth. Inoculates were prepared in this medium from 24-h broth cultures of bacteria (10(7) CFU/mL). Microtiter plates were incubated at 37°C and the MICs were recorded after 24 h of incubation. Two susceptibility endpoints were recorded for each isolate. The crude methanol extract presented moderate activity against the Gram-positive bacteria B. subtilis (MIC = 250 µg/mL) and S. aureus (MIC = 500 µg/mL), and was inactive against the Gram-negative bacteria E. coli and P. aeruginosa (MIC > 1000 µg/mL). Fractions I and II were inactive against standard strains at concentrations of <=1000 µg/mL and fraction III displayed moderate antibacterial activity against B. subtilis (MIC = 500 µg/mL) and S. aureus (MIC = 250 µg/mL). Fraction IV showed high activity against B. subtilis and S. aureus (MIC = 15.6 µg/mL) and moderate activity against E. coli and P. aeruginosa (MIC = 250 µg/mL). Fraction V presented high activity against B. subtilis (MIC = 15.6 µg/mL) and S. aureus (MIC = 31.3 µg/mL) and was inactive against Gram-negative bacteria (MIC > 1000 µg/mL). Fractions III, IV and V were then submitted to bioassay-guided fractionation by silica gel column chromatography, yielding individual purified ramiflorines A and B. Both ramiflorines showed significant activity against S. aureus (MIC = 25 µg/mL) and E. faecalis (MIC = 50 µg/mL), with EC50 of 8 and 2.5 µg/mL for ramiflorines A and B, respectively, against S. aureus. These results are promising, showing that these compounds are biologically active against Gram-positive bacteria.

Antibacterial activity of indole alkaloids from Aspidosperma ramiflorum.

We evaluated the antibacterial activities of the crude methanol extract, fractions (I-V) obtained after acid-base extraction and pure compounds from the stem bark of Aspidosperma ramiflorum. The minimum inhibitory concentration (MIC) was determined by the microdilution technique in Mueller-Hinton broth. Inoculates were prepared in this medium from 24-h broth cultures of bacteria (10(7) CFU/mL). Microtiter plates were incubated at 37 masculineC and the MICs were recorded after 24 h of incubation. Two susceptibility endpoints were recorded for each isolate. The crude methanol extract presented moderate activity against the Gram-positive bacteria B. subtilis (MIC = 250 microg/mL) and S. aureus (MIC = 500 microg/mL), and was inactive against the Gram-negative bacteria E. coli and P. aeruginosa (MIC > 1000 microg/mL). Fractions I and II were inactive against standard strains at concentrations of < or =1000 microg/mL and fraction III displayed moderate antibacterial activity against B. subtilis (MIC = 500 microg/mL) and S. aureus (MIC = 250 microg/mL). Fraction IV showed high activity against B. subtilis and S. aureus (MIC = 15.6 microg/mL) and moderate activity against E. coli and P. aeruginosa (MIC = 250 microg/mL). Fraction V presented high activity against B. subtilis (MIC = 15.6 microg/mL) and S. aureus (MIC = 31.3 microg/mL) and was inactive against Gram-negative bacteria (MIC > 1000 microg/mL). Fractions III, IV and V were then submitted to bioassay-guided fractionation by silica gel column chromatography, yielding individual purified ramiflorines A and B. Both ramiflorines showed significant activity against S. aureus (MIC = 25 microg/mL) and E. faecalis (MIC = 50 microg/mL), with EC50 of 8 and 2.5 microg/mL for ramiflorines A and B, respectively, against S. aureus. These results are promising, showing that these compounds are biologically active against Gram-positive bacteria.

Antiprotozoal and molluscicidal activities of five Brazilian plants

Leishmaniasis, Chagas' disease and schistosomiasis (bilharzia) are parasitic diseases with wide distribution on the American continent, affecting millions of people. In the present study, biological assays for antiprotozoal and molluscicidal activities were carried out with ethanolic extracts of plant species from the Brazilian part of the Upper Paraná River. Crude extracts were obtained by percolation with absolute ethanol from the leaves of Cayaponia podantha Cogn., Nectandra falcifolia (Nees) Castiglioni and Paullinia elegans Cambess., as well as from the aerial parts of Helicteres gardneriana St. Hil. & Naud. and Melochia arenosa Benth., all belonging to genera used in folk medicine. Trypanocidal activity of plants was assayed on epimastigote cultures in liver infusion tryptose. Anti-leishmanial activity was determined over cultures of promastigote forms of the parasite in Schneider's Drosophila medium. Microscopic countings of parasites, after their incubation in the presence of different concentrations of the crude extracts, were made in order to determine the percentage of growth inhibition. C. podantha and M. arenosa, at a concentration of 10 æg/mL, showed 90.4 ± 11.52 and 88.9 ± 2.20 percent growth inhibition, respectively, of epimastigote forms of Trypanosoma cruzi, whereas N. falcifolia demonstrated an LD50 of 138.5 æg/mL against promastigote forms of Leishmania (Viannia) braziliensis. Regarding molluscicidal activity, the acute toxicity of the extracts on Biomphalaria glabrata was evaluated by a rapid screening procedure. M. arenosa was 100 percent lethal to snails at 200 æg/mL and showed an LD50 of 143 æg/mL. Screening of plant extracts represents a continuous effort to find new antiparasitic drugs.

Antiprotozoal and molluscicidal activities of five Brazilian plants.

Leishmaniasis, Chagas' disease and schistosomiasis (bilharzia) are parasitic diseases with wide distribution on the American continent, affecting millions of people. In the present study, biological assays for antiprotozoal and molluscicidal activities were carried out with ethanolic extracts of plant species from the Brazilian part of the Upper Paraná River. Crude extracts were obtained by percolation with absolute ethanol from the leaves of Cayaponia podantha Cogn., Nectandra falcifolia (Nees) Castiglioni and Paullinia elegans Cambess., as well as from the aerial parts of Helicteres gardneriana St. Hil. & Naud. and Melochia arenosa Benth., all belonging to genera used in folk medicine. Trypanocidal activity of plants was assayed on epimastigote cultures in liver infusion tryptose. Anti-leishmanial activity was determined over cultures of promastigote forms of the parasite in Schneider's Drosophila medium. Microscopic countings of parasites, after their incubation in the presence of different concentrations of the crude extracts, were made in order to determine the percentage of growth inhibition. C. podantha and M. arenosa, at a concentration of 10 microg/mL, showed 90.4 +/- 11.52 and 88.9 +/- 2.20% growth inhibition, respectively, of epimastigote forms of Trypanosoma cruzi, whereas N. falcifolia demonstrated an LD50 of 138.5 microg/mL against promastigote forms of Leishmania (Viannia) braziliensis. Regarding molluscicidal activity, the acute toxicity of the extracts on Biomphalaria glabrata was evaluated by a rapid screening procedure. M. arenosa was 100% lethal to snails at 200 microg/mL and showed an LD50 of 143 microg/mL. Screening of plant extracts represents a continuous effort to find new antiparasitic drugs.

Neolignanas e análise do óleo essencial das folhas de Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck / Neolignans and the analysis of the essential oil of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck leaves

O óleo essencial das folhas de Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck Piparaceae, coletadas no Horto de Plantas Medicinais da Universidade Estadual de Maringá, foi obtido por hidrodestilação. Uma análise preliminar por CG/EM e RMN 13C foi realizada. O b-mirceno (70 por cento) foi identificado como componente majoritário através da comparação dos espectros de massa e RMN 13C com dados da literatura. Quatro neolignanas foram isoladas do extrato hidroetanólico das folhas e identificadas: eupomatenóide-6, eupomatenóide-5, eupomatenóide-3 e conocarpano. As estruturas dessas substâncias foram estabelecidas por meio de estudos de RMN ¹H e 13C, ¹H x ¹H - COSY, HETCOR, HMBC, gNOE e EM.

The essential oil of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck Piparaceae leaves, which were collected at a tree farm named Horto de Plantas Medicinais of the Universidade Estadual de Maringá, was obtained by hydrodistillation. A preliminary analysis by GC/MS was carried out. b-mirceno (70 percent) was identified as the main constituent by comparing MS and 13C NMR with the literature data. Four neolignans were isolated from the leaves and identified: eupomatenoid-6, eupomatenoid-5, eupomatenoid-3 and conocarpan. Their structures were established by extensive ¹H and 13C NMR, ¹H x ¹H - COSY, HETCOR, HMBC, gNOE and MS spectral studies.

Evaluation of the antioedematogenic, free radical scavenging and antimicrobial activities of aerial parts of Tillandsiastreptocarpa Baker-Bromeliaceae.

The crude methanolic extract of the aerial parts of Tillandsiastreptocarpa was investigated for their acute toxicity and antioedematogenic, antioxidant and antimicrobial activities. Also, the antioedematogenic activity of the hexane fraction resulting from the partition of the crude methanolic extract was evaluated. The methanolic extract and the hexane fraction showed significant (P < 0.05) inhibition of ear oedema, observed at 2 mg/ear in the croton oil-induced mice ear oedema test. In the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging test, a high reactivity and potent antioxidant effect (IC(50) = 0.0056%, w/v) were observed for the methanolic extract. The antimicrobial activity assay showed that the crude methanolic extract was inactive toward Escherichiacoli, Staphylococcusaureus, Pseudomonasaeruginosa, Bacillussubtilis, Candidaalbicans, C. parapsilosis, C. krusei and C. tropicalis (MIC > 500 microg/ml). The methanolic extract showed no toxic effect on mice at a single dose of 2000 mg/kg (p.o). Common side effects including mild diarrhoea, loss of weight and depression were not recorded. The compounds cycloartenol, 4',5-dihydroxy-3',7-dimethoxyflavanone and a mixture of the steroids stigmasterol, beta-sitosterol and campesterol, were isolated from the hexane fraction and identified by spectroscopic methods.

Avaliação da atividade antibacteriana e antifúngica de extratos de plantas utilizados na medicina popular

Foram selecionados extratos de 13 plantas utilizadas na medicina popular brasileira para avaliar a atividade antimicrobiana. Destes, 10 extratos apresentaram níveis variados de atividade antibacteriana. Cinco dos extratos testados, apresentaram compostos com valores de Rf similares a de compostos antibacterianos visíveis na bioautografia. Três destas plantas pertencem à família Compositae indicando que o mesmo composto pode ser responsável pela atividade antibacteriana destas plantas. Atividade anticandida foi observada em 9 extratos de plantas. Os resultados podem explicar o uso etnobotânico das espécies estudadas para o tratamento de várias doenças infecciosas.

Extracts of 13 Brazilian medicinal plants were screened for their antimicrobial activity against bacteria and yeast. Of these, 10 plant extracts showed varied levels of antibacterial activity. Five of the plant extracts presented compounds with Rf values similar to the antibacterial compounds visible on bioautogram. Of these, three plants belong to the Compositae family. This may mean that the same compounds are responsible for the antibacterial activity in these plants. Anticandidal activity was detected in 9 plant extracts. The results might explain the ethnobotanical use of the studied species for the treatment of various infectious diseases.

Antibacterial activity of Ocimum gratissimum L. essential oil.

The essential oil (EO) of Ocimum gratissimum inhibited Staphylococcus aureus at a concentration of 0.75 mg/ml. The minimal inhibitory concentrations (MICs) for Shigella flexineri, Salmonella enteritidis, Escherichia coli, Klebsiella sp., and Proteus mirabilis were at concentrations ranging from 3 to 12 microg/ml. The endpoint was not reached for Pseudomonas aeruginosa (>=24 mg/ml). The MICs of the reference drugs used in this study were similar to those presented in other reports. The minimum bactericidal concentration of EO was within a twofold dilution of the MIC for this organism. The compound that showed antibacterial activity in the EO of O. gratissimum was identified as eugenol and structural findings were further supported by gas chromatography/mass spectra retention time data. The structure was supported by spectroscopic methods.