In vitro antiproliferative activity of alkaloids isolated from Tabernaemontana catharinensis A.DC (Apocynaceae).

In this work, four alkaloids from the stem bark of T. catharinensis were isolated, namely: voacangine (1); ethyl apovincaminate (2); affinisine (3) and voachalotine (4). The alkaloids were tested in vitro for antiproliferative capacity in eight tumor cell lines: U251 (glioma), MCF-7 (breast), NCI-ADR/RES (drug resistant ovary), 786-0 (kidney), NCI-H460 (lung), HT-29 (colon), K562 (leukemia) and PC-3 (prostate) and a non-tumor keratinocyte cell line (HaCat). Antiproliferative activity was observed after 48 hours and results expressed as the concentration needed to induce 50% growth inhibition (GI50) in µM. The chemotherapy drug Doxorubicin was used as a standard. The alkaloid affinisine (3) was the most promising, showing moderate inhibition rates in addition to the cytotoxic and cytocidal effect against all strains tested. It also proved to be a very promising compound, showing high selectivity rates when compared to the non-tumor keratinocyte cell line (HaCat).

Antifungal and antibacterial activity of extracts produced from Diaporthe schini.

The aim of this work was the production of bioactive metabolites by submerged fermentation from the fungus Diaporthe schini, followed by their extraction, separation and characterization. Different solvents (methanol, dichloromethane and hexane) were used for the extraction of metabolites from the fermentation broth and the extracts obtained were evaluated by in vitro antibacterial and antifungal activity. The separation and characterization of the extract from the hexane extraction was performed by column chromatography and GC-MS, respectively. The extracts had a great inhibitory action on the Gram-positive bacteria Staphylococcus epidermidis and Staphylococcus aureus, on the Gram-negative bacteria Enterobacter aerogenes and Klebsiella pneumoniae and on the fungus Candida krusei. The main metabolites produced were: 13-docosenamide, (Z)-; 2-hexadecene, 3,7,11,15-tetramethyl; 9-octadecenamide and 11-octadecenoic acid. Studies related to the antibacterial and antifungal activities of metabolites extracted from microorganisms are found in the literature. However, works about the identification of metabolites produced by submerged fermentation from Diaporthe schini were not found until the present moment. This work is an initial study where the conditions of the process can be optimized by looking for the production of a specific compound and can be a promising source for obtaining new drugs.

Antibacterial activity of Discaria americana Gillies ex Hook (Rhamnaceae).

ETHNOPHARMACOLOGY RELEVANCE: Discaria americana Gillies ex Hook (sin. Discaria febrifuga and Discaria longispina) (Rhamnaceae) is a plant native from Rio Grande do Sul (Southern Brazil), Uruguay and Argentine, and has been used in Brazilian traditional medicine as antipyretic agent, and for stomach disorders. In Rio Grande do Sul, Uruguay and Argentine, the roots, in decoction, are used as tonic and febrifuge. Although it is a plant widely used by the population, there are no studies proving this popular use. MATERIAL AND METHODS: The crude neutral methanol extract, and pure isolated alkaloids, were investigated in vitro for antimicrobial activities against four Gram-positive bacteria: Staphylococcus aureus, Bacillus subtillis, Bacillus cereus, Enterococcus faecium; and five Gram-negative bacteria: Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa. RESULTS: The crude neutral methanol (CME) extract of the root bark of Discaria americana showed antibacterial activity, ranging from 62.5 to 250 µg mL-1 (MIC), against the tested bacteria. From the fractions obtained from the crude extract, the basic ethereal fraction (BEF) showed to be more effective, with MICs between 31.5 and 125 µg mL-1 against the tested bacteria. The bioassay-guided fractionation of the ethyl ether basic fraction yielded eight cyclopeptide alkaloids: frangufoline (1), frangulanine (2), adouetine Y' (3), discarine A (4) discarine B (5), discarine C (6), discarene C (7) and discarine D (8). When evaluated against the Gram-positive bacteria Enterococcus faecium, discarine B (5) proved to be the most active alkaloid with a MIC/MLC = 0.77/1.55 µg mL-1, near the most active antibacterial agent levofloxacin (MIC/MLC = 0.77/0.77 µg mL-1). Moreover, discarine C (6) was the more active alkaloid against Salmonella enterica serovar Typhimurium, with a MIC/MLC = 3.1/6.2 µg mL-1, the same observed for the antibacterial agent azithromycin. Kinetic measurements of the bacteriolytic activities of discarine B (5) against Enterococcus faecium (Gram-positive), and of discarine C (6) against Salmonella enterica serovar Typhimurium (Gram-negative) were determined by optical density based on real time assay, suggesting that both mode of action are partially bacteriolytic. CONCLUSION: In conclusion, five 14-membered cyclopeptide alkaloids isolated from Discaria americana Gillies ex Hook (Rhamnaceae) showed promising antibacterial activity, making this metabolites a class of scientific interest. The good activity presented by the extract and the alkaloids against the Gram-positive bacteria Enterococcus faecium and against the Gram-negative bacteria Salmonella enterica serovar Typhimurium, Enterobacter. aerogenes and Escherichia coli, corroborate with the popular use of this plant for stomach disorders and as antifebrile.

Root bark of Discaria americana attenuates pain: A pharmacological evidence of interaction with opioidergic system and TRP/ASIC channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Discaria americana (Rhamnaceae) root bark infusion have been used in traditional medicine as antipyretic, tonic, ameliorative of stomach and skin diseases and diabetes. This study was designed to investigate whether the methanolic extract of the root bark of Discaria americana (MEDa) exhibits antinociceptive effects in mice. Furthermore, it was investigated the involvement of the opioidergic system in MEDa mechanism of action as well the interactions with TRP/ASIC channels in its effect. MATERIALS AND METHODS: The antinociceptive effect of intra-gastric gavage (i.g.) of MEDa (0.3-300 mg/kg) was evaluated in mice subjected to acute chemical (acetic-acid, formalin, glutamate, capsaicin, cinnamaldehyde, and acidified saline) or thermal (hot plate) tests of pain. The involvement of opioid system was evaluated in the formalin test. A nonspecific effect of MEDa was observed by measuring locomotor activity and exploratory behavior in open field test. RESULTS: MEDa significantly reduced the number of writhing induced by acetic acid and inhibited the nociception in the two phases of formalin. These effects were inhibited by pretreatment with naloxone. The nociception induced by hot plate and intraplantar injection of glutamate, capsaicin, cinnamaldehyde and acidified saline were significantly inhibited by MEDa. Only the dose of 300 mg/kg altered the locomotor activity. CONCLUSIONS: Our results demonstrated, for the first time, that the methanolic extract of the root bark of Discaria americana presents antinociceptive effect in chemical and thermal stimuli and its analgesic properties can be due activation of the opioidergic system. These results support the use of Discaria americana in traditional medicine and demonstrate that this plant presents a therapeutic potential for the development of phytomedicines with antinociceptive profile.

In vitro effects of Blepharocalyx salicifolius (H.B.K.) O. Berg on the viability of Echinococcus ortleppi protoscoleces.

Scolicidal agents are important in the treatment of cystic echinococcosis. This study evaluated the scolicidal activity of the plant Blepharocalyx salicifolius (H.B.K.) Berg against Echinococcus ortleppi protoscoleces. The parasite species was identified by amplifying a fragment of the gene cytochrome c oxidase subunit 1 (COX 1). B. salicifolius crude extract at concentrations of 100, 200, 300 and 400 mg/mL was analyzed at different times (5, 10, 15, 30, 45 and 60 min). N-butanol and ethyl acetate fractions (100 and 200 mg/ mL) were also analyzed at 5, 10, 15 and 30 min. Both fractions showed 100% scolicidal activity at the concentration of 200 mg/mL at 5 min. Gallic acid, identified as the major compound of the ethyl acetate fraction- was responsible for the observed scolicidal activity. The results showed that crude extract and fractions of B. salicifolius have scolicidal effect against E. ortleppi protoscoleces.

Oral treatment with methanolic extract of the root bark of Condalia buxifolia Reissek alleviates acute pain and inflammation in mice: Potential interactions with PGE2, TRPV1/ASIC and PKA signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: The Condalia buxifolia root bark infusion is used in traditional medicine in Brazil as antipyretic, anti-inflammatory and anti-dysentery. Previous data from our group showed that methanolic extract of Condalia buxifolia (MECb) produced a marked antinociceptive effect in animal models of acute pain. The purpose of this study was to investigate the mechanisms of MECb-induced antinociception as measured by nocifensive behavior in pain induced by endogenous (prostaglandin E2) or exogenous (TRPs and ASIC agonist, and protein kinase A and C activators) chemical stimuli, and the potential role of PKA signaling and capsaicin-sensitive central C-fiber afferents. MATERIALS AND METHODS: The effect of MECb administered orally (0.1-300mg/kg, i.g.) to mice on nociception induced by capsaicin (TRPV1 agonist), cinnamaldehyde (TRPA1 agonist), menthol (TRPM8 agonist), acidified saline (ASIC agonist), PMA (protein kinase C activator), PGE2 and forskolin (protein kinase A activator) was assessed. Moreover, this study also investigated the role of C-fibers desensitizing mice with a high dose of intrathecal capsaicin. Furthermore, this study performed the western blot to PKA phosphorylated on nocifensive behavior induced by forskolin. RESULTS: MECb was able to reduce the nociception and paw edema induced by capsaicin, acidified saline, PMA, PGE2 and forskolin, but not by cinnamaldehyde or menthol. Western blot analyses showed that MECb reduced the levels of PKA phosphorylation induced by forskolin in hind paws. Finally, ablating central afferent C-fibers abolished MECb antinociception. CONCLUSION: In accordance with its use in traditional medicine, these findings provide new evidence indicating that Condalia buxifolia reduces the acute painful behavior of animals caused by chemical stimuli. The precise mechanism of MECb antinociceptive activity is not completely understood but the results suggest involvement of PGE2, TRPV1/ASIC and PKA signaling pathways, and require integrity of the capsaicin-sensitive central C-fiber afferents.

The Pavonia xanthogloea (Ekman, Malvaceae): Phenolic compounds quantification, anti-oxidant and cytotoxic effect on human lymphocytes cells.

INTRODUCTION: Pavonia xanthogloea is traditionally used as an antimicrobial and anti-tumour medicine in Southern Brazilian region. However, investigations about this species are still incipient. HYPOTHESIS TESTED: The study postulated that P. xanthologea specie present some phenolic compound and present some biological properties as anti-oxidant and cytoprotective effect against oxidative stress. MATERIALS AND METHODS: The content of eight phenolic molecules in the crude ethanolic extract of the aerial part of P. xanthogloea and its five fractions (hexane, dichloromethane, ethyl-acetate, n-butanol, and water) was determined by heterotrophic plate count method. The anti-oxidant capacity of the extract and the fractions was determined by 1,1-diphenyl-2-picryl-hydrazyl assay. The potential anti-oxidant and cytoprotective effect was also analyzed in human lymphocyte culture treated with extract/fractions at different concentrations with and without oxidative stress generated by hydrogen peroxide (H2O2) and sodium nitroprusside (SNP) exposition. RESULTS: Tiliroside was the molecule detected in all extract. Water and ethyl-acetate fractions showed the highest radical-scavenging activity. The crude extract, hexane, water, and n-butanol reversed the higher reactive oxygen specie levels generated by H2O2 and SNP to levels similar to those observed in the control group. In addition, crude extract, hexane, ethyl-acetate and n-butanol did not caused cytotoxicity, whereas water fraction was cytotoxic at higher concentration tested here (300 µg/mL). The cytotoxicity reversion caused by SNP exposition was concentration-dependent of the extract and fractions. However, dichloromethane fraction increased cell mortality in all concentrations investigated and was not able to decrease cell death in the lymphocytes exposed to SNP. CONCLUSION: The results suggest potential medicine use of this species.

The in vitro genotoxic effect of Tucuma (Astrocaryum aculeatum), an Amazonian fruit rich in carotenoids.

Tucuma (Astrocaryum aculeatum) is an Amazonian fruit that presents high levels of carotenoids and other bioactive compounds such as quercetin. The extracts of tucuma peel and pulp present strong antioxidant activity which illustrate an elevated concentration that causes cytotoxic effects in human peripheral blood mononuclear cells (PBMCs). This study performed additional investigations to analyze the potential genotoxic effects of the tucuma extracts on PBMCs. The genotoxicity was evaluated by DNA fragmentation, Comet assay, and chromosomal instability G-band assays. The acute tucuma extract treatment showed genoprotective effects against DNA denaturation when compared with untreated PBMC cells. However, in the experiments with 24 and 72 h treatments to tucuma treatments, we observed low genotoxicity through a concentration of 100 µg/mL, some genotoxic effects related to intermediary concentrations (100-500 µg/mL), and more pronounced genotoxic effects on higher tucuma extract concentrations. After 24 h of treatment, the reactive oxygen species were similar among treatments and PBMC control groups. However, the caspase-1 activity related to the apoptosis and pyroptosis process increased significantly in higher tucuma concentrations. In summary, tucuma extracts, despite their higher antioxidant content and antioxidant activity, would present PBMCs genotoxic effects that are dependent on concentration and time exposition. These results need to be considered in future in vitro and in vivo studies of tucuma effects.

Efficacy of eugenol and the methanolic extract of condalia buxifolia during the transport of the silver catfish Rhamdia quelen

This study evaluated extracts of Condalia buxifolia as anesthetics for the silver catfish Rhamdia quelen. The effectiveness of eugenol and of the methanolic extract (ME) of C. buxifolia during the transport of this species was also assessed. Fish of two different weights (1.50±0.02 g and 165.70±22.50 g) were transferred to aquaria containing water with the C. buxifolia ME or with fractions obtained from the ME, such as the n-hexane, dichloromethane, ethyl acetate, n-butane and aqueous fractions, at concentrations from 0-300 °L L-1. The C. buxifolia ME in the 0.5-120 °L L-1 range caused only light sedation, and the fractions did not have an effect on the fish. In the second experiment, another group of fish was transported for 12 h in 15 plastic bags. The fish were divided into five groups: control, 1 or 2.5 °L L-1 eugenol and 25 or 50 °L L-1 C. buxifolia ME. The non-ionized ammonia levels were lower at the end of transport in the groups with the compounds than in that with water alone. Moreover, both compounds decreased the Na+, Cl-, and K+ net effluxes; therefore, their addition to the water during transport is advisable because they reduce fish mortality and ion loss.

Este estudo investigou extratos de Condalia buxifolia como anestésico para jundiá Rhamdia quelen, e também a eficiência do eugenol e do extrato metanólico (EM) de C. buxifolia para utilização durante o transporte dessa espécie. Peixes de dois diferentes pesos (1,50±0,02 g e 165,70±22,50 g) foram transferidos para aquários contendo água com o EM de C. buxifolia ou frações obtidas a partir do EM (n-hexano, acetato de diclorometano, etil n- butano e aquoso, em concentrações na faixa de 0 - 300 °L L-1. O EM de C. buxifolia em concentrações na faixa de 0,5 - 120 °L L-1 causou somente uma sedação leve e as frações não tiveram efeito. No segundo experimento outro grupo de peixes foi transportado por 12 h em 15 sacos plásticos divididos em cinco tratamentos: controle, 1 ou 2,5 °L L-1 de eugenol e 25 ou 50 °L L-1 de EM de C. buxifolia. Os níveis de amônia não-ionizada foram menores nos tratamentos com ambos compostos em relação à água (controle). Além disso, ambos compostos diminuíram os efluxos líquidos de Na+, Cl- e K+ e, portanto, sua adição na água de transporte é aconselhável, pois reduzem a mortalidade e a perda de íons dos peixes.

Estudo morfoanatômico das folhas de Cuphea glutinosa Cham. & Schltdl., Lythraceae / Morpho-anatomical study of the leaves of Cuphea glutinosa Cham. et Schltdl. (Lythraceae)

Adeterminação de características macro e microscópicas das folhas de Cuphea glutinosa Cham. & Schltdl., Lythraceae, foi o objetivo deste trabalho. Esta planta é utilizada tradicionalmente como medicinal. As características macroscópicas das folhas como a forma, a textura e a consistência, bem como o ápice e a base da lâmina foliar são relevantes na determinação da autenticidade de insumos farmacêuticos desta espécie. Microscopicamente a presença de formações epicuticulares, cristais de oxalato de cálcio na forma de drusas, tricomas tectores e glandulares, estômatos do tipo diacítico, células mucilaginosas e floema interxilemático são características úteis no controle botânico de qualidade desta planta como insumo farmacêutico, quando analisados em conjunto.

The determination of macroscopic and microscopic characteristics of Cuphea glutinosa Cham. & Schltdl., Lythraceae, leaves were the aim of this work. This plant is traditionally used as medicinal. The macroscopic characteristics of the leaves as the shape, texture and consistency as well as the apex and the base of the leaf are relevant to determinate pharmaceutical insumes autenticity of this plant. In the leaves microscopy there are epicuticular formations, crystals of calcium oxalate in the form of druses, and glandular trichomes, xerophytytic stoma of the diacytic type, mucilaginous cells, and interxilematic phloem are important characteristics in the botanical quality control of this plant as pharmaceutical insume.

Morfonatomia do caule de Cuphea glutinosa Cham. & Schltdl. (Lythraceae) / Morpho-anatomy of the stem of Cuphea glutinosa Cham & Schltdl. (Lythraceae)

Foram determinados parâmetros morfo-anatômicos do caule de Cuphea glutinosa cham. & Schltdl, planta reconhecida tradicionalmente como medicinal, objetivando sua autenticidade como insumo farmacêutico. As características macroscópicas do caule são insuficientes para a determinação da autenticidade dos insumos farmacêuticos desta planta, havendo necessidade de verificar a presença de sementes não aladas e de vesículas infraestaminais nas flores. Microscopicamente a presença de formações epicuticulares, cristais de oxalato de cálcio na forma de drusas, tricomas tectores e glandulares, estômatos xerofíticos, células mucilaginosas e floema interxilemático, são características significativas no controle botânico de qualidade desta espécie para a indústria farmacêutica, quando analisadas em conjunto.

Morpho-anatomical parameters of Cuphea glutinosa cham. & Schltdl, stem were determined aiming its authenticity as a pharmaceutical drug. This plant is traditionally known as medicinal. The macroscopic characteristics of the stem are insuficient to determine the autenticity of this plant pharmaceutics input, it requires checking out the presence of not winged seeds and vesicles placed under the stamens of the flowers. Microscopically, the presence of epicuticular formations, crystals of calcium oxalato in druses form, glandular and non-glandular thricomes, xerophytic stomatas, mucilages cells and interxilematic phloem are meaningful characteristics in the botanical quality control of this plant for the pharmaceutical industry, when analyzed together.

Antinociceptive effects of 14-membered cyclopeptide alkaloids.

The analgesic potential of six 14-membered-ring cyclopeptide alkaloids, namely, franganine (1), discarine B (2), scutianines B (3), C (4), and D (5), and adouetine X (6), have been investigated. Among the compounds tested, only franganine (1) and adouetine X (6) produced antinociceptive effects in a mouse model of acute pain, without inducing undesirable side effects. Furthermore, compound 6 also exhibited a pronounced analgesic effect in a chronic neuropathic pain model in mice. It has been found that adouetine X (6) can decrease the activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase in vitro. Thus, the present findings have demonstrated that adouetine X (6) is a promising analgesic agent.

Antioxidant effects of different extracts from Melissa officinalis, Matricaria recutita and Cymbopogon citratus.

Considering the important role of oxidative stress in the pathogenesis of several neurological diseases, and the growing evidence of the presence of compounds with antioxidant properties in the plant extracts, the aim of the present study was to investigate the antioxidant capacity of three plants used in Brazil to treat neurological disorders: Melissa officinalis, Matricaria recutita and Cymbopogon citratus. The antioxidant effect of phenolic compounds commonly found in plant extracts, namely, quercetin, gallic acid, quercitrin and rutin was also examined for comparative purposes. Cerebral lipid peroxidation (assessed by TBARS) was induced by iron sulfate (10 microM), sodium nitroprusside (5 microM) or 3-nitropropionic acid (2 mM). Free radical scavenger properties and the chemical composition of plant extracts were assessed by 1'-1' Diphenyl-2' picrylhydrazyl (DPPH) method and by Thin Layer Chromatography (TLC), respectively. M. officinalis aqueous extract caused the highest decrease in TBARS production induced by all tested pro-oxidants. In the DPPH assay, M. officinalis presented also the best antioxidant effect, but, in this case, the antioxidant potencies were similar for the aqueous, methanolic and ethanolic extracts. Among the purified compounds, quercetin had the highest antioxidant activity followed by gallic acid, quercitrin and rutin. In this work, we have demonstrated that the plant extracts could protect against oxidative damage induced by various pro-oxidant agents that induce lipid peroxidation by different process. Thus, plant extracts could inhibit the generation of early chemical reactive species that subsequently initiate lipid peroxidation or, alternatively, they could block a common final pathway in the process of polyunsaturated fatty acids peroxidation. Our study indicates that M. officinalis could be considered an effective agent in the prevention of various neurological diseases associated with oxidative stress.

Óleo de citronela no controle do carrapato de bovinos / Citronella oil on the control of catle ticks

O objetivo deste estudo foi avaliar o efeito in vivo e in vitro do óleo de citronela (Cymbopogon nardus (L.) Rendle) no carrapato de bovinos (Boophilus microplus). O óleo foi obtido de folhas frescas pelo processo de destilação e o rendimento foi de 0,7 por cento. Para as experimentações in vitro foram usadas sete (0; 0,5; 1,0; 10,0; 25,0; 50,0; 100,0 por cento) e nove (0,1; 0,25; 0,5; 1,0; 2,0; 10,0; 25,0; 50,0; 100 por cento) concentrações de óleo de citronela, sendo testadas em fêmeas ingurgitadas. A eficácia observada foi de 0; 44,2; 92,1; 85,6; 87,8; 87,0; 88,9 e de 0,7; 2,8; 51,6; 79,3; 81,0; 87,1; 86,7; 89,5 por cento, respectivamente. Os resultados sugerem que o óleo de citronela pode ser usado no controle do carrapato bovino.

This research was aimed at evaluating in vitro and in vivo effects of citronella (Cymbopogon nardus (L.) Rendle) oil on catle ticks. The volatile oil was obtained from de fresh leaves of citronella grass by steam destilation and the yelds was 0.7 percent. Seven (0; 0.5; 1.0; 10.0; 25.0; 50.0; 100.0 percent) and nine (0.1; 0.25; 0.5; 1.0; 2.0; 10.0; 25.0; 50.0; 100.0 percent) concentrations of citonella oil were used on in vitro trials with engorged female ticks. The efficacy control ticks was 0; 44.2; 92.1; 85.6; 87.8; 87.0; 88.9 and 0; 0.7; 2.8; 51.6; 79.3; 81.0; 87.1; 86.7 89.5 percent, respectively. These sugest that the citronella oil can be practical used for controlling of catle ticks.

Composition and antibacterial activity of the essential oils from Zanthoxylum rhoifolium.

The essential oils from the aerial parts of leaves, fruits and flowers of Zanthoxylum rhoifolium of Southern Brazil (Rio Grande do Sul), were analysed by GC, GC/MS, and chiral phase gas chromatography (CPGC). Forty-eight compounds were identified from the essential oils. The major constituents of the essential oil of the leaves were germacrene D (34 %) and bicyclogermacrene (23 %) and of the fruits, menth-2-en-1-ol (46.2 %), beta-myrcene (30.2 %), (-)-linalool (15 %) and (-)-alpha-terpineol (8.45 %). beta-Myrcene (65 %) and menth-2-en-1-ol (5.4 %) dominate the essential oil of the flowers. The oils of the leaves and fruits were bioactive with antibacterial activity against Staphylococcus aureus (Gram positive), and Klebsiella pneumoniae and Salmonella setubal bacteria (Gram negative) microorganisms, while the essential oil of the flowers was inactive.

Diterpenos isolados de Humirianthera ampla. Miers.

From Humirianthera ampla, Icacinaceae, were isolated a phthalate, lupeol, â-sitosterol, glycosyl- sitosterol, one known annonalide diterpene and two new diterpenes named Humirianthol and Acrenol. Humirianthol and Acrenol were determined by 1D and 2D NMR spectroscopic techniques to be 3 â, 20:14 â, 16-diepoxy-3 á, 15 á-dihydroxy-7-pimaren-19, 6 â-olide and 3 â, 20-epoxi-3 á, 15, 16-trihydroxy-7-pimaren-19, 6 â-olide, respectively. Acrenol has antimicrobial activity.