Antitumor effect of depsidones from lichens on tumor cell lines and experimental murine melanoma

Abstract Lichens have exhibited numerous biological activities, including growth inhibition of tumor cells. This study evaluated the antiproliferative activity of hypostictic and salazinic acids against tumor cell lines (B16-F10, PC-03, MCF7, HT-29, HEP-G2, K562 and 786-0) by the SRB assay in vitro and antitumor activity in experimental murine melanoma in vivo. Activation of caspase-3 was quantified by flow cytometry. The murine experimental melanoma model B16-F10 was used in BALB/c mice for evaluation of antitumor activity. Hypostictic acid showed significant antiproliferative activity in K562 cells (GI50 2.20 µM), B16-F10 (GI50 13.78 µM) and 786-0 (GI50 14.24 µM), whereas salazinic acid was more active against K562 cells (GI50 64.36 µM), HT-29 (GI50 67.91 µM) and B16-F10 (GI5078.64 µM). Quantification of capase-3 revealed that the test compounds did not increase the expression of that enzyme. In the in vivo antitumor evaluation in B16-F10 melanoma, the isolated compounds inhibited tumor growth in relation to weight and volume. Hypostictic acid (16.7 mg/kg) inhibited 72% and salazinic acid 88% of tumor volume (p < 0.05). The results indicated that, both in the in vitro and in vivo models, the compounds evaluated showed antiproliferative and antitumor activities.

Inhibition of Mushroom Tyrosinase Activity by Orsellinates.

Several applications have been proposed for tyrosinase inhibitors in the pharmaceutical, food bioprocessing, and environmental industries. However, only a few compounds are known to serve as effective tyrosinase inhibitors. This study evaluated the tyrosinase-related activity of resorcinol (1), orcinol (2) lecanoric acid (3), and derivatives of this acid (4-15). Subjected to alcoholysis, lecanoric acid (3), a depside isolated from the lichen Parmotrema tinctorum, produces orsellinic acid (2,4-dihydroxy-6-methylbenzoic acid) (4) and orsellinates (2,4-dihydroxy-6-methyl benzoates) (5-15). At 0.50 mM, methyl (5), ethyl (6), n-propyl (7), tert-butyl (11), and n-cetyl orsellinates (15) acted as tyrosinase activators, whereas n-butyl (8), iso-propyl (9), sec-butyl (10), n-pentyl (12), n-hexyl (13), and n-octyl orsellinates (14) behaved as inhibitors. Tyrosinase inhibition rose with chain elongation-n-butyl (8)

Phytotoxicity of Perlatolic Acid and Derivatives

ABSTRACT Perlatolic acid (1), methyl perlatolate (2), and the products of perlatolic acid alcoholysis-namely, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl 2-hydroxy-4-methoxy-6-n-pentylbenzoate (3-11)-were evaluated for their herbicidal potential on Lactuca sativa L. (lettuce) and Allium cepa L. (onion) seeds. The compounds exhibited low phytotoxicity on L. sativa germination. Perlatolic acid (1) proved the most active compound (20%). Activity was lowest for n-hexyl 2-hydroxy-4-methoxy-6-n-pentylbenzoate (11) (3.5%). The esters iso-propyl and sec-butyl 2-hydroxy-4-methoxy-6-n-pentylbenzoate (6 and 8, respectively) inhibited root (35% e 43%, respectively) and hypocotyl growth (59% e 56%, respectively). The esters n-butyl, n-pentyl, and n-hexyl 2-hydroxy-4-methoxy-6-n-pentylbenzoate (7, 10, and 11, respectively) proved phytotoxic to A. cepa, delaying and reducing seed germination (27%), while n-pentyl 2-hydroxy-4-methoxy-6-n-pentylbenzoate (10) was the most inhibitory for root (42%) and coleoptile growth (24%). The behaviors of iso-propyl, sec-butyl, and n-pentyl 2-hydroxy-4-methoxy-6-n-pentylbenzoate (6, 8, and 10, respectively) suggest the potential utility of these esters as natural herbicides. The esters iso-propyl and sec-butyl 2-hydroxy-4-methoxy-6-n-pentylbenzoate (6 and 8, respectively) may serve as model molecules in the investigation of potential herbicides for dicotyledon control, while n-pentyl 2-hydroxy-4-methoxy-6-n-pentylbenzoate (10) may serve the same function for monocotyledon species.

Increment of antimycobaterial activity on lichexanthone derivatives.

A new dihydropyranexanthone derived from the natural xanthone lichexanthone (1) was synthesised and, together with other 18 derivatives including ω-bromo and ω-aminoalkoxylxanthones (containing methyl, ethyl, propyl, tertbutylamino and piperidinyl moieties), were tested against Mycobacterium tuberculosis. Nine ω-aminoalkoxylxanthones showed good antimycobacterial activity, and their in vitro cytotoxicity was determined using VERO cells in order to calculate the selectivity index (SI). One of these nitrogenated xanthone derivatives showed very promising results, with MIC of 2.6 µM and SI of 48. This MIC is comparable to values found in "first and second line" drugs commonly used to treat TB. In order to understand better about this compound, it was evaluated together with two other ones that showed good SI, against resistant clinical strains of M. tuberculosis to verify the existence of cross-resistance. A chemometrical approach was useful to establish a pattern of antitubercular activity among the group of ω-aminoalkoxylxanthones, according to some structural and chemical features.

Cytotoxic evaluation of phenolic compounds from lichens against melanoma cells.

Atranorin, lichexanthone, and the (+)-usnic, diffractaic, divaricatic, perlatolic, psoromic, protocetraric, and norstictic acids isolated from the lichens Parmotrema dilatatum (VAIN.) HALE, Usnea subcavata MOTYKA, Usnea sp., Ramalina sp., Cladina confusa (SANT.) FOLMM. & AHTI, Dirinaria aspera HÄSÄNEN, and Parmotrema lichexanthonicum ELIASARO & ADLER were evaluated against UACC-62 and B16-F10 melanoma cells and 3T3 normal cells. Sulforhodamine B assay revealed significant cytotoxic activity in protocetraric, divaricatic, and perlatolic acids on UACC-62 cells (50% growth inhibitory concentration (GI(50)) 0.52, 2.7, and 3.3 µg/mL, respectively). Divaricatic and perlatolic acids proved the most active on B16-F10 cells (GI(50) 4.4, 18.0 µg/mL, respectively) and the most cytotoxic to 3T3 normal cells. Diffractaic, usnic, norstictic, and psoromic acids were cytotoxic to UACC-62 cells in the 24.7 to 36.6 µg/mL range, as were protocetraric and diffractaic acids to B16-F10 cells (GI(50) 24.0, 25.4 µg/mL, respectively). Protocetraric acid was highly selective (selectivity index (SI*) 93.3) against UACC-62 cells, followed by norstictic, perlatolic, psoromic, and divaricatic acids, while norstictic and divaricatic acids were more selective against B16-F10 cells. The high SI* value obtained for protocetraric acid on UACC-62 cells makes it a potential candidate for the study of melanomas in experimental models. Chemometric analysis was performed to evaluate the general behavior of the compounds against the cell lines tested.

Phenolic compounds and antioxidant, antimicrobial and antimycobacterial activities of Serjania erecta Radlk. (Sapindaceae)

Serjania erecta Radlk.(Sapindaceae) is a medicinal plant traditionally used in Brazil. We assayed the ethanolic extract of leaves and roots against seven microorganisms. The REMA (Resazurin Microtiter Assay) assay was used to measure the biological activity in vitro against Mycobacterium tuberculosis and our results showed moderate activity of the ethanolic extract. On the other hand S. aureus, P. aeruginosa, S. setubal, C. albicans, S. cerevisiae and E. coli revealed that the leaves and roots of S. erecta inhibited the growth of all microorganisms. The ethanolic extracts of leaves and roots showed low values of antioxidant activities. The ethanolic extracts of leaves and roots were analyzed by chromatographic and spectrometric methods. (-)-Epicatechin, kaempferol aglycone and five glycoside derivates were isolated: kaempferol-3-O-α-L-rhamnopyranoside, kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside from the roots and kaempferol, kaempferol 3,7-di-O-α-L-rhamnopyranoside, vitexin, isovitexin and (-)-epicatechin in the leaves. This is the first chemical study reported in the literature about this specie.

Serjania erecta Radlk. (Sapindaceae) é uma planta medicinal utilizada no Brasil. Analisamos o extrato etanólico de folhas e raízes para sete microorganismos. O REMA (Ensaio de Microtitulação Resazurina) foi utilizado para medir a atividade biológica in vitro contra o Mycobacterium tuberculosis e nossos resultados mostraram atividade moderada do extrato etanólico. Por outro lado, ensaios com S. aureus, P. aeruginosa, S. setubal, C. albicans, S. cerevisiae e E. coli revelaram que as folhas e raízes de S. erecta inibiram crescimento em todos os microorganismos. Os extratos etanólicos de folhas e raízes apresentaram valores baixos de atividade antioxidante. Os extratos etanólicos de folhas e raízes foram analisados por métodos cromatográficos e espectroscópicos. (-)-Epicatequina, canferol aglicona e cinco derivados glicosídicos foram isolados: canferol -3-O-α-L-ramnopiranosídeo, canferol-3-O-α-L-ramnopiranosil-(1→6)-β-D-glucopiranosídeo a partir das raízes e canferol, canferol 3,7-di-O-α-L-ramnopiranosídeo, vitexina, isovitexina e (-) epicatequina nas folhas. Este é o primeiro estudo químico na literatura sobre esta espécie.

Antimicrobial activity of the extracts and fractions of hexanic fruits of Campomanesia species (Myrtaceae).

The antimicrobial activity of the hexanic extracts and fractions of fruits of Campomanesia pubescens pubescens (D.C) O. Berg and Campomanesia adamantium (Cambess.) O. Berg were assayed against six microorganisms. The hexanic extracts of C. pubescens and C. adamantium fruits were also investigated in their composition by gas chromatography-mass spectrometry and high-performance liquid chromatography with diode array detection. The results of this study showed the presence of 34 volatile compounds and flavonoids in the hexanic extract of C. pubescens and 33 volatile compounds and flavonoids in the hexanic extract of C. adamantium.

Chemical composition and antioxidant and antimycobacterial activities of Bromelia balansae (Bromeliaceae).

Bromelia balansae (Family Bromeliaceae) is a medicinal plant commonly used in the central region of Brazil as a cough syrup and also eaten roasted. The methanolic extract of ripe fruits was analyzed by chromatographic methods and spectrometrically. Four glycoside flavonols were isolated: kaempferol-3-O-α-L-rhamnopyranoside (1), kaempferol-3-O-α-L-rhamnopyranosyl-(1-->6)-ß-D-glucopyranoside (2), quercetin-3-O-α-L-rhamnopyranosyl-(1-->6)-ß-d-glucopyranoside (3), and kaempferol 3,7-di-O-α-L-rhamnopyranoside (4). The resazurin microtiter assay was used to measure the biological activity in vitro against Mycobacterium tuberculosis. The results showed a moderate activity of the methanolic extract with a minimal inhibitory concentration of 128 µg/mL. Antioxidant activity was evaluated as free radical scavenging capacity and inhibition of peroxidation. Free radical scavenging capacity was assessed by measuring the scavenging activity of methanolic extract and methanolic fraction on 2,2-diphenyl-1-picrylhydrazyl radical. The methanolic extract showed low values of antioxidant activities, whereas the methanolic fraction exhibited free radical scavenging activity ranging from 20.2% to 91.1%, and the inhibition of peroxidation values ranging from 5.6% to 27.5%. This is the first chemical study reported in the literature about this species.

In vitro antitumour activity of orsellinates.

Lichen phenolic compounds exhibit antioxidant, antimicrobial, antiproliferative, and cytotoxic activities. The purpose of this study was to evaluate the anticancer activity of lecanoric acid, a secondary metabolite of the lichen Parmotrema tinctorum, and its derivatives, orsellinates, obtained by structural modification. A cytotoxicity assay was carried out in vitro with sulforhodamine B (SRB) using HEp-2 larynx carcinoma, MCF7 breast carcinoma, 786-0 kidney carcinoma, and B16-F10 murine melanoma cell lines, in addition to a normal (Vero) cell line in order to calculate the selectivity index of the compounds. n-Butyl orsellinate was the most active compound, with IC50 values (the concentration that inhibits 50% of growth) ranging from 7.2 to 14.0 microg/mL, against all the cell lines tested. The compound was more active (IC50 = 11.4 microg/mL) against B16-F10 cells than was cisplatin (12.5 microg/mL). Conversely, lecanoric acid and methyl orsellinate were less active against all cell lines, having an IC50 value higher than 50 microg/mL. Ethyl orsellinate was more active against HEp-2 than against MCF7, 786-0, or B16-F10 cells. The same pattern was observed for n-propyl and n-butyl orsellinates. n-Pentyl orsellinate was less active than n-propyl or n-butyl orsellinates against HEp-2 cells. The orsellinate activity increased with chain elongation (from methyl to n-butyl), a likely consequence of an increase in lipophilicity. The results revealed that the structural modification of lecanoric acid increases the cytotoxic activity of the derivatives tested.

Gas Chromatography-Mass Spectrometry (GC-MS) and evaluation of antioxidant and antimicrobial activities of essential oil of Campomanesia adamantium (Cambess.) O. Berg (Guavira) / Cromatografia Gasosa-Espectrometria de Massas (CG-EM) e avaliação da atividade antioxidante e antimicrobiana do óleo essencial de Campomanesia adamantium (Cambess.) O. Berg (guavira)

The essential oils from Campomanesia adamantium (Cambess.) O. Berg leaves, collected in the reproductive (flowering and fruit-bearing) and vegetative stages, were characterized by GC-MS (Gas Chromatography-Mass Spectrometry). A total of 95 compounds of the essential oils were identified. In the reproductive stage (flowering) the major constituents were monoterpenes (limonene, α-pinene and β-pinene) while during the vegetative stage the major constituents were the sesquiterpenes (bicyclogermacrene and globulol). The essential oil of the reproductive stage shows high antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, and all show moderate activity against Escherichia coli. The essential oils were also evaluated for their radical-scavenging activity by DPPH. The chemogeographical variations of the oil composition from the four distinct localities studied all contained α-pinene, β-pinene, limonene, linalool, β-caryophyllene, germacrene D and bicyclogermacrene, however the samples from Jardim city contained neither limonene nor linalool.

Os óleos essenciais obtidos das folhas de Campomanesia adamantium foram caracterizados através da combinação de CG-EM e índice de retenção, sendo identificado um total de 95 compostos. Na floração as substâncias majoritárias foram monoterpenos (limoneno, α-pineno e β-pineno) e durante o estágio vegetativo as substâncias majoritárias foram sesquiterpenos (biciclogermacreno e globulol). Os óleos essenciais obtidos da floração e frutificação mostraram alta atividade contra Staphylococcus aureus, Pseudomonas aeruginosa e Candida albicans e moderada contra Escherichia coli em todos os estágios. Foi avaliada a atividade antioxidante dos óleos essenciais usando o método do DPPH. O óleo essencial das 4 cidades mostrou a presença de α-pineno, β-pineno, limoneno, linalol, β-cariofileno, germacreno D e biciclogermacreno, mas a amostra da cidade de Jardim não apresentou limoneno e linalol.

Allelopathic potential of orsellinic acid derivatives

Environmental degradation caused by the excessive use of pesticides has motivated studies aimed at the discovery of new formulations for pest control. The bioassays carried out under laboratory conditions for the present study involved the germination and seedling growth of Lactuca sativa cv. Grand Rapids and Allium cepa cv. Baia Periforme treated with lecanoric acid or one of seven esters obtained from the alcoholysis of the acid. Each substance was tested at concentrations of 10-3, 10-5, 10-7 M, and 10-9 M. The results revealed that, except for lecanoric acid and methyl orsellinate, all other compounds exhibited significant differences in their action when compared with the controls. Also, A. cepa was more sensitive to the compounds assayed, at the concentrations tested. Nearly all the esters exhibited significant effects, a feature that could makes them promising for the development of new natural herbicides.

A degradação do meio ambiente ocasionada pelo uso excessivo de agrotóxicos impulsiona pesquisas que visam a descoberta de novos pesticidas. Os bioensaios conduzidos em condições de laboratório no presente estudo, envolveu a germinação e o crescimento de plântulas de Lactuca sativa cv. Grand Rapids e Allium cepa cv. Baia Periforme tratadas com ácidos lecanórico ou um dos sete ésteres obtidos pela alcoólise deste ácido. Cada substância foi ensaiada nas concentrações 10-3 M, 10-5 M, 10-7 M, e 10-9 M. Os resultados indicaram que exceto o ácido lecanórico e o orselinato de metila, todos os demais apresentaram diferenças significativas em relação ao controle. Além disso, observou-se que A. cepa foi mais sensível aos compostos ensaiados, nas quatro concentrações. Praticamente todos os ésteres avaliados apresentaram efeitos significativos, o que os torna compostos potenciais na descoberta de novos herbicidas naturais.

Gastroprotective effect of Serjania erecta Radlk (Sapindaceae): involvement of sensory neurons, endogenous nonprotein sulfhydryls, and nitric oxide.

The present study reveals the pharmacological action of Serjania erecta Radlk. (Family Sapindaceae), an important medicinal plant species used in the Brazilian Pantanal against gastric pain. The methanolic (Me) and chloroformic (Se) extracts obtained from leaves of S. erecta were challenged by a very strong necrotizing agent in rodents, absolute ethanol. Se was also confronted with a nitric oxide synthase inhibitor (N(G)-nitro-L-arginine methyl ester), a capsaicin cation channel transient receptor potential vanilloid type 1 antagonist (ruthenium red), or a sulfhydryl-blocker (N-ethylmaleimide) to evaluate the participation of these cytoprotective factors in gastroprotection. In an in vivo experimental model, Me and Se presented several degrees of gastroprotective action without signs of acute toxicity. The best gastroprotective effect was restricted to all doses of Se. The mechanisms involving the gastroprotective action of Se are related to an augmented defense mechanism of the gastrointestinal mucosa consisting of sensory neurons, nitric oxide, and sulfhydryl groups that prevent and attenuate the ulcer process. The presence of polyisoprenoids in the Se explains the potent gastroprotective action of this medicinal species. Effective gastroprotective action and the absence of acute toxicity indicate this species may be a promising herbal drug against gastric disease.

Radical-scavenging activity of orsellinates.

Lichens are an important source of phenolic compounds and have been intensively investigated for their biological and pharmacological activities. Lecanoric acid (1), a lichen depside, was isolated from a Parmotrema tinctorum specimen and treated with alcohols to produce orsellinic acid (2) and orsellinates (3) to (9) (2,4-dihydroxy-6-n-methyl benzoates). Free radical scavenging activity of methyl (3), ethyl (4), n-propyl (5), n-butyl (6), iso-propyl (7), sec-butyl (8), tert-butyl (9) orsellinates was evaluated using 2,2'-diphenyl-1-picrylhydrazyl (DPPH) method. Results showed that chain elongation of methyl (3) to n-butyl (6) causes a rise in the antioxidant activity. However, iso-propyl (7) and tert-butyl (9) were more active than the correspondent linear compounds, although sec-butyl (8) was less active among the chain ramified compounds. All the orsellinates were less active than lecanoric acid (1) and orsellinic acid (2). Orcinol (10) and resorcinol (11) were also determined for comparison with activities of orsellinates. Gallic acid (12) was used as control.

Nanoencapsulation of usnic acid: An attempt to improve antitumour activity and reduce hepatotoxicity.

Despite the recognised antiproliferative and antitumour properties of usnic acid, its therapeutic application has yet to be introduced. In fact, the high hepatotoxicity and low water solubility of usnic acid have somewhat restricted its practical use in anticancer therapy. The aim of this study was therefore to investigate the antitumour activity of usnic acid encapsulated into nanocapsules prepared with lactic co-glycolic acid polymer. Usnic acid-loaded nanocapsules were obtained using the interfacial deposition of a preformed polymer. The antitumour activity was confirmed on an ascitic tumour (Sarcoma-180) implanted in Swiss mice and estimated by means of the tumour inhibition. The results of antitumour activity confirmed that the encapsulation of usnic acid into PLGA-nanocapsules produced a 26.4% increase in tumour inhibition as compared with the standard free usnic acid treatment. Vacuolization of hepatocytes and a mild lymphocytic infiltration in portal spaces were observed in animals treated with free usnic acid. However, this hepatotoxicity was substantially reduced when animals were treated with usnic acid-loaded nanocapsules. No histological changes were noticed in the kidneys or spleen of animals treated either with usnic acid or usnic acid-loaded nanocapsules. These results suggest that nanoencapsulation may be a way of enabling usnic acid to be used in chemotherapy.

Avaliação do perfil cromatográfico em espécies de Polygonum e amostras comercializadas como "erva-de-bicho" / Evaluation of the chromatographic profile of species of Polygonum and samples of the "erva-de-bicho"

As espécies Polygonum punctatum, Polygonum hydropiperoides, Polygonum ferrugineum, Polygonum acuminatum foram analisadas quanto ao perfil cromatográfico e comparadas com amostras comercializadas como "erva-de-bicho". As análises foram realizadas empregando cromatografia líquida de alta eficiência. Verificou-se que as amostras analisadas exibem perfil cromatográfico distinto. O perfil cromatográfico das amostras comercializadas como "erva-de-bicho" foram semelhantes aos obtidos para as amostra de Polygonum acuminatum ou Polygonum ferrugineum ou não apresentaram picos nos cromatogramas. Foram monitorados na determinação do perfil cromatográfico os comprimentos de onda de 254 nm e 362 nm.

The species Polygonum punctatum, Polygonum hydropiperoides, Polygonum ferrugineum, Polygonum acuminatum were analyzed and compared with commercialized samples of "erva-de-bicho". This analysis was performed using high performance liquid chromatography. It was verified that the analyzed samples show a distinct. The chromatographic profile of the samples commercialized as "erva-de-bicho" was similar to the Polygonum acuminatum or Polygonum ferrugineum profiles, or did not present peaks in the chromatograms. The chromatographic profiles were monitored in the 254 nm and 362 nm.

Quantitative determination of jatrophone in "cachaça" prepared with Jatropha elliptica.

A method for sample preparation and analysis by high performance liquid chromatography with UV detection (HPLC-UV) was developed for analysis of jatrophone in "cachaça" prepared with Jatropha elliptica, administered orally, employed in Brazil for the treatment of venomous snake bites. The linearity, accuracy, precision of the procedure was evaluated. Analytical curve for jatrophone was linear in the range of 16.24-81.20 microg ml(-1). The recovery of the jatrophone in the samples analyzed was 98.99-99.89%. The percent coefficient of variation for the quantitative analysis of the "cachaça" in the analyses was under 2%.

A method for quantitative determination of furanocoumarins in capsules and tablets of phytochemical preparations.

A method for sample preparation and analysis by high-performance liquid chromatography with UV detection (HPLC-UV) was developed for analysis of psoralen, bergapten and 5-[3-(4,5-dihydro-5,5-dimethyl-4-oxo-2-furanyl)-butoxy]-7H-furo[3-2-g][1]benzopyran-7-one in capsules and tablets employed in Brazil for certain illnesses. The linearity, accuracy, the inter- and intra-day precision of the procedure were evaluated. Analytical curves for furanocoumarins were linear in the range of 1.0-50.0 microg/ml. The recoveries of the furanocoumarins in the products analyzed were 97.3-99.5%, and the percent coefficient of variation for the quantitative analysis of the furanocoumarins in the analyses was under 5%. For inter-equipment study gas chromatography (GC) was employed.

Antibacterial activity of orsellinates

Para obter novos compostos com atividade antibacteriana, foram preparados os derivados 2,4-diidróxi-6-metilbenzoatos (orselinatos) e o ácido 2,4-diidróxi-6-metilbenzóico (ácido orselínico) através de reacões de alcoólise do ácido lecanórico. Todas as substâncias foram testadas contra bactérias Gram-positivas e Gram-negativas pelo método da microdiluicão. Staphylococcus aureus, Xanthomonas campestris var. vesicatoria and Ralstonia solanacearum foram mais sensíveis ao 2,4-diidróxi-6-metilbenzoato de n-propila, 2,4-diidróxi-6-metilbenzoato de n-pentila e 2,4-diidróxi-6-metilbenzoato de n-hexila com valores de CIM (Concentracão Inibitória Mínima) variando de 62,5 a 7,8 µg.mL1. Esses resultados mostraram que a homologacão da cadeia carbônica pode conduzir a compostos mais ativos.