Induction and growth pattern of callus from Piper permucronatum leaves / Indução e padrão de crescimento de calos de folhas de Piper permucronatum

ABSTRACT Piper permucronatum is a perennial shrub, a medicinal plant native to the Amazon Rainforest. Traditionally, the tea of its leaves is used to combat menstrual and intestinal cramps, stomach pain, digestive problems, diarrhea, hemorrhage, and nausea. Its leaf’s essential oil is effective against Aedes aegypti larvae; its flavones and flavanones have a fungicidal effect against Clamidosporium cladosporioides and C. sphaerospermum; its hexanic extract is effective against Leishmania amazonensis. The objective of this study was to provide a protocol for callus induction from P. permucronatum leaves and an identification of the callus growth pattern, focusing on the deceleration phase, when the callus cells must be subcultured into liquid medium in order to produce a cell suspension cultures. Leaf explants were inoculated in a solid MS medium supplemented with factorial combinations of 2,4-D, BA, NAA and GA3. Callus formation was evaluated weekly until the 49th day. Subsequently, new explants were inoculated at the hormonal combination that resulted in the highest callus cell proliferation and, every seven days during a period of 70 days, samples were dried and weighed to determine the callus growth pattern. NAA and GA3 were not effective for callus induction. Combinations of 2,4-D and BA resulted in callus induction and proliferation. The highest percentage of callus induction was observed with the combination of 4.52 µM 2,4-D and 4.44 µM BA. The calluses thereby produced were friable and whitish. The callus growth pattern followed a sigmoid shape. The deceleration phase started on the 56th day of culture.

RESUMO Indução e padrão de crescimento de calos de folhas de Piper permucronatum. Piper permucronatum é um arbusto perene, uma planta medicinal native da Floresta Amazônica. Tradicionalmente, o chá de suas folhas é usado em casos de cólicas menstruais e intestinais, dores de estômago, problemas digestivos, diarreia, hemorragia e náusea. O óleo essencial das folhas é efetivo contra a larva de Aedes aegypti; suas flavonas e flavanonas têm efeito fungicida contra Clamidosporium cladosporioides e C. sphaerospermum; seu extrato hexânico é efetivo contra Leishmania amazonensis. O objetivo deste trabalho foi determinar um protocolo para indução de calos em folhas de P. permucronatum e identificar o padrão de crescimento dos calos, com foco na fase de desaceleração, quando as células de calo devem ser subcultivadas em meio líquido para produzir culturas de células em suspensão. Explantes foliares foram inoculados em meio MS sólido suplementado com combinações fatoriais de 2,4-D, BAP, ANA e GA3. A formação de calos foi avaliada semanalmente até o 49º dia. Posteriormente, novos explantes foram inoculados na combinação hormonal que resultou na maior proliferação de células de calo e, a cada sete dias durante 70 dias, amostras foram secas e pesadas para determinar o padrão de crescimento dos calos. ANA e GA3 não foram efetivas para a indução de calos. Combinações de 2,4-D e BAP resultaram em indução e proliferação de calos. A maior porcentagem de indução de calos foi observada com a combinação de 4,52 µM de 2,4-D e 4,44 µM de BAP. Os calos produzidos eram friáveis e esbranquiçados. O crescimento dos calos seguiu um padrão sigmoide. A fase de desaceleração iniciou no 56º dia de cultivo.

Medicinal plants used in Rondônia, Western Amazon, Brazil / Plantas medicinais usadas em Rondônia, Amazônia Ocidental, Brasil

This study refers to the use of medicinal plants by populations in the Western Amazon and provides information that can be used in phytochemical studies. It draws upon the traditional knowledge regarding the use of medicinal plants in five regions of the state of Rondônia, in the Brazilian Amazon, focusing on native species. The field research was carried out in five municipalities of the state of Rondônia: Ariquemes, Buritis, Candeias do Jamari, Cujubim and Itapoa do Oeste, characterized by primary economic sectors: agriculture, cattle farming, plant extraction and mineral exploration. Structured interviews were applied to 227 persons chosen because of their prestige in the communities in relation to the knowledge and use of medicinal plants, identifying the therapeutic purpose, parts of the plant used and methods of preparation. The species were taxonomically identified. The ethnobotanic knowledge (inferred by the number of uses of medicinal plants per person) was correlated with the Brazilian region of origin, age, and gender of the interviewees. According to the collected data, 34 botanical families and 53 native species were identified. Of the 53 species, only 7 occur exclusively in the Amazon Forest: Theobroma grandiflorum (Willd. ex Spreng.) K. Schum., Psidium densicomum Mart. ex DC, Piper cavalcantei Yunck., Pilocarpus microphyllus Stapf ex Wardlew., Euterpe oleracea Mart., Croton cajucara Benth., Baccharis altimontana G. Heiden. The most common disorders treated with the plants were kidney problems, influenza, generalized infections and inflammations, malaria and high blood pressure. Leaves were the most used parts in preparations. Barks, fruits, roots, flowers, stems, seeds, oils, buds, tubercles, and rhizomes were also mentioned. Thirteen forms of preparations were recorded, and infusion and decoction were the most used. Syrups, juices, flour, sap, oil and parts of the plant blended with milk, honey and coffee or flamed, macerated and in the form of poultice, were also found. Persons from the Southeast region had more information compared with persons from the other regions; persons in the age groups between 50-59, 60-69 and 70-79 had more information in comparison with the other age groups; and the female interviewees mentioned significantly more uses of medicinal plants than the male ones. The relatively small number of native Amazon species identified can be the result of the loss of knowledge about medicinal plants in the Amazon because of internal migration, extinction of local indigenous groups, increasing urbanization and consequent globalization of the lifestyles.

Este estudo se refere ao uso de plantas medicinais por populações da Amazônia Ocidental, fornece informações passíveis de utilização em estudos fitoquímicos e resgata o conhecimento tradicional a respeito do uso de plantas medicinais em cinco regiões do estado de Rondônia na Amazônia Brasileira com foco nas espécies nativas. A pesquisa de campo foi realizada nos cinco municípios de Rondônia: Ariquemes, Buritis, Candeias do Jamari, Cujubim e Itapoã do Oeste, caracterizados por atividades econômicas primárias: agricultura, pecuária e extrativismo vegetal e mineral. Entrevistas estruturadas foram aplicadas a 227 pessoas escolhidas por seu prestígio nas comunidades em relação ao conhecimento e uso de plantas medicinais, identificando a finalidade terapêutica, as partes das plantas utilizadas, e os métodos de preparação. As espécies foram taxonomicamente identificadas. O conhecimento etnobotânico (inferido a partir do número de citações por entrevistado) foi correlacionado com a região de origem, idade e gênero dos entrevistados. De acordo com os dados coletados, 34 famílias botânicas e 53 espécies nativas foram identificadas. Dentre as 53 espécies, apenas sete ocorrem exclusivamente na Amazônia: Theobroma grandiflorum (Willd. ex Spreng.) K.Schum., Psidium densicomum Mart. ex DC, Piper cavalcantei Yunck., Pilocarpus microphyllus Stapf ex Wardlew., Euterpe oleracea Mart., Croton cajucara Benth., Baccharis altimontana G. Heiden. As afecções mais comuns tratadas com plantas foram: problemas de fígado, gripe, infecções e inflamações generalizadas, malária, e hipertensão arterial. As folhas são as partes mais utilizadas nas preparações. Também foram mencionadas cascas, frutos, raízes, flores, caules, sementes, óleos, brotos, tubérculos e rizomas. Treze formas de preparação foram registradas sendo mais comuns as infusões e decoctos. Outras formas de preparação observadas foram xarope, suco, farinha, seiva, óleo, partes vegetais misturadas com leite, mel ou café, flambada, macerada e em forma de cataplasma. Pessoas da região Sudeste apresentaram maior número de informações do que as outras regiões; faixas etárias entre 50-59, 60-69 e 70-79 apresentaram maior número de citações que os outros grupos etários; e as mulheres mencionaram significativamente mais usos medicinais em comparação com os homens. O número relativamente pequeno de espécies nativas amazônicas identificadas pode ser um resultado da perda de conhecimento sobre plantas medicinais na Amazônia, devido à migração interna, extinção dos grupos indígenas locais, crescente urbanização e consequente globalização dos estilos de vida.

Avaliação da atividade antibacteriana do óleo essencial de Lippia origanoides frente à Staphylococcus sp. isolados de alimentos de origem animal / Assessment of the antibacterial activity of the essential oil of Lippia origanoides in relation to Staphylococcus sp. isolated from food of animal origin

Objetivou-se no trabalho avaliar a ação antibacteriana do óleo essencial de Lippia origanoides frente à isolados de Staphylococcus sp. de alimentos de origem animal. Para tanto, realizou-se análise química da composição do óleo, teste de sensibilidade das bactérias frente a dez antibióticos de uso terapêutico e ao óleo essencial de Lippia origanoides, além da determinação da concentração inibitória mínima (CIM) e da concentração bactericida mínima (CBM). A análise cromatográfica do óleo apresentou o timol como composto majoritário (48,70%), além de pequena quantidade de carvacrol (1,14%). No teste de sensibilidade frente aos antibióticos, 75% dos isolados apresentaram resistência a, no mínimo, três antibióticos. Em relação ao óleo essencial, os isolados de Staphylococcus sp oriundos de leite bovino mostraram-se mais resistentes e os isolados de carne ovina apresentaram-se mais sensíveis. A CIM foi maior para os Staphylococcus sp. isolados de leite bovino e leite ovino (60µL/mL). Enquanto que para os isolados de carcaça de ovinos e queijo, a CIM foi de 15µL/mL e 30µL/mL respectivamente. A CBM, consequentemente, foi maior para os isolados de leite bovino e leite ovino, sendo de 240µL/mL e 120µL/mL respectivamente. O óleo essencial de Lippia origanoides apresenta atividade antimicrobiana sobre Staphylococcus sp. isolados de alimentos.

The aim of this study was to evaluate the antibacterial activity of the essential oil of Lippia origanoides in relation to Staphylococcus sp. isolated from food of animal origin. For this purpose, we performed the chemical analysis to determine the oil composition, evaluated the bacteria sensibility to ten antibiotics of therapeutic use and determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The chromatographic analysis showed that tymol was the main compound (48.70%) and that carvacrol was present in a small amount (1.14%). In the test of antibiotic susceptibility, 75% of the microorganism isolates were resistant to at least three of the ten antibiotics tested. The Staphylococcus sp isolated from bovine milk was more resistant to the essential oil of Lippia origanoides, and the microorganism isolated from sheep meat was more susceptible to this oil. The Staphylococcus sp. isolated from bovine or sheep milk showed a MIC of 60µL/mL and for those isolated from sheep carcass and cheese the MIC was 15µL/mL and 30µL/mL, respectively. Consequently, the MBC was higher for isolates from cow's (240µL/mL) and sheep's milk (120µL/mL) when compared with other food sources. These results suggest that the essential oil of Lippia origanoides has antimicrobial activity against staphylococcus sp. isolated from food.

Callogenesis in leaves of Kalanchoe pinnata Lam. by 2, 4-D and BA action / Calogênese em folhas de Kalanchoe pinnata Lam. pela ação de 2, 4-D e BAP

The Kalanchoe pinnata Lam. is a bush species of the Crassulaceae that is distinguished by its important medicinal properties. Its leaves are used as cataplasm to treat headaches and wounds. There is evidence for a hypotensive and anti-inflammatory effect. Techniques of plant tissue culture have been applied to plant species that produce substances likely to be explored in pharmacology, cell suspension being the main technique. At the industrial level, this method utilizes bioreactors in order to produce secondary metabolites on a large scale. The objective of this study was to evaluate the effects of in vitro combinations of 2,4-dichlorophenoxiacetic acid (2,4-D) and benzylaminopurine (BA) on callus induction in leaf explants of K. pinnata. Leaf fragments were inoculated in MS medium supplemented with 3.0% sucrose, 0.8% agar and factorial combinations of 2,4-D (0.00, 4.52, 9.06, 18.12 µM) and BA (0.00, 4.44, 8.88, 17.76 µM). The cultures were kept in the darkness at 24±2ºC for 50 days. The percentage of callus induction and the area of explants covered by callus cells were evaluated. In the absence of growth regulators, callus induction did not occur, with necrosis of all explants. The highest percentage of callus induction was 100%, obtained with the combination of 9.06 µM 2,4-D and 8.88 µM BA, but the calluses covered only 25% of the leaf area. The most efficient combination was 4.52 µM 2,4-D and 8.88 µM BA, resulting in 91% callus induction with 50 to 100% of the explants being covered by callus cells.

Kalanchoe pinnata Lam. é uma espécie arbustiva da família Crassulaceae que apresenta interessantes propriedades medicinais. Suas folhas são utilizadas em cataplasma para tratar enxaqueca e ferimentos. Há evidência de seu efeito como hipotensiva e anti-inflamatória. Técnicas de cultura de tecidos vegetais têm sido aplicadas para espécies que possuem substâncias passíveis de exploração na farmacologia, sendo a suspensão celular a principal técnica utilizada. A nível industrial, este método utiliza biorreatores para produzir metabólitos secundários em larga escala. Este estudo teve como objetivo avaliar os efeitos in vitro de combinações do ácido 2,4-diclorofenoxiacético (2,4-D) e de benzilaminopurina (BAP) na indução de calos em explantes foliares de K. pinnata. Fragmentos foliares foram inoculados em meio MS contendo 3% de sacarose, 0,8% de ágar e combinações fatoriais de 2,4-D (0,00; 4,52; 9,06 e 18,12 µM) e BAP (0,00; 4,44; 8,88 e 17,76 µM). Os cultivos foram mantidos no escuro, a 24±2ºC por 50 dias. A porcentagem de indução de calos e a área dos explantes coberta por células de calos foram avaliadas. Na ausência de reguladores de crescimento não ocorreu indução de calos, com necrose de todos os explantes. A porcentagem mais alta de indução de calos foi de 100%, obtida com a combinação de 9,06 µM de 2,4-D e 8,88 µM de BAP, mas estes calos cobriram apenas 25% da área foliar. A combinação mais eficiente foi de 9,06 µM de 2,4-D e 8,88 µM de BAP, que resultou em 91% de indução e 50 a 100% da área dos explantes coberta por células de calos.

Composição química e atividade inseticida do óleo essencial de Schinus terebinthifolius Raddi (Anacardiaceae) sobre a broca-do-café (Hypothenemus hampei) Ferrari / Chemical composition and insecticidal activity of the essential oil of Schinus terebinthifolius Raddi (Anacardiaceae) on coffee berry borer (Hypothenemus hampei) Ferrari

O óleo essencial das folhas de Schinus terebinthifolius foi obtido por hidrodestilação e analisado por CG-EM. O rendimento do óleo foi de 0,8%, sendo identificados 37 constituintes químicos. Os componentes principais foram germacreno D (25,0%), (E)-ß-cariofileno (17,5%) e δ -elemeno (10,5%). O óleo essencial foi diluído em acetona nas concentrações de 10-2 a 10-8 e aplicado aos insetos (Hypothenemus hampei) por aplicação tópica e exposição em superfície contaminada. As taxas de mortalidade foram avaliadas após 24 e 48 horas do início do experimento. A aplicação em superfície contaminada resultou em 25% de mortalidade no controle, enquanto nas diluições de 10-2 a 10-8 foram observados 100,0 a 30,0% de mortalidade. Na aplicação tópica, observou-se 27,5% de mortalidade no controle e 97,5 a 77,5% nas diluições de 10-2 a 10-8.

The essential oil from the leaves of Schinus terebinthifolius was obtained by hydrodistillation and was analyzed by GC-MS. The oil yield was 0.8%, and 37 chemical constituents were identified. The main components were germacrene D (25.0%), (E)-ß-cariophyllene (17.5%) and δ-elemene (10.5%). The essential oil was diluted in acetone in concentrations of 10-2 to 10-8 and it was applied to insects (Hypothenemus hampei) by topical application and exposure to contaminated surface. Mortality rates were evaluated after 24 and 48 hours. The contaminated surface application resulted in 25.0% of mortality in control while the dilutions of 10-2 to 10-8 resulted in 100% to 30.0% of mortality respectively. In the topical application, it was observed 27.5% of mortality in control and 97.5 to 77.5% of mortality in dilutions of 10-2 to 10-8

Perfil de suscetibilidade de leveduras do gênero Candida isoladas de animais ao óleo essencial de Rosmarinus officinalis L / Susceptibility profile of Candida spp. isolated from animals to the essential oil of Rosmarinus officinalis L

Neste trabalho foram avaliadas a atividade antifúngica e composição química do óleo essencial de Rosmarinus officinalis (alecrim). O óleo essencial foi obtido, através da hidrodestilação, das partes aéreas da planta, o qual foi analisado por cromatografia gasosa com detectores de ionização em chama (GC/FID) e espectrometria de massas (GC/MS). Esse óleo foi testado em isolados de C. albicans da mucosa vaginal de fêmeas caninas, casos clínicos de candidíase em animais cepas padrões e espécies não-albicans, usando a técnica de microdiluição em caldo (CLSI-M27A2). A análise cromatográfica do óleo permitiu identificar como principais compostos cânfora, verbenona e 1,8-cineol. Os valores de CIM do óleo para os isolados padrões de C. albicans variaram entre 1,25 e 2,5 µL mL-1 e de 2,5 a 5,0 µL mL-1 para CFM, enquanto que para os isolados não-albicans observou-se CIM entre 1,25 e 5,0 µL mL-1 e CFM de 2,5 a 10,0 µL mL-1. C. albicans isolada de animais apresentou valores de 2,5 a >10,0 µL mL-1 para CIM e de 5,0 a >10,0 µL mL-1 para CFM. Os resultados demonstraram atividade fungicida e fungistática do óleo essencial de alecrim em isolados de Candida spp. provenientes de animais.

In this work the antifungal activity and chemical composition of the Rosmarinus officinalis (rosemary) essential oil against Candida spp. were evaluated. The essential oil was obtained by hydrodistillation of aerial parts, and analyzed by gas chromatography with a flame ionization detector (GC/FID) and gas chromatography coupled with mass spectrometry (GC/MS). This oil was tested against fourteen isolates of Candida sp. (ten C. albicans and four no-albicans species) by the microdillution broth assay (CLSI-M27A2). Chromatography analyses of the essential oil showed compounds, as camphor, verbenone and 1,8-cineole as major constituents. MIC and MFC values for C. albicans standard strains were 1.25 - 2.5 µL mL-1 and 2.5 - 5.0 µL mL-1, respectively. MIC and MFC for the non-albicans species were 1.25 - 5.0 µL mL-1 and 2.5 - 10.0 µL mL-1, respectively. C. albicans isolates obtained from animals exhibited MIC and MFC values of 2.5 - > 10.0 µL mL-1 and 5.0 - > 10.0 µL mL-1, respectively. According to the results, the essential oil of rosemary presented fungicidal and fungistatic activities against Candida spp.

High prevalence of hepatitis C in patients with thalassemia and patients with liver diseases in Myanmar (Burma).

We conducted Myanmar-Japan cooperation studies on hepatitis B and hepatitis C virus markers in patients with thalassemias and those with liver diseases. Among the 102 patients with liver diseases, 92% had a history of hepatitis B virus infection (antibody to hepatitis B core antigen positive), 35% were hepatitis B surface antigen positive, 39% were positive for anti-HCV. Among 28 patients with hepatocellular carcinoma, 46% had hepatitis B surface antigen, 21.4% had antibody to hepatitis C virus, and 7% were positive for both hepatitis B surface antigen and anti hepatitis C virus. The history of HCV infection among blood recipients at the Haematology Department of the Yangon General Hospital and at the Yangon Children's Hospital was found to be 55.5% and 46.7%, respectively, which is comparable to the history of hepatitis B infection (66.7% and 46.7%, respectively). This preliminary survey also encountered 2 cases positive for anti-HCV among 34 voluntary blood donors. This survey is the first one to report that hepatitis C is at the epidemic stage in Myanmar. As there is no effective treatment for hepatitis C in this country, a screening program for blood used in transfusion should be started immediately.

HIV transmission in sexual partners of persons with HIV/AIDS attending the Infectious Diseases Hospital, Yangon.

A study was conducted in the Infectious Diseases Hospital, Yangon, for one year from August 1996 to 1997, to assess the extent and the factors related to HIV transmission among sexual partners of HIV/AIDS cases. It was a cross-sectional comparative study on 67 (61 males, 6 females) HIV positive individuals with or without AIDS, and their sexual partners. Separate interviews of index cases and partners were done, and clinical examination and laboratory tests for HIV and sexually transmitted diseases (STDs) were performed. HIV transmission was found in 41.8% of the partners. Male-to-female transmission was 39.3% (n=61) and female to male transmission was 66.7% (n=6), Seven and one half percent of the partners were suffering from AIDS. There were 4 (6.6%) pregnant mothers and 1 (25%) was HIV positive. Though 75% of the partners did not use condoms during their married life, HIV transmission was significantly reduced in the condom users (odds ratio (OR) = 0.18, 95% confidence interval (CI) 0.02-0.98 p = < 0.05). The seropositive men who were less than 30 years of age had greater HIV transmission (OR = 5.67, 95% CI 1.13-36.46). However, socio-demographic factors, number of marital partners and age of first sex partners, duration of marriage, number of sexual relationships between these couples, duration of HIV positivity and AIDS infection, immunological status of the index group and STD positivity among partners had no significant association with the transmission of HIV. This study demonstrated that the transmission of HIV was high among the sexual partners of persons with HIV infection. It also highlighted the requirement of effective counseling and preventive measures against HIV infection among the couples.

Genetic and serologic characterization of HIV type 1 prevailing in Myanmar (Burma).

To study the molecular epidemiology of HIV-1 spread in Myanmar and the interplay with the epidemic in surrounding Southeast Asian countries, we determined the HIV-1 subtypes prevailing in Myanmar. Thirty HIV-positive blood specimens were sampled in the capital city, Yangon, and an additional 459 sera were collected nationwide in 1995. Genetic subtyping based on the env C2/V3 sequence and serologic data, using a V3 peptide enzyme immunoassay (PEIA), revealed three patterns of HIV spread in different geographic regions in Myanmar: (1) in the capital city, Yangon, HIV-1 subtype B' ("Thai-B" cluster within subtype B) predominated both in IDUs and heterosexuals; (2) in the cities near the border with Thailand, including Tachelaik and Kawthaung, where heterosexual transmission is a major pathway of HIV-1 spread, HIV-1 subtype E was predominantly distributed among the commercial sex workers and heterosexuals; (3) in central and northeast Myanmar, both HIV-1 subtypes B' and E occurred in a mixed distribution, without showing any significant segregation by risk group. In addition, the PEIA data implied the occurrence of other subtype(s) in these areas. The interperson nucleotide sequence variations in env C2/V3 regions of B' and E, prevailing in Yangon, were 6.7 +/- 2.1 and 7.1 +/- 0.7%, respectively. They were similar to those levels observed in Thailand. These findings are consistent with the view that HIV spread in Myanmar might have taken place at about the same time as that in Thailand, and that multiple entries and exchanges of HIV-1 with neighboring countries are important factors contributing to the current distribution of subtypes in Myanmar.

Cardiac abnormalities in the thalassaemia syndromes.

Cardiac complications are the leading cause of death in patients with the thalassaemia syndromes. The cardiac abnormalities may be the result of chronic anaemia or iron overload. The cardiac status was evaluated in 30 patients with thalassaemia to determine whether the structural and functional changes could be related to the degree of anaemia or to other factors suggesting iron overload. Clinical, radiological, electrocardiographic and echocardiographic methods were employed. The study provided evidence that cardiac abnormalities were due to undertransfusion. Echocardiography provides a reliable, noninvasive means of assessing cardiac abnormalities in patients with thalassaemia.