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2262 J. Med. Plants Res. Table 1. Species of Brazilian plants with their correspondent families and popular names. Species Family Popular name Bauhinia blakeana Dunn Caesalpiniaceae Pata de vaca Begonia fruticosa A.DC. Begoniaceae Begônia Bumelia sartorum Mart. Sapotaceae Quixaba Cassia ferruginea Schrad Caesalpiniaceae Canafístula Chenopodium ambrosioides L. Chenopodiaceae Erva de Santa Maria Citrus limon (L.) Burm. f. Rutaceae Limão Coffea arabica L. Rubiaceae Café Endopleura uchi (Huber) Cuatrec. Humiriaceae Uxi amarelo Ficus enormis Miq Moraceae Figueira Impatiens walleriana Hook. f. Balsaminaceae Maria sem vergonha Leandra purpurescens Cogn. Melastomataceae Pixirica Ligustrum lucidum W.T. Aiton Oleaceae Ligustro Morus nigra L. Moraceae Amora Origanum vulgare L. Lamiaceae Orégano Pittosporum undulatum Vent. Pittosporaceae Pau – incenso Plectranthus barbatus Andrews Lamiaceae Boldo brasileiro Pothomorphe umbellata (L.) Miq. Piperaceae Pariparoba Prunus myrtifolia (L.) Urban Rosaceae Pessegueiro bravo Psidium cattleianum Sabine Myrtaceae Araçá Rhynchelytrum repens (Willd.) C.E.Hubb. Poaceae Capim favorito Ricinus communis (L.) Euphhorbiaceae Mamona Solidago chinesis (Osbeck) Merr. Asteraceae Arnica Symphyopappus compressus (Gardner) B.L.Rob. Asteraceae not known Tabebuia impetiginosa (Mart. ex DC.) Standl Bignoniaceae Ipê roxo Tibouchina granulosa Cogn. Melastomataceae Quaresmeira Tibouchina mutabilis Cogn. Melastomataceae Manacá da serra Uncaria tomentosa (Willd. ex Roem. & Schult.) DC. Rubiaceae Unha de gato lyophilized. For the assays, the lyophilized extracts were dissolved in equal parts of distilled water and Eagle's minimum essential medium (MEM) (Cultilab®, Campinas/SP, Brazil) for final concentration of 4,000 µg/ml. Virus and cell line Strains Nova Prata of SuHV-1 and Los Angeles of BoHV-1 were propagated in cell line MDBK (ATCC-CCL 22), and were maintained in MEM with 10% fetal calf serum. Cytotoxicity assays The cytotoxicity of the plant extracts was based on cellular morphologic alterations using 96-well plates with 30,000 cells/well (Simoni et al., 2007; Gomes et al., 2008). Briefly, MDBK cells were exposed to concentrations of extracts, ranging from 31.25 to 2.000 µg/ml, in triplicate. Daily, the cell morphologies were checked to determine the maximum non-cytoxic concentration (MNCC) of each extract used in the antiviral tests. Cells incubated only with MEM were used as controls. Antiviral assays The antiviral activity of the extracts was determined by the reduction of virus titers that was calculated by the method of Reed and Muench (1938) and expressed in 50% tissue culture infective dose (TCID50). The difference of viral titer between treated and untreated control cultures is expressed as viral inhibition index (VII) and was considered significant when the value is greater or equal to 1.5 (Simoni et al., 2007; Silveira et al., 2009). Briefly, cell monolayers were treated with the extracts at their respective MNCC for I h and were inoculated with the logarithmic dilutions (10 -1 to 10 -7 ) of each correspondent virus. Controls consists of untreated infected (virus titer), treated non-infected (cytotoxicity control) and untreated noninfected (cell control) cells. RESULTS The 27 plant species screened in this study and their respective family and popular name are listed in Table 1. They covered 19 families and 26 genera.
(a) (b) (c) Figure 1. (a) Normal cells MDBK; (b) infected cells with SuHV-1; (c) infected cells with BoHV-1; (×200). The appearance of normal cells MDBK is as shown in Figure 1a. Figure 1b and c shows the cytopathic effect of suid and bovine herpesviruses type 1, respectively. It is characterized by cell rounding and ballooning. The cytotoxic and antiviral activities of these plant extracts against BoHV-1 and SuHV-1 are as shown in Table 2. Most of them showed low cytotoxicity to cells with MNCC ranging from 1.000 to 125 µg/ml, but some of them were more cytotoxic with MNCC between 62.5 and 7.8 µg/ml. The extracts of Bumelia sertorum, C. arabica, Endopleura uchi, Leandra purpurescens, Psidium cattleianum and Uncaria tomentosa presented VII greater or equal to 1.5 for both viruses. The extracts of Prunus myrtifolia and Symphyopappus compressus were positive for BoHV-1, while the extracts of Bauhinia blakeana, Origanum vulgare, R. communis and Tibouchina mutabilis were positive for SuHV-1. DISCUSSION Although, the collect of the plants was based on random study of natural flora of the region/country; majority of the plants after identification were found to be medicinal and some of them are also part of human feeding (Cordeiro et al., 2002). Considering the plants with antiviral effect for at least one of the viruses, all of them have use in Brazilian folk medicine except S. compressus. There is no information about it in literature. These data corroborated the importance of ethnopharmacology as an efficient strategy for selecting a plant for drug development and antiviral studies (Vlietinck and Vanden Berghe, 1991; Rates, 2001; Jassim and Naji, 2003; Cos et al., 2006). Plant extracts should be firstly assayed for toxicity evaluation since the safety of a therapeutic agent is of paramount importance (Harbell et al., 1997; Rates, 2001; Veiga et al., 2005). Cytotoxicity tests based on the cell Fernandes et al. 2263 morphological alterations, although qualitative and more subjective can initially be used in in vitro antiviral screening programs when evaluating many plants. Furthermore, with the use of the extracts in their respective MNCC in the antiviral tests can distinguish the antiviral effects of the possible toxic effects of the extracts. Studies with some of these extracts have already demonstrated their antiviral activity as E. uchi (Plantamed, 2009). Simões et al. (1999) reported the antiviral effect of P. cattleianum against herpes simplex (HSV) type 1 and 2, but using hydroalcoholic extracts from leaves. Our extract is aqueous and then probably the active compound could probably be a polar chemical substance since both preparations presented activity. Barks and roots of U. tomentosa have a broad therapeutic potential including treatment of viral infections (Aquino et al., 1989; Willians, 2001). Otherwise, among the diverse properties and uses of C. arabica, L. purpurescens and P. cattleianum, an antibacterial activity is also included (Almeida et al., 2006; Coelho de Souza et al., 2004; Plantamed, 2009). However, in the studies of Mccutcheon et al. (1995), P. myrtifolia did not present antiviral activity against other viruses including HSV type 1. Kudi and Myint (1999) showed the antiviral activity of B. blakeana against other viruses as equine herpesvirus; the essential oils from O, vulgare presented antibacterial and antifungicidal activities among others (Bozin et al., 2006). Semple et al. (1998) and Kudi and Myint (1999) described the antiviral activities of Pittosporum undulatum and Cassia ferruginea, respectively, against a range of viruses, although these plants were not active in this study for the two herpesviruses. Researches for antiviral effects of plant extracts against these two herpesviruses have continuously been made (Ahmad et al., 1996; Simoni et al., 1996; Summerfield et al., 1997; Barrio and Parra, 2000; Felipe et al., 2006; Simoni et al., 2007). The positive plants for the BoHV-1
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Figure 2. Contd. at 2.0 mg/L yellow
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Journal of Medicinal Plants Researc
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Table 1. Biochemical parameters in
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Figure 3. Bcl-xL reactions in inter
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ACKNOWLEDGMENTS The authors would l
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Figure 3. The most widely included
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Figure 4. Lithographic print “St.
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Table 1. List of plants included in
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14 7 1 15 1 35 17 16 Imported plant
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Academy of Science Publishing House
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et al., 1996; van Wyk and Gericke,
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DPPH inhibition (%) DPPH % inhibiti
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% Mortality Concentration (µg/ml)
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information on plants used for the
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Table 1. Levels of independent vari
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Arbutin (mg/g) Co-solvent Figure 1.
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Arbutin (mg/g) Extraction pressure
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Table 3. Arbuitn content of Asian p
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Arbutin (mg/g) Extraction pressure
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Darjazi 2369 Table 2. Statistical a
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Figure 2. Effect of different alcoh
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Table 6. Results of ANOVA. Nie et a
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Table 2. Plant data and MIC values
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exhibiting the polar extracts of P.
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Figure 1. Map of Ladakh region of I
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Warghat et al. 2391 Table 3. Summar
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Table 5. Inter-Population genetic i
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Excoffier L, Smouse PE, Quattro JM
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ice residue is an ideal raw materia
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DPPH• scavenging ratio(%) OD valu
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scavenging effect on free radical a
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our new CL system. Fenton reaction
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1 2 Time (s) Figure 2. Kinetic curv
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Wong et al., 2006). It can be seen
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Pan YM, Liang Y, Wang HS, Liang M (
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UPCOMING CONFERENCES 15th European
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