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Plants used to treat snakebites in Santarém, western Pará, Brazil: an assessment of their effectiveness in inhibiting hemorrhagic activity induced by Bothrops jararaca venom Valéria Mourão de Moura, Luciana A. Freitas de Sousa, Maria Cristina Dos-Santos, Juliana Divina Almeida Raposo, Aline Evangelista Lima, Ricardo Bezerra de Oliveira, Milton Nascimento da Silva, Rosa Helena Veras Mourão
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S0378-8741(14)00901-5 http://dx.doi.org/10.1016/j.jep.2014.12.020 JEP9194
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Journal of Ethnopharmacology
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Received date: 11 October 2014 Revised date: 7 December 2014 Accepted date: 14 December 2014 Cite this article as: Valéria Mourão de Moura, Luciana A. Freitas de Sousa, Maria Cristina Dos-Santos, Juliana Divina Almeida Raposo, Aline Evangelista Lima, Ricardo Bezerra de Oliveira, Milton Nascimento da Silva, Rosa Helena Veras Mourão, Plants used to treat snakebites in Santarém, western Pará, Brazil: an assessment of their effectiveness in inhibiting hemorrhagic activity induced by Bothrops jararaca venom, Journal of Ethnopharmacology, http://dx.doi. org/10.1016/j.jep.2014.12.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Plants used to treat snakebites in Santarém, western Pará, Brazil: an assessment of their effectiveness in inhibiting hemorrhagic activity induced by Bothrops jararaca venom
Valéria Mourão de Mouraa,b*, Luciana A. Freitas de Sousaa, Maria Cristina Dos-Santosb, Juliana Divina Almeida Raposoa, Aline Evangelista Limaa , Ricardo Bezerra de Oliveiraa, Milton Nascimento da Silvad, Rosa Helena Veras Mourãoa a
Programa de Pós-Graduação em Recursos Naturais da Amazônia, Laboratório de
Bioprospecção e Biologia Experimental, Universidade Federal do Oeste do Pará - UFOPA, Rua Vera Paz, s/n, 68035-110, Santarém, PA, Brazil; b
Programa Multi-Institucional de Pós-Graduação em Biotecnologia, Laboratório de
Imunologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas- UFAM, Av. Rodrigo Octávio Ramos, 3000, Manaus, AM, Brazil; d
Programa de Pós-Graduação em Química, Universidade Federal do Pará – UFPA, Rua
Augusto Corrêa, 66075-110, Belém, PA, Brazil.
*Corresponding author: Tel.: +5509281641596; E-mail – [email protected] Programa Multi-institucional de Pós-graduação em Biotecnologia, Laboratório de Imunologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Av. General Octávio Jordão Ramos, 3000, Japiim, CEP 69057-000.
Abstract Ethnopharmacological importance: The poor distribution and limited availability of antivenoms in Brazil has led to greater use of plants to treat snakebites. Very often such plants are the only alternative available to riverside communities. Material and methods: Direct questionnaire-based interviews were conducted with members of the Cucurunã, São Pedro and Alter do Chão communities in Santarém, Pará, Brazil. For each of the twelve most frequently mentioned species aqueous extracts were prepared and the phytochemical profiles determined by thin layer chromatography. The concentrations of phenolic compounds (tannins and flavonoids) in the aqueous extracts were determined by colorimetric assays. To assess inhibition of the hemorrhagic activity of Bothrops jararaca venom, solutions containing the venom mixed with aqueous extracts in the ratios 1:12 and 1:48 were tested (w/w). SDS-PAGE and Western blot were used to assess the action of the extracts on B. jararaca venom. Results: In all, 24 plants belonging to 19 families were mentioned in the survey as being used to treat snakebites. Leaves (84%), seeds (60.9%) and inner bark (53%) were cited as the most frequently used parts in folk remedies, which were usually prepared in the form of a decoction (62.5%), tincture (45%) or maceration (22.5%). Hemorrhage induced by B. jararaca venom was completely inhibited by aqueous extracts of Bellucia dichotoma, Connarus favosus, Plathymenia reticulata and Philodendron megalophyllum, which had a high phenolic content and contained condensed and hydrolyzable tannins. The results of SDS-PAGE showed that some venom protein bands were not visible when the venom was preincubated with the extracts that had completely inhibited hemorrhagic activity of the venom. Western blot showed that the extracts did not have any enzymatic action on the proteins in the venom as it failed to detect low-molecular-weight bands, which are indicative of possible enzymatic cleavage.
Conclusion: Traditional use of plants to treat snakebites is common practice in the western region of Pará, Brazil. Our findings show that some plant extracts were able to inhibit snake venom-induced hemorrhage in vitro. In vivo studies are being carried out to validate the traditional use of these species to treat snakebites. Keywords: Ethnobotany, plant extracts, snakebite envenomation, anti-snakebite plants, hemorrhage.
1. Introduction According to official estimates, snakebites affect around 240,000 people worldwide and account for 20,000 deaths. However, if undernotification is taken into consideration, these figures could reach 1,841,000 and 94,000, respectively (Kasturiratne et al., 2008). In light of these data, the World Health Organization granted snakebite envenomation neglected-disease status in 2009. In Brazil, approximately 90% of snakebites reported to the Ministry of Health were caused by snakes of genus Bothrops sp. (Ministério da Saúde do Brasil, 2010). The Northern region of the country has the highest incidence, and Pará is the state with highest number of reported cases (Bochner and Struchiner, 2003; Araújo et al., 2003). Of all the municipalities in the state, Santarém has the greatest number of reported cases, with an annual average of 755 cases between 2011 and 2013 (Sistema de Informações de Agravos de NotificaçãoSINAN, 2013). These numbers may in reality be higher because of undernotification as a result of the difficulty snakebite victims have getting medical attention and the precarious nature of the system for reporting notifiable diseases. Bothrops jararaca is the species that causes the greatest number of snakebites in the Southeast of Brazil (Ministério da Saúde do Brasil, 2005). Envenomation by snakes of the genus Bothrops causes immediate local effects such as pain, edema, hemorrhage and
myonecrosis, as well as systemic reactions, most notably coagulant and hemorrhagic effects (Cardoso et al., 2003). Hemorrhage is one of the most serious symptoms of envenomation by snakes from genus Bothrops and is induced by metalloproteinases. These hemorrhagic toxins play an important role in vascular damage and the subsequent generation of ischemic zones, which are the main cause of necrosis of local tissue, potentially leading to amputation of the affected member (Gutiérrez, 1998; Warrell, 1992). A major problem in this type of envenomation is that the local lesion, once induced, cannot be reversed by standard Bothrops antivenom (Baldo et al., 2010). Hence, it is important to study natural inhibitors that could be used in combination with serum therapy to treat the local effects of snakebites. In the Amazon region, the use of plant preparations to treat snakebites is quite common and indeed very often the only option. Some medicinal plants are a source of bioactive compounds with the potential to directly inhibit some of the activities induced by snake venom. Many of these plants have been studied in order to validate their use in popular medicine (Borges et al., 1996; Magalhães et al., 2011; Sousa et al., 2013; Moura et al., 2014) or to isolate and characterize these bioactive components (Mors et al. 2000, Ambikabothy et al., 2011; Krishnan et al., 2014). The vast majority of studies of plants with anti-snakebite properties are based on folk medicine. Although there are some studies on the ethnobotany of species used to treat snakebites, few scientific studies to date have validated the anti-snakebite properties of aqueous extracts or compounds from these plant species. This study therefore sought not only to recover and preserve traditional knowledge about the use of plants in the treatment of snakebites in the western region of Pará, Brazil, but also to evaluate the extent to which aqueous extracts of such plants can inhibit the hemorrhagic activity induced by B. jararaca venom as well as to determine the phytochemical profile of these extracts by thin layer chromatography.
2. Materials and Methods 2.1. Ethnobotanical survey An ethnobotanical survey was carried out into the use of medicinal plants with antisnakebite properties in the following communities in Santarém in western Pará, Brazil, with a high incidence of snakebite accidents: São Pedro (02º32´08.9” S and 54º54´23.9” W), Cucurunã (02º27´21.0” S and 54º47´45.7” W), Fazenda Experimental Carauá (02º33´99.3” S and 54º36´61.2” W) and Alter do Chão (02030´53.3” S and 54057´00.1” W) (Figure 1). Collections points were identified with the aid of a global positioning system (GPS – GARMIN, GPSMap 60CSx, Kansas, USA). Between January and July 2010, an open-ended questionnaire on the use of plants with anti-snakebite properties was applied in direct interviews. Interviewees were selected by the residents of the communities themselves and were generally people who worked as plant healers and faith healers or were knowledgeable about the plants used in the region. They were told about the importance of the study, and those who agreed to take part signed a voluntary informed-consent form. The questions in the questionnaire were about the frequency of snakebite accidents in the interviewees' communities and treatment of snakebites with plant extracts, with a particular emphasis on the part of the plant used, the form in which it is used (such as decoction and maceration) and the provenance of the plant.
Fig. 1. Study Area. A) Location of Brazil in South America; B) Political map of Brazil showing the state of Pará in red; C) Political map of Pará showing the western region of the state and the municipalities in this region; D) Map of Santarém. Prepared by Fabio Guerra Santos, Forestry Engineer at the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA).
2.2. Collection and identification of plant material The most frequently mentioned species were collected and identified. After drying, extracts were prepared so that their phytochemistry and their effectiveness in inhibiting hemorrhage induced by B. jararaca venom could be studied. Dried specimens were also deposited in the Embrapa Western Amazonia Herbarium, Belém, with the following (IAN)
codes: Plathymenia reticulata Benth. (Fabaceae) 185215; Connarus favosus Planch. (Connaraceae) 185216; Philodendron megalophyllum Schott (Araceae) 184899; Aniba fragrans Ducke (Lauraceae) 184897; Bellucia dichotoma Cogn. (Melastomataceae) 185213; Dipteryx odorata (Aubl.) Willd (Fabaceae) 184694; Annona montana Macfad. (Annonaceae) 185214; Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz var. ferrea (Fabaceae) 185211; Justicia pectoralis Jacq. (Acanthaceae) 184526; Cassia fistula, L. (Fabaceae) 184691; Kalanchoe brasiliensis Camb. (Crassulaceae) 184695 and Crataeva benthamii. (Capparaceae) 184823.
2.3. Preparation of the aqueous extracts Aqueous extracts were prepared from the part of the plant which the initial survey indicated was most frequently used by the population. After being cleaned and dried in a forced-air incubator at 40 °C (Solab, SL – 102/336, Piracicaba, SP, Brazil), the plants were comminuted with a cutting mill. The plant powders obtained were extracted with distilled water using a 1:5 ratio (w/v) at 70 °C for 2 h under constant shaking at 1,250 rpm and then filtered and lyophilized. The yield for each extract was calculated based on the moisture-free biomass.
2.4. Analysis of the phytochemical profile of the extracts using thin layer chromatography Phytochemical screening of the aqueous extracts was carried out using thin layer chromatography (TLC) as described by Marini-Bettolo et al. (1981). Specific color reagents and standards such as rutin, esculin and thymol were used to detect the following classes of secondary metabolites: fatty acids, anthraquinones, flavonoids, terpenoids, tannins and coumarins.
2.4. Colorimetric assays Total phenolic content in the aqueous extracts was determined by measuring absorbance at 510 nm in a Quimis Q 108U2VL spectrophotometer using FeCl3 (Vetec) as the chromogenic agent and a standard curve based on a serial dilution of tannic acid (Sigma) with concentrations ranging from 0.05 to 0.8 mg/mL (R=0.99) (Mole and Waterman, 1987). Total tannin content was measured by the Hagerman and Butler method (Mole and Waterman, 1987) after precipitation with bovine serum albumin (BSA, Sigma) in sodium acetate buffer pH 4.9; FeCl3 was used to develop the color. Absorbance was read at 510 nm using a standard curve (tannic acid 0.2 to 0.8 mg/mL, R=0.99). The concentration of hydrolyzable tannins was determined by complexing them with potassium iodate (Vetec), as described by Willis and Allen (1998). Absorbance was read at 550 nm, and tannic acid was used for the standard curve (0.05 to 0.6 mg/mL, R=0.99). The concentration of condensed tannins was determined following the method described by Sun et al. (1998) using vanillin (Vetec) in an acid medium to develop the color. Hydrated catechin (Sigma) was used for the standard curve (0.1 to 1.0 mg/mL, R=0.99), and absorbance was read at 500 nm. The test to determine the concentration of flavonoids was based on complexation of these compounds with AlCl3 (Vetec), as described by Chabariberi et al. (2009). Hydrated rutin (Sigma) was used for the standard curve (5 to 50 mg/mL, R= 0.99), and absorbance was read at 425 nm. All colorimetric assays were performed in triplicate. 2.5. Animals and venom The lyophilized B. jararaca venom was supplied by the Instituto Butantan, São Paulo, SP, Brazil. Male and female Swiss mice (34 to 41 g) were obtained from the breeding colony at the Universidade Federal do Oeste do Pará, Santarém, Pará, Brazil. The animals were kept under standard conditions (22 ± 1 °C, 12h/12h light/dark cycle) in standard cages with ad libitum food and water.
The experiments complied with the guidelines specified in federal law 11.794 and were approved by the Animal Ethics Committee at the Universidade do Estado do Pará under reference number 43/11.
2.6. Protocol to assess inhibition of the effects of venom by plant extracts Crude B. jararaca venom (1 mg/mL) was dissolved in saline solution (NaCl, 0.9%), and its protein concentration was determined by the Bradford method (1976). Venom solutions containing a fixed amount of protein were mixed in two venom-to-plant extract ratios [1:12 and 1:48 (w/w)]. All the mixtures were incubated at 37 °C for 30 minutes in the following groups: crude venom + saline solution (positive control), crude venom + plant extract (test) and plant extract + saline solution (negative control), n=4 per group.
2.7 Hemorrhagic activity Hemorrhage induced by B. jararaca venom was assessed following Kondo et al. (1960) with some modifications. Minimum hemorrhagic dose (MHD) was defined as the smallest amount of venom that could induce a 10 mm-diameter hemorrhagic lesion. To assess the inhibitory action of the plant extracts, mice were injected intradermally in the dorsal region with 50 µL of aqueous solution containing 10 µg of B. jararaca venom (2 x MHD) preincubated with the plant extracts at ratios of 1:12 and 1:48 (w/w). One hour after being injected the animals were sacrificed and the dorsal tissue removed and photographed. The images were digitalized, saved as RGB files and processed following Dougherty (2002) using a Matlab script (Gonzalez, 2009). Image components were selected with a threshold device (Guerra et al., 2011), and a morphological image processor
was used to obtain the haloes. The area (in mm2) and greatest diameter (in mm) of the haloes were measured following Dougherty and Lotufo (2003).
2.8 Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot Interaction between the plant extracts and the proteins in the venom was assessed by SDS-PAGE as described by Laemmli (1970) using a 12.5% resolving gel and 5% stacking gel. Aliquots of B. jararaca venom and plant extracts were incubated for 30 minutes at 37 °C in a venom-to-extract ratio of 1:48 (w/w) and analyzed under non-reducing conditions. After electrophoresis, one gel was used for Western blot and the other was stained with Coomassie Brilliant Blue (Bio-Rad Laboratories, CA, USA). For the Western blot, the proteins in the gel were transferred to a 0.45 µm nitrocellulose membrane (Bio-Rad Laboratories, CA, USA) in the Mini Trans-Blot® Cell system (Bio-Rad Laboratories, CA, USA) under a constant 200 mA current and refrigeration for 3 hours. Immediately after the transfer, the membrane was immersed overnight in blocking solution [TBS buffer containing 5% skim milk (Molico Nestlé, SP)] at 4 °C. Next, the membrane was incubated with Bothrops antivenom produced in horses (SAB, Instituto Butantan, São Paulo, SP, Brazil) at a 1:1000 dilution in TBS for 1 hour at room temperature under constant shaking. The membrane was then washed five times with saline solution and incubated with peroxidase-labeled rabbit anti-horse IgG antibody (Sigma-Aldrich, Israel) at a dilution of 1:1000 in TBS for 1 h at room temperature. Color was developed with DAB (3,3’- diaminobenzidine) and 30% H2O2. The reaction was stopped by successive washes with distilled water.
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September 15, 2014. Table 1. Plants used by residents of the São Pedro, Cucurunã and Alter do Chão communities in Santarém, PA, Brazil, to treat snakebite victims. Plant Common name no. 1 2
Açaí Algodão roxo
3
Amor crescido
4
Araticum
5 6
Canafístula Catauari
7 Cipó de Tracuá*
8
Corama*
9
Cumaru
10
Erva paracari*
11
Falso Boldo
12
Japana
13
Jucá
14 Macacaporanga* 15 Maniva de veado
16 17
Marrequinha Mutuquinha
18
Muúba*
Scientific name
Family
Arecaceae Gossypium Malvaceae hirsutum L. Portulaca Portulacaceae pilosa L. Annona Annonaceae montana Macfad. Cassia fístula Fabaceae Crataeva Capparaceae benthamii Eichler. Philodendron Araceae megalophyllum Schott Kalonchoe Crassulaceae brasiliensis Camb. Dipteryx Fabaceae odorata (Aubl.)Willd Marsypianthes Lamiaceae chamaedrys (Vahl) Kuntze Lamiaceae
Eupatorium Asteraceae triplinerve Vahl. Libidibia ferrea Fabaceae (Mart. ex Tul.) L.P Queiroz var. ferrea Aniba fragrans Lauraceae Ducke Manihot Euphorbiaceae esculenta Crantz Salvinia sp. Salvinaceae Justicia Acanthaceae pectoralis Jacq. Bellucia Melastomataceae dichotoma
Origin
Native
Part used
Method of preparation
Method of administration
Maceration
Topical
Naturalized
Unripe fruit Leaves
Juice
Oral
Native
Leaves
Decoction/infusion
Oral
Native
Leaves
Juice
Oral
Exotic Native
Seeds Leaves
Decoction Tincture
Oral Topical
Native
Vine
Maceration
Oral
Nativa
Leaves
Paste
Topical
Native
Seeds
Decoction
Oral
Native
Leaves
Maceration
Oral
Naturalized
Leaves
Decoction
Oral
Native
Leaves
Decoction
Oral
Native
Seeds
Decoction
Oral
Native
Bark
Decoction
Oral
Native
Leaves
Decoction
Oral
Exotic Native
Leaves Leaves
Decoction Infusion
Oral Oral
Native
Bark
Decoction
Oral
Cogn. Tabebuia Bignoniaceae barbata (E. Mey.) Sandwith 20 Perpétua do Alternanthera Amaranthaceae mato brasiliana (L.) Kuntze var. brasiliana Lippia grandis Verbenaceae 21 Salva de Marajó Schau. 22 Sarabatucu Machaerium Fabaceae ferox (Mart.ex Benth.)Ducke 23 Verônica* Connarus Connaraceae favosus Planch. 24 Vinhático* Plathymenia Fabaceae reticulata Benth. * Species with anti-snakebite properties tested in previous studies 19
Pau d’arco
Native
Leaves
Decoction
Oral
Native
Flower
Infusion
Oral
Native
Leaves
Decoction
Oral
Native
Leaves
Tincture
Topical
Native
Inner bark Bark
Tincture and Decoction Tincture
Oral
Native
Oral
Table 2. Phytochemical analysis of aqueous extracts of the twelve species selected. BD (Bellucia dichotomabark), AF (Aniba fragrans- bark), AM (Anonna montana- leaf), CF1 (Connarus favosus-bark), JP (Justicia pectoralis- leaf), PR (Plathymenia reticulata- bark), PM (Philodendron megalophyllum- vine), CF2 (Cassia fistula- seed), LF (Libidibia ferrea- seed), CB (Crataeva benthamii- leaf), KB (Kalanchoe brasiliensis- leaf and DO (Dipterix odorata- seed).
− − − − − − − − − − − − − −
− − − − − − − − − − − − − − − − − − − − − − − − + (positive); − (negative)
Table 3
Table 3. Total phenolics, total tannin, hydrolyzable tannin, condensed tannin and total flavonoid content in aqueous extracts of the twelve most frequently mentioned plant species.
Plant species
Total phenolics
Total tannins
Hydrolyzable tannins
Condensed tannins
Total flavonoids
Bellucia dichotoma Aniba fragrans Anonna montana Connarus favosus Justicia pectoralis Plathymenia reticulata Philodendron megalophyllum Cassia fistula Libidibia ferrea Crataeva benthamii Kalanchoe brasiliensis Dipterix odorata
17.74 ± 0.43 16.02 ± 0.70 5.20 ± 0.26 25.83 ± 0.45 8.55 ± 0.37 41.49 ± 2.16 13.96 ± 1.05
17.58 ± 0.59 14.89 ± 0.77 2.25 ± 0.06 24.40 ± 2.90 2.44 ± 0.11 32.75 ± 1.62 13.42 ± 1.12
35.32 ± 0.61 15.00 ± 0.08 ˂ LQ* 27.16 ± 2.95 < LQ* < LQ* 15.61 ± 1.1
54.97 ± 3.17 29.56 ± 1.13 ˂ LQ* 28.51 ± 3.16 ˂ LQ* 68.79 ± 0.49 29.57 ± 2.51
0.14 ± 0.03 0.50 ± 0.076 ˂ LQ* ˂ LQ* 2.21 ± 0.05 ˂ LQ* 1.68 ± 0.051
˂ LQ* ˂ LQ* ˂ LQ* ˂ LQ* < LQ*
0.63 ± 0.07 ˂ LQ* 3.53 ± 0.25 2.23 ± 0.48 ˂ LQ*
1.49 ± 0.07 1.51 ± 0.029 < LQ* 2.38 ± 0.19 1.60 ± 0.04 0.67 ± 0.02 8.60 ± 0.27 3.31 ± 0.08 < LQ* 1.83 ± 0.14 1.86 ± 0.01 < LQ* 2.48 ± 0.15 1.59 ± 0.19 1.24 ± 0.24 Results expressed in g per 100 g, dry basis. Mean ± standard deviation (n=3). * Concentration below the quantification limit of the method (0.03 mg/mL).
Table 4
Table 4: Inhibition by aqueous extracts of hemorrhage induced by B. jararaca venom. m
Values are given as mean ± SD, n=4 per group.
Diameter of the hemorrhagic lesion (mm)m
Inhibition of hemorrhage (%)m
1:12 (w/w)
1:12 (w/w)
1:48 (w/w)
100* 21 ± 2* 5.6 ± 0.9 100* 8.2 ± 0.8 100* 100* 12.9 ± 1* 6.6 ± 0.3 0.8 ± 2 40.5 ±2.8* 1.7 ± 2
100* 59 ± 0.4* 5.5 ± 2 100* 20.3* 100* 100* 20.9 ± 1* 9.3 ± 1 0 57 ± 0.6* 0.2 ± 4
Groups 1:48 (w/w)
B. jararaca + saline 10.21 ±0.13 10.21 ±0.13 B. dichotom + B. jararaca 0 0 A. fragrans + B. jararaca 8.07 ± 0.25 4.21 ± 0.04 A. montana + B. jararaca 9.64 ± 0.09 9.65 ± 0.29 C. favosus + B. jararaca 0 0 J. pectoralis + B. jararaca 9.37 ± 0.08 8.14 ± 0.16 P. reticulata + B. jararaca 0 0 P. megalophyllum + B. jararaca 0 0 C. fistula + B. jararaca 8.89 ± 0.11 8.07 ± 0.14 L. ferrea + B. jararaca 9.53 ± 0.03 9.26 ±0.10 C.benthamii + B. jararaca 10.12 ± 0.25 10.21 ± 0.33 K. brasiliensis + B. jararaca 6.08 ± 0.29 4.39 ± 0.06 D. odorata + B. jararaca 10.09 ± 0.20 10.19 ± 0.40 Dunnett’s test *p ˂ 0.05 vs. control (B. jararaca venom).
The venom and extract were preincubated for 30 minutes at 37 °C at venom-to-extract ratios of 1:12 and 1:48.
Graphical Abstract (for review)
Plants used to treat snakebites in Santarém, western Pará, Brazil: an assessment of their effectiveness in inhibiting hemorrhagic activity induced by Bothrops jararaca venom Valéria Mourão de Moura, Luciana A. Freitas de Sousa, Maria Cristina Dos-Santos, Juliana Divina Almeida Raposo, Aline Evangelista Lima, Ricardo Bezerra de Oliveira, Milton Nascimento da Silva, Rosa Helena Veras Mourão
PII: DOI: Reference:
S0378-8741(14)00901-5 http://dx.doi.org/10.1016/j.jep.2014.12.020 JEP9194
To appear in:
Journal of Ethnopharmacology
www.elsevier.com/locate/jep
Received date: 11 October 2014 Revised date: 7 December 2014 Accepted date: 14 December 2014 Cite this article as: Valéria Mourão de Moura, Luciana A. Freitas de Sousa, Maria Cristina Dos-Santos, Juliana Divina Almeida Raposo, Aline Evangelista Lima, Ricardo Bezerra de Oliveira, Milton Nascimento da Silva, Rosa Helena Veras Mourão, Plants used to treat snakebites in Santarém, western Pará, Brazil: an assessment of their effectiveness in inhibiting hemorrhagic activity induced by Bothrops jararaca venom, Journal of Ethnopharmacology, http://dx.doi. org/10.1016/j.jep.2014.12.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Plants used to treat snakebites in Santarém, western Pará, Brazil: an assessment of their effectiveness in inhibiting hemorrhagic activity induced by Bothrops jararaca venom
Valéria Mourão de Mouraa,b*, Luciana A. Freitas de Sousaa, Maria Cristina Dos-Santosb, Juliana Divina Almeida Raposoa, Aline Evangelista Limaa , Ricardo Bezerra de Oliveiraa, Milton Nascimento da Silvad, Rosa Helena Veras Mourãoa a
Programa de Pós-Graduação em Recursos Naturais da Amazônia, Laboratório de
Bioprospecção e Biologia Experimental, Universidade Federal do Oeste do Pará - UFOPA, Rua Vera Paz, s/n, 68035-110, Santarém, PA, Brazil; b
Programa Multi-Institucional de Pós-Graduação em Biotecnologia, Laboratório de
Imunologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas- UFAM, Av. Rodrigo Octávio Ramos, 3000, Manaus, AM, Brazil; d
Programa de Pós-Graduação em Química, Universidade Federal do Pará – UFPA, Rua
Augusto Corrêa, 66075-110, Belém, PA, Brazil.
*Corresponding author: Tel.: +5509281641596; E-mail – [email protected] Programa Multi-institucional de Pós-graduação em Biotecnologia, Laboratório de Imunologia, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Amazonas, Av. General Octávio Jordão Ramos, 3000, Japiim, CEP 69057-000.
Abstract Ethnopharmacological importance: The poor distribution and limited availability of antivenoms in Brazil has led to greater use of plants to treat snakebites. Very often such plants are the only alternative available to riverside communities. Material and methods: Direct questionnaire-based interviews were conducted with members of the Cucurunã, São Pedro and Alter do Chão communities in Santarém, Pará, Brazil. For each of the twelve most frequently mentioned species aqueous extracts were prepared and the phytochemical profiles determined by thin layer chromatography. The concentrations of phenolic compounds (tannins and flavonoids) in the aqueous extracts were determined by colorimetric assays. To assess inhibition of the hemorrhagic activity of Bothrops jararaca venom, solutions containing the venom mixed with aqueous extracts in the ratios 1:12 and 1:48 were tested (w/w). SDS-PAGE and Western blot were used to assess the action of the extracts on B. jararaca venom. Results: In all, 24 plants belonging to 19 families were mentioned in the survey as being used to treat snakebites. Leaves (84%), seeds (60.9%) and inner bark (53%) were cited as the most frequently used parts in folk remedies, which were usually prepared in the form of a decoction (62.5%), tincture (45%) or maceration (22.5%). Hemorrhage induced by B. jararaca venom was completely inhibited by aqueous extracts of Bellucia dichotoma, Connarus favosus, Plathymenia reticulata and Philodendron megalophyllum, which had a high phenolic content and contained condensed and hydrolyzable tannins. The results of SDS-PAGE showed that some venom protein bands were not visible when the venom was preincubated with the extracts that had completely inhibited hemorrhagic activity of the venom. Western blot showed that the extracts did not have any enzymatic action on the proteins in the venom as it failed to detect low-molecular-weight bands, which are indicative of possible enzymatic cleavage.
Conclusion: Traditional use of plants to treat snakebites is common practice in the western region of Pará, Brazil. Our findings show that some plant extracts were able to inhibit snake venom-induced hemorrhage in vitro. In vivo studies are being carried out to validate the traditional use of these species to treat snakebites. Keywords: Ethnobotany, plant extracts, snakebite envenomation, anti-snakebite plants, hemorrhage.
1. Introduction According to official estimates, snakebites affect around 240,000 people worldwide and account for 20,000 deaths. However, if undernotification is taken into consideration, these figures could reach 1,841,000 and 94,000, respectively (Kasturiratne et al., 2008). In light of these data, the World Health Organization granted snakebite envenomation neglected-disease status in 2009. In Brazil, approximately 90% of snakebites reported to the Ministry of Health were caused by snakes of genus Bothrops sp. (Ministério da Saúde do Brasil, 2010). The Northern region of the country has the highest incidence, and Pará is the state with highest number of reported cases (Bochner and Struchiner, 2003; Araújo et al., 2003). Of all the municipalities in the state, Santarém has the greatest number of reported cases, with an annual average of 755 cases between 2011 and 2013 (Sistema de Informações de Agravos de NotificaçãoSINAN, 2013). These numbers may in reality be higher because of undernotification as a result of the difficulty snakebite victims have getting medical attention and the precarious nature of the system for reporting notifiable diseases. Bothrops jararaca is the species that causes the greatest number of snakebites in the Southeast of Brazil (Ministério da Saúde do Brasil, 2005). Envenomation by snakes of the genus Bothrops causes immediate local effects such as pain, edema, hemorrhage and
myonecrosis, as well as systemic reactions, most notably coagulant and hemorrhagic effects (Cardoso et al., 2003). Hemorrhage is one of the most serious symptoms of envenomation by snakes from genus Bothrops and is induced by metalloproteinases. These hemorrhagic toxins play an important role in vascular damage and the subsequent generation of ischemic zones, which are the main cause of necrosis of local tissue, potentially leading to amputation of the affected member (Gutiérrez, 1998; Warrell, 1992). A major problem in this type of envenomation is that the local lesion, once induced, cannot be reversed by standard Bothrops antivenom (Baldo et al., 2010). Hence, it is important to study natural inhibitors that could be used in combination with serum therapy to treat the local effects of snakebites. In the Amazon region, the use of plant preparations to treat snakebites is quite common and indeed very often the only option. Some medicinal plants are a source of bioactive compounds with the potential to directly inhibit some of the activities induced by snake venom. Many of these plants have been studied in order to validate their use in popular medicine (Borges et al., 1996; Magalhães et al., 2011; Sousa et al., 2013; Moura et al., 2014) or to isolate and characterize these bioactive components (Mors et al. 2000, Ambikabothy et al., 2011; Krishnan et al., 2014). The vast majority of studies of plants with anti-snakebite properties are based on folk medicine. Although there are some studies on the ethnobotany of species used to treat snakebites, few scientific studies to date have validated the anti-snakebite properties of aqueous extracts or compounds from these plant species. This study therefore sought not only to recover and preserve traditional knowledge about the use of plants in the treatment of snakebites in the western region of Pará, Brazil, but also to evaluate the extent to which aqueous extracts of such plants can inhibit the hemorrhagic activity induced by B. jararaca venom as well as to determine the phytochemical profile of these extracts by thin layer chromatography.
2. Materials and Methods 2.1. Ethnobotanical survey An ethnobotanical survey was carried out into the use of medicinal plants with antisnakebite properties in the following communities in Santarém in western Pará, Brazil, with a high incidence of snakebite accidents: São Pedro (02º32´08.9” S and 54º54´23.9” W), Cucurunã (02º27´21.0” S and 54º47´45.7” W), Fazenda Experimental Carauá (02º33´99.3” S and 54º36´61.2” W) and Alter do Chão (02030´53.3” S and 54057´00.1” W) (Figure 1). Collections points were identified with the aid of a global positioning system (GPS – GARMIN, GPSMap 60CSx, Kansas, USA). Between January and July 2010, an open-ended questionnaire on the use of plants with anti-snakebite properties was applied in direct interviews. Interviewees were selected by the residents of the communities themselves and were generally people who worked as plant healers and faith healers or were knowledgeable about the plants used in the region. They were told about the importance of the study, and those who agreed to take part signed a voluntary informed-consent form. The questions in the questionnaire were about the frequency of snakebite accidents in the interviewees' communities and treatment of snakebites with plant extracts, with a particular emphasis on the part of the plant used, the form in which it is used (such as decoction and maceration) and the provenance of the plant.
Fig. 1. Study Area. A) Location of Brazil in South America; B) Political map of Brazil showing the state of Pará in red; C) Political map of Pará showing the western region of the state and the municipalities in this region; D) Map of Santarém. Prepared by Fabio Guerra Santos, Forestry Engineer at the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA).
2.2. Collection and identification of plant material The most frequently mentioned species were collected and identified. After drying, extracts were prepared so that their phytochemistry and their effectiveness in inhibiting hemorrhage induced by B. jararaca venom could be studied. Dried specimens were also deposited in the Embrapa Western Amazonia Herbarium, Belém, with the following (IAN)
codes: Plathymenia reticulata Benth. (Fabaceae) 185215; Connarus favosus Planch. (Connaraceae) 185216; Philodendron megalophyllum Schott (Araceae) 184899; Aniba fragrans Ducke (Lauraceae) 184897; Bellucia dichotoma Cogn. (Melastomataceae) 185213; Dipteryx odorata (Aubl.) Willd (Fabaceae) 184694; Annona montana Macfad. (Annonaceae) 185214; Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz var. ferrea (Fabaceae) 185211; Justicia pectoralis Jacq. (Acanthaceae) 184526; Cassia fistula, L. (Fabaceae) 184691; Kalanchoe brasiliensis Camb. (Crassulaceae) 184695 and Crataeva benthamii. (Capparaceae) 184823.
2.3. Preparation of the aqueous extracts Aqueous extracts were prepared from the part of the plant which the initial survey indicated was most frequently used by the population. After being cleaned and dried in a forced-air incubator at 40 °C (Solab, SL – 102/336, Piracicaba, SP, Brazil), the plants were comminuted with a cutting mill. The plant powders obtained were extracted with distilled water using a 1:5 ratio (w/v) at 70 °C for 2 h under constant shaking at 1,250 rpm and then filtered and lyophilized. The yield for each extract was calculated based on the moisture-free biomass.
2.4. Analysis of the phytochemical profile of the extracts using thin layer chromatography Phytochemical screening of the aqueous extracts was carried out using thin layer chromatography (TLC) as described by Marini-Bettolo et al. (1981). Specific color reagents and standards such as rutin, esculin and thymol were used to detect the following classes of secondary metabolites: fatty acids, anthraquinones, flavonoids, terpenoids, tannins and coumarins.
2.4. Colorimetric assays Total phenolic content in the aqueous extracts was determined by measuring absorbance at 510 nm in a Quimis Q 108U2VL spectrophotometer using FeCl3 (Vetec) as the chromogenic agent and a standard curve based on a serial dilution of tannic acid (Sigma) with concentrations ranging from 0.05 to 0.8 mg/mL (R=0.99) (Mole and Waterman, 1987). Total tannin content was measured by the Hagerman and Butler method (Mole and Waterman, 1987) after precipitation with bovine serum albumin (BSA, Sigma) in sodium acetate buffer pH 4.9; FeCl3 was used to develop the color. Absorbance was read at 510 nm using a standard curve (tannic acid 0.2 to 0.8 mg/mL, R=0.99). The concentration of hydrolyzable tannins was determined by complexing them with potassium iodate (Vetec), as described by Willis and Allen (1998). Absorbance was read at 550 nm, and tannic acid was used for the standard curve (0.05 to 0.6 mg/mL, R=0.99). The concentration of condensed tannins was determined following the method described by Sun et al. (1998) using vanillin (Vetec) in an acid medium to develop the color. Hydrated catechin (Sigma) was used for the standard curve (0.1 to 1.0 mg/mL, R=0.99), and absorbance was read at 500 nm. The test to determine the concentration of flavonoids was based on complexation of these compounds with AlCl3 (Vetec), as described by Chabariberi et al. (2009). Hydrated rutin (Sigma) was used for the standard curve (5 to 50 mg/mL, R= 0.99), and absorbance was read at 425 nm. All colorimetric assays were performed in triplicate. 2.5. Animals and venom The lyophilized B. jararaca venom was supplied by the Instituto Butantan, São Paulo, SP, Brazil. Male and female Swiss mice (34 to 41 g) were obtained from the breeding colony at the Universidade Federal do Oeste do Pará, Santarém, Pará, Brazil. The animals were kept under standard conditions (22 ± 1 °C, 12h/12h light/dark cycle) in standard cages with ad libitum food and water.
The experiments complied with the guidelines specified in federal law 11.794 and were approved by the Animal Ethics Committee at the Universidade do Estado do Pará under reference number 43/11.
2.6. Protocol to assess inhibition of the effects of venom by plant extracts Crude B. jararaca venom (1 mg/mL) was dissolved in saline solution (NaCl, 0.9%), and its protein concentration was determined by the Bradford method (1976). Venom solutions containing a fixed amount of protein were mixed in two venom-to-plant extract ratios [1:12 and 1:48 (w/w)]. All the mixtures were incubated at 37 °C for 30 minutes in the following groups: crude venom + saline solution (positive control), crude venom + plant extract (test) and plant extract + saline solution (negative control), n=4 per group.
2.7 Hemorrhagic activity Hemorrhage induced by B. jararaca venom was assessed following Kondo et al. (1960) with some modifications. Minimum hemorrhagic dose (MHD) was defined as the smallest amount of venom that could induce a 10 mm-diameter hemorrhagic lesion. To assess the inhibitory action of the plant extracts, mice were injected intradermally in the dorsal region with 50 µL of aqueous solution containing 10 µg of B. jararaca venom (2 x MHD) preincubated with the plant extracts at ratios of 1:12 and 1:48 (w/w). One hour after being injected the animals were sacrificed and the dorsal tissue removed and photographed. The images were digitalized, saved as RGB files and processed following Dougherty (2002) using a Matlab script (Gonzalez, 2009). Image components were selected with a threshold device (Guerra et al., 2011), and a morphological image processor
was used to obtain the haloes. The area (in mm2) and greatest diameter (in mm) of the haloes were measured following Dougherty and Lotufo (2003).
2.8 Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot Interaction between the plant extracts and the proteins in the venom was assessed by SDS-PAGE as described by Laemmli (1970) using a 12.5% resolving gel and 5% stacking gel. Aliquots of B. jararaca venom and plant extracts were incubated for 30 minutes at 37 °C in a venom-to-extract ratio of 1:48 (w/w) and analyzed under non-reducing conditions. After electrophoresis, one gel was used for Western blot and the other was stained with Coomassie Brilliant Blue (Bio-Rad Laboratories, CA, USA). For the Western blot, the proteins in the gel were transferred to a 0.45 µm nitrocellulose membrane (Bio-Rad Laboratories, CA, USA) in the Mini Trans-Blot® Cell system (Bio-Rad Laboratories, CA, USA) under a constant 200 mA current and refrigeration for 3 hours. Immediately after the transfer, the membrane was immersed overnight in blocking solution [TBS buffer containing 5% skim milk (Molico Nestlé, SP)] at 4 °C. Next, the membrane was incubated with Bothrops antivenom produced in horses (SAB, Instituto Butantan, São Paulo, SP, Brazil) at a 1:1000 dilution in TBS for 1 hour at room temperature under constant shaking. The membrane was then washed five times with saline solution and incubated with peroxidase-labeled rabbit anti-horse IgG antibody (Sigma-Aldrich, Israel) at a dilution of 1:1000 in TBS for 1 h at room temperature. Color was developed with DAB (3,3’- diaminobenzidine) and 30% H2O2. The reaction was stopped by successive washes with distilled water.
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September 15, 2014. Table 1. Plants used by residents of the São Pedro, Cucurunã and Alter do Chão communities in Santarém, PA, Brazil, to treat snakebite victims. Plant Common name no. 1 2
Açaí Algodão roxo
3
Amor crescido
4
Araticum
5 6
Canafístula Catauari
7 Cipó de Tracuá*
8
Corama*
9
Cumaru
10
Erva paracari*
11
Falso Boldo
12
Japana
13
Jucá
14 Macacaporanga* 15 Maniva de veado
16 17
Marrequinha Mutuquinha
18
Muúba*
Scientific name
Family
Arecaceae Gossypium Malvaceae hirsutum L. Portulaca Portulacaceae pilosa L. Annona Annonaceae montana Macfad. Cassia fístula Fabaceae Crataeva Capparaceae benthamii Eichler. Philodendron Araceae megalophyllum Schott Kalonchoe Crassulaceae brasiliensis Camb. Dipteryx Fabaceae odorata (Aubl.)Willd Marsypianthes Lamiaceae chamaedrys (Vahl) Kuntze Lamiaceae
Eupatorium Asteraceae triplinerve Vahl. Libidibia ferrea Fabaceae (Mart. ex Tul.) L.P Queiroz var. ferrea Aniba fragrans Lauraceae Ducke Manihot Euphorbiaceae esculenta Crantz Salvinia sp. Salvinaceae Justicia Acanthaceae pectoralis Jacq. Bellucia Melastomataceae dichotoma
Origin
Native
Part used
Method of preparation
Method of administration
Maceration
Topical
Naturalized
Unripe fruit Leaves
Juice
Oral
Native
Leaves
Decoction/infusion
Oral
Native
Leaves
Juice
Oral
Exotic Native
Seeds Leaves
Decoction Tincture
Oral Topical
Native
Vine
Maceration
Oral
Nativa
Leaves
Paste
Topical
Native
Seeds
Decoction
Oral
Native
Leaves
Maceration
Oral
Naturalized
Leaves
Decoction
Oral
Native
Leaves
Decoction
Oral
Native
Seeds
Decoction
Oral
Native
Bark
Decoction
Oral
Native
Leaves
Decoction
Oral
Exotic Native
Leaves Leaves
Decoction Infusion
Oral Oral
Native
Bark
Decoction
Oral
Cogn. Tabebuia Bignoniaceae barbata (E. Mey.) Sandwith 20 Perpétua do Alternanthera Amaranthaceae mato brasiliana (L.) Kuntze var. brasiliana Lippia grandis Verbenaceae 21 Salva de Marajó Schau. 22 Sarabatucu Machaerium Fabaceae ferox (Mart.ex Benth.)Ducke 23 Verônica* Connarus Connaraceae favosus Planch. 24 Vinhático* Plathymenia Fabaceae reticulata Benth. * Species with anti-snakebite properties tested in previous studies 19
Pau d’arco
Native
Leaves
Decoction
Oral
Native
Flower
Infusion
Oral
Native
Leaves
Decoction
Oral
Native
Leaves
Tincture
Topical
Native
Inner bark Bark
Tincture and Decoction Tincture
Oral
Native
Oral
Table 2. Phytochemical analysis of aqueous extracts of the twelve species selected. BD (Bellucia dichotomabark), AF (Aniba fragrans- bark), AM (Anonna montana- leaf), CF1 (Connarus favosus-bark), JP (Justicia pectoralis- leaf), PR (Plathymenia reticulata- bark), PM (Philodendron megalophyllum- vine), CF2 (Cassia fistula- seed), LF (Libidibia ferrea- seed), CB (Crataeva benthamii- leaf), KB (Kalanchoe brasiliensis- leaf and DO (Dipterix odorata- seed).
− − − − − − − − − − − − − −
− − − − − − − − − − − − − − − − − − − − − − − − + (positive); − (negative)
Table 3
Table 3. Total phenolics, total tannin, hydrolyzable tannin, condensed tannin and total flavonoid content in aqueous extracts of the twelve most frequently mentioned plant species.
Plant species
Total phenolics
Total tannins
Hydrolyzable tannins
Condensed tannins
Total flavonoids
Bellucia dichotoma Aniba fragrans Anonna montana Connarus favosus Justicia pectoralis Plathymenia reticulata Philodendron megalophyllum Cassia fistula Libidibia ferrea Crataeva benthamii Kalanchoe brasiliensis Dipterix odorata
17.74 ± 0.43 16.02 ± 0.70 5.20 ± 0.26 25.83 ± 0.45 8.55 ± 0.37 41.49 ± 2.16 13.96 ± 1.05
17.58 ± 0.59 14.89 ± 0.77 2.25 ± 0.06 24.40 ± 2.90 2.44 ± 0.11 32.75 ± 1.62 13.42 ± 1.12
35.32 ± 0.61 15.00 ± 0.08 ˂ LQ* 27.16 ± 2.95 < LQ* < LQ* 15.61 ± 1.1
54.97 ± 3.17 29.56 ± 1.13 ˂ LQ* 28.51 ± 3.16 ˂ LQ* 68.79 ± 0.49 29.57 ± 2.51
0.14 ± 0.03 0.50 ± 0.076 ˂ LQ* ˂ LQ* 2.21 ± 0.05 ˂ LQ* 1.68 ± 0.051
˂ LQ* ˂ LQ* ˂ LQ* ˂ LQ* < LQ*
0.63 ± 0.07 ˂ LQ* 3.53 ± 0.25 2.23 ± 0.48 ˂ LQ*
1.49 ± 0.07 1.51 ± 0.029 < LQ* 2.38 ± 0.19 1.60 ± 0.04 0.67 ± 0.02 8.60 ± 0.27 3.31 ± 0.08 < LQ* 1.83 ± 0.14 1.86 ± 0.01 < LQ* 2.48 ± 0.15 1.59 ± 0.19 1.24 ± 0.24 Results expressed in g per 100 g, dry basis. Mean ± standard deviation (n=3). * Concentration below the quantification limit of the method (0.03 mg/mL).
Table 4
Table 4: Inhibition by aqueous extracts of hemorrhage induced by B. jararaca venom. m
Values are given as mean ± SD, n=4 per group.
Diameter of the hemorrhagic lesion (mm)m
Inhibition of hemorrhage (%)m
1:12 (w/w)
1:12 (w/w)
1:48 (w/w)
100* 21 ± 2* 5.6 ± 0.9 100* 8.2 ± 0.8 100* 100* 12.9 ± 1* 6.6 ± 0.3 0.8 ± 2 40.5 ±2.8* 1.7 ± 2
100* 59 ± 0.4* 5.5 ± 2 100* 20.3* 100* 100* 20.9 ± 1* 9.3 ± 1 0 57 ± 0.6* 0.2 ± 4
Groups 1:48 (w/w)
B. jararaca + saline 10.21 ±0.13 10.21 ±0.13 B. dichotom + B. jararaca 0 0 A. fragrans + B. jararaca 8.07 ± 0.25 4.21 ± 0.04 A. montana + B. jararaca 9.64 ± 0.09 9.65 ± 0.29 C. favosus + B. jararaca 0 0 J. pectoralis + B. jararaca 9.37 ± 0.08 8.14 ± 0.16 P. reticulata + B. jararaca 0 0 P. megalophyllum + B. jararaca 0 0 C. fistula + B. jararaca 8.89 ± 0.11 8.07 ± 0.14 L. ferrea + B. jararaca 9.53 ± 0.03 9.26 ±0.10 C.benthamii + B. jararaca 10.12 ± 0.25 10.21 ± 0.33 K. brasiliensis + B. jararaca 6.08 ± 0.29 4.39 ± 0.06 D. odorata + B. jararaca 10.09 ± 0.20 10.19 ± 0.40 Dunnett’s test *p ˂ 0.05 vs. control (B. jararaca venom).
The venom and extract were preincubated for 30 minutes at 37 °C at venom-to-extract ratios of 1:12 and 1:48.
Graphical Abstract (for review)
