Comparison of two Jatropha species (Euphorbiaceae) used popularly to treat snakebites in Northeastern Brazil: Chemical profile, inhibitory activity against Bothrops erythromelas venom and antibacterial activity

Author’s Accepted Manuscript Comparison of two Jatropha species (Euphorbiaceae) used popularly to treat snakebites in Northeastern Brazil: Chemical profile, inhibitory activity against Bothrops erythromelas venom and antibacterial activity Juliana Félix-Silva, Jacyra A.S. Gomes, Júlia M. Fernandes, Angela K.C. Moura, Yamara A.S. Menezes, Elizabeth C.G. Santos, Denise V. Tambourgi, Arnóbio A. Silva-Junior, Silvana M. Zucolotto, Matheus F. Fernandes-Pedrosa

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To appear in: Journal of Ethnopharmacology Received date: 23 August 2017 Revised date: 1 November 2017 Accepted date: 2 November 2017 Cite this article as: Juliana Félix-Silva, Jacyra A.S. Gomes, Júlia M. Fernandes, Angela K.C. Moura, Yamara A.S. Menezes, Elizabeth C.G. Santos, Denise V. Tambourgi, Arnóbio A. Silva-Junior, Silvana M. Zucolotto and Matheus F. Fernandes-Pedrosa, Comparison of two Jatropha species (Euphorbiaceae) used popularly to treat snakebites in Northeastern Brazil: Chemical profile, inhibitory activity against Bothrops erythromelas venom and antibacterial activity, Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2017.11.002 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.

1 Comparison of two Jatropha species (Euphorbiaceae) used popularly to treat snakebites in Northeastern Brazil: chemical profile, inhibitory activity against Bothrops erythromelas venom and antibacterial activity

Juliana Félix-Silvaa,b,1, Jacyra A. S. Gomesa,b,1, Júlia M. Fernandesc, Angela K. C. Mouraa,b, Yamara A. S. Menezesa,b, Elizabeth C. G. Santosb, Denise V. Tambourgid, Arnóbio A. Silva-Juniora, Silvana M. Zucolottoc, Matheus F. Fernandes-Pedrosaa,b,*

a

Laboratório de Tecnologia & Biotecnologia Farmacêutica (TecBioFar), Faculdade de Farmácia, Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN, Brazil b Laboratório de Biotecnologia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN, Brazil c Grupo de Pesquisa em Produtos Naturais Bioativos (PNBio), Laboratório de Farmacognosia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN, Brazil d Laboratório de Imunoquímica, Instituto Butantan, São Paulo, SP, Brazil

* Corresponding author. Av. Gal. Cordeiro de Farias, s/n, Petrópolis, CEP 59012-570, Natal, RN, Brazil. Tel: + 55 84 33429820. Fax: + 55 84 33429822. E-mail addresses: [email protected] (J. Félix-Silva), [email protected] (J. A. S. Gomes), [email protected] (J. M. Fernandes), [email protected] (A. K. C. Moura), [email protected] (Y. A. S. Menezes), [email protected] (E. C. G. Santos), [email protected] (D. V. Tambourgi), [email protected] (A. A. Silva-Junior), [email protected] (S. M. Zucolotto), [email protected] (M. F. FernandesPedrosa). 1 These authors contributed equally to this work.

2 ABSTRACT Ethnopharmacological relevance: Jatropha species (Euphorbiaceae) are largely used in traditional medicine to treat different pathologies in Africa, Asia and Latin America. In Northeastern Brazilian folk medicine, several Jatropha species, such as Jatropha gossypiifolia L. and Jatropha mollissima (Pohl) Baill., are indistinctly used to treat snakebites. Aim of the study: To compare two of the Brazilian most used Jatropha species for snakebites (J. gossypiifolia and J. mollissima), in relation to their ability to inhibit local edematogenic activity of Bothrops erythromelas snake venom in mice, their in vitro antibacterial activity and phytochemical profile. Material and methods: Aqueous leaf extracts of J. gossypiifolia (AEJg) and J. mollissima (AEJm) were prepared by decoction. AEJg and AEJm were compared chemically, by thin layer chromatography (TLC) and high-performance liquid chromatography with diode array detection (HPLC-DAD) analysis. They were also pharmacologically compared, using the mouse model of paw edema induced by Bothrops erythromelas snake venom (BeV), and in vitro by broth microdilution and agar dilution antimicrobial tests. Results: Flavonoids were detected as the major compounds in both extracts. However, AEJg and AEJm showed quantitatively different chemical profiles by HPLC-DAD. AEJg presented fewer peaks of flavonoids than AEJm, however, when the intensity of peaks were analyzed, these compounds were at high concentration in AEJg, even using the same concentration of both extracts. Differences were also observed in the biological activity of the two extracts. While no difference was observed when the extracts were administered by oral route (P>0.05), by the intraperitoneal route AEJg presented anti-edematogenic activity significantly (P<0.001) higher than AEJm. In antimicrobial assays, only AEJg presented antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and Bacillus cereus. Conclusions: Although used indistinctly by folk medicine, our results suggested that AEJg is more active than AEJm in relation to its antiedematogenic and antibacterial activities. Significant differences were observed in their phytochemical profiles, especially a higher content of Cglycosylated flavonoids in the most active species, which could justify the different biological effects observed. These findings strengthen the potentiality of J. gossypiifolia species for use as complementary treatment for local effects induced by Bothrops venoms and could be helpful for distinction of the species and control quality assessment of future herbal medicines based on Jatropha plants. Keywords: Euphorbiaceae; Jatropha gossypiifolia; Jatropha mollissima; Bothrops erythromelas; antiophidic activity; antibacterial activity; Northeastern Brazil folk medicine; flavonoids. Chemical compounds studied in this article: Isoorientin (PubChem CID: 114776); Isovitexin (PubChem CID: 162350); Orientin (PubChem CID: 5281675); Vitexin (PubChem CID: 5280441). Abbreviations: AEJg, aqueous extract of Jatropha gossypiifolia; AEJm, aqueous extract of Jatropha mollissima; ATCC, American type culture collection; ANOVA, analysis of variance; AUC0-120 min, area under the time-course curves after 120 min; BeV, Bothrops erythromelas venom; CGEN, Brazilian Genetic Heritage Management Council; CLSI, Clinical and Laboratory Standards Institute; CIOMS, Council of International Organizations of Medical Sciences; CONCEA, National Council for the Control of Animal Experimentation of Brazil; HEK-293, human embryonic kidney 293 cells; HPLCDAD, high-performance liquid chromatography with diode array detection; HPLC-MS, high

3 performance liquid chromatography coupled to mass spectrometry; i.p., intraperitoneal route; mAU, miliabsorbance units; MIC, minimum inhibitory concentration; MPO, myeloperoxidase; PBS, phosphate buffered saline; p.o., oral route; RENISUS, National List of Medicinal Plants of Interest to Brazilian Public Health System; SEM, standard error of mean; SISBIO, Brazilian Biodiversity Authorization and Information System; TLC, thin layer chromatography; Rt, retention time.

4 1. Introduction Euphorbiaceae is considered one of the largest families of Angiosperms, covering about 7,800 species distributed in approximately 300 genera and 5 subfamilies around the world. Among the main genera belonging to this family, there is Jatropha L., which belongs to the Crotonoideae subfamily, Jatropheae tribe and is represented by about 200 species, widely distributed in tropical and subtropical regions of Africa and Americas (Félix-Silva et al., 2014a; Webster, 1994). In traditional medicine, Jatropha species are frequently used to treat various clinical conditions in Africa, Asia and Latin America (Sabandar et al., 2013; Sharma and Singh, 2012). In fact, the name “Jatropha” is derived from the Greek words “jatros”, which means “doctor”, and “trophe”, meaning “food”, which could be associated with its popular medicinal uses (Sabandar et al., 2013). Several species from this genus have been studied in relation to its medicinal uses, chemical constituents and biological activities, such as Jatropha gossypiifolia, Jatropha mollissima, Jatropha curcas, Jatropha elliptica, among others (Félix-Silva et al., 2014a; Gomes et al., 2016; Sabandar et al., 2013; Sharma and Singh, 2012). In Brazil, especially in the Northeastern Region, several Jatropha plants are popularly known as “pinhão”, being this genus one of the richest in number of species in this Region (Albuquerque et al., 2007). Jatropha species are often indicated in Brazilian traditional medicine from this Region as antidotes for snakebites, for treatment of both animals and humans, being often used orally and/or applied topically at the site of the bite (Agra et al., 2008; Albuquerque and Andrade, 2002; Albuquerque et al., 2007; Félix-Silva et al., 2017b). Interestingly, the popular use of Jatropha species is a known example of Zoopharmacognosy, since it is common the existence of reports by local people about the behavior of reptiles that ingest Jatropha plants before facing snakes, what the population associates with possible beneficial effects of these plants as antidotes for snakebites (Albuquerque and Andrade, 2002). Moreover, it is interesting to mention that these species are indicated as antiophidic not only in the Northeast of Brazil, but also in other regions of the Country and even in other continents, such as Asia and Africa (Coelho-Ferreira, 2009; Dharmadasa et al., 2016; Di Stasi and Hiruma-Lima, 2002; Félix-Silva et al., 2017b; Kadir et al., 2015; Sharma and Devi, 2013). Snake envenoming is a serious public health problem, especially in tropical and subtropical regions of the world. In Brazil, almost 90% of the snakebites are provoked by Bothrops snakes (Brasil, 2016). Bothropic envenomation is mainly characterized by immediate and intense development of local tissue damage, which may occasionally lead to permanent incapacitations of the affected members of the victims (Brasil, 2010; Gutiérrez and Lomonte, 1989). In addition, bacterial infection secondary to snakebites is a common complication in the victims, which can lead to the formation of abscesses, and it is, therefore, an important risk factor for amputations (Hearn et al., 2015; Jorge and Ribeiro, 1997). The specific treatment for bothropic envenoming is the intravenous administration of antibothropic serum, which is produced in Brazil through the hyperimmunization of horses with a pool of Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops alternatus and Bothrops newiedi snake venoms, in order to neutralize all snakes of the genus Bothrops distributed in Brazil, given the expected cross reactivity between the toxins of different bothropic venoms (Brasil, 2010). However, some previous studies have shown pre-clinical evidence that this antivenom may not fully neutralize the toxic activities induced by all bothropic venoms, suggesting that new strategies should be developed for the preparation of a universal bothropic antivenom (Félix-Silva et al., 2017a; Muniz et al., 2000; Queiroz et al., 2008). This situation is particularly relevant in Brazilian Northeastern Region, since the main Bothrops snake responsible for snakebites in this region is Bothrops erythromelas (Amaral, 1923), a venomous species popularly

5 known as Caatinga lancehead, which is not included in the antigenic mixture used for production of bothropic antivenom distributed around the country (Félix-Silva et al., 2017a; Lira-Da-Silva et al., 2009). Furthermore, the antivenom is able to neutralize venom toxins, but it is not able to reverse tissue damage eventually caused by endogenous mediators released in response to venom components, which makes this treatment, in most cases, little effective against local tissue damage caused by snakebite (Gutiérrez and Lomonte, 1989; Gutiérrez et al., 2006). In addition to the low inhibition of local tissue damage, there is a certain risk of developing adverse immune reactions, high production costs and very difficult access in some regions (Gutiérrez et al., 2011; Silva et al., 2015). Thus, the search for complementary therapies to antivenom serum therapy becomes relevant, and in this context, the medicinal plants stand out as strong candidates (Félix-Silva et al., 2017b; Mors et al., 2000; Santhosh et al., 2013). Jatropha plants have been extensively studied in our research group aiming their potential application as antiophidic agents, especially the species J. gossypiifolia and J. mollissima, which demonstrated significant inhibitory activity against bothropic venoms (Félix-Silva et al., 2017a; FélixSilva et al., 2014d; Gomes et al., 2016). Considering that these species are used indistinctly in the traditional medicine of Northeast Brazil to treat snakebites, and that the popular nomenclature of these plants is often confusing and may cause errors in medicinal use, the present work aimed to compare these Brazilian Jatropha species, in relation to their ability to inhibit the local edematogenic effect of B. erythromelas snake venom in a murine model, their in vitro antibacterial activity and phytochemical profile.

6 2. Material and methods 2.1. Chemicals and reagents 2,3,5-triphenyltetrazolium chloride, hexadecyltrimethylammonium bromide, isoorientin (≥ 98%), isovitexin (≥ 98%), NP reagent (2-aminoethyl diphenyl borate), orientin (≥ 97%), o-dianisidine and vitexin (≥ 95%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen peroxide was purchased from Merck (Darmstadt, HE, Germany). Sodium thiopental was purchased from Cristália (Itapira, SP, Brazil). All other reagents and solvents used were of analytical or HPLC grade. The water used was purified by reverse osmosis. Phosphate buffered saline (PBS) pH 7.4 was employed and contained 137 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4 and 10 mM Na2HPO4. 2.2. Plant collection and botanical authentication Jatropha gossypiifolia L. leaves were collected in Carnaubais, a municipality in Rio Grande do Norte State, Brazil (5.27ºS, 36.8ºW), in April 2012. Leaves of Jatropha mollissima (Pohl) Baill. were collected in Rafael Godeiro, another municipality in the State of Rio Grande do Norte, Brazil (6.04ºS, 7.42ºW), in January 2014. The collection of the plant material was conducted under authorization of Brazilian Biodiversity Authorization and Information System (SISBIO) (Process no. 35017) and Brazilian Genetic Heritage Management Council (CGEN) (Process no. 010844/2013-9). The botanical identification of the material was performed at the Herbarium in the Centro de Biociências of Universidade Federal do Rio Grande do Norte, by Msc. Alan de Araújo Roque and PhD Jomar Gomes Jardim, where authentic samples of J. gossypiifolia and J. mollissima are deposited under voucher numbers UFRN 12561 and UFRN 16879, respectively. Plant names were checked with http://www.theplantlist.org (access in Aug, 2017). The leaves were dried at room temperature, triturated and stored in hermetically sealed bottles protected from light and humidity until use for extract preparation. 2.3. Extract preparation Dried leaves of each plant species were extracted by decoction with purified water to the ratio 1:10 (w/v) for 15 min at 100ºC, to obtain the aqueous leaf extracts of J. gossypiifolia (named AEJg) and J. mollissima (named AEJm). The method of extraction was chosen based on previous literature that indicates that this is one of the main form of utilization in folk medicine (tea decoction) and on previous research by our group (Félix-Silva et al., 2014a; Félix-Silva et al., 2014d; Gomes et al., 2016). The aqueous extracts obtained after vacuum filtration were freeze-dried and stored at 20ºC. The yields of AEJg and AEJm were, respectively, 13.6 and 12.5%, relative to dry plant. 2.4. Phytochemical analysis 2.4.1. Thin layer chromatography (TLC) First, in order to facilitate the chromatographic analysis of compounds, the crude extracts were fractionated by liquid-liquid partition with solvents of increasing polarity in order to obtain the dichloromethane, ethyl acetate, n-butanol and residual aqueous fractions. Analyzes were conducted in silica gel F254 (Merck, Darmstadt, HE, Germany) as stationary phase, employing two different mobiles phases: (1) ethyl acetate: formic acid: water (8:1:1 v/v/v), and (2) toluene: ethyl acetate: formic acid (5:5:0.5 v/v/v). After chromatograms development, the plates were dried and the compounds observed under UV light (254 and 365 nm). Then, plates were sprayed with specific

7 chromogenic reagents according to the class of compounds investigated (sulfuric vanillin + heating, NP reagent + UV 365 nm, ferric chloride and Dragendorff reagent). The retention factors, color and behavior of the spots were compared with chromatographic profiles of reference substances in the literature (Wagner and Bladt, 2001). Additionally, standard samples of flavonoids were employed for co-TLC analysis. 2.4.2. High-performance liquid chromatography with diode array detection (HPLC-DAD) For confirmation of TLC results, the crude extracts were analyzed by high-performance liquid chromatography with diode array detection (HPLC-DAD). Analyses were conducted in a liquid chromatography system from Merck-Hitachi®, model Chromaster, coupled to a DAD detector with quaternary pump, oven column and auto injector. The chromatographic analyses were performed using a Thermo Scientific® RP-18 column (250 x 4.6 mm, 5 µm particle size). The eluents used were: 0.5% formic acid pH 2.37 (A) and acetonitrile (B). The following gradient (v/v) was applied (70 minutes total analysis time): 10-17% B, 0-10 min; 17% B, 10-60 min; 17-20% B, 60-70 min. Flow elution was kept constant at 0.5 mL/min and 20 µL of each sample was injected. The temperature of the column oven was kept in 23ºC. The lyophilized extracts were resuspended in water (final concentration: 2 mg/mL) and standards were resuspended in methanol: water (1:1, v/v) (final concentration: 50 µg/mL). Samples and solvents were filtered through a 0.45 μm membrane. All analyses were performed in triplicate. Retention times (Rt) and ultraviolet spectra were obtained from chromatogram peaks at 254 and 340 nm, with the acquisition of UV spectra in the range of 200 to 400 nm. The identification of flavonoids was based on comparison of Rt, UV spectrum of the major peaks, and observation of the increase in the peak area after co-injection of extracts with standards. 2.5. Inhibition of edematogenic activity of Bothrops erythromelas snake venom (BeV) 2.5.1. Snake venom Venom from Bothrops erythromelas (BeV) snake was kindly supplied by Instituto Butantan, SP, Brazil. Venom was obtained by manual extraction from adult specimens and then lyophilized and kept at -20ºC until used. Venom solutions were prepared with PBS at time of use. The amount of venom was expressed by protein content, quantified by Bradford protein assay using albumin as standard (Bradford, 1976). The scientific use of the venom was approved by the Brazilian Genetic Heritage Management Council (CGEN) (Process number 010844/2013-9). 2.5.2. Animals Swiss albino mice, with 25-30 g, 6-8 weeks old, from both sexes, supplied by the animal facility of Centro de Ciências da Saúde from Universidade Federal do Rio Grande Norte, were used. Animals were housed in standard polypropylene cages and maintained under controlled temperature (22 ± 2ºC) in a 12 h light/ dark cycle. Mice were fed with standard laboratory extruded food and water ad libitum. At the end of the experiments, the animals were euthanized by sodium thiopental overdose (100 mg/kg) by intraperitoneal (i.p.) route. Procedures involving animals were approved by the Ethics Committee on Animal Use from Universidade Federal do Rio Grande do Norte (protocols no. 004/2013 and 053/2014), and performed in agreement with the recommendations of the Brazilian National Council for the Control of Animal Experimentation (CONCEA) and the International Guiding Principles for Biomedical Research Involving Animals of the Council of International Organizations of Medical Sciences (CIOMS).

8 2.5.3. Paw edema induction The edematogenic activity of BeV was evaluated using the paw edema assay, as previously described in the literature with a few modifications (Félix-Silva et al., 2017a). First, groups of 5 animals were treated (p.o. or i.p.) with AEJg or AEJm (200 mg/kg), dexamethasone (5 mg/kg, antiinflammatory drug standard) or PBS (10 mL/kg, vehicle control). Dose and via of administration of extracts were chosen based on pilot assays and papers previously published in the literature (FélixSilva et al., 2014d; Gomes et al., 2016). After 1 h, the animals were injected subcutaneously into the right hind paw with BeV (1 µg/ 50 µL of PBS). BeV dose was chosen based on pilot assays, where the selected dose induced significant paw edema without producing paw hemorrhage. Individual right hind paw thickness was measured immediately before venom injection (basal value) and at selected time intervals after edema induction (15, 30, 60, 90 and 120 min), using a digital caliper (Digimess, São Paulo, SP, Brazil). Edema was expressed as the percentage difference between the thickness of the paw after (at respective time points) and before (basal values) venom injection. In addition, the area under the time-course curve after 120 min (AUC0-120 min) was calculated for each experimental group. A group of animals that received only subplantar injection of PBS was used as negative control. 2.5.4. Determination of myeloperoxidase (MPO) activity After 120 min of BeV injection, animals were euthanized and their right hind paws were collected for quantification of myeloperoxidase (MPO) enzyme activity, as a biochemical marker of neutrophil migration to the inflammation site, as previously described (Alves-Filho et al., 2006; Bradley et al., 1982). Briefly, paw skin tissues were weighed, chopped and homogenated in PBS (1 mL of buffer for each 50 mg of tissue). Then, the samples were centrifuged at 10,000 g at 4 ºC for 10 min. The supernatants were discarded and the pellets were homogenated in 50 mM potassium phosphate pH 6.0 containing 0.5% hexadecyltrimethylammonium bromide (1 mL of solution for each 50 mg of original mass of tissue). The samples were then sonicated in an ice bath for 30 s, submitted to three freeze-thaw cycles, and finally sonicated for 30 seconds once more, for MPO enzyme extraction. The supernatant obtained after centrifugation at 10,000 g for 10 minutes at 4ºC was used for MPO activity determination, by mixing 20 µL of each supernatant with 200 µL of 50 mM potassium phosphate pH 6.0 containing 0.0005% hydrogen peroxide and 0.167 mg/mL o-dianisidine. The measurement of the activity was carried out at 460 nm using a microplate reader (Epoch-Biotek, Winooski, VT, USA), through kinetic reading at 1 min intervals, during 3 min. One unit of MPO was defined as the equivalent to the consumption of 1 μmol of hydrogen peroxide per minute, considering that 1 μmol of hydrogen peroxide gives a change in absorbance of 1.13 × 10-2 per minute (Posadas et al., 2004). Samples of each animal were analysed in triplicate and the mean of these determinations was used to express the result as mean ± standard error of mean (SEM), with n=5 animals per group. 2.5.5. Statistical analysis Results are presented as mean ± SEM. One-way ANOVA followed by Tukey’s test or two-way ANOVA followed by Bonferroni’s test were performed using GraphPad Prism version 5.00 (San Diego, CA, USA). P values less than 0.05 were considered significant. 2.6. In vitro antimicrobial activity 2.6.1. Microorganisms For antimicrobial assays, the following strains of Gram-positive were used: Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC12228), Enterococcus faecalis (ATCC

9 29212) and Bacillus cereus (ATCC 11778). The Gram-negative strains used were: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 10031) and Enterobacter cloacae (ATCC 13047). All microorganisms were obtained from Clinical Microbiology Laboratory from UFRN and maintained in nutrient agar at 4ºC, or stored in beads at 80ºC. 2.6.2. Antibacterial screening For antibacterial screening, the agar dilution method was used. Solutions of AEJg and AEJm were previously prepared and filtered on a 0.22 μm membrane, for further incorporation in MuellerHinton agar medium at a final concentration of 10 μg/μL. As positive growth control, plaques containing only medium (absence of extracts) were prepared. To each well of the Steers replicator were added 200 μL of the microbial suspensions (corresponding to 0.5 of the McFarland scale), equivalent to 1 × 105 colony forming units (CFU) per spot. Microorganisms that grew on the plate in the absence of extracts and presented growth inhibition in the plates containing the extracts incorporated in the agar were considered sensitive. Sensitivity tests were performed in duplicate. 2.6.3. Minimal inhibitory concentration (MIC) determination The minimum inhibitory concentration (MIC) was determined according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) using the broth microdilution method (Cockerill et al., 2012). Briefly, in 96-well microplates, 100 μL of bacterial suspension (1 × 108 CFU/mL) were added to 100 μL of extracts at different concentrations (0.023 – 12 μg/μL). The plates were incubated at 35 ± 2ºC for 24 h under continuous shaking (200 rpm) in a humid atmosphere. Positive growth controls were inoculated as described above, without the addition of extracts. Sterile medium with and without extract was used as negative growth controls, and vancomycin was used as control for growth inhibition. MIC was defined as the lowest concentration capable to inhibit microbial growth, based on the optical density at 595 nm. After reading, 100 µL of 1% 2,3,5triphenyltetrazolium chloride dye were added to each well, as indicative of microbial growth by changing the coloration of the medium to red.

10 3. Results 3.1. Phytochemical analysis TLC analyses, using different spray reagents, suggested the presence of alkaloids, phenolic compounds, steroids and/or terpenoids, flavonoids and tannins in AEJg, and phenolic compounds, flavonoids and saponins in AEJm, according to the color spots compared with literature. In both extracts, among the compounds detected, flavonoids can be considered as major compounds, judging by the number and intensity of the spots. By TLC, in general, the flavonoid profile of both extracts observed when the chromatoplates were revealed with NP reagent was qualitatively similar. By co-TLC, it was possible to observe the presence of the flavonoids orientin and isoorientin (orange spots), vitexin (green spot) and luteolin (orange spot) in both extracts. In addition, the flavonoids isovitexin (green spot) and apigenin (yellow spot) were identified only in AEJg and AEJm, respectively. To confirm the presence of these substances, additional analyzes were performed by HPLC-DAD. The HPLC-DAD chromatograms allowed to visualize and to distinguish the flavonoid profile of the extracts (Figure 1). Analyzing the chromatographic profiles, it is possible to observe that the two species have similar chemical profiles, but with marked differences in concentration of the major compounds, even analyzing the extracts at the same concentration (2 mg/mL). Furthermore, AEJm showed two compounds (peaks 1, Rt 19.07 min, and 2, Rt 22.16 min) that could not be detected in AEJg. The chromatographic profile of AEJg (Figure 1-A) exhibited at least four major peaks, showing the peaks 3 and 4 UV spectra similar to C-glycosylated flavonoid derived from luteolin and the peaks 5 and 6 showing UV spectra similar to C-glycosylated flavonoid derived from apigenin (Mabry et al., 1970). By the other hand, the chromatographic profile of AEJm (Figure 1-B) exhibited at least 6 major peaks, also with UV spectrum similar to flavonoids derived from luteolin (peaks 3 and 4) and apigenin (peaks 1, 2, 5 and 6) (Mabry et al., 1970), of which peak 2 (absent in AEJg) is the peak with the highest intensity in AEJm.

11

Fig. 1. HPLC-DAD chromatograms of aqueous extracts of J. gossypiifolia (AEJg) (A) and J. mollissima (AEJm) (B). Detection at 340 nm.

To confirm the identity of the peaks, co-injections of the extracts with flavonoid standards were performed, being possible to observe the increase in the area of the peak of each chromatographic standard analyzed at the same Rt. Peaks 3, 4, 5 and 6 had their identity confirmed as, respectively, isoorientin, orientin, vitexin and isovitexin (Figure 2). Rt values and UV spectra can be seen in Table 1.

12

Fig. 2. Chemical structure of C-glycosylated flavonoids in common in the aqueous extracts of J. gossypiifolia (AEJg) (A) and J. mollissima (AEJm) (B) detected by HPLC-DAD analysis. Table 1 Major peaks identified in the aqueous extracts of J. gossypiifolia (AEJg) and J. mollissima (AEJm) by HPLC-DAD analysis. Extract Peak Rt (min) Maximum UV (nm) Identified compound AEJg 3 23.00 269 and 348 Isoorientin 4 24.08 256 (sh), 267 and 248 Orientin 5 29.61 268 and 337 Vitexin 6 31.63 269 and 337 Isovitexin AEm 1 19.07 271 and 336 2 22.16 271 and 335 3 22.97 268 and 347 Isoorientin 4 24.13 267 and 345 Orientin 5 29.58 268 and 337 Vitexin 6 31.62 269 and 367 Isovitexin Rt: Retention time.

3.2. Evaluation of inhibitory activity against BeV in vivo By oral route, AEJg and AEJm significantly inhibited (P<0.001) the edematogenic activity of BeV (around 50% inhibition), with intensity similar to dexamethasone (Table 2 and Figure 3). By this route, there was no significant difference between the inhibitory capacity of the extracts (P>0.05). By intraperitoneal route, however, AEJg presented anti-edematogenic effect (76.4% inhibition) significantly higher (P<0.001 when compared to each other, at all times analyzed) than AEJm (31.9% inhibition) (Table 2 and Figure 3). Interestingly, when administered intraperitoneally, AEJg was more effective in reducing edema induced by BeV than dexamethasone (P<0.001 when compared to each other, at all times analyzed). AEJm, by intraperitoneal route, started to inhibit the edematogenic activity of BeV only after 60 min, while AEJg and dexamethasone, by the same route, were active as fast as 15 min after venom injection.

13 Table 2 Inhibition percentages of aqueous extracts of J. gossypiifolia (AEJg) and J. mollissima (AEJm) against paw edema induced by B. erythromelas venom (BeV). Treatment Route AUC0-120 min MPO AEJg p.o. 49.2 ± 9.5*** 69.5 ± 8.4*** i.p. 76.4 ± 3.7*** 78.2 ± 4.1*** AEm p.o. 56.1 ± 9.3*** 61.5 ± 17.5** i.p. 31.9 ± 4.3** 69.5 ± 2.6*** Dexamethasone p.o. 55.1 ± 18.5*** 83.9 ± 5.7*** i.p. 44.9 ± 7.1*** 57.3 ± 8.6*** AUC0-120 min: area under time-course curve after 120 min. MPO: myeloperoxidase. p.o.: oral route. i.p.: intraperitoneal route. Data showed as mean ± SEM (n=5/group). **P<0.01 and ***P<0.001, when compared to vehicle control group in one-way ANOVA followed by Tukey’s test. Inhibition percentage calculated as follows: [1 – (%Activitytest – %Activitynegative control mean) ÷ (%Activityvechicle control mean – %Activitynegative control mean)] × 100.

Fig. 3. Inhibition of the edematogenic activity of B. erythromelas venom (BeV) by aqueous extracts of J. gossypiifolia (AEJg) and J. mollissima (AEJm). A and C: Time-course of paw edema. **P<0.01 and ***P<0.001, when compared to the BeV (vehicle control) group in two-way ANOVA followed by Bonferroni’s test. B and D: Area under the time-course curves after 120 min (AUC0-120 min) values. **P<0.01 and ***P<0.001, when compared to BeV (vehicle control) group in one-way ANOVA followed by Tukey’s test. Data showed as mean ± SEM (n=5/group). p.o.: oral route. i.p.: intraperitoneal route.

In the evaluation of the myeloperoxidase (MPO) enzyme extracted from mice paws, no significant difference (P>0.05) was detected between the inhibitory potentials of the two plant species. Both AEJg and AEJm inhibitory potential were similar to dexamethasone, either oral and intraperitoneally (Table 2 and Figure 4).

14

Fig. 4. Effect of aqueous extracts of J. gossypiifolia (AEJg) and J. mollissima (AEJm) on mieloperoxidase (MPO) activity in mice paws injected with B. erythromelas venom (BeV). A: Treatment (with vehicle, dexamethasone or extracts) by oral route (p.o.). B: Treatment (with vehicle, dexamethasone or extracts) by intraperitoneal route (i.p). **P<0.01 and ***P<0.001, when compared to BeV (vehicle control) group in one-way ANOVA followed by Tukey’s test. Data showed as mean ± SEM (n=5/group).

3.3. Evaluation of antibacterial activity in vitro Significant differences were also observed in the antibacterial activity of the extracts. In the initial screening assay, using a fixed concentration of each extract incorporated in the culture medium, it was observed that AEJg was able to inhibit the growth of S. epidermidis, S. aureus and B. cereus, whereas no microorganisms were sensitive to AEJm at the concentration tested. Additional experiments were performed to determine the MIC of each extract against sensitive microorganisms. The broth microdilution method using the 2,3,5-triphenyltetrazolic chloride dye showed a significant inhibitory activity of AEJg against S. epidermidis, S. aureus and B. cereus, whereas AEJm was ineffective at the concentrations tested, reinforcing previous result of the initial screening (Table 3). Table 3 Minimum inhibitory concentration (MIC) of aqueous extracts of J. gossypiifolia (AEJg) and J. mollissima (AEJm) by broth microdilution Microorganisms AEJg (µg/µL) AEJm (µg/µL) Vancomycin (µg/mL) Staphylococcus aureus (ATCC 29213) 6.0 >12.0 0.5 Staphylococcus epidermidis (ATCC 12228) 6.0 >12.0 0.5 Bacillus cereus (ATCC 11778) 6.0 >12.0 0.5

15 4. Discussion Several species of the genus Jatropha are frequently used interchangeable in traditional medicine, especially in the Northeast region of Brazil, as antidotes for snakebites (Albuquerque and Andrade, 2002; Albuquerque et al., 2007). However, in traditional medicine, there is no obvious distinction between the uses of different species of this genus. In addition, the popular names of different species are confusing (several species with the same popular name, or the same species presents several popular names, according to the region), as well some species have leaves morphologically similar, leading to the possibility of incorrect popular use of vegetal species. Considering that, eventually, some of these species may present different levels of efficacy and/or safety, a study comparing them becomes interesting. Thus, the objective of this work was to compare two of the Jatropha species most popularly used in the Brazilian Northeast region (J. gossypiifolia and J. mollissima), both in terms of efficacy and phytochemical constitution, in order to contribute to the differentiation and future quality control of derivatives of these medicinal plants. Studies comparing the flavonoid profile of species of the genus Jatropha are not found in the literature. Thus, the analysis of the chemical constitution of these extracts is important to suggest which compounds are related to the inhibitory activity of these species, as well as in the attempt to obtain chemical parameters to differentiate them. From the analysis by TLC, a qualitatively similar flavonoid profile was observed in the vegetal species, mainly composed of C-glycosylated flavonoids derived from luteolin and apigenin. In the plant species studied, these flavonoids can still be pointed out as major compounds, judging by the number, intensity and size of bands revealed with NP reagent, a specific spray reagent for this class of compounds. Previous studies demonstrated the presence of C-glycosylated flavonoids in the leaves of J. gossypiifolia (Félix-Silva et al., 2014c; Pilon et al., 2013; Subramanian et al., 1971) and J. mollissima (Gomes et al., 2016), as well as the presence of these and other types of flavonoids in other species of the genus Jatropha (Zhang et al., 2009). For the confirmation of the presence of these substances and to distinguish the flavonoid profile of the species, additional analyzes were performed by HPLC-DAD, as shown in Figure 1. The analysis of the UV spectra of the predominant peaks in the extracts revealed the presence of compounds with profile compatible with flavonoids derived from luteolin and apigenin, which corroborates with the findings obtained by TLC. Analysis with the co-injection of flavonoid standards confirmed the identity of these compounds as isoorientin (peak 3), orientin (peak 4), vitexin (peak 5) and isovitexin (peak 6), present in both species (Figure 2). Despite the similarity between the flavonoid profiles of the two extracts, the compounds common to both species are at higher concentration in AEJg, as can be seen by the area of the peaks in Figure 1. In addition, AEJm has two compounds apparently absent in AEJg, one of them being the major one in J. mollissima (peak 2, Rt 22.26 min), whose identities could not be confirmed by co-injection. However, Gomes et al. (2016) identified, through high performance liquid chromatography coupled to mass spectrometry (HPLC-MS), two glycosylated flavonoids derived from apigenin named schaftoside and isoschaftoside, whose UV spectra are similar to those from peaks 1 and 2 observed in the present work. Thus, it can be concluded that the species have a similar qualitative flavonoid profile, but can be quantitatively differentiated, especially in relation to the major flavonoids of each species that are quantitatively different. Comparisons were made between AEJg and AEJm in relation to their ability to inhibit the edematogenic response to PBe. Considering the widespread use of these species in the Brazilian Northeast region, we evaluated the efficacy of these species against B. erythromelas snake, since it is the most frequent species of Bothrops of this Brazilian region (Lira-Da-Silva et al., 2009). The paw edema model was chosen since bothropic envenoming is characterized by the rapid development of an inflammatory process at the injection site (Gutiérrez and Lomonte, 1989). In addition, the in vitro

16 antimicrobial activity of the two plant species was compared in view of the possible complications that could affect the victims of snake envenoming due to secondary bacterial infections (Jorge and Ribeiro, 1997). Bothropic envenoming is characterized by the rapid development of an inflammatory process at the site of venom inoculation. The pathophysiology of the edematogenic process is multifactorial, involving the direct action of venom components in the microvasculature and the effect of endogenous inflammatory mediators released by components of the venom (Gutiérrez and Lomonte, 1989; Gutiérrez et al., 2009). This response often leads to severe edema, ischemia and neural compression, which can result in a compartment syndrome that can lead to permanent tissue loss or amputation of the affected site due to necrosis (Teixeira et al., 2009). In the present study, as shown in Table 2, both AEJg and AEJm were able to inhibit the edematogenic effect produced by BeV, both orally and intraperitoneally, demonstrating the inhibitory potential of the two plant species under study. However, although the effect of the extracts was similar orally (P>0.05), AEJg was significantly (P<0.001) more active than AEJm when the evaluation was done intraperitoneally (Figure 3 and Table 2). As shown in Table 2, by intraperitoneal route, AEJg was more active than dexamethasone (P<0.001). This is relevant since previous studies have shown that anti-inflammatory and/or antioxidant drugs may inhibit the inflammatory response produced by snake venoms (Barreto et al., 2017; Patrão-Neto et al., 2013; Sunitha et al., 2015). In fact, recent studies with the aqueous extract of the leaves of J. gossypiifolia have shown significant anti-inflammatory and antioxidant activity of this species (Félix-Silva et al., 2014b; Félix-Silva et al., 2014c). In addition, previous studies demonstrate the inhibitory potential of this plant species against classes of toxins relevant to the genesis of local tissue damage induced by bothropic venoms, such as metalloproteinases, phospholipases A2 and hyaluronidases (Félix-Silva et al., 2017a; Félix-Silva et al., 2014d). Thus, it may be suggested that possibly the best inhibitory action of AEJg is due to its indirect action, as an anti-inflammatory and antioxidant agent to reverse the endogenous inflammatory mediators released by venom toxins, as well as to a direct inhibitory action on these toxins, which dexamethasone is not able to perform. This is one of the contexts that points out that the use of antiophidic plants as adjuvant in the antivenom therapy is interesting, due to its potential ability to neutralize a wide spectrum of toxins and to inhibit the local tissue damage efficiently by an indirect action against endogenous inflammatory mediators (Félix-Silva et al., 2017a; Félix-Silva et al., 2017b). Flavonoids, main compounds identified in the studied Jatropha species, are able to promote strong hydrogen bonds with amides of protein chains and exhibit metal chelating activity, which highlight the potentiality of this class of compound as interesting enzymatic inhibitors (Mors et al., 2000). Previous studies showed the antiophidic potential of these Jatropha species, showing their activity against local effects induced by Bothrops venoms such as edema, hemorrhage and myotoxicity (Félix-Silva et al., 2017a; Félix-Silva et al., 2014d; Gomes et al., 2016). The effectiveness of an aqueous extract of J. gossypiifolia was evaluated against local toxicity and enzymatic effects of B. erythromelas and B. jararaca snake venoms (Félix-Silva et al., 2017a; FélixSilva et al., 2014d). Interestingly, the extract was active when it was administered both before (pretreatment) or after venom injection (post-envenomation protocol), thus showing the great potentiality of the extract of this species in the complementary treatment of snakebites (Félix-Silva et al., 2017a). J. gossypiifolia was able to inhibit significantly almost 73% of edema and 43% of hemorrhage caused by B. erythromelas (Félix-Silva et al., 2017a), while it was able to reduce about 50% of hemorrhage and almost 100% of edema and myotoxicity induced by B. jararaca snake venom (Félix-Silva et al., 2014d). The aqueous extract of J. mollissima was also evaluated previously against local toxicity induced by B. erythromelas and B. jararaca snake venoms, showing

17 promising inhibitory result against edema, hemorrhage and myotoxicity induced by these Bothrops venoms (Gomes et al., 2016). J. mollissima was not able to inhibit significantly hemorrhage induced by B. erythromelas, but reduced in about 46% and 80% the edematogenic and myotoxic activities of this venom, respectively. By the other hand, this plant species were significantly active against hemorrhage (44% of inhibition), edema (around 25% of inhibition) and myotoxicity (about 72% of inhibition) induced by B. jararaca venom. So, in general, our results corroborate with previous ones using different protocols and models. Bacterial infections secondary to snakebites are a common complication in victims, which can lead to abscess formation and is therefore an important risk factor for amputations (Hearn et al., 2015; Jorge and Ribeiro, 1997; Saravia-Otten et al., 2007). The main source of microorganisms is the oral cavity of the snakes, but the microbiota in the different layers of the skin of the victims or even microorganisms coming from their clothes can participate in these secondary infections (Dehghani et al., 2016). A large number of bacteria, including gram-negative bacilli and grampositive cocci, can be inoculated through snakebites and have been isolated from abscesses of envenoming victims (Jorge and Ribeiro, 1997; Saravia-Otten et al., 2007). Microorganisms such as Staphylococcus, Pseudomonas, Salmonella, Escherichia, Providencia, Proteus, Enterococcus and Bacillus have been identified in the oral cavity of some venomous snakes (Dehghani et al., 2016). The use of antimicrobial drugs is often therapeutically recommended to avoid possible complications due to infections (Jorge and Ribeiro, 1997; Palappallil, 2015). In this context, medicinal plants exhibiting antimicrobial activity may be useful as a complement to serum therapy for the treatment and prevention of local tissue damage (Silva et al., 2016). As shown in Table 3, AEJg was able to inhibit the growth of S. epidermidis, S. aureus and B. cereus, whereas no microorganism was sensitive to AEJm at the concentrations tested. This is an interesting finding, as it points to a possible additional benefit of AEJg in the treatment of local effects of snake venoms. In fact, it is often discussed in the literature that the presence of tranquilizing, antioxidant, antimicrobial, immunostimulatory and/or anti-inflammatory activity in certain plants may be of great interest for the relief of symptoms of snake envenoming (Houghton and Osibogun, 1993; Shenoy et al., 2013). Thus, the present study contributes with additional evidence regarding the potentiality of the species J. gossypiifolia for the adjuvant treatment of bothropic envenomation. Regarding safety, it is important to mention that in vitro hemolytic tests were performed, in which it was observed that none of the extracts presented hemolytic activity at the concentrations used in the antimicrobial assays, which suggests their low toxicity (data not shown). Previous studies evaluating the cytotoxicity of AEJg against human embryonic kidney cells (HEK-293) also demonstrated absence of cytotoxicity (Félix-Silva et al., 2014c). These are particularly interesting findings for AEJg, as they demonstrate a certain specificity of the bioactive compounds against bacterial cells, instead to cells of animal origin. Based in our results, it can be suggested that the different quantitative chemical profile may justify the different biological effects observed. Compounds present in a higher concentration in AEJg (orientin, isoorientin, vitexin and isovitexin) may be involved, at least partially, in the effects of the Jatropha species investigated. Previous studies have demonstrated different biological activities of luteolin and apigenin flavonoids, as well as their C-glycosylated derivatives. For example, apigenin and its analogous synthetic molecules were able to inhibit a number of toxic effects induced by metalloproteinases, which is a class of toxins extremely involved in the genesis of local effects induced by several snake venoms (Srinivasa et al., 2014). The flavonoid luteolin, as previously described in literature, presents inhibitory action against hyaluronidase activity of Crotalus adamenteus snake venom, another class of toxins highly related with toxicity of snake venoms (Kuppusamy and Das, 1993). Other studies demonstrate other biological activities of

18 orientin and isoorientin (luteolin-derived C-glycosylated flavonoids) and vitexin and isovitexin (apigenin-derived C-glycosylated flavonoids), such as anti-inflammatory, antioxidant and antimicrobial activity (Bae, 2015; Das et al., 2016; Lv et al., 2016; Rosa et al., 2016; Sumalatha et al., 2015). Studies are being carried out in our laboratory in order to isolate and evaluate the bioactivity of these compounds, especially with regard to their antiophidic potential. However, it is relevant to emphasize that, considering that plant extracts are a complex mixture of miscellaneous chemical groups, many other active constituents in these extracts besides flavonoids may be involved, acting by different mechanisms against several different toxins in snake venoms. In fact, in many cases, the whole herbal extracts are more powerful than the isolated herbal compounds (Fernandes et al., 2016; Gomes et al., 2010). So, further studies are also needed to verify the potential role of minor compounds from these species in their antiophidic activity. Interestingly, it is important to mention that J. gossypiifolia, due to its ethnopharmacological relevance, is included in the National List of Medicinal Plants of Interest to Brazilian Public Health System (RENISUS), which is a report published by the Brazilian Health Ministry that includes 71 species of medicinal plants with the potential to generate pharmaceutical products of interest in the Brazilian public health system (Brasil, 2009). So, the promising pharmacological results observed in the present study justify the inclusion of J. gossypiifolia in this list, reinforcing its potentiality for the generation of pharmaceutical products. Thus, in conclusion, this approach demonstrates that although used indistinctly in traditional medicine, the species J. gossypiifolia is shown to be significantly more effective than J. mollissima in relation to its inhibitory action against B. erythromelas edematogenic activity and antibacterial activities, which in turn may be justified by different chemical composition among plant species, especially with respect to a higher content of C-glycosylated flavonoids in the most active species. With this study, more scientific evidence on the potentiality of the species J. gossypiifolia as a complementary treatment against the local effects produced by bothropic venoms is presented. The results obtained may be useful for the species distinction and quality control of future herbal products based on plants of the genus Jatropha.

19 Author contributions JFS, JASG, JMF, SMZ and MFFP designed all the experiments. JFS and JASG conducted the collection of plant material, preparation of plant extracts and in vivo experiments. JFS, JASG and JMF conducted the phytochemical analysis of plant extracts. AKCM and YASM conducted the antibacterial tests of plant extracts. JFS, JASG, JMF, AKCM, YASM, ECGS, SMZ and MFFP analyzed the data. ECGS, AASJ, DVT, SMZ and MFFP contributed with reagents, materials and/or analysis tools. JFS, JASG, JMF, AKCM and YASM contributed in manuscript preparation. ECGS, AASJ, DVT, SMZ and MFFP refined the manuscript for publication. All authors read and approved the final manuscript.

20 Declaration of interest The authors have no conflict of interest to disclose.

21 Acknowledgements This research was supported by grants from CAPES (grant number: 23038000814/2011-83) and FAPERN (grant number: PRONEM/2011). M. F. Fernandes-Pedrosa and D. V. Tambourgi are CNPq fellowship-honored researchers. J. Félix-Silva, J. A. S. Gomes, J. M. Fernandes and Y. A. S. Menezes thank CAPES for PhD Scholarships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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