Research Article Aqueous Leaf Extract of Jatropha ...downloads.hindawi.com › journals › bmri ›...

Research ArticleAqueous Leaf Extract of Jatropha mollissima (Pohl) BailDecreases Local Effects Induced by Bothropic Venom

Jacyra Antunes dos Santos Gomes1 Juliana Feacutelix-Silva1 Juacutelia Morais Fernandes2

Juliano Geraldo Amaral34 Norberto Peporine Lopes3

Eryvaldo Soacutecrates Tabosa do Egito5 Arnoacutebio Antocircnio da Silva-Juacutenior1

Silvana Maria Zucolotto2 and Matheus de Freitas Fernandes-Pedrosa1

1TECBIOFAR Department of Pharmacy Federal University of Rio Grande do Norte (UFRN) Natal RN Brazil2PNBIO Department of Pharmacy Federal University of Rio Grande do Norte (UFRN) Natal RN Brazil3NPPNS Faculty of Pharmaceutical Sciences of Ribeirao Preto University of Sao Paulo Ribeirao Preto SP Brazil4Federal University of Bahia Multidisciplinary Institute in Health Vitoria da Conquista BA Brazil5LASID Department of Pharmacy Federal University of Rio Grande do Norte (UFRN) Natal RN Brazil

Correspondence should be addressed to Matheus de Freitas Fernandes-Pedrosa mpedrosa31uolcombr

Received 28 June 2016 Revised 3 September 2016 Accepted 29 September 2016

Academic Editor Michele Rechia Fighera

Copyright copy 2016 Jacyra Antunes dos Santos Gomes et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Snakebites are a serious worldwide public health problem In Brazil about 90 of accidents are attributed to snakes from theBothrops genus The specific treatment consists of antivenom serum therapy which has some limitations such as inability toneutralize local effects difficult access in some regions risk of immunological reactions and high cost Thus the search foralternative therapies to treat snakebites is relevant Jatropha mollissima (Euphorbiaceae) is a medicinal plant popularly used in folkmedicine as an antiophidic remedy Therefore this study aims to evaluate the effect of the aqueous leaf extract from J mollissimaon local effects induced by Bothrops venoms High Performance Liquid Chromatography with Diode Array Detection analysisand Mass Spectrometry analysis of aqueous leaf extract confirmed the presence of the flavonoids isoschaftoside schaftosideisoorientin orientin vitexin and isovitexin This extract at 50ndash200mgkg doses administered by intraperitoneal route showedsignificant inhibitory potential against local effects induced by Bothrops erythromelas and Bothrops jararaca snake venoms Localskin hemorrhage local edema leukocyte migration and myotoxicity were significantly inhibited by the extract These resultsdemonstrate that J mollissima extract possesses inhibitory potential especially against bothropic venoms suggesting its potentialas an adjuvant in treatment of snakebites

1 Introduction

Snakebites represent a serious worldwide public health andsocial problem because of their high frequency morbi-mortality and sequelae left in the victims Moreover acci-dents caused by snakes are considered a neglected diseasemainly in Africa Latin America Asia and Oceania [1 2]Data indicate that worldwide more than 5 million peoplesuffer snakebites every year resulting in 25000 to 125000deaths and leaving approximately 400000 people with per-manent disabilities [1] In Brazil an estimated number of

25000 snakebites occur per year [3] Most of these accidentsare caused by snake species of the Bothrops genus which isresponsible for about 90of the cases in Latin America [3 4]The main representative species of the Bothrops genus inBrazil are Bothrops jararaca (South and Southeast) Bothropserythromelas (Northeastern) Bothrops atrox (North) andBothrops moojeni (Center-West) [3] The Bothrops snakeshave high complexity and variation in the protein compo-sition of their venom This variation is due to factors suchas diet age seasonal variation sexual dimorphism and geo-graphical origin which occurs within the species interfamily

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 6101742 13 pageshttpdxdoiorg10115520166101742

2 BioMed Research International

intergenus interspecies intersubspecies and intraspecies [56] The pathophysiological process of the snake envenomingis complex and includes the combined action of severaltoxins such as snake venom metalloproteinases (SVMPs)snake venom serine proteinases (SVSPs) phospholipasesA2 (PLA2) hyaluronidases bradykinin-releasing enzymeslectins L-amino oxidases and pharmacological mediators[7 8] Bothrops envenomation causes mainly immediate localtissue damage (including pain edema local hemorrhage andmyonecrosis) and systemic effects (cardiovascular alterationscoagulation and renal alterations) [9 10]

Currently the intravenously antivenom serum therapy isthe only specific treatment for snakebites The antivenomhowever has some limitations such as difficult access insome regions risk of immunological reactions (includingserum sickness) high cost and limited effectiveness in theimprovement of the local tissue damage [11 12] Adminis-tration of the antivenom may prevent death but does notprevent local tissue damage and resultant disabilities Thelow inhibition of local effects (due to the delay of receivingthe serum or due to the low efficacy) is the leading cause ofamputations which can lead to serious social economic andhealth negative impacts given that most victims live in ruralareas [13] So the search for new complementary therapiesto treat snakebites has become of utmost importance In thisscenario the use of many medicinal plants has been an oldpractice in folk medicine against snakebites especially intropical and subtropical regions such as Africa Asia andSouth America [14 15]

The Jatropha L genus (Euphorbiaceae) belongs to thesubfamily Crotonoideae and Jatropheae tribe consisting ofmore than 300 species and is widely distributed in the tropicaland subtropical regions of Africa and the Americas [16]The name Jatropha is derived from the Greek words ldquojatrosrdquo(doctor) and ldquotropherdquo (food) which could be correlated withthe medicinal properties of plants from this genus [17] InAfrica Asia and Latin America the Jatropha species are usednot only in traditional medicine to cure various ailmentsbut also as ornamental plants and energy crops [17] Severalknown species from genus Jatropha have been reported fortheir medicinal uses chemical constituents and biologicalactivities such as Jatropha curcas Jatropha elliptica Jatrophagossypifolia and Jatropha mollissima [17 18]

Jatropha mollissima (Pohl) Bail (see Figure S1 in Sup-plementary Material available online at httpdxdoiorg10115520166101742) is a medicinal plant popularly knownin Brazil as ldquopinhao-bravordquo [16] This plant is endemic in thesemiarid region of Northeastern Brazil In folk medicine itis widely used for various purposes especially as antiophidic[19ndash21] and anti-inflammatory [22] remedies healing [20]veterinary vermifuge [19] and treatment of renal inflam-mation and loss of appetite [20] Some studies have shownthat this species has antioxidant [23] antimicrobial [24] andantihelmintic activities [25]Thus the aim of this work was toevaluate the ability of the aqueous leaf extract of J mollissimato inhibit the local effects induced by B erythromelas andB jararaca snake venoms in mice Our results indicate thatJ mollissima aqueous leaf extract has significant inhibitoryaction against hemorrhagic inflammatory and myotoxic

local activities of the Bothrops venoms studied suggesting thepotential of this species as a source of bioactive moleculesagainst bothropic venom

2 Materials and Methods

21 Chemicals and Reagents The flavonoids apigenin(ge99) luteolin (ge98) luteolin-7-O-glucoside (ge98)orientin (ge97) isoorientin (ge98) vitexin (ge95) isovi-texin (ge98) and vitexin-2-O-rhamnoside (ge98) (Sigma-Aldrich) were purchased from Sigma-Aldrich (St LouisMO USA) All other reagents and solvents used were ofanalytical grade The water used was purified by reverseosmosis

22 Plant Material Leaves from Jatropha mollissima werecollected in the city of ldquoRafael Godeirordquo 6∘0410158404010158401015840S7∘4210158405410158401015840W RN Brazil in January 2014 The collection ofthe plant material was conducted under authorization of theBrazilian Authorization and Biodiversity Information System(SISBIO) (Process number 35017) and the Brazilian AccessAuthorization and Dispatch Component of the GeneticPatrimony (CGEN) (Process 0108442013-9) The botanicalidentification of the material was performed by Dr JomarGomes Jardim and a voucher specimen was deposited at theHerbarium of the Bioscience of the Federal University of RioGrande do Norte Brazil (UFRN 16879) After identificationand confirmation of the plant species the leaves were dried atroom temperature triturated with an industrial blender andstored in hermetically sealed bottles until used for aqueousextract preparation

23 Snake Venom Lyophilized B erythromelas and B jara-raca snake venoms were used in this work B erythromelaswas generously supplied by the Instituto Butantan SP BrazilB jararaca was purchased from Sigma-Aldrich (St LouisMO USA) (product number V5625)The scientific use of thematerial was approved by the Brazilian Access Authorizationand Dispatch Component of Genetic Patrimony (CGEN)(Process 0108442013-9) The venom was weighed and dis-solved with phosphate buffer saline (PBS) and the proteincontent quantified by the Bradford method [26]

24 Animals Male and female Swiss albino mice (30ndash35 g)6ndash8 weeks of age used in this study were maintained understandard environmental conditions with free access to waterand food On the day of the experiment the animals wereplaced in the experimental room for at least one hour priorto tests for acclimation All animals were euthanized bysodium thiopental overdose associated with 2 lidocaine byintraperitoneal (ip) route at the end of the experimentsThe experimental protocols using animals were performedin agreement with the National Council for the Controlof Animal Experimentation of Brazil (CONCEA) and theInternational Guiding Principles for Biomedical ResearchInvolving Animals of the Council of International Organiza-tions of Medical Sciences (CIOMS) The animal experimentswere approved by the Ethics Committee on Animal Use from

BioMed Research International 3

the UFRN (protocol number 0532014) The total number ofanimals used was 230

25 Preparation of the Aqueous Extract from the Leaves of Jmollissima Dried leaves were submitted to decoction (10wv plant water) for 15min at a temperature of around 100∘Cto obtain the aqueous leaf extract of J mollissima (yield 125relative to dry plant) The aqueous extract obtained aftervacuum filtration was freeze-dried and dissolved in PBS atadequate concentrations for the biological assays

26 Phytochemical Analysis of the Aqueous Leaf Extract ofJ mollissima

261 TLC Profile The extract was fractionated by liquid-liquid partition in order to obtain the dichloromethane(CH2Cl2) ethyl acetate (AcOEt) n-butanol (BuOH) andresidual aqueous fractions respectively Then the extractand fractions were analyzed by Thin Layer Chromatog-raphy (TLC) using aluminum precoated sheets with sil-ica gel F254 (Merck Darmstadt Germany) as adsorbentTwo different mobile phases were used ethyl acetate formicacid water (8 1 1 vvv) and toluene ethyl acetate formicacid (5 5 05 vvv) The chromatograms were analyzedunder 365 nmUV light and then sprayed with specific chro-mogenic agents according to the class of compounds investi-gated (Dragendorff reagent natural reagent A ferric chlorideand sulfuric vanillin and heating) The retention factorbehavior and color of the spots were comparedwith the chro-matographic profiles of the reference substances Accordingto the results standard samples of the flavonoids apigenin(ge99) luteolin (ge98) luteolin-7-O-glucoside (ge98)orientin (ge97) isoorientin (ge98) vitexin (ge95) isovi-texin (ge98) and vitexin-2-O-rhamnoside (ge98) (Sigma-Aldrich) were comparedwith the extract and fractions Whennecessary to confirm the presence of any substance in extractandor fractions they were analyzed by co-TLC

262 HPLC-DAD Profile The analyses were performedusing anHPLCMerck-Hitach (Hichrommodel) instrumentequipped with a diode array detector (DAD) quaternarypump oven column and autoinjector The chromatographicanalyses were performed using a Phenomenex Luna RP-18 column (250 times 46mm 5 120583m particle size) and theeluents were (A) acetic acid 03 and (B) acetonitrile Thefollowing gradient (vv) was applied 10ndash15 B 0minus5min 15-16 B 5ndash50min 16ndash20 B 50ndash55min 20 55ndash75min 75minutes total analysis time Flow elution was 07mLminand 20 120583L of each sample was injected The aqueous leafextract of J mollissima was compared against standards ofcompounds vitexin (ge95) isovitexin (ge98) vitexin-2-O-rhamnoside (ge98) orientin (ge97) isoorientin (ge98)and luteolin (ge98) (Sigma-Aldrich) The lyophilized aque-ous extract of J mollissima and standards were resuspendedin methanol water 1 1 (vv) The final concentration of theextract was 25mgmL For standards the final concentrationwas 100120583gmL Their retention time and ultraviolet spectrawere obtained for chromatogram peaks at 254 and 340 nm

with the acquisition of UV spectra in the range of 200 to400 nm In this technique the identification of flavonoidswas based on comparison of retention time UV spectrumof the major peaks and coinjection of standards + extractby the observation of the increase in the peak area Theextract was prepared in triplicate and analyzed To confirmthe presence of the standards in the aqueous extract ofJ mollissima coinjections of the extract with standardswere performed Acetonitrile HPLC grade purchased fromPanreac (Brazil) was used Acetic acid was provided byVetec (Brazil) Water was purified with a Milli-Q system(Millipore) After total dissolution and prior to analysisthe samples and solvents were filtered through a 045 120583mmembrane (MillexTM Merck)

263 HPLC-DAD-MSMS Analysis To identify the chro-matographic signals 1 and 2 and to confirm the signals 34 5 and 6 the aqueous leaf extract of J mollissima wasanalyzed using a Shimadzu (Kyoto Japan) High PerformanceLiquid Chromatography (HPLC) coupled to an amaZon-SL ion trap (IT) Bruker Daltonics (Billerica USA) TheHPLC comprises a LC-20AD solvent pump unit a CTO-20A column oven a DGU-20A3 online degasser a CBM-20Asystem controller and a SPD-M20A (200 to 400 nm) diodearray detector Injections were performed automatically(20120583L) through a 100 120583L loop SIL-20A HT The separationof compounds was performed at 25∘C using a PhenomenexLuna C18 column (250mm times 460mm internal diameter5120583m particle size) The mobile phase was comprised ofacetic acid 03 (solvent A) and acetonitrile (solvent B) andwas pumped at a flow rate of 07mLminminus1 The gradientelution (vv) program was performed as follows 10ndash15 B0minus5min 15-16B 5ndash50min 16ndash20B 50ndash55min and 2055ndash75min

The mass spectrometer source parameters were set asfollows capillary voltage at 30 kV and end plate offset at500V Nitrogen (N2) was used as nebulizing (60 psi) anddrying gas (10 L minminus1 320∘C) Full-scan MS and MSMSspectra were obtained by scanning mz from 50 to 1300The electrospray ionization (ESI) source was operated inthe positive and negative ionization mode The data wereacquired using amplitudes of 07 V (MS2) and 10 V (MS3)The data were processed through Bruker Compass DataAnalysis 41 software (Bremen Germany)

27 Inhibition of the Local Hemorrhagic Activity The hem-orrhagic activity of B erythromelas and B jararaca venomswas induced using the in vivomodel of local hemorrhage aspreviously described in the literature with few modifications[27] Groups of 5 animals were treated with different dosesof the extract (50ndash200mgkg ip) After 30min the animalsreceived a subcutaneous (sc) injection of 25120583g of bothvenoms (in 100120583L of PBS) in the dorsal region 3 h laterthe animals were sacrificed and had the inner surface of theskin exposed After photo documentation of the producedhemorrhagic halos the hemorrhagic skin was removed andweighedThegroup inwhich animals received sc injection ofvenom and ip treatment of PBS was used as control (venom


control) Another group that received sc injection and iptreatment of PBS was used as negative control (PBS control)

28 Inhibition of the Edematogenic Activity The edemato-genic activity of B erythromelas and B jararaca venomswas induced using the in vivo model of paw edema aspreviously described in the literature with few modifications[28] Groups of 5 animals were treated with different dosesof J mollissima extract (50 100 and 200mgkg ip) ordexamethasone (2mgkg ip) After 30min the animalsreceived an intraplantar (ipl) injection of 1 120583g or 05 120583g ofB erythromelas or B jararaca venoms respectively in 50 120583Lof PBS in the right hind paw The individual right hindpaw thickness was measured immediately before injectionof the venoms (basal values) and at different time intervals(30 60 90 and 120 minutes) after injection of the venomsusing a digital caliper (Digimess Sao Paulo SP Brazil) Agroup of animals that received ipl injection of venoms andip treatment of PBS was used as control (venom control)Another group that received ipl injection and ip treatmentof PBS was used as negative control (PBS control)

29 Inhibition of the Cell Migration into Peritoneal CavityThe ability of the B erythromelas and B jararaca venoms toinduce migration of leukocytes into the peritoneal cavity wasevaluated as described in the literaturewith fewmodifications[29] Groups of 5 animals were treated with different doses ofJ mollissima extract (50 100 and 200mgkg ip) or dexam-ethasone (2mgkg ip) After 30min the animals received byip route an injection of 5 120583g or 25120583g of B erythromelas or Bjararaca venoms respectively in 500120583L of PBS After 6 hours(B erythromelas venom) or 4 hours (B jararaca venom)the animals were sacrificed and the peritoneal exudates werecollected through abdominal laparotomy for total leukocytecount To facilitate the collection all animals received aninjection of 3mL of heparinized PBS (5 IUmL) and hadthe abdomen massaged to release the adhered cells Thelavage fluid was centrifuged (at 392 g for 5min) and thecell button resuspended in 500120583L of PBS The samples werethen diluted 1 20 in Turk solution (acetic acid and crystalviolet 1) for total leukocyte count in Neubauerrsquos chamberCytospin preparations were stained with Leishmanrsquos stain forthe differential cell counts A group of animals that receivedip injection of venoms and ip treatment of PBS was usedas control (venom control) Another group that received ipinjection and ip treatment of PBS was used as negativecontrol (PBS control)

210 Inhibition of theMyotoxic Activity Themyotoxic activityof B erythromelas and B jararaca venoms was induced usingthe serum creatine kinase (CK) level as previously describedin the literature with few modifications [28] Groups of 5animals were treated with different doses of J mollissimaextract (50ndash200mgkg ip) or dexamethasone (2mgkgip) After 30 min all the animals received an intramuscular(im) injection of 25120583g of both venoms (in 50 120583L of PBS)in the right thigh 3 h later the animals were anesthetizedwith sodium thiopental and the blood was collected The

blood samples were incubated for 10min at 37∘C and thencentrifuged at 10000 g for 10min to obtain the serum Theserum CK activity was determined using a commercial kitaccording to the manufacturerrsquos protocol adapted for readingin themicroplate reader (Epoch-BiotekWinooski VTUSA)A group in which animals received im injection of venomand ip treatment of PBS was used as control (venomcontrol) Another group that received sc injection and iptreatment of PBS was used as negative control (PBS control)

211 Statistical Analysis All results were presented as meanplusmn standard error of mean (SEM) One-way ANOVA withTukeyrsquos posttest and regression analysis were performedusing GraphPad Prism version 500 (San Diego CA USA)p values less than 005 were considered significant

3 Results

31 Phytochemical Analysis of the Aqueous Extract ofJ mollissima

311 Thin Layer Chromatography (TLC) Profile For phy-tochemical analysis by TLC the aqueous extract was frac-tionated by liquid-liquid partition to obtain fractions withdifferent polarities thus facilitating the chromatographicanalysis of the compounds Moreover the TLC analysis wasperformed with different developers in order to identify theclasses of compounds presented in the aqueous extract of Jmollissima The chromatograms show spots suggestive of thepresence of phenolic compounds flavonoids and saponins(Figure S2) By co-TLC analysis it was possible to observethe presence of the flavonoids apigenin (Rf 064 fluorescentgreen color) luteolin (Rf 064 fluorescent yellow color) ori-entin (Rf 064 fluorescent yellow color) isoorientin (Rf 064fluorescent yellow color) and vitexin (Rf 064 fluorescentgreen color) in the extract (Figure S3) For confirmation ofpresence of such substances analyses were performed byHPLC

312 High Performance Liquid Chromatography (HPLC-DAD) Profile The chromatographic fingerprint obtained byHPLC-DAD of aqueous extract of J mollissima is depicted(Figure 1) It is possible to observe that J mollissima exhibitsat least six major peaks (1ndash6) Among them most have UVspectra similar to glycosylated flavonoid derivatives fromapigenin (267 nm II band and 336 nm I band) and luteolin(253 and 267 nm II band and 349 nm I band) [30] Glyco-sylated derivatives from these two flavonoids orientin andisoorientin (derived from luteolin) and vitexin and isovitexin(derived from apigenin) have similar absorption II bands forthese aglycones differing mainly by the maximum absorp-tion of the I bandThereafter by the analysis of standards andcoinjection of extract + standard it was possible to observethe increase in peak area of each standard analyzedThepeaks3 4 5 and 6 had their identity confirmed as respectivelyisoorientin (tR 2495min UV 269 and 349 nm) orientin (tR2796min UV 256 and 348 nm) vitexin (tR 398min UV267 and 336 nm) and isovitexin (tR 4135min UV 269 and


OH

OHOH

OH

OH

OH

OHOH

OHOH

OH

OH

OH

OHOH

OH

OH

OH OH

OH

OHOH

HO

HO

HO

HO

HO

HO

HO

HO

O

O

OO

O

O

O

O

5

2

431

6

OO

O

O

605030 40 700 10 20(Minutes)

0

20

40

(mAU

)

Figure 1 High Performance Liquid Chromatography of the aqueous extract of J mollissima SP Phenomenex Luna C18 column (250 times46mm 5120583m) MPACN gradient acetic acid 03 flow rate 07mLmin detection 340 nm Four compounds were identified as isoorientin(peak 3) orientin (peak 4) vitexin (peak 5) and isovitexin (peak 6)

Table 1 Identification de C-glycosyl and di-C-glycosyl flavones and relative abundances of ions obtained from the respective ions [MndashH]minusby MSMS

Peak Identification [MminusH]minus MSMS [MminusH]minus

minus18 minus60 minus90 minus120 A + 113 A + 831 Isoschaftoside 563 545(12) 503(39) 473(100) 443(88) 383(51) 353(68)

2 Schaftoside 563 545(19) 503(40) 473(84) 443(100) 383(40) 353(54)

3 Isoorientin 447 429(24) mdash 357(87) 327(100) mdash mdash4 Orientin 447 mdash mdash 357(47) 327(100) mdash mdash5 Vitexin 431 mdash mdash 341(6) 311(100) mdash mdash6 Isovitexin 431 413(6) mdash 341(31) 311(100) mdash mdash

337nm) Peaks 1 (tR 249min UV 271 and 335nm) and 2(tR 26min UV 269 and 347 nm) could not be identified bycoinjection

313 HPLC-DAD-MSMS Analysis Mass Spectrometryanalysis of signals 1 and 2 showed the same ion formationat mz 5632 [MminusH]minus in negative mode and mz 5652[M+H]+ in positive mode The MSMS spectra exhibitedfragmentation patterns of the isomers apigenin-di-C-gly-coside by the presence of ions [Mndash90]+minus for C-pento-sides and [Mminus120]+minus for C-hexosides and fragment ions[Agl+83113]minus which for di-C-glycosides represent the agly-cone and the residues of the sugars that remained linkedto it identified the type of aglycone that is [311341]minus and[353383]minus for apigenin and [327357]minus and [369399]minus forluteolin mono-C and di-C-glycosides respectively [31]Applying the systematic analyses carried out by Ferreres andcoworkers [31 32] it was possible to identify the compoundsas schaftoside and isoschaftoside flavonoid respectivelyAccording to such report preferential fragmentation is ofthe sugar moiety at the 6-C rather than the 8-C position

Observed in the present study was the formation of ionsmz 473 [(MminusH)minus90]minus as the base peak of chromatographicsignal 1 and mz 473 [(MminusH)minus120]minus as the base peakof chromatographic signal 2 (Table 1) Considering thepreferential fragmentation at the C-6 position chromato-graphic signal 1 corresponded to the isoschaftoside flavonoidand chromatographic signal 2 corresponded to the schafto-side flavonoid Furthermore peaks 3 4 5 and 6 were con-firmed as isoorientin orientin vitexin and isovitexin assuggested by the coinjection performed with these standards(see Section 312) All the signals present in Table 1 are in fullagreement with the previous published data Figure 2 showsflavonoids identified by HPLC-DAD-MSMS for the speciesJ mollissima

32 Inhibition of the Local Hemorrhagic Activity The subcu-taneous injection of B jararaca and B erythromelas venomsinduced severe hemorrhagic damage surrounding the ventralregion after 3 h compared to the subcutaneous injection ofPBS (119901 lt 005) (Figures 3(a) and 3(b)) The B jararacavenom proved to be more potent in causing hemorrhage in


R1 R2 R3

OH

OH

HO O

O

R3

R2

R1

1 Isoschaftoside C-arab C-gluc H

2 Schaftoside C-gluc C-arab H

3 Isoorientin C-gluc H OH

4 Orientin H C-gluc OH

5 Vitexin H C-gluc H

6 Isovitexin C-gluc H H

Figure 2 Flavonoids from J mollissima

relation to the B erythromelas venom using equal doses peranimal All doses of the J mollissima extract (50ndash200mgkg)inhibited the local hemorrhagic activity caused by the Bjararaca venom (119901 lt 005) as shown in Figure 3(a)Themax-imum inhibition by extract was 44 at a dose of 200mgkgfor 3 hours This result can be seen with the decrease inhemorrhagic halo weight and with the visual decreased halodiameter in each dose tested On the other hand for Berythromelas it was observed that the hemorrhage caused bythis venom was reduced by the extract particularly at dosesof 50 and 100mgkg but this decrease was not statisticallysignificant (119901 gt 005) (Figure 3(b))

33 Inhibition of the Edematogenic Activity B erythromelasandB jararaca venoms showed amarked edematogenic effectfor 120min after intraplantar injection compared to PBS(119901 lt 005) (Figure 4) The B jararaca venom proved to bemore potent in causing edema in relation to the B erythrome-las venom since a lower dose of B jararaca (05120583gpaw)produced an effect similar to that of the B erythromelasvenom (10 120583gpaw) A lower dose of the B jararaca venomwas used since this venom is very hemorrhagic and weintended to evaluate edema dissociated from hemorrhageThe treatment with dexamethasone significantly reduced theedema induced by both venoms It could be observed that theJ mollissima extract (50ndash200mgkg) administered half anhour before the injection of the B erythromelas (Figure 4(a))and the B jararaca (Figure 4(b)) venoms inhibited theedematogenic activity after 120min of the venom injection(119901 lt 005) For B erythromelas the maximum inhibition byextract was 4641 at a dose of 50mgkg for 120min whileforB jararaca themaximum inhibition by extract was 2519at a dose of 200mgkg for 120min Maximum inhibition for

dexamethasone was 5758 for B erythromelas and 3065for B jararaca

34 Total Number of Leukocytes in the Peritoneal CavityLeukocyte infiltration was evaluated in the peritoneal cavityat the time of 4 hours after the intraperitoneal injection ofthe venoms The group with the injected venoms showed anincrease in the number of white cells compared to the controlPBS group count (119901 lt 005) The B jararaca venom provedto be more potent in causing leukocyte infiltration in relationto the B erythromelas venom Figures 5(a) and 5(b) showthe total number of leukocytes in the peritoneal cavity of theanimals treated with the J mollissima extract half an hourbefore the intraperitoneal injection of the B erythromelasvenom and the B jararaca venom respectively In both casesall tested doses of the J mollissima extract (50ndash200mgkg)and dexamethasone were able to reduce the leukocyte influxcompared to the control group (which received only venom)(119901 lt 005)

The differential count of the leukocyte infiltration wasevaluated J mollissima extract (50ndash200mgkg) or dexam-ethasone significantly reduced the influx of mononuclear(Figures 6(a) and 6(c)) and polymorphonuclear (Figures6(b) and 6(d)) cells induced by both venoms (119901 lt 005)All the results show that the animals treated with the Jmollissima extract or dexamethasone half an hour beforethe intraperitoneal injection of the venoms significantlyinhibited the migration of these cells into the peritonealcavity compared to the control group (which received onlythe venoms) (119901 lt 005) For B erythromelas the maximuminhibition by extract was 80 at a dose of 100mgkg while forB jararaca themaximum inhibition by extractwas 8018 at adose of 200mgkg Maximum inhibition for dexamethasonewas 8727 for B erythromelas and 6697 for B jararaca

35 Inhibition of the Myotoxic Activity The intramuscularinjection of both venoms induced a significant increase inthe CK in serum after 3 h compared to PBS (119901 lt 005)(Figures 7(a) and 7(b)) Treatment with dexamethasonereduced serum CK induced by these venoms (119901 lt 005) Inthe same way treatment with extract (50ndash200mgkg) showedsignificant reduction in the serum CK levels induced by theB erythromelas (Figure 7(a)) and the B jararaca (Figure 7(b))venoms (119901 lt 005) For B erythromelas the maximuminhibition by extract was 8170 at a dose of 200mgkgwhile for B jararaca the maximum inhibition by extractwas 7273 at a dose of 100mgkg Maximum inhibition fordexamethasone was 9266 for B erythromelas and 9610for B jararaca

Table 2 summarizes the maximum percentage of inhibi-tion of the J mollissima extract in the hemorrhagic edemato-genic inflammatory and myotoxic activities induced by Berythromelas and B jararaca venoms

4 Discussion

Currently the only available specific treatment for snakebitesis the antivenom serum therapy which has some limitations


Jm (mgkg)200PBS Bja 50 100

10 mm

PBS BjaJm (mgkg)

50 100 200lowastlowastlowast

lowast lowast lowast

00

02

04

06

08

Hem

orrh

agic

hal

o m

ass (

g)

(a)

Jm (mgkg)200PBS Ber 50 100

10 mm

Jm (mgkg)

lowastlowastlowast

PBS Ber 50 100 20000

02

04

06

Hem

orrh

agic

hal

o m

ass (

g)

(b)

Figure 3 Inhibition of the hemorrhagic activity of B jararaca and B erythromelas venoms by aqueous extract of J mollissima (Jm) (a) Bjararaca (Bja) and (b) B erythromelas (Ber) venoms were injected sc in the ventral region of the animals treated with different ip doses ofJm 3 h later the inner surface skin was exposed photo documented and the halos weighed Values were expressed as mean plusmn standard errorof mean with 119899 = 5 lowast119901 lt 005 and lowastlowastlowast119901 lt 0001 when compared to the venom control group (Tukeyrsquos test one-way ANOVA)

such as reduced effectiveness against local effects risk ofimmunological reactions high cost and difficult access insome regions [11 12] Even though the antivenom causesthe inhibition of systemic effects the neutralization of thelocal tissue damage is much more difficult [4 28] Given

these limitations it is important to find alternative treatmentsandor complementary therapies In this context the useof medicinal plants could be highlighted since many ofthem could be able to neutralize a broad spectrum of toxins(including the local tissue damage) [28 33] Indeed several


PBSBerDexa

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowast

lowastlowast

Jm 200 mgkgJm 100 mgkgJm 50 mgkg

60 30 90 1200Time (min)

0

20

40

60

80Ed

ema (

)

(a)

PBSBjaDexa Jm 200 mgkg

Jm 100 mgkgJm 50 mgkg

lowastlowastlowast

lowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowastlowast


60 30 90 1200Time (min)

0

20

40

60

80

Edem

a (

)

(b)

Figure 4 Inhibition of the edematogenic activity of B erythromelas (a) and B jararaca (b) by aqueous leaf extract of J mollissima anddexamethasone Values were expressed as mean plusmn standard error of mean with 119899 = 5 lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 whencompared to the venom control group (Bonferronirsquos test two-way ANOVA)

PBS BerJm mgkg

Dexa 20050 100


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

5

10

15

20

(times106m

L)

(a)

PBS BjaJm mgkg

Dexa 20050 100



lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

10

20

30

40

50

(times106m

L)

(b)

Figure 5 Evaluation of the inhibition by the J mollissima extract of the cell migration induced by (a) B erythromelas and (b) B jararacavenoms Evaluation of the total leukocyte influx into peritoneal cavity after venom injection Values were expressed as mean plusmn standard errorof mean with 119899 = 5 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 when compared to the venom control group (ANOVA)

medicinal plants are rich sources of natural inhibitors andhave pharmacologically active componentsThey are also ableto be stable at room temperature having easy access and lowcost [34]

In this work a phytochemical study of J mollissimawas conducted for better comprehension of the chemicalcompounds presented in the aqueous extract of leaves ofthis species The TLC analysis identified the presence offlavonoids that could be suggested as major compoundsjudging by the number size and intensity of spots whenrevealed with Reagent A Natural (specific spray reagentfor this class of compounds) By the HPLC-DAD andHPLC-DAD-MSMS analysis the presence of six flavonoids(isoschaftoside schaftoside isoorientin orientin vitexin

and isovitexin) was confirmed These flavonoids have beendescribed in the literature for this species and some others ofthe Jatropha genus [16 35ndash37] These results are interestingsince several studies show that flavonoids have significantinhibitory activities against some snake venom enzymatictoxins such as PLA2 and hyaluronidases [38 39] Thesecompounds could inhibit snake venom toxins directly actingas enzyme inhibitors and chemical inactivators or indirectlyas immunomodulators interacting with biological targets[38] Additionally these phenolic compounds are capableof chelating metal ions which are essential for the activityof toxins such as SVMPs and PLA2 [34 38] Among theflavonoids detected in J mollissima aqueous leaf extractthe presence of glycosylated derivatives from luteolin could


PBS BerJm mgkg

Dexa 20050 100

Mon

onuc

lear

cells

lowastlowastlowastlowastlowastlowast lowastlowastlowast


0

2

4

6

8

10(times

106m

L)

(a)

PBS BerJm mgkg

Dexa 20050 100

Poly

mor

phon

ucle

ar ce

lls

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

0

5

10

15

(times106m

L)

(b)

PBS Bja Dexa 50 100 200Jm mgKg

lowastlowast

lowastlowast

lowastlowastlowast

lowast

0

5

10

15

Mon

onuc

lear

cells

(times10

6 mL)

(c)

Bja Dexa 50 100 200Jm mgKg

PBS

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

0

5

10

15

20

25

Poly

mon

onuc

lear

cells

(times1

06 mL)

(d)

Figure 6 Evaluation of the inhibition by the J mollissima extract of the cell migration induced by B erythromelas (a b) and B jararacavenoms (c d) (a c) Mononuclear cell count (b d) Polymorphonuclear cell count Evaluation of the leukocyte influx after treatment withthe extract of J mollissima (Jm) (50 100 and 200mgkg ip) and dexamethasone (Dexa) (2mgkg ip) Values were expressed as mean plusmnstandard error of mean with 119899 = 5 lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 when compared with venom control group (ANOVA)

PBS Ber 50 100 200Jm (mgkg)

Dexa



lowastlowast

0

100

200

300

400

CK (U

L)

(a)

PBS Bja 50 100 200Jm (mgkg)

Dexa


lowastlowastlowast


0

100

200

300

400

CK (U

L)

(b)

Figure 7 Inhibition of the myotoxic activity of B erythromelas (a) and B jararaca (b) by aqueous leaf extract of J mollissima B erythromelas(Ber) and B jararaca venoms (Bja) were injected im in the left thigh of animals treated with different ip doses of the J mollissima (Jm)extract 3 h later the blood was collected for creatine kinase (CK) determination Values were expressed as mean plusmn standard error of meanwith 119899 = 5 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 when compared with venom control group (ANOVA)


Table 2 Maximum percentage of inhibition of J mollissima againstlocal effects produced by Bothrops erythromelas and B jararacavenoms Percentage of inhibition was calculated as [1 ndash ( activityof test mean ndash activity of PBS control mean)( activity of Ber orBja control mean ndash activity of PBS control mean)] times 100

Activity Dose Inhibition()

Bothrops erythromelasEdematogeniclowast 50mgkg 4641Inflammatory 100mgkg 8181Myotoxic 200mgkg 8170

Bothrops jararaca

Hemorrhagic 200mgkg 4400Edematogeniclowast 200mgkg 2519Inflammatory 200mgkg 8018Myotoxic 100mgkg 7273

lowastThe edematogenic activity was calculated using the area under the curve

be highlighted as these compounds have been shown topossess antiophidic properties [40 41] Therefore a plausiblehypothesis is that the major compounds of the J mollissimaextract could be responsible for the inhibitory propertiesexperimentally observed in this work To confirm suchassumption the isolation of these compounds is currentlyunderway in our group

Local hemorrhage is one of the main symptoms ofbothropic envenomation [42] The hemorrhagic SVMPs(hemorrhagins) are the main compounds responsible forthis effect causing proteolysis of basal lamina componentsof the microvasculature leading to a rupture of the bloodvessels with the appearance of fissures which will result inleakage of blood to the exterior [43] Additionally SVMPs areresponsible for hydrolyzing proteins in the cell membranesuch as integrins cadherins collagen type IV laminin andfibronectin [42] Indeed SVMPs are the key enzymes thatcontribute to the toxicity of Bothrops genus as it has beenestimated that they comprise at least 30 of the totalprotein content [7] SVMPs are zinc-dependent enzymeswithmolecular masses that range from 20 to 100 kDa and playdifferent functions in snake envenoming such as proteolyticdegradation alterations in blood coagulation proinflamma-tory activity inhibition of platelet aggregation and myotoxicand myonecrotic activity The SVMPs are divided into threeclasses and several subclasses PI SVMPs (no hemorrhagicactivity) PII SVMPs (disintegrin domain with proteolyticactivity) and PIII SVMPs (disintegrin domain and cysteine-rich domain being the most hemorrhagic) [7 44 45] Todate several SVMPs from PI PII and PIII classes have beenpurified andor characterized in B jararaca venom [7 44]while in B erythromelas venom only a very few SVMPs havealready been characterized being PI and PIII SVMPs [46]This study revealed that the J mollissima extract was ableto inhibit the hemorrhage caused by the B jararaca venomIn fact through the external appearance of the hemorrhagichalos a decrease in the hemorrhage according to the increaseof the extract dose was observed (Figure 3(a)) So this resultcould indicate an inhibitory action upon the SVMP actionOn the other hand the J mollissima extract was also able

to inhibit the hemorrhage caused by the B erythromelasvenoms but this decrease was not significant (Figure 3(b))This may be due to the different composition of SVMPs inboth venoms and the possible selectivity of the extract againstSVMPs from the B jararaca venom

Edema is one of the first effects caused by the bothropicenvenoming Various toxins may be responsible for edemato-genic activity produced by bothropic venoms including Asp49 or Lys 49 PLA2 and hemorrhagic or nonhemorrhagicSVMPs [47 48] This activity is the result of combinedaction of various toxins found in Bothrops venoms actingrapidly in the connective and muscle tissue inducing therelease of various endogenous inflammatory mediators Thisis the reason for the decrease in the efficacy of conventionalantivenom serum therapy against these local inflammatoryreactions [4 49] In fact this therapy is able to neutralizethe toxins but cannot neutralize the effects produced by theendogenous inflammatory mediators [49 50] Moreover theinjection of the B jararaca venom in mice paws inducesedema which is mainly mediated by the metabolites of thearachidonic acid and the involvement in a low level ofhistamine serotonin and platelet-activating factor [51 52]SVMPs also have an important role in the inflammatoryresponse by degrading the extracellular matrix an effectthat can affect wound healing and tissue regeneration [48]The results obtained in this study revealed that J mollis-sima extract efficiently inhibited the edematogenic activityproduced by B erythromelas (Figure 4(a)) and B jararaca(Figure 4(b)) venoms after 120 minutes This result was sim-ilar to that produced by dexamethasone which is a steroidalanti-inflammatory drug widely used in medical practicesDexamethasone inhibits the PLA2 and consequently there isa decrease in the production of the products derived from thearachidonic acid which is generated by the cyclooxygenaseand the lipoxygenase route [49] Previous studies show thatdexamethasone decreased the acute inflammatory responseinduced by the Bothrops moojeni in mice because of its abilityto decrease the formation of eicosanoids in the presence ofthe venom [49 50] So in this context two explanationscould be addressed for the inhibitory effect presented by theJ mollissima extract it could be directly inhibiting the toxinsinvolved in the inflammatory effect produced by the venomsandor it could be acting as a potent anti-inflammatoryagent

After the formation of the edema the next local reactionis the recruitment of leukocytes which selectively migrateto the site of the inflammation [53] In the acute inflam-matory response there is a predominant accumulation ofneutrophils These cells represent the first line of defense inthe body and have a phagocytic capacity for the removal ofthe aggressor agent In the later stages of the inflammatoryresponse mononuclear cells are observed [54] Previousstudies have shown that the metalloproteinases present inthe B asper venom when injected into the peritoneal cavityof mice induced an increase in the IL-1 levels followedby an increased expression of adhesion molecules Thesemetalloproteases were also responsible for the activationof the complement system resulting in an increase inthe cell migration [55 56] Bothrops venom also possesses


PLA2 toxins which are important for the induction of theleukocyte migration most likely by inducing more potentlythe release of proinflammatory mediators [57 58] In thisstudy an increase in the leukocytes was observed in theperitoneal cavity after 6 hours and 4 hours induced by Berythromelas and B jararaca venoms respectively Similar tothe dexamethasone J mollissima extract efficiently inhibitedthe number of total leukocytes (Figures 5(a) and 5(b)) andthe number of mononuclear and polymorphonuclear cellsthat migrated into the peritoneal cavity induced by the Berythromelas and B jararaca venoms at all tested doses(Figures 6(a) 6(b) 6(c) and 6(d)) Therefore it can besuggested that the J mollissima extract is inhibiting the Asp49 andor the Lys 49 PLA2 or that the J mollissima extract hasa potent inhibitory effect against the endogenous chemicalmediators released by the action of the toxins Anotherpossibility may be an inhibitory action upon SVMPs sincethe J mollissima extract also presented an antihemorrhagiceffect (Figure 3)

Damage to the muscle tissue (myonecrosis) is a seriouslocal effect of the bothropic envenomation since it can leadto permanent loss of tissue disability and even amputation[59 60] The myotoxicity may be due to a direct actionof the Asp 49 or the Lys 49 PLA2 which directly injuresskeletal muscle cells affecting the integrity of their plasmaticmembrane The PLA2 damages the sarcolemma resulting ina loss of calcium permeability and consequently causingrupture of this membrane leading to a rapid release of thecytosolic markers such as lactate dehydrogenase (LDH) andcreatine kinase (CK) [55 61] SVMPs can lead to myotoxicityby an indirect action due to the ischemia caused by thevascular disorders resulting from hemorrhagic action whichcan lead to muscle necrosis and the consequent release ofCK [4 61] The inflammatory reaction induced by the snakevenoms contributes to further development of muscle dam-age [62 63] Patrao-Neto et al (2013) [49] demonstrated thatdexamethasone decreased the late myotoxicity triggered bybothropic venom since this compound has significant anti-inflammatory properties In this present work J mollissimaextractwas able to decreaseCK levels in animals injectedwithB erythromelas and B jararaca venoms as shown in Figures7(a) and 7(b) respectively This result was similar to the anti-inflammatory drug dexamethasone which possibly inhibitsthe inflammatory effects produced by Bothrops venoms Thisresult suggests that the extract possesses antimyotoxic actionpossibly by inhibiting inflammation induced by the venomsince inflammation is an important finding in the local mus-cle damage Furthermore a possible inhibition of the directaction of PLA2 could be suggested in addition to this anti-inflammatory effect Additionally the action of the extract onthe indirect myotoxic action of SVMPs could be supportedsince the extract also presented an antihemorrhagic effect

5 Conclusions

Overall these results demonstrate the potential of the J mol-lissima extract in the treatment of the local effects producedby bothropic venoms It could be concluded that probably

the aqueous extract from the leaves of J mollissima hassubstances that can inhibit or inactivate the toxins presentedin the B erythromelas and the B jararaca venoms as well asacting indirectly upon endogenous mediators Therefore theset of results provides scientific evidence of the potentiality ofthe J mollissima extract Other experimental models shouldbe tested by our research group in order to suggest theusefulness of this plant as a future potential adjuvant inthe treatment of local effects due to snakebites along withantivenom therapy

Competing Interests

The authors declare that there is no actual or potentialconflict of interests including any financial personal or otherrelationships with other people or organizations

Acknowledgments

The authors acknowledge all participants for their valuabletime and commitment to the study and the Faculdade deFarmacia (UFRN) for providing the facilities that made thisstudy possible The authors also wish to thank Kathleen Fer-nandes Grego from the Butantan Institute who generouslysupplied the B erythromelas venom the CAPES and CNPQfor the financial support and Glenn Hawes MEd (Masterof English Education University of Georgia) for editing thismanuscript

References

[1] J M Gutierrez T Burnouf R A Harrison et al ldquoA call forincorporating social research in the global struggle againstsnakebiterdquo PLoS Neglected Tropical Diseases vol 9 no 9 articlee0003960 2015

[2] L F Chaves T Chuang M Sasa and J M GutierrezldquoSnakebites are associated with poverty weather fluctuationsandEl Ninordquo Science Advances vol 1 no 8 Article ID e15002492015

[3] Brasil Guia de Vigilancia Epidemiologica Ministerio da SaudeBrasılia Brazil 2009

[4] J M Gutierrez and B Lomonte ldquoLocal tissue damage inducedby Bothrops snake venoms A reviewrdquo Memorias do InstitutoButantan vol 51 no 4 pp 211ndash223 1989

[5] G P Queiroz L A Pessoa F C V Portaro M D F DFurtado andDV Tambourgi ldquoInterspecific variation in venomcomposition and toxicity of Brazilian snakes from Bothropsgenusrdquo Toxicon vol 52 no 8 pp 842ndash851 2008

[6] J-P Chippaux V Williams and J White ldquoSnake venom vari-ability methods of study results and interpretationrdquo Toxiconvol 29 no 11 pp 1279ndash1303 1991

[7] T S Kang D Georgieva N Genov et al ldquoEnzymatic toxinsfrom snake venom structural characterization and mechanismof catalysisrdquo The FEBS Journal vol 278 no 23 pp 4544ndash45762011

[8] J M Gutierrez ldquoComprendiendo los venenos de serpientes 50Anos de investigaciones en America LatinardquoRevista de BiologiaTropical vol 50 no 2 pp 377ndash394 2002


[9] J M Gutierrez T Escalante and A Rucavado ldquoExperimentalpathophysiology of systemic alterations induced by Bothropsasper snake venomrdquo Toxicon vol 54 no 7 pp 976ndash987 2009

[10] J M Gutierrez A Rucavado F Chaves C Dıaz and TEscalante ldquoExperimental pathology of local tissue damageinduced by Bothrops asper snake venomrdquo Toxicon vol 54 no7 pp 958ndash975 2009

[11] HADe Silva NM Ryan andH J De Silva ldquoAdverse reactionsto snake antivenom and their prevention and treatmentrdquoBritish Journal of Clinical Pharmacology vol 81 no 3 pp 446ndash452 2016

[12] L Scheske J Ruitenberg and B Bissumbhar ldquoNeeds andavailability of snake antivenoms relevance and application ofinternational guidelinesrdquo International Journal of Health Policyand Management vol 4 no 7 pp 447ndash457 2015

[13] J M Gutierrez D A Warrell D J Williams et al ldquoThe needfor full integration of snakebite envenoming within a globalstrategy to combat the neglected tropical diseases the wayforwardrdquo PLoS Neglected Tropical Diseases vol 7 no 6 ArticleID e2162 2013

[14] M Molander C H Saslis-Lagoudakis A K Jager and NRoslashnsted ldquoCross-cultural comparison of medicinal floras usedagainst snakebitesrdquo Journal of Ethnopharmacology vol 139 no3 pp 863ndash872 2012

[15] A Shabbir M Shahzad P Masci and G C Gobe ldquoProtectiveactivity of medicinal plants and their isolated compoundsagainst the toxic effects from the venomofNaja (cobra) speciesrdquoJournal of Ethnopharmacology vol 157 pp 222ndash227 2014

[16] C K A Leal and M F Agra ldquoEstudo farmacobotanico com-parativo das folhas de Jatropha molissima (Pohl) Baill e Jat-ropha ribifolia (Pohl) Baill (Euphorbiaceae)rdquoActa FarmaceuticaBonaerense vol 24 no 1 pp 5ndash13 2005

[17] C W Sabandar N Ahmat F M Jaafar and I Sahidin ldquoMedic-inal property phytochemistry and pharmacology of severalJatropha species (Euphorbiaceae) a reviewrdquo Phytochemistryvol 85 pp 7ndash29 2013

[18] J Felix-Silva R B Giordani A A D Silva-Jr S M Zucolottoand M D F Fernandes-Pedrosa ldquoJatropha gossypiifolia L(Euphorbiaceae) a review of traditional uses phytochem-istry pharmacology and toxicology of this medicinal plantrdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 369204 32 pages 2014

[19] M F Agra G S Baracho K Nurit I J L D Basılio and VP M Coelho ldquoMedicinal and poisonous diversity of the floraof lsquoCariri Paraibanorsquo Brazilrdquo Journal of Ethnopharmacology vol111 no 2 pp 383ndash395 2007

[20] U P Albuquerque P M Medeiros A L S Almeida et alldquoMedicinal plants of the caatinga (semi-arid) vegetation of NEBrazil a quantitative approachrdquo Journal of Ethnopharmacologyvol 114 no 3 pp 325ndash354 2007

[21] J C Vilar C M D Carvalho and M D F D Furtado ldquoEffectsof the aqueous extracts of plants of the genera Apodanthera(Cucurbitaceae) and Jatropha (Euphorbiaceae) on the lethalityof the venom of Bothrops jararaca (serpentes Viperidae)rdquoBiologia Geral Experimental vol 7 no 2 pp 32ndash39 2007

[22] D H Lyra L S Sampaio D A Pereira A P Silva and C L FAmaral ldquoPollen viability and germination in Jatropha ribifoliaand Jatropha mollissima (Euphorbiaceae) specieswith potentialfor biofuel productionrdquoAfrican Journal of Biotechnology vol 10no 3 pp 368ndash374 2011

[23] J G Melo T A Sousa Araujo V T N Almeida Castro etal ldquoAntiproliferative activity antioxidant capacity and tannin

content in plants of semi-arid northeastern Brazilrdquo Moleculesvol 15 no 12 pp 8534ndash8542 2010

[24] F A Rocha and L I Dantas ldquoAtividade antimicrobiana invitro do latex do aveloz (Euphorbia tirucalli l) pinhao bravo(Jatropha mollissima l) e pinhao roxo (Jatropha gossypiifolia l)sobre microrganismos patogenicosrdquoHolos vol 4 2009

[25] A R Ribeiro F D Andrade M C Medeiros et al ldquoEstudoda atividade anti-helmıntica do extrato etanolico de Jatrophamollissima (Pohl) Baill (Euphorbiaceae) sob Haemonchus con-tortus em ovinos no semiarido paraibanordquo Pesquisa VeterinariaBrasileira vol 34 no 11 pp 1051ndash1055 2014

[26] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[27] A R Roodt J A Dolab J C Dokmetjian S Litwin L Segreand J C Vidal ldquoA comparison of differentmethods to assess thehemorrhagic activity of Bothrops venomsrdquo Toxicon vol 38 no6 pp 865ndash873 2000

[28] J Felix-Silva T Souza Y A S Menezes et al ldquoAqueous leafextract of Jatropha gossypiifolia L (Euphorbiaceae) inhibitsenzymatic and biological actions of Bothrops jararaca snakevenomrdquo PLoS ONE vol 9 no 8 Article ID e104952 2014

[29] AMagalhaes G BD SantosMC D S Verdam et al ldquoInhibi-tion of the inflammatory and coagulant action of Bothrops atroxvenom by the plant speciesMarsypianthes chamaedrysrdquo Journalof Ethnopharmacology vol 134 no 1 pp 82ndash88 2011

[30] T J Mabry K R Markham and M B ThomasThe SystematicIdentification of Flavonoids Springer NewYork NY USA 1970

[31] F Ferreres A Gil-Izquierdo P B Andrade P Valentao andF A Tomas-Barberan ldquoCharacterization of C-glycosyl flavonesO-glycosylated by liquid chromatography-tandem mass spec-trometryrdquo Journal of Chromatography A vol 1161 no 1-2 pp214ndash223 2007

[32] F Ferreres B M Silva P B Andrade R M Seabra and MA Ferreira ldquoApproach to the study of C-glycosyl flavones byion trapHPLC-PAD-ESIMSMS application to seeds of quince(Cydonia oblonga)rdquo Phytochemical Analysis vol 14 no 6 pp352ndash359 2003

[33] A C Patino J C Quintana J M Gutierrez A RucavadoD M Benjumea and J A Pereanez ldquoExtracts of Renealmiaalpinia (rottb) MAAS protect against lethality and systemichemorrhage induced by Bothrops asper venom insights froma model with extract administration before venom injectionrdquoToxins vol 7 no 5 pp 1532ndash1543 2015

[34] A Gomes R Das S Sarkhel et al ldquoHerbs and herbal con-stituents active against snake biterdquo Indian Journal of Experimen-tal Biology vol 48 no 9 pp 865ndash878 2010

[35] J Felix-Silva J A S Gomes L M Q Barbosa et al ldquoSystemicand local anti-inflammatory activity of aqueous leaf extractfrom Jatropha gossypiifolia L (Euphorbiaceae)rdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 6 no 6pp 142ndash145 2014

[36] J Felix-Silva T Souza R B A G Camara et al ldquoIn vitroanticoagulant and antioxidant activities of Jatropha gossypiifoliaL (Euphorbiaceae) leaves aiming therapeutical applicationsrdquoBMC Complementary and Alternative Medicine vol 14 article405 2014

[37] A C Pilon R L Carneiro F Carnevale Neto V S Bolzaniand I Castro-Gamboa ldquoInterval multivariate curve resolutionin the dereplication of HPLC-DAD data from Jatropha gossypi-foliardquo Phytochemical Analysis vol 24 no 4 pp 401ndash406 2013


[38] W B Mors M C Nascimento B M Ruppelt Pereira and NAlvares Pereira ldquoPlant natural products active against snakebitemdashthe molecular approachrdquo Phytochemistry vol 55 no 6pp 627ndash642 2000

[39] M S Santhosh M Hemshekhar K Sunitha et al ldquoSnakevenom induced local toxicities plant secondary metabolites asan auxiliary therapyrdquoMini-Reviews inMedicinal Chemistry vol13 no 1 pp 106ndash123 2013

[40] B M A Carvalho J D L Santos B M Xavier et al ldquoSnakevenom PLA2s inhibitors isolated from Brazilian plants syn-thetic and natural moleculesrdquo BioMed Research Internationalvol 2013 Article ID 153045 8 pages 2013

[41] M S Santhosh M S Sundaram K Sunitha K Kemparajuand K S Girish ldquoViper venom-induced oxidative stress andactivation of inflammatory cytokines a therapeutic approachfor overlooked issues of snakebite managementrdquo InflammationResearch vol 62 no 7 pp 721ndash731 2013

[42] T Escalante A Rucavado J W Fox and J M GutierrezldquoKey events in microvascular damage induced by snake venomhemorrhagicmetalloproteinasesrdquo Journal of Proteomics vol 74no 9 pp 1781ndash1794 2011

[43] C Baldo C Jamora N Yamanouye T M Zorn and A MMoura-da-Silva ldquoMechanisms of vascular damage by hemor-rhagic snake venommetalloproteinases tissue distribution andin situ hydrolysisrdquo PLoS Neglected Tropical Diseases vol 4 no6 article e727 2010

[44] J W Fox and S M T Serrano ldquoInsights into and specula-tions about snake venom metalloproteinase (SVMP) synthesisfolding and disulfide bond formation and their contribution tovenom complexityrdquoThe FEBS Journal vol 275 no 12 pp 3016ndash3030 2008

[45] S Takeda H Takeya and S Iwanaga ldquoSnake venom metal-loproteinases structure function and relevance to the mam-malian ADAMADAMTS family proteinsrdquo Biochimica et Bio-physica Acta (BBA)mdashProteins and Proteomics vol 1824 no 1pp 164ndash176 2012

[46] R J B Jorge H S A Monteiro L Goncalves-Machado et alldquoVenomics and antivenomics of Bothrops erythromelas fromfive geographic populations within the Caatinga ecoregion ofnortheastern Brazilrdquo Journal of Proteomics vol 114 pp 93ndash1142015

[47] J M Gutierrez and B Lomonte ldquoPhospholipase A2 myotoxinsfrom Bothrops snake venomsrdquo Toxicon vol 33 no 11 pp 1405ndash1424 1995

[48] J M Gutierrez and A Rucavado ldquoSnake venom metallopro-teinases their role in the pathogenesis of local tissue damagerdquoBiochimie vol 82 no 9-10 pp 841ndash850 2000

[49] F C Patrao-Neto M A Tomaz M A Strauch et al ldquoDexam-ethasone antagonizes the in vivo myotoxic and inflammatoryeffects of Bothrops venomsrdquo Toxicon vol 69 pp 55ndash64 2013

[50] S DAraujo A De Souza F P B Nunes and L R C GoncalvesldquoEffect of dexamethasone associated with serum therapy ontreatment of Bothrops jararaca venom-induced paw edema inmicerdquo Inflammation Research vol 56 no 10 pp 409ndash413 2007

[51] Y Cury C F P Teixeira and L S Sudo ldquoEdematogenicresponses induced by Bothrops jararaca venom in rats role oflymphocytesrdquo Toxicon vol 32 no 11 pp 1425ndash1431 1994

[52] H A Trebien and J B Calixto ldquoPharmacological evaluation ofrat paw oedema induced by Bothrops jararaca venomrdquo Agentsand Actions vol 26 no 3-4 pp 292ndash300 1989

[53] S R Zamuner J M Gutierrez M N Muscara S A Teixeiraand C F P Teixeira ldquoBothrops asper and Bothrops jararaca

snake venoms trigger microbicidal functions of peritonealleukocytes in vivordquo Toxicon vol 39 no 10 pp 1505ndash1513 2001

[54] SMAlbelda CW Smith andPAWard ldquoAdhesionmoleculesand inflammatory injuryrdquo FASEB Journal vol 8 no 8 pp 504ndash512 1994

[55] C F P Teixeira E C T Landucci E Antunes M Chacurand Y Cury ldquoInflammatory effects of snake venom myotoxicphospholipases A2rdquo Toxicon vol 42 no 8 pp 947ndash962 2003

[56] C D F P Teixeira C M Fernandes J P Zuliani and S FZamuner ldquoInflammatory effects of snake venom metallopro-teinasesrdquo Memorias do Instituto Oswaldo Cruz vol 100 no 1pp 181ndash184 2005

[57] J P Zuliani C M Fernandes S R Zamuner J M Gutierrezand C F P Teixeira ldquoInflammatory events induced by Lys-49 and Asp-49 phospholipases A2 isolated from Bothrops aspersnake venom role of catalytic activityrdquo Toxicon vol 45 no 3pp 335ndash346 2005

[58] J M Gutierrez and B Lomonte ldquoPhospholipases A2 unveilingthe secrets of a functionally versatile group of snake venomtoxinsrdquo Toxicon vol 62 pp 27ndash39 2013

[59] C Herrera J K Macedo A Feoli et al ldquoMuscle tissue damageinduced by the venom of bothrops asper identification of earlyand late pathological events through proteomic analysisrdquo PLOSNeglectedTropical Diseases vol 10 no 4 article e0004599 2016

[60] B Lomonte and J M Gutierrez ldquoPhospholipases A2 fromviperidae snake venoms how do they induce skeletal muscledamagerdquo Acta Chimica Slovenica vol 58 no 4 pp 647ndash6592011

[61] J M A Gutierrez and C L Ownby ldquoSkeletal muscle degener-ation induced by venom phospholipases A2 insights into themechanisms of local and systemicmyotoxicityrdquoToxicon vol 42no 8 pp 915ndash931 2003

[62] S H P Farsky L R C Goncalves and Y Cury ldquoCharacteriza-tion of local tissue damage evoked by Bothrops jararaca venomin the rat connective tissue microcirculation An IntravitalMicroscopic Studyrdquo Toxicon vol 37 no 7 pp 1079ndash1083 1999

[63] S H P Farsky J Walber M Costa-Cruz Y Curry and C FP Teixeira ldquoLeukocyte response induced by Bothrops jararacacrude venom in vivo and in vitro studiesrdquo Toxicon vol 35 no2 pp 185ndash193 1997

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


intergenus interspecies intersubspecies and intraspecies [56] The pathophysiological process of the snake envenomingis complex and includes the combined action of severaltoxins such as snake venom metalloproteinases (SVMPs)snake venom serine proteinases (SVSPs) phospholipasesA2 (PLA2) hyaluronidases bradykinin-releasing enzymeslectins L-amino oxidases and pharmacological mediators[7 8] Bothrops envenomation causes mainly immediate localtissue damage (including pain edema local hemorrhage andmyonecrosis) and systemic effects (cardiovascular alterationscoagulation and renal alterations) [9 10]

Currently the intravenously antivenom serum therapy isthe only specific treatment for snakebites The antivenomhowever has some limitations such as difficult access insome regions risk of immunological reactions (includingserum sickness) high cost and limited effectiveness in theimprovement of the local tissue damage [11 12] Adminis-tration of the antivenom may prevent death but does notprevent local tissue damage and resultant disabilities Thelow inhibition of local effects (due to the delay of receivingthe serum or due to the low efficacy) is the leading cause ofamputations which can lead to serious social economic andhealth negative impacts given that most victims live in ruralareas [13] So the search for new complementary therapiesto treat snakebites has become of utmost importance In thisscenario the use of many medicinal plants has been an oldpractice in folk medicine against snakebites especially intropical and subtropical regions such as Africa Asia andSouth America [14 15]

The Jatropha L genus (Euphorbiaceae) belongs to thesubfamily Crotonoideae and Jatropheae tribe consisting ofmore than 300 species and is widely distributed in the tropicaland subtropical regions of Africa and the Americas [16]The name Jatropha is derived from the Greek words ldquojatrosrdquo(doctor) and ldquotropherdquo (food) which could be correlated withthe medicinal properties of plants from this genus [17] InAfrica Asia and Latin America the Jatropha species are usednot only in traditional medicine to cure various ailmentsbut also as ornamental plants and energy crops [17] Severalknown species from genus Jatropha have been reported fortheir medicinal uses chemical constituents and biologicalactivities such as Jatropha curcas Jatropha elliptica Jatrophagossypifolia and Jatropha mollissima [17 18]

Jatropha mollissima (Pohl) Bail (see Figure S1 in Sup-plementary Material available online at httpdxdoiorg10115520166101742) is a medicinal plant popularly knownin Brazil as ldquopinhao-bravordquo [16] This plant is endemic in thesemiarid region of Northeastern Brazil In folk medicine itis widely used for various purposes especially as antiophidic[19ndash21] and anti-inflammatory [22] remedies healing [20]veterinary vermifuge [19] and treatment of renal inflam-mation and loss of appetite [20] Some studies have shownthat this species has antioxidant [23] antimicrobial [24] andantihelmintic activities [25]Thus the aim of this work was toevaluate the ability of the aqueous leaf extract of J mollissimato inhibit the local effects induced by B erythromelas andB jararaca snake venoms in mice Our results indicate thatJ mollissima aqueous leaf extract has significant inhibitoryaction against hemorrhagic inflammatory and myotoxic

local activities of the Bothrops venoms studied suggesting thepotential of this species as a source of bioactive moleculesagainst bothropic venom

2 Materials and Methods

21 Chemicals and Reagents The flavonoids apigenin(ge99) luteolin (ge98) luteolin-7-O-glucoside (ge98)orientin (ge97) isoorientin (ge98) vitexin (ge95) isovi-texin (ge98) and vitexin-2-O-rhamnoside (ge98) (Sigma-Aldrich) were purchased from Sigma-Aldrich (St LouisMO USA) All other reagents and solvents used were ofanalytical grade The water used was purified by reverseosmosis

22 Plant Material Leaves from Jatropha mollissima werecollected in the city of ldquoRafael Godeirordquo 6∘0410158404010158401015840S7∘4210158405410158401015840W RN Brazil in January 2014 The collection ofthe plant material was conducted under authorization of theBrazilian Authorization and Biodiversity Information System(SISBIO) (Process number 35017) and the Brazilian AccessAuthorization and Dispatch Component of the GeneticPatrimony (CGEN) (Process 0108442013-9) The botanicalidentification of the material was performed by Dr JomarGomes Jardim and a voucher specimen was deposited at theHerbarium of the Bioscience of the Federal University of RioGrande do Norte Brazil (UFRN 16879) After identificationand confirmation of the plant species the leaves were dried atroom temperature triturated with an industrial blender andstored in hermetically sealed bottles until used for aqueousextract preparation

23 Snake Venom Lyophilized B erythromelas and B jara-raca snake venoms were used in this work B erythromelaswas generously supplied by the Instituto Butantan SP BrazilB jararaca was purchased from Sigma-Aldrich (St LouisMO USA) (product number V5625)The scientific use of thematerial was approved by the Brazilian Access Authorizationand Dispatch Component of Genetic Patrimony (CGEN)(Process 0108442013-9) The venom was weighed and dis-solved with phosphate buffer saline (PBS) and the proteincontent quantified by the Bradford method [26]

24 Animals Male and female Swiss albino mice (30ndash35 g)6ndash8 weeks of age used in this study were maintained understandard environmental conditions with free access to waterand food On the day of the experiment the animals wereplaced in the experimental room for at least one hour priorto tests for acclimation All animals were euthanized bysodium thiopental overdose associated with 2 lidocaine byintraperitoneal (ip) route at the end of the experimentsThe experimental protocols using animals were performedin agreement with the National Council for the Controlof Animal Experimentation of Brazil (CONCEA) and theInternational Guiding Principles for Biomedical ResearchInvolving Animals of the Council of International Organiza-tions of Medical Sciences (CIOMS) The animal experimentswere approved by the Ethics Committee on Animal Use from


















3 Results





OH

OHOH

OH

OH

OH

OHOH

OHOH

OH

OH

OH

OHOH

OH

OH

OH OH

OH

OHOH

HO

HO

HO

HO

HO

HO

HO

HO

O

O

OO

O

O

O

O

5

2

431

6

OO

O

O

605030 40 700 10 20(Minutes)

0

20

40

(mAU

)





2 Schaftoside 563 545(19) 503(40) 473(84) 443(100) 383(40) 353(54)







R1 R2 R3

OH

OH

HO O

O

R3

R2

R1















4 Discussion




10 mm

PBS BjaJm (mgkg)



00

02

04

06

08

Hem

orrh

agic

hal

o m

ass (

g)

(a)


10 mm

Jm (mgkg)

lowastlowastlowast

PBS Ber 50 100 20000

02

04

06

Hem

orrh

agic

hal

o m

ass (

g)

(b)





PBSBerDexa



lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowast


60 30 90 1200Time (min)

0

20

40

60

80Ed

ema (

)

(a)



lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowastlowast



60 30 90 1200Time (min)

0

20

40

60

80

Edem

a (

)

(b)


PBS BerJm mgkg

Dexa 20050 100


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

5

10

15

20

(times106m

L)

(a)

PBS BjaJm mgkg

Dexa 20050 100



lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

10

20

30

40

50

(times106m

L)

(b)






PBS BerJm mgkg

Dexa 20050 100

Mon

onuc

lear

cells



0

2

4

6

8

10(times

106m

L)

(a)

PBS BerJm mgkg

Dexa 20050 100

Poly

mor

phon

ucle

ar ce

lls

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

5

10

15

(times106m

L)

(b)


lowastlowast

lowastlowast

lowastlowastlowast

lowast

0

5

10

15

Mon

onuc

lear

cells

(times10

6 mL)

(c)


PBS

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

0

5

10

15

20

25

Poly

mon

onuc

lear

cells

(times1

06 mL)

(d)



Dexa



lowastlowast

0

100

200

300

400

CK (U

L)

(a)


Dexa


lowastlowastlowast


0

100

200

300

400

CK (U

L)

(b)






Bothrops jararaca











5 Conclusions



Competing Interests


Acknowledgments


References








































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


















3 Results





OH

OHOH

OH

OH

OH

OHOH

OHOH

OH

OH

OH

OHOH

OH

OH

OH OH

OH

OHOH

HO

HO

HO

HO

HO

HO

HO

HO

O

O

OO

O

O

O

O

5

2

431

6

OO

O

O

605030 40 700 10 20(Minutes)

0

20

40

(mAU

)





2 Schaftoside 563 545(19) 503(40) 473(84) 443(100) 383(40) 353(54)







R1 R2 R3

OH

OH

HO O

O

R3

R2

R1















4 Discussion




10 mm

PBS BjaJm (mgkg)



00

02

04

06

08

Hem

orrh

agic

hal

o m

ass (

g)

(a)


10 mm

Jm (mgkg)

lowastlowastlowast

PBS Ber 50 100 20000

02

04

06

Hem

orrh

agic

hal

o m

ass (

g)

(b)





PBSBerDexa



lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowast


60 30 90 1200Time (min)

0

20

40

60

80Ed

ema (

)

(a)



lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowastlowast



60 30 90 1200Time (min)

0

20

40

60

80

Edem

a (

)

(b)


PBS BerJm mgkg

Dexa 20050 100


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

5

10

15

20

(times106m

L)

(a)

PBS BjaJm mgkg

Dexa 20050 100



lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

10

20

30

40

50

(times106m

L)

(b)






PBS BerJm mgkg

Dexa 20050 100

Mon

onuc

lear

cells



0

2

4

6

8

10(times

106m

L)

(a)

PBS BerJm mgkg

Dexa 20050 100

Poly

mor

phon

ucle

ar ce

lls

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

5

10

15

(times106m

L)

(b)


lowastlowast

lowastlowast

lowastlowastlowast

lowast

0

5

10

15

Mon

onuc

lear

cells

(times10

6 mL)

(c)


PBS

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

0

5

10

15

20

25

Poly

mon

onuc

lear

cells

(times1

06 mL)

(d)



Dexa



lowastlowast

0

100

200

300

400

CK (U

L)

(a)


Dexa


lowastlowastlowast


0

100

200

300

400

CK (U

L)

(b)






Bothrops jararaca











5 Conclusions



Competing Interests


Acknowledgments


References








































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


OH

OHOH

OH

OH

OH

OHOH

OHOH

OH

OH

OH

OHOH

OH

OH

OH OH

OH

OHOH

HO

HO

HO

HO

HO

HO

HO

HO

O

O

OO

O

O

O

O

5

2

431

6

OO

O

O

605030 40 700 10 20(Minutes)

0

20

40

(mAU

)





2 Schaftoside 563 545(19) 503(40) 473(84) 443(100) 383(40) 353(54)







R1 R2 R3

OH

OH

HO O

O

R3

R2

R1















4 Discussion




10 mm

PBS BjaJm (mgkg)



00

02

04

06

08

Hem

orrh

agic

hal

o m

ass (

g)

(a)


10 mm

Jm (mgkg)

lowastlowastlowast

PBS Ber 50 100 20000

02

04

06

Hem

orrh

agic

hal

o m

ass (

g)

(b)





PBSBerDexa



lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowast


60 30 90 1200Time (min)

0

20

40

60

80Ed

ema (

)

(a)



lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowastlowast



60 30 90 1200Time (min)

0

20

40

60

80

Edem

a (

)

(b)


PBS BerJm mgkg

Dexa 20050 100


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

5

10

15

20

(times106m

L)

(a)

PBS BjaJm mgkg

Dexa 20050 100



lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

10

20

30

40

50

(times106m

L)

(b)






PBS BerJm mgkg

Dexa 20050 100

Mon

onuc

lear

cells



0

2

4

6

8

10(times

106m

L)

(a)

PBS BerJm mgkg

Dexa 20050 100

Poly

mor

phon

ucle

ar ce

lls

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

5

10

15

(times106m

L)

(b)


lowastlowast

lowastlowast

lowastlowastlowast

lowast

0

5

10

15

Mon

onuc

lear

cells

(times10

6 mL)

(c)


PBS

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

0

5

10

15

20

25

Poly

mon

onuc

lear

cells

(times1

06 mL)

(d)



Dexa



lowastlowast

0

100

200

300

400

CK (U

L)

(a)


Dexa


lowastlowastlowast


0

100

200

300

400

CK (U

L)

(b)






Bothrops jararaca











5 Conclusions



Competing Interests


Acknowledgments


References








































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


R1 R2 R3

OH

OH

HO O

O

R3

R2

R1















4 Discussion




10 mm

PBS BjaJm (mgkg)



00

02

04

06

08

Hem

orrh

agic

hal

o m

ass (

g)

(a)


10 mm

Jm (mgkg)

lowastlowastlowast

PBS Ber 50 100 20000

02

04

06

Hem

orrh

agic

hal

o m

ass (

g)

(b)





PBSBerDexa



lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowast


60 30 90 1200Time (min)

0

20

40

60

80Ed

ema (

)

(a)



lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowastlowast



60 30 90 1200Time (min)

0

20

40

60

80

Edem

a (

)

(b)


PBS BerJm mgkg

Dexa 20050 100


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

5

10

15

20

(times106m

L)

(a)

PBS BjaJm mgkg

Dexa 20050 100



lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

10

20

30

40

50

(times106m

L)

(b)






PBS BerJm mgkg

Dexa 20050 100

Mon

onuc

lear

cells



0

2

4

6

8

10(times

106m

L)

(a)

PBS BerJm mgkg

Dexa 20050 100

Poly

mor

phon

ucle

ar ce

lls

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

5

10

15

(times106m

L)

(b)


lowastlowast

lowastlowast

lowastlowastlowast

lowast

0

5

10

15

Mon

onuc

lear

cells

(times10

6 mL)

(c)


PBS

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

0

5

10

15

20

25

Poly

mon

onuc

lear

cells

(times1

06 mL)

(d)



Dexa



lowastlowast

0

100

200

300

400

CK (U

L)

(a)


Dexa


lowastlowastlowast


0

100

200

300

400

CK (U

L)

(b)






Bothrops jararaca











5 Conclusions



Competing Interests


Acknowledgments


References








































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



10 mm

PBS BjaJm (mgkg)



00

02

04

06

08

Hem

orrh

agic

hal

o m

ass (

g)

(a)


10 mm

Jm (mgkg)

lowastlowastlowast

PBS Ber 50 100 20000

02

04

06

Hem

orrh

agic

hal

o m

ass (

g)

(b)





PBSBerDexa



lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowast


60 30 90 1200Time (min)

0

20

40

60

80Ed

ema (

)

(a)



lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowastlowast



60 30 90 1200Time (min)

0

20

40

60

80

Edem

a (

)

(b)


PBS BerJm mgkg

Dexa 20050 100


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

5

10

15

20

(times106m

L)

(a)

PBS BjaJm mgkg

Dexa 20050 100



lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

10

20

30

40

50

(times106m

L)

(b)






PBS BerJm mgkg

Dexa 20050 100

Mon

onuc

lear

cells



0

2

4

6

8

10(times

106m

L)

(a)

PBS BerJm mgkg

Dexa 20050 100

Poly

mor

phon

ucle

ar ce

lls

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

5

10

15

(times106m

L)

(b)


lowastlowast

lowastlowast

lowastlowastlowast

lowast

0

5

10

15

Mon

onuc

lear

cells

(times10

6 mL)

(c)


PBS

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

0

5

10

15

20

25

Poly

mon

onuc

lear

cells

(times1

06 mL)

(d)



Dexa



lowastlowast

0

100

200

300

400

CK (U

L)

(a)


Dexa


lowastlowastlowast


0

100

200

300

400

CK (U

L)

(b)






Bothrops jararaca











5 Conclusions



Competing Interests


Acknowledgments


References








































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


PBSBerDexa



lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowast


60 30 90 1200Time (min)

0

20

40

60

80Ed

ema (

)

(a)



lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowastlowast



60 30 90 1200Time (min)

0

20

40

60

80

Edem

a (

)

(b)


PBS BerJm mgkg

Dexa 20050 100


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

5

10

15

20

(times106m

L)

(a)

PBS BjaJm mgkg

Dexa 20050 100



lowastlowastlowast

Perit

onea

l leu

kocy

tes

0

10

20

30

40

50

(times106m

L)

(b)






PBS BerJm mgkg

Dexa 20050 100

Mon

onuc

lear

cells



0

2

4

6

8

10(times

106m

L)

(a)

PBS BerJm mgkg

Dexa 20050 100

Poly

mor

phon

ucle

ar ce

lls

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

5

10

15

(times106m

L)

(b)


lowastlowast

lowastlowast

lowastlowastlowast

lowast

0

5

10

15

Mon

onuc

lear

cells

(times10

6 mL)

(c)


PBS

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

0

5

10

15

20

25

Poly

mon

onuc

lear

cells

(times1

06 mL)

(d)



Dexa



lowastlowast

0

100

200

300

400

CK (U

L)

(a)


Dexa


lowastlowastlowast


0

100

200

300

400

CK (U

L)

(b)






Bothrops jararaca











5 Conclusions



Competing Interests


Acknowledgments


References








































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


PBS BerJm mgkg

Dexa 20050 100

Mon

onuc

lear

cells



0

2

4

6

8

10(times

106m

L)

(a)

PBS BerJm mgkg

Dexa 20050 100

Poly

mor

phon

ucle

ar ce

lls

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


0

5

10

15

(times106m

L)

(b)


lowastlowast

lowastlowast

lowastlowastlowast

lowast

0

5

10

15

Mon

onuc

lear

cells

(times10

6 mL)

(c)


PBS

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

0

5

10

15

20

25

Poly

mon

onuc

lear

cells

(times1

06 mL)

(d)



Dexa



lowastlowast

0

100

200

300

400

CK (U

L)

(a)


Dexa


lowastlowastlowast


0

100

200

300

400

CK (U

L)

(b)






Bothrops jararaca











5 Conclusions



Competing Interests


Acknowledgments


References








































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Bothrops jararaca











5 Conclusions



Competing Interests


Acknowledgments


References








































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




5 Conclusions



Competing Interests


Acknowledgments


References








































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Aqueous Leaf Extract of Jatropha ...downloads.hindawi.com › journals › bmri ›...

Documents

Transcript of Research Article Aqueous Leaf Extract of Jatropha ...downloads.hindawi.com › journals › bmri ›...