Antiophidian properties of the aqueous extract of Mikania glomerata

Journal of Ethnopharmacology 102 (2005) 364–370

Antiophidian properties of the aqueous extract ofMikania glomerata

Victor A. Maioranoa, Silvana Marcussib,c, Maristela A.F. Dahera, Clayton Z. Oliveiraa,c,Lucelio B. Coutod, Odair A. Gomesd, Suzelei C. Franc¸aa,

Andreimar M. Soaresc,∗, Paulo S. Pereiraa,∗a Unidade de Biotecnologia, Universidade de Ribeirao Preto (UNAERP), Ribeirao Preto, SP, Brazil

b Departamento de Bioquımica e Imunologia, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo (USP), Ribeirao Preto, SP, Brazilc Departamento de Analises Clınicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto (FCFRP),

Universidade Sao Paulo (USP), Ribeirao Preto, SP, Brazild Curso de Medicina, Universidade de Ribeirao Preto (UNAERP), Ribeirao Preto, SP, Brazil

Received 24 September 2004; received in revised form 15 June 2005; accepted 18 June 2005Available online 3 August 2005

Abstract

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Aqueous extracts, prepared from dried or fresh roots, stems or leaves ofMikania glomerata, a plant found in Mata Atlantica in SoutheasternBrazil, were able to efficiently neutralize different toxic, pharmacological, and enzymatic effects induced by venoms fromBothrops andCrotalus snakes. Phospholipase A2 activity and the edema induced byCrotalus durissus terrificus venom were inhibited around 100 an∼40%, respectively, although this inhibition was only partial forBothrops venoms. The hemorrhagic activity ofBothrops venoms (Bothropsaltenatus, Bothrops moojeni, Bothrops neuwiedi, andBothrops jararacussu) was significantly inhibited by this vegetal species, while tclotting activity of Crotalus durissus terrificus, Bothrops jararacussu, andBothrops neuwiedi venoms was totally inhibited. Although, thmechanism of action ofMikania glomerata extract is still unknown, the finding that no visible change was detected in the electrophpattern of snake venom after incubation with the extract excludes proteolytic degradation as a potential mechanism. Since the extractMikaniaglomerata significantly inhibited the studied snake venoms, it may be used as an alternative treatment to serumtherapy and, in ada rich source of potential inhibitors of PLA2s, metalloproteases and serineproteases, enzymes involved in several physiopathologicaand animal diseases.© 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Mikania glomerata; Snake venoms; Antiophidian activity; Anti-snake venoms; Antitoxins; Natural inhibitors

Abbreviations: AEDS, aqueous extract of the dried stem fromMikaniaglomerata; AEDL, aqueous extract of the dried leaves fromMikania glom-erata; AEDR, aqueous extract of the dried root fromMikania glomerata;AEFR, aqueous extract of the fresh root fromMikania glomerata; AEFS,aqueous extract of the fresh stem fromMikania glomerata; AEFL, aqueousextract of the fresh leaves fromMikania glomerata; Balt, Bothrops alterna-tus venom; Bjussu,Bothrops jararacussu venom; Bmooj,Bothrops moojenivenom; Bneuw,Bothrops neuwiedi venom; Cdt,Crotalus durissus terri-flcus venom; MCD, minimum coagulant dose; MHD, minimum hemorrhagicdose; MiHD, minimum indirect hemolytic dose; PBS, phosphate-bufferedsaline solution; PLA2, phospholipase A2

∗ Corresponding authors. Tel.: +55 16 3967 3572; fax: +55 16 633 1936.E-mail addresses: [email protected] (A.M. Soares),

[email protected] (P.S. Pereira).

1. Introduction

Animal venoms, including snake venoms, embody a coplex mixture of toxic enzymes and proteins, as phosphpases A2, myotoxins, hemorrhagic metalloproteases, clottserineproteases, neurotoxins, cytotoxins and others. In BrBothrops and Crotalus snakes are responsible for mostthe envenomations which induce mainly local tissue dam(as hemorrhage, necrosis and edema) and systemic efas alterations in blood clotting, neurotoxicity, and sho(Ownby, 1990; Braud et al., 2000).

Envenomation by snakes is often treated by partheral antiophidian serum administration, obtained frhyperimmunized equine serum, often inducing adve

0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.jep.2005.06.039

V.A. Maiorano et al. / Journal of Ethnopharmacology 102 (2005) 364–370 365

reactions. During serumtherapy, neutralization of toxic sys-temic effects is usually reached, but neutralization oflocal tissue damage usually does not occur (Cardoso etal., 2003). Thus, it is important to search novel venominhibitors, be it synthetic or natural, able to complementserumtherapy, in order to neutralize mainly local damages ofenvenomation.

Vegetal extracts constitute an alternative for treatment, dis-playing a large diversity of chemical compounds with severalpharmacological activities of medical-scientific interest. Alarge number of extracts were already prepared and showedexcelent antivenom activities (Martz, 1992; Melo et al., 1994;Mors et al., 2000; Izidoro et al., 2003; Soares et al., 2004; daSilva et al., 2005; Oliveira et al., 2005).

Mikania glomerata (Asteraceae) popularly known as“guaco”, shows many well-known pharmacological activ-ities, among them antifungal, antimicrobian, bronchodila-tor, anti-allergic and anti-inflammatory (Fierro et al.,1999; Holetz et al., 2002). Ruppelt et al. (1991)identi-fied anti-inflammatory activity againstBothrops jararacavenom, in the infusion ofMikania glomerata. This isa species scarcely studied chemically, but several com-pounds were already therefrom isolated, chiefly cumarins,diterpenes, and essential oils (Veneziani et al., 1999;Cabral et al., 2001; Celeghini et al., 2001). The presentstudy shows the ability of aqueous extracts, from driedo lav

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Filho of the Instituto de Biologia, UNICAMP. Two hun-dred grams of each part collected (leaves, stems, and roots)were submitted to extraction by boiling water (1 L) andmacerated for 24 h. The remaining material was dried ina ventilated stove at 60◦C and then extracted with boil-ing water (150 g/L) and macerated for 24 h. Following thisextraction period, a vacuum filtration was performed andthe extract was lyophilized. Extracts from fresh (AEFS,AEFL, and AEFR; yield: 2.0, 2.6, and 6.5% (w/w)) anddried (AEDS, AEDL, and AEDR; yield: 8.8, 11.7, and 7.7%(w/w)) material was so obtained. The dried extracts werekept at 4◦C. A chromatographic procedure was performedwith all extracts by TLC and HPLC at 20 mg/mL. The TLCsystem consisted of silica gel plates (10 cm× 10 cm) andn-butanol:acetic acid:water (4:1:5; upper phase) as mobilephase, detection at 254 and 366 nm, using vanilin or NP-PEG reagent. The reverse phase HPLC system included:Supelcosil C18 column (4.6 mm× 250 mm, 5�m) usinga linear gradient of methanol:water:0.1% acetic acid asmobile phase, at 10–100% methanol during 50 min andUV detection (210, 280, and 340 nm), a flow rate of1 mL/min. Chromatograms and its spectral data were com-pared with spectra of standards and those from literature.Protein concentration was determined by the Micro-biuretmethod.

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r fresh Mikania glomerata to inhibit pharmacologicand enzymatic activities ofBothrops and Crotalus snakeenoms.

. Materials and methods

.1. Venoms and animals

Bothrops jararacussu, Bothrops moojeni, Bothrops alter-atus, Bothrops neuwiedi, and Crotalus durissus terri-cus venoms were purchased from Serpentario Bioagentsatatais, SP. Groups of six male Swiss mice (18–22 g)btained from the Central Bioterium of USP in Ribeiaoreto. Animals were housed in standard cages and m

ained under standard conditions (12 h light/12 h dark ct room temperature). Mice feed and tap water were provd libitum. All experimental procedures were performeccordance with the guidelines of the institutional aniare and use commitee of the UNAERP in accordancehe legislation on animal care.

.2. Preparation of plant extract

Leaves, stems, and roots ofMikania glomerata SprengeAsteraceae) were collected from Unidade de Biotecnia of Universidade de Ribeirao Preto (UNAERP), Ribeiraoreto, SP, Brazil. A voucher specimen (no. 24) of the planeen deposited at the Unidade de Biotecnologia, UNAnd authenticated by Professor Hermogenes de Freitas Leitao

.3. Inhibition of the venom

Desiccated venoms were weighed and dissolved int 10 or 2�g/�L. For inhibition experiments, solutions co

aining a fixed amount of venom were mixed with varymounts of AEF and AED from leaves, stems, and roorder to obtain various ratios (w/w) venom:inhibitor. All m

ures were incubated for 30 min at 37◦C and aliquots werssayed in the different assay systems.

.4. Inhibition of phospholipase A2 activity

Indirect hemolytic activity was assayed using agaroseolk–erythrocyte gels as substrate. A minimum indiemolytic dose (MiHD) was defined for each venom asmount of enzyme that produces halos of 10 mm, as descy Gutierrez et al. (1988). AEF or AED from leaves, stemnd roots were assayed after incubation with all crudems at ratios 1:10, 1:50, 1:100, and 1:200 (w/w). Enzymctivity was expressed as a percentage, 100% inhibitionesponding to the absence of activity of the venom incubith the aqueous extract. Each experiment was express

he mean± S.D. (n = 6).

.5. Edema-inducing activity

Edema was evaluated after subplantar injection, inight footpad of male Swiss mice (n = 6, 18–22 g), ofBothropsararacussu andCrotalus durissus terriflcus venoms. Inhibiion studies were performed after preincubation of ve

366 V.A. Maiorano et al. / Journal of Ethnopharmacology 102 (2005) 364–370

with AEF or AED from leaves, stems and roots at 1:50 (w/w)ratio. Control animals received an injection of PBS underidentical conditions. The progression of edema was evaluatedwith a low-pressure pachymeter (Mtutoyo, Japan) at varioustime intervals after injection (Soares et al., 2000). Activitywas expressed as the mean of percent edema values inducedby the snake venoms in the absence and presence of the plantextracts.

2.6. Hemorrhagic activity

A minimum hemorrhagic dose (MHD), evaluated only forcrudeBothrops venoms, is the amount of venom which pro-duces a hemorrhagic halo of 10 mm (Nikai et al., 1984). Swissmale mice (n = 6, 18–22 g) were injected intradermically inthe back with dose of 1:50 (w/w), lMHDvenom/AEF or AEDfrom leaves, stems, and roots). Control mice received PBSalone. Two hours after injection, mice were killed, and thediameter of the hemorrhage zone in the skin was measured.Hemorrhagic activity was expressed by the mean (in mm) ofthe hemorrhagic halos induced by the venoms in the absenceand presence of the plant extracts.

2.7. Coagulant activity

thea 0 s(w orA :100( at3 dedp ne.C oag-u encea

2.8. Polyacrylamide gel electrophoresis (SDS–PAGE)

SDS–PAGE was performed on a 12% (w/v) polyacry-lamide gel, in Tris buffer at pH 8.1, at 15 mA, for 2 h, follow-ing the method ofLaemmli (1970)for denaturant proteins.

2.9. Statistical analysis

Results are presented as the mean± S.D. obtained withthe indicated number of tested animals. The statistical sig-nificance of differences between groups was evaluated usingStudent’s unpairedt-test. Ap value <0.05 was considered toindicate significance.

3. Results and discussion

Vegetal extracts constitute an excelent alternative sourceof novel antiophidian agents. In many countries, vegetalextracts have been traditionally used in the treatment ofenvenomations evoked by snakebites (Mors, 1991), but inmost cases, the use of these plants need scientific evidenceof their claimed pharmacological activities. Several plantsshowed already to have antivenom activity (Gowda, 1997;Mors et al., 2000; Mahanta and Mukherjee, 2001; Soares eta

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A minimum coagulant dose (MCD) was defined asmount ofBothrops venom which clots 0.2 mL plasma in 6Gene et al., 1989). Briefly, aliquots of 0.2 mL plasma (n = 6)ere incubated with 50�L of each venom or venom/AEFED from leaves, stems, and roots at 1:10, 1:50, and 1

w/w, lMCD venom/AEF or AED), incubated for 30 min7◦C and clotting times recorded. Control tubes inclulasma incubated wilh PBS plus calcium or AEF/AED alooagulant activity was expressed by the mean time of clation (in min) induced by the snake venoms in the absnd presence of plant extracts.

able 1nhibition of PLA2 activity of snake venoms by aqueous extracts fromt differents ratios for 30 min at 37◦C

amples Inhibition of phospholip

Without AE 1:

othrops jararacussu 0.0 –othrops jararacussu + AEFR 0.0 1othrops jararacussu + AEFS 0.0 1othrops jararacussu + AEFL 0.0 2

rotalus durissus terrificus – 0rotalus durissus terrificus + AEFR 0.0 5rotalus durissus terrificus + AEFS 0.0 3rotalus durissus terrificus + AEFL 0.0 8

othrops jararacussu 0.0 –othrops jararacussu + AEDR 0.0

rotalus durissus terrificus 0.0 –rotalus durissus terrificus + AEDR 0.0 4a Enzymatic activity was expressed as a percentage. 100% of inhibitach experiment represents the mean± S.D. (n = 6).

l., 2004).PLA2 activity induced byCrotalus durissus terrificus

enom was totally inhibited by the aqueous extracts fresh or dried roots and stems ofMikania glomerata, while,or Bothrops jararacussu venom, no significative inhibitioas observed (Table 1). Among the extracts from drieources, only that from the root (AEDR) showed an inhion of 25% againstBothrops jararacussu venom and 100%gainstCrotalus durissus terrificus venom (Table 1). Aque-us extracts fromMandevilla velutina, Mandevilla illustris,nd Casearia sylvestris neutralized 75–100% of the PLA2

ED) and fresh (AEF) (root, stem, and leaves)Mikania glomerata, preincubate

tivity (%)a

) 1:50 (w/w) 1:100 (w/w) 1:200 (w/w

– – –20.0 25.0 30.015.0 15.0 15.03.0 10.0 10.0

– – –95.0 100.0 100.075.0 85.0 100.025.0 45.0 80.0

– – –15.0 18.0 25.0

– – –95.0 100.0 100.0

responding to the absence of activity of venom incubated with the aquract.

V.A. Maiorano et al. / Journal of Ethnopharmacology 102 (2005) 364–370 367

Fig. 1. Inhibition of edema-inducing activity of crude snake venoms by aqueous extracts from fresh (A) and dried (B) ofMikania glomerata, preincubated withratio at 1:50 (w/w) for 30 min at 37◦C. Each experiment represents the mean± S.D. (n = 6).

activity of Crotalus durissus terrificus venom, respectively(Borges et al., 2000; Biondo et al., 2003, 2004). Therefore,theMikania glomerata extract is also a good source of power-ful natural PLA2 inhibitors, mainly neurotoxic PLA2s foundin Crotalus durissus terrificus venom.

Regarding edema, only AEDR or AEFR inhibited∼40%of this effect induced byCrotalus durissus terrificus venom,at 1:50 (w/w) ratio (Fig. 1), while the other extracts didnot show any anti-edema activity against the different snakevenoms.Soares de Moura et al. (2002)showed that frac-tion MG1, a dichloromethane fraction and that contains 11%of coumarin, fromMikania glomerata, decreased 30% theedema produced byBothrops jararaca venom.Biondo etal. (2003)also noted thatCrotalus durissus terrificus andBothrops alternatus venoms, at 1:30 (w/w) ratio withMan-devilla velutina extract, had 46 and 30%, respectively, of theiredema inducing activitity inhibited. Regarding the extractsof Mikania glomerata, we suggest that its natural inhibitorsmight be acting against the PLA2s in rattlesnake venom,similarly as it was observed byBorges et al. (2000)usingCasearia sylvestris extract and PLA2s isolated from snakevenoms.

Several works have explored vegetal extracts andinhibitors with antihemorrhagic activity (Borges et al., 2001;Soares et al., 2004; Januario et al., 2004). Our results showedthat the hemorrhagic activity ofBothrops alternatus venomw R,r lso8 ni opro-t entialm

B -i

ata (Table 2). Fresh and dried root extracts also inhibitedtotally Crotalus andBothrops venoms (Table 2). Borges et al.(2001)evidenced the ability of vegetal extracts fromCaseariasylvestris to inhibit proteases isolated from severalBoth-rops snakes.Melo et al. (1994)identified antiproteolytic andantihemorrhagic components in the crude extract ofEcliptaProstrata.

These envenomations usually produce prolonged hemor-rhages due to a considerable degradation of fibrinogen andother clotting factors, avoiding clot formation (Markland,

F yae tr

as 50 and 80% inhibited by AEFS and AEFR or AEDespectively. The extracts AEDR or AEFR inhibited a0–95% of otherBothrops venoms (Fig. 2). This suggests a

nteraction between the extract components and metalleases, involving catalytic sites of these enzymes or essetal ions, thus, inhibiting their hemorrhagic activities.The clotting activity induced byBothrops jararacussu,

othrops neuwiedi andBothrops moojeni venoms were inhibted by the different extracts from driedMikania glomer-

ig. 2. Inhibition of hemorrhagic activity of crudeBothrops snake venom bqueous extracts of dried and fresh (roots, stems, and leaves)Mikania glom-rata, preincubated at 1:50 (w/w) ratio for 30 min at 37◦C. Each experimenepresents the mean± S.D. (n = 8).

368 V.A. Maiorano et al. / Journal of Ethnopharmacology 102 (2005) 364–370

Table 2Inhibition of the coagulant activity of snake venoms (l0�g) by aqueous extracts from dried (AED) and fresh (AEF) (root, stem and leaves)Mikania glomerata,preincubated at differents ratios for 30 min at 37◦C

Samples Coagulant activity (min)a

Without AE 1:10 (w/w) 1:50 (w/w) 1:100 (w/w)

PBS + Ca2+ 5 min 15 s± 0.02 – – –

Bothrops jararacussu l min 15 s± 0.01 – – –Bothrops jararacussu + AEFR – 18 min 30 s± 0.02 >45 min 00 s± 0.02 >45 min 00 s± 0.02Bothrops jararacussu + AEFS – 5 min 00 s± 0.02 12 min 00 s± 0.02 33 min 40 s± 0.02Bothrops jararacussu + AEFL – 2 min 00 s± 0.02 3 min 30 s± 0.02 4 min 00 s± 0.02

Crotalus durissus terrificus l min 48 s± 0.01 – – –Crotalus durissus terrificus + AEFR – 27 min 40 s± 0.02 >45 min 00 s± 0.02 >45 min 00 s± 0.02Crotalus durissus terrificus + AEFS – 8 min 00 s± 0.02 22 min 00 s± 0.02 30 min 10 s± 0.02Crotalus durissus terrificus + AEFL – 5 min 00 s± 0.02 8 min 30 s± 0.02 9 min 00± 0.02

Bothrops moojeni 45 s± 0.01 – – –Bothrops moojeni + AEFR – 3 min 00 s± 0.02 12 min 00 s± 0.02 >45 min 0 s± 0.02Bothrops moojeni + AEFS – 3 min 00 s± 0.02 13 min 00 s± 0.02 23 min 00 s± 0.02Bothrops moojeni + AEFL – 2 min 00 s± 0.02 4 min 30 s± 0.02 3 min 30 s± 0.02

Bothrops neuwiedi 48 s± 0.01 – – –Bothrops neuwiedi + AEFR – 2 min 00 s± 0.02 11 min 00 s± 0.02 >45 min 00 s± 0.02Bothrops neuwiedi + AEFS – 2 min 00 s± 0.02 8 min 00 s± 0.02 35 min 00 s± 0.02Bothrops neuwiedi + AEFL – 2 min 00 s± 0.02 6 min 30 s± 0.02 6 min 30 s± 0.02

Bothrops jararacussu 1 min 15 s± 0.01 – – –Bothrops jararacussu + AEDR – 15 min 00 s± 0.02 >45 min 00 s± 0.02 >45 min 00 s± 0.02Crotalus durissus terrificus 1 min48 s± 0.01 – – –Crotalus durissus terrificus + AEDR – 18 min 30 s± 0.02 >45 min 00 s± 0.02 >45 min 00 s± 0.02

Bothrops moojeni 45 s± 0.01 – – –Bothrops moojeni + AEDR – 3 min 00 s± 0.02 15 min 00 s± 0.02 >45 min 00 s± 0.02

Bothrops neuwiedi 48 s± 0.01 – – –Bothrops neuwiedi + AEDR – 2 min 00 s± 0.02 14 min 00 s± 0.02 >45 min 00 s± 0.02

a Each experiment represents the mean± S.D. (n = 6).

1998). Results withMikania glomerata showed that itsextracts act as powerful inhibitors of the clotting activity,probably due to interaction with thrombin-like enzymes.The action mechanism ofMikania glomerata is howeverunknown, but, as shown inFig. 3, no proteolytic degrada-tion is evident.

Concluding,Mikania glomerata extracts were able to con-siderably inhibit most of the activities of the assayed venoms.TLC and HPLC profile showed a similarity between freshand dried extracts composition. The presence of coumarinand other non-polar compounds was verified in leaves andstem extracts, but coumarin concentration decreases in thestem extract and it is lower in the dried extract. A highercontent of total protein was observed in the aqueous extractfrom roots (15–30%) and lower in the leave and stem extracts(<8%). The inhibitory ability against isolated toxins shouldalso be later investigated. Some studies point to coumarinsas main components ofMikania glomerata showing severalactivities. However, pharmacological studies should still beperformed using new extract fractions, in order to isolate andcharacterize the active principle responsible for the antio-phidian activity, thus providing insights to the elucidation ofthe action mechanism of the corresponding toxins and devel-

Fig. 3. PAGE to show interaction between snake venoms and plantMikaniaglomerata extracts. Samples containing venoms and extracts were incubatedfor 30 min at 37◦C in ratios 1:50 (w/w). 1, MW markers; 2,Bothrops jarara-cussu venom (20�g); 3,Bothrops jararacussu venom + AEFR; 4,Bothropsjararacussu venom + AEFS; 5,Crotalus durissus terriflcus venom (20�g);6, Crotalus durissus terriflcus venom + AEDR; 7,Crotalus durissus terri-flcus venom + AEDS; 8, AEDS (1000�g); 9, AEDL (1000�g); 10, AEDR(1000�g).

V.A. Maiorano et al. / Journal of Ethnopharmacology 102 (2005) 364–370 369

opment of future therapeutic agents for treatment of ophidianaccidents.

Acknowledgements

The authors are grateful to the financial support byFundac¸ao de Pesquisa do Estado de Sao Paulo (FAPESP) andUniversidade de Ribeirao Preto (UNAERP), and the technicalassistence of all students and technicians (Vanessa C. Fernan-des and Eliandra G. Silva, TT-II, FAPESP) who collaboratedin this work.

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