Antiophidian properties of the aqueous extract of Mikania glomerata

Antiophidian properties of the aqueous extract of Mikania glomerata

Journal of Ethnopharmacology 102 (2005) 364–370 Antiophidian properties of the aqueous extract of Mikania glomerata Victor A. Maiorano a , Silvana Ma...

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Journal of Ethnopharmacology 102 (2005) 364–370

Antiophidian properties of the aqueous extract of Mikania glomerata Victor A. Maiorano a , Silvana Marcussi b,c , Maristela A.F. Daher a , Clayton Z. Oliveira a,c , Luc´elio B. Couto d , Odair A. Gomes d , Suzelei C. Franc¸a a , Andreimar M. Soares c,∗ , Paulo S. Pereira a,∗ a

b

Unidade de Biotecnologia, Universidade de Ribeir˜ao Preto (UNAERP), Ribeir˜ao Preto, SP, Brazil Departamento de Bioqu´ımica e Imunologia, Faculdade de Medicina de Ribeir˜ao Preto, Universidade de S˜ao Paulo (USP), Ribeir˜ao Preto, SP, Brazil c Departamento de An´ alises Cl´ınicas, Toxicol´ogicas e Bromatol´ogicas, Faculdade de Ciˆencias Farmacˆeuticas de Ribeir˜ao Preto (FCFRP), Universidade S˜ao Paulo (USP), Ribeir˜ao Preto, SP, Brazil d Curso de Medicina, Universidade de Ribeir˜ ao Preto (UNAERP), Ribeir˜ao Preto, SP, Brazil Received 24 September 2004; received in revised form 15 June 2005; accepted 18 June 2005 Available online 3 August 2005

Abstract Aqueous extracts, prepared from dried or fresh roots, stems or leaves of Mikania glomerata, a plant found in Mata Atlˆantica in Southeastern Brazil, were able to efficiently neutralize different toxic, pharmacological, and enzymatic effects induced by venoms from Bothrops and Crotalus snakes. Phospholipase A2 activity and the edema induced by Crotalus durissus terrificus venom were inhibited around 100 and ∼40%, respectively, although this inhibition was only partial for Bothrops venoms. The hemorrhagic activity of Bothrops venoms (Bothrops altenatus, Bothrops moojeni, Bothrops neuwiedi, and Bothrops jararacussu) was significantly inhibited by this vegetal species, while the clotting activity of Crotalus durissus terrificus, Bothrops jararacussu, and Bothrops neuwiedi venoms was totally inhibited. Although, the mechanism of action of Mikania glomerata extract is still unknown, the finding that no visible change was detected in the electrophoretic pattern of snake venom after incubation with the extract excludes proteolytic degradation as a potential mechanism. Since the extract of Mikania glomerata significantly inhibited the studied snake venoms, it may be used as an alternative treatment to serumtherapy and, in addition, as a rich source of potential inhibitors of PLA2 s, metalloproteases and serineproteases, enzymes involved in several physiopathological human and animal diseases. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Mikania glomerata; Snake venoms; Antiophidian activity; Anti-snake venoms; Antitoxins; Natural inhibitors

1. Introduction Abbreviations: AEDS, aqueous extract of the dried stem from Mikania glomerata; AEDL, aqueous extract of the dried leaves from Mikania glomerata; AEDR, aqueous extract of the dried root from Mikania glomerata; AEFR, aqueous extract of the fresh root from Mikania glomerata; AEFS, aqueous extract of the fresh stem from Mikania glomerata; AEFL, aqueous extract of the fresh leaves from Mikania glomerata; Balt, Bothrops alternatus venom; Bjussu, Bothrops jararacussu venom; Bmooj, Bothrops moojeni venom; Bneuw, Bothrops neuwiedi venom; Cdt, Crotalus durissus terriflcus venom; MCD, minimum coagulant dose; MHD, minimum hemorrhagic dose; MiHD, minimum indirect hemolytic dose; PBS, phosphate-buffered saline 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). 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.06.039

Animal venoms, including snake venoms, embody a complex mixture of toxic enzymes and proteins, as phospholipases A2 , myotoxins, hemorrhagic metalloproteases, clotting serineproteases, neurotoxins, cytotoxins and others. In Brazil, Bothrops and Crotalus snakes are responsible for most of the envenomations which induce mainly local tissue damage (as hemorrhage, necrosis and edema) and systemic effects, as alterations in blood clotting, neurotoxicity, and shock (Ownby, 1990; Braud et al., 2000). Envenomation by snakes is often treated by parentheral antiophidian serum administration, obtained from hyperimmunized equine serum, often inducing adverse

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reactions. During serumtherapy, neutralization of toxic systemic effects is usually reached, but neutralization of local tissue damage usually does not occur (Cardoso et al., 2003). Thus, it is important to search novel venom inhibitors, be it synthetic or natural, able to complement serumtherapy, in order to neutralize mainly local damages of envenomation. Vegetal extracts constitute an alternative for treatment, displaying a large diversity of chemical compounds with several pharmacological activities of medical-scientific interest. A large number of extracts were already prepared and showed excelent antivenom activities (Martz, 1992; Melo et al., 1994; Mors et al., 2000; Izidoro et al., 2003; Soares et al., 2004; da Silva et al., 2005; Oliveira et al., 2005). Mikania glomerata (Asteraceae) popularly known as “guaco”, shows many well-known pharmacological activities, among them antifungal, antimicrobian, bronchodilator, anti-allergic and anti-inflammatory (Fierro et al., 1999; Holetz et al., 2002). Ruppelt et al. (1991) identified anti-inflammatory activity against Bothrops jararaca venom, in the infusion of Mikania glomerata. This is a species scarcely studied chemically, but several compounds were already therefrom isolated, chiefly cumarins, diterpenes, and essential oils (Veneziani et al., 1999; Cabral et al., 2001; Celeghini et al., 2001). The present study shows the ability of aqueous extracts, from dried or fresh Mikania glomerata to inhibit pharmacological and enzymatic activities of Bothrops and Crotalus snake venoms.

2. Materials and methods 2.1. Venoms and animals Bothrops jararacussu, Bothrops moojeni, Bothrops alternatus, Bothrops neuwiedi, and Crotalus durissus terriflcus venoms were purchased from Serpent´ario Bioagents, Batatais, SP. Groups of six male Swiss mice (18–22 g) were obtained from the Central Bioterium of USP in Ribeir˜ao Preto. Animals were housed in standard cages and maintained under standard conditions (12 h light/12 h dark cycle at room temperature). Mice feed and tap water were provided ad libitum. All experimental procedures were performed in accordance with the guidelines of the institutional animal care and use commitee of the UNAERP in accordance with the legislation on animal care. 2.2. Preparation of plant extract Leaves, stems, and roots of Mikania glomerata Sprengel (Asteraceae) were collected from Unidade de Biotecnologia of Universidade de Ribeir˜ao Preto (UNAERP), Ribeir˜ao Preto, SP, Brazil. A voucher specimen (no. 24) of the plant has been deposited at the Unidade de Biotecnologia, UNAERP and authenticated by Professor Herm´ogenes de Freitas Leit˜ao

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Filho of the Instituto de Biologia, UNICAMP. Two hundred grams of each part collected (leaves, stems, and roots) were submitted to extraction by boiling water (1 L) and macerated for 24 h. The remaining material was dried in a ventilated stove at 60 ◦ C and then extracted with boiling water (150 g/L) and macerated for 24 h. Following this extraction period, a vacuum filtration was performed and the extract was lyophilized. Extracts from fresh (AEFS, AEFL, and AEFR; yield: 2.0, 2.6, and 6.5% (w/w)) and dried (AEDS, AEDL, and AEDR; yield: 8.8, 11.7, and 7.7% (w/w)) material was so obtained. The dried extracts were kept at 4 ◦ C. A chromatographic procedure was performed with all extracts by TLC and HPLC at 20 mg/mL. The TLC system consisted of silica gel plates (10 cm × 10 cm) and n-butanol:acetic acid:water (4:1:5; upper phase) as mobile phase, detection at 254 and 366 nm, using vanilin or NPPEG reagent. The reverse phase HPLC system included: Supelcosil C18 column (4.6 mm × 250 mm, 5 ␮m) using a linear gradient of methanol:water:0.1% acetic acid as mobile phase, at 10–100% methanol during 50 min and UV detection (210, 280, and 340 nm), a flow rate of 1 mL/min. Chromatograms and its spectral data were compared with spectra of standards and those from literature. Protein concentration was determined by the Micro-biuret method. 2.3. Inhibition of the venom Desiccated venoms were weighed and dissolved in PBS at 10 or 2 ␮g/␮L. For inhibition experiments, solutions containing a fixed amount of venom were mixed with varying amounts of AEF and AED from leaves, stems, and roots in order to obtain various ratios (w/w) venom:inhibitor. All mixtures were incubated for 30 min at 37 ◦ C and aliquots were assayed in the different assay systems. 2.4. Inhibition of phospholipase A2 activity Indirect hemolytic activity was assayed using agarose–egg yolk–erythrocyte gels as substrate. A minimum indirect hemolytic dose (MiHD) was defined for each venom as the amount of enzyme that produces halos of 10 mm, as described by Guti´errez et al. (1988). AEF or AED from leaves, stems, and roots were assayed after incubation with all crude venoms at ratios 1:10, 1:50, 1:100, and 1:200 (w/w). Enzymatic activity was expressed as a percentage, 100% inhibition corresponding to the absence of activity of the venom incubated with the aqueous extract. Each experiment was expressed by the mean ± S.D. (n = 6). 2.5. Edema-inducing activity Edema was evaluated after subplantar injection, in the right footpad of male Swiss mice (n = 6, 18–22 g), of Bothrops jararacussu and Crotalus durissus terriflcus venoms. Inhibition studies were performed after preincubation of venom

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with AEF or AED from leaves, stems and roots at 1:50 (w/w) ratio. Control animals received an injection of PBS under identical conditions. The progression of edema was evaluated with a low-pressure pachymeter (Mtutoyo, Japan) at various time intervals after injection (Soares et al., 2000). Activity was expressed as the mean of percent edema values induced by the snake venoms in the absence and presence of the plant extracts. 2.6. Hemorrhagic activity A minimum hemorrhagic dose (MHD), evaluated only for crude Bothrops venoms, is the amount of venom which produces a hemorrhagic halo of 10 mm (Nikai et al., 1984). Swiss male mice (n = 6, 18–22 g) were injected intradermically in the back with dose of 1:50 (w/w), lMHDvenom/AEF or AED from leaves, stems, and roots). Control mice received PBS alone. Two hours after injection, mice were killed, and the diameter of the hemorrhage zone in the skin was measured. Hemorrhagic activity was expressed by the mean (in mm) of the hemorrhagic halos induced by the venoms in the absence and presence of the plant extracts. 2.7. Coagulant activity A minimum coagulant dose (MCD) was defined as the amount of Bothrops venom which clots 0.2 mL plasma in 60 s (Gen´e et al., 1989). Briefly, aliquots of 0.2 mL plasma (n = 6) were incubated with 50 ␮L of each venom or venom/AEF or AED from leaves, stems, and roots at 1:10, 1:50, and 1:100 (w/w, lMCD venom/AEF or AED), incubated for 30 min at 37 ◦ C and clotting times recorded. Control tubes included plasma incubated wilh PBS plus calcium or AEF/AED alone. Coagulant activity was expressed by the mean time of coagulation (in min) induced by the snake venoms in the absence and presence of plant extracts.

2.8. Polyacrylamide gel electrophoresis (SDS–PAGE) SDS–PAGE was performed on a 12% (w/v) polyacrylamide gel, in Tris buffer at pH 8.1, at 15 mA, for 2 h, following the method of Laemmli (1970) for denaturant proteins. 2.9. Statistical analysis Results are presented as the mean ± S.D. obtained with the indicated number of tested animals. The statistical significance of differences between groups was evaluated using Student’s unpaired t-test. A p value <0.05 was considered to indicate significance.

3. Results and discussion Vegetal extracts constitute an excelent alternative source of novel antiophidian agents. In many countries, vegetal extracts have been traditionally used in the treatment of envenomations evoked by snakebites (Mors, 1991), but in most cases, the use of these plants need scientific evidence of their claimed pharmacological activities. Several plants showed already to have antivenom activity (Gowda, 1997; Mors et al., 2000; Mahanta and Mukherjee, 2001; Soares et al., 2004). PLA2 activity induced by Crotalus durissus terrificus venom was totally inhibited by the aqueous extracts from fresh or dried roots and stems of Mikania glomerata, while, for Bothrops jararacussu venom, no significative inhibition was observed (Table 1). Among the extracts from dried sources, only that from the root (AEDR) showed an inhibition of 25% against Bothrops jararacussu venom and 100% against Crotalus durissus terrificus venom (Table 1). Aqueous extracts from Mandevilla velutina, Mandevilla illustris, and Casearia sylvestris neutralized 75–100% of the PLA2

Table 1 Inhibition of PLA2 activity of snake venoms 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

Inhibition of phospholipase A2 activity (%)a Without AE

1:10 (w/w)

1:50 (w/w)

1:100 (w/w)

1:200 (w/w)

Bothrops jararacussu Bothrops jararacussu + AEFR Bothrops jararacussu + AEFS Bothrops jararacussu + AEFL

0.0 0.0 0.0 0.0

– 10.0 15.0 2.0

– 20.0 15.0 3.0

– 25.0 15.0 10.0

– 30.0 15.0 10.0

Crotalus durissus terrificus Crotalus durissus terrificus + AEFR Crotalus durissus terrificus + AEFS Crotalus durissus terrificus + AEFL

– 0.0 0.0 0.0

0.0 50.0 35.0 8.0

– 95.0 75.0 25.0

– 100.0 85.0 45.0

– 100.0 100.0 80.0

Bothrops jararacussu Bothrops jararacussu + AEDR

0.0 0.0

– 5.0

– 15.0

– 18.0

– 25.0

Crotalus durissus terrificus Crotalus durissus terrificus + AEDR

0.0 0.0

– 45.0

– 95.0

– 100.0

– 100.0

a Enzymatic activity was expressed as a percentage. 100% of inhibition corresponding to the absence of activity of venom incubated with the aqueous extract. Each experiment represents the mean ± S.D. (n = 6).

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Fig. 1. Inhibition of edema-inducing activity of crude snake venoms by aqueous extracts from fresh (A) and dried (B) of Mikania glomerata, preincubated with ratio 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, the Mikania glomerata extract is also a good source of powerful natural PLA2 inhibitors, mainly neurotoxic PLA2 s found in Crotalus durissus terrificus venom. Regarding edema, only AEDR or AEFR inhibited ∼40% of this effect induced by Crotalus durissus terrificus venom, at 1:50 (w/w) ratio (Fig. 1), while the other extracts did not show any anti-edema activity against the different snake venoms. Soares de Moura et al. (2002) showed that fraction MG1, a dichloromethane fraction and that contains 11% of coumarin, from Mikania glomerata, decreased 30% the edema produced by Bothrops jararaca venom. Biondo et al. (2003) also noted that Crotalus durissus terrificus and Bothrops alternatus venoms, at 1:30 (w/w) ratio with Mandevilla velutina extract, had 46 and 30%, respectively, of their edema inducing activitity inhibited. Regarding the extracts of Mikania glomerata, we suggest that its natural inhibitors might be acting against the PLA2 s in rattlesnake venom, similarly as it was observed by Borges et al. (2000) using Casearia sylvestris extract and PLA2 s isolated from snake venoms. Several works have explored vegetal extracts and inhibitors with antihemorrhagic activity (Borges et al., 2001; Soares et al., 2004; Janu´ario et al., 2004). Our results showed that the hemorrhagic activity of Bothrops alternatus venom was 50 and 80% inhibited by AEFS and AEFR or AEDR, respectively. The extracts AEDR or AEFR inhibited also 80–95% of other Bothrops venoms (Fig. 2). This suggests an interaction between the extract components and metalloproteases, involving catalytic sites of these enzymes or essential metal ions, thus, inhibiting their hemorrhagic activities. The clotting activity induced by Bothrops jararacussu, Bothrops neuwiedi and Bothrops moojeni venoms were inhibited by the different extracts from dried Mikania glomer-

ata (Table 2). Fresh and dried root extracts also inhibited totally Crotalus and Bothrops venoms (Table 2). Borges et al. (2001) evidenced the ability of vegetal extracts from Casearia sylvestris to inhibit proteases isolated from several Bothrops snakes. Melo et al. (1994) identified antiproteolytic and antihemorrhagic components in the crude extract of Eclipta Prostrata. These envenomations usually produce prolonged hemorrhages due to a considerable degradation of fibrinogen and other clotting factors, avoiding clot formation (Markland,

Fig. 2. Inhibition of hemorrhagic activity of crude Bothrops snake venom by aqueous extracts of dried and fresh (roots, stems, and leaves) Mikania glomerata, preincubated at 1:50 (w/w) ratio for 30 min at 37 ◦ C. Each experiment represents the mean ± S.D. (n = 8).

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Table 2 Inhibition 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 Bothrops jararacussu + AEFR Bothrops jararacussu + AEFS Bothrops jararacussu + AEFL

l min 15 s ± 0.01 – – –

– 18 min 30 s ± 0.02 5 min 00 s ± 0.02 2 min 00 s ± 0.02

– >45 min 00 s ± 0.02 12 min 00 s ± 0.02 3 min 30 s ± 0.02

– >45 min 00 s ± 0.02 33 min 40 s ± 0.02 4 min 00 s ± 0.02

Crotalus durissus terrificus Crotalus durissus terrificus + AEFR Crotalus durissus terrificus + AEFS Crotalus durissus terrificus + AEFL

l min 48 s ± 0.01 – – –

– 27 min 40 s ± 0.02 8 min 00 s ± 0.02 5 min 00 s ± 0.02

– >45 min 00 s ± 0.02 22 min 00 s ± 0.02 8 min 30 s ± 0.02

– >45 min 00 s ± 0.02 30 min 10 s ± 0.02 9 min 00 ± 0.02

Bothrops moojeni Bothrops moojeni + AEFR Bothrops moojeni + AEFS Bothrops moojeni + AEFL

45 s ± 0.01 – – –

– 3 min 00 s ± 0.02 3 min 00 s ± 0.02 2 min 00 s ± 0.02

– 12 min 00 s ± 0.02 13 min 00 s ± 0.02 4 min 30 s ± 0.02

– >45 min 0 s ± 0.02 23 min 00 s ± 0.02 3 min 30 s ± 0.02

Bothrops neuwiedi Bothrops neuwiedi + AEFR Bothrops neuwiedi + AEFS Bothrops neuwiedi + AEFL

48 s ± 0.01 – – –

– 2 min 00 s ± 0.02 2 min 00 s ± 0.02 2 min 00 s ± 0.02

– 11 min 00 s ± 0.02 8 min 00 s ± 0.02 6 min 30 s ± 0.02

– >45 min 00 s ± 0.02 35 min 00 s ± 0.02 6 min 30 s ± 0.02

Bothrops jararacussu Bothrops jararacussu + AEDR Crotalus durissus terrificus Crotalus durissus terrificus + AEDR

1 min 15 s ± 0.01 – 1 min48 s ± 0.01 –

– 15 min 00 s ± 0.02 – 18 min 30 s ± 0.02

– >45 min 00 s ± 0.02 – >45 min 00 s ± 0.02

– >45 min 00 s ± 0.02 – >45 min 00 s ± 0.02

Bothrops moojeni Bothrops moojeni + AEDR

45 s ± 0.01 –

– 3 min 00 s ± 0.02

– 15 min 00 s ± 0.02

– >45 min 00 s ± 0.02

Bothrops neuwiedi Bothrops neuwiedi + AEDR

48 s ± 0.01 –

– 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 with Mikania glomerata showed that its extracts act as powerful inhibitors of the clotting activity, probably due to interaction with thrombin-like enzymes. The action mechanism of Mikania glomerata is however unknown, but, as shown in Fig. 3, no proteolytic degradation is evident. Concluding, Mikania glomerata extracts were able to considerably inhibit most of the activities of the assayed venoms. TLC and HPLC profile showed a similarity between fresh and dried extracts composition. The presence of coumarin and other non-polar compounds was verified in leaves and stem extracts, but coumarin concentration decreases in the stem extract and it is lower in the dried extract. A higher content of total protein was observed in the aqueous extract from roots (15–30%) and lower in the leave and stem extracts (<8%). The inhibitory ability against isolated toxins should also be later investigated. Some studies point to coumarins as main components of Mikania glomerata showing several activities. However, pharmacological studies should still be performed using new extract fractions, in order to isolate and characterize the active principle responsible for the antiophidian activity, thus providing insights to the elucidation of the action mechanism of the corresponding toxins and devel-

Fig. 3. PAGE to show interaction between snake venoms and plant Mikania glomerata extracts. Samples containing venoms and extracts were incubated for 30 min at 37 ◦ C in ratios 1:50 (w/w). 1, MW markers; 2, Bothrops jararacussu venom (20 ␮g); 3, Bothrops jararacussu venom + AEFR; 4, Bothrops jararacussu venom + AEFS; 5, Crotalus durissus terriflcus venom (20 ␮g); 6, Crotalus durissus terriflcus venom + AEDR; 7, Crotalus durissus terriflcus venom + AEDS; 8, AEDS (1000 ␮g); 9, AEDL (1000 ␮g); 10, AEDR (1000 ␮g).

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opment of future therapeutic agents for treatment of ophidian accidents.

Acknowledgements The authors are grateful to the financial support by Fundac¸a˜ o de Pesquisa do Estado de S˜ao Paulo (FAPESP) and Universidade de Ribeir˜ao Preto (UNAERP), and the technical assistence of all students and technicians (Vanessa C. Fernandes and Eliandra G. Silva, TT-II, FAPESP) who collaborated in this work.

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