Inhibition of enzymatic and pharmacological activities of some snake venoms and toxins by Mandevilla velutina (Apocynaceae) aqueous extract

Biochimie 85 (2003) 1017–1025 www.elsevier.com/locate/biochi

Inhibition of enzymatic and pharmacological activities of some snake venoms and toxins by Mandevilla velutina (Apocynaceae) aqueous extract Ronaldo Biondo, Ana Maria S. Pereira, Silvana Marcussi, Paulo S. Pereira, Suzelei C. França, Andreimar M. Soares * Unidade de Biotecnologia, Universidade de Ribeirão Preto UNAERP, Bloco A, Avenida Costábile Romano 2201, CEP 14096 380 Ribeirão Preto, SP, Brazil Received 24 June 2003; accepted 18 July 2003

Abstract Phospholipases A2 (PLA2) are multifunctional proteins which exhibit varied biological activities correlated to the structural diversities of the sub-classes. The crude aqueous extract from subterranean system of Mandevilla velutina, a plant found in Brazilian savanna, was assayed for its ability to inhibit biological activities of several snake venoms and isolated PLA2s. The extract induced total inhibition of the phospholipase activity of Crotalus durissus terrificus venom and only partial inhibition of Bothrops venoms. When assayed against purified toxins, the highest efficacy was detected against CB and crotoxin, while almost ineffective against PLA2s from the genus Bothrops. Although M. velutina crude extract significantly inhibited the myotoxic activity of C. d. terrificus venom and CB, it produced only partial inhibition of either Bothrops jararacussu venom or its main myotoxins BthTX-I (basic Lys49), BthTX-II (basic Asp49) and BthA-I-PLA2 (acidic Asp49). The extract exhibited also full inhibition of hemorrhage caused by Bothrops alternatus, Bothrops moojeni and Bothrops pirajai snake venoms, but partial inhibition (90%) of that induced by B. jararacussu venom. The extract was ineffective to inhibit the fibrinogenolytic activity of B. moojeni, B. alternatus and B. pirajai crude venoms, while their caseinolytic activity was only partially inhibited. No inhibition of the anticoagulant activity, although partial reduction of the edema-inducing activity of C. d. terrificus and B. alternatus crude venoms, CB, PrTX-I, BthTX-I and crotoxin was observed. Besides extending survival of mice injected with lethal doses of C. d. terrificus and B. jararacussu venoms, M. velutina extract decreased to 50% the lethality of mice. Extracts of 18 month old micropropagated plants were able to partially neutralize the effect of the crude venoms and toxins. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Mandevilla velutina; Snake venoms; Edema-inducing activity; Myotoxins; Phospholipases A2; Anti-ophidian activity; Micropropagated plants

1. Introduction Animal venoms are complex mixtures of enzymatic and toxic proteins including phospholipases A2, myotoxins, hemAbbreviations: PLA2, phospholipase A2; Cdt, Crotalus durissus terrificus; CB, C. d. terrificus phospholipase A2; Bjussu, Bothrops jararacussu; BthTX-I and II, B. jararacussu bothropstoxins I and II; BthA-I-PLA2, B. jararacussu acidic phospholipase A2; Bpir, Bothrops pirajai; PrTX-I and III, B. pirajai piratoxins I and III; Bmooj, Bothrops moojeni; Balt, Bothrops alternatus; PBS, phosphate-buffered saline solution; CK, creatine kinase; SSE, crude aqueous extract from subterranean system of M. velutina; LSE, crude aqueous extract from leaves and stem of M. velutina; SSE-SC, crude aqueous extract from subterranean system of M. velutina collected in São Carlos, São Paulo, Brazil; SSE-Pd, crude aqueous extract from subterranean system of M. velutina collected in Pedregulho, São Paulo, Brazil; SSi, crude aqueous extract of subterranean system of M. velutina micropropagated plants; GSP, global system positionament; MiHD, minimum indirect hemolytic dose; MHD, minimum hemorrhagic dose; MCD, minimum coagulant dose; LD100, dose for 100% lethality. * Corresponding author. Tel.: +55-16-603-6706; fax: +55-16-603-7030. E-mail address: [email protected] (A.M. Soares). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0300-9084(03)00138-X

orrhagic metalloproteases and other proteolytic enzymes, coagulant components, neurotoxins, cytotoxins and cardiotoxins, among others [1]. Ophidian envenomations are characterized by prominent local tissue damage, i.e. hemorrhage, necrosis and edema, alterations in the blood coagulation system as well as systemic neurotoxic effect. Additive or synergistic effects of active enzymes and toxins present in the venoms are responsible for this complex pathological picture [2–4]. The search for natural venom inhibitors is substantial to complement the traditional therapy, particularly regarding neutralization of local tissue damage. Plant extracts, rich source of natural inhibitors and pharmacologically active compounds, have been shown to antagonize the activity of some venoms and toxins. Casearia sylvestris (Flacourtiaceae), Eclipta prostata (Asteraceae), Mimosa pudica (Fabaceae), Tabernaemontana catharinensis (Apocynaceae) and others are known to inhibit a variety of snake venom activities [5–9].

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In some regions of Brazil, venomous snakebites and common inflammatory processes have been traditionally treated with infusions of Mandevilla velutina (Apocynaceae) rhizomes [10]. The anti-inflammatory action of M. velutina aqueous extract was investigated and it appears to selectively antagonize bradykinin [11–14] probably due to the triterpene velutinol [15]. The inhibition of phospholipase A2 and phospholipase C from Naja naja crude venom by the aqueous extract of Mandevilla velutina has already been reported [16]. Mandevilla velutina is a perennial endemic plant of the Brazilian savanna, running the risk of extinction due to extractivism, which interrupts the natural propagation process. Recently, this plant was considered indispensable for environmental preservation [17] since the Brazilian savanna has been considered a hotspot area with about 80% of its territory devastated [18]. Investigations on the anatomy of the subterranean system of M. velutina revealed that it is comprised of xylopodium and tuberous root [19]. The production of plants in vitro has been encouraged focusing the preservation and multiplication of elite genotypes. The acknowledged competence to synthesize secondary compounds and genetic stability of clones permit the production of plants identical to the parent plant, preserving the pharmacological effects [20–22]. The aim of this study was to evaluate the ability of the crude aqueous extract of M. velutina to inhibit enzymatic and pharmacological activities of both snake venoms and toxic PLA2s isolated from these venoms. 2. Materials and methods 2.1. Venoms and toxins Venoms of Bothrops jararacussu (Bjussu), Bothrops moojeni (Bmooj), Bothrops alternatus (Balt), Bothrops pirajai (Bpir) and C. d. terrificus (Cdt) were a gift from L.H.A. Pedrosa (FMRP-USP). Toxins BthTX-I, BthTX-II, BthA-IPLA2, PrTX-I, PrTX-III and CB were purified as previously described [23–26]. 2.2. Preparation of plant extract Mandevilla velutina plants were collected in São Carlos (SC) and Pedregulho (Pd), two cities located in the State of São Paulo, Brazil. The site of collection was marked using a global system of position measurement (GSP—Garmin legend model—79728002). M. velutina leaves and stems were separated from the subterranean system, washed, homogenized separately using deionized water (500 g/l) in a warring blender at room temperature and then filtered through a fine filter. The aqueous extracts (LSE and SSE) from either plants collected in SC or in Pd were further centrifuged at 10,000 × g during 10 min, the supernatants were freeze-dried and stored at –20 °C. The subterranean systems extracts (SSE-SC and SSE-Pd) were processed as above. Before use, the LSE and SSE were weighed, dissolved in PBS and stock solutions (250 µg/µl) were stored at –2 °C.

2.3. Inhibition of crude venoms and toxin activities Crude venoms and freeze-dried toxins were weighed and dissolved in PBS (10 µg/µl). For inhibition experiments, solutions containing a fixed amount of venoms or toxins were mixed with different volumes of stock solutions, in order to obtain different proportions extracts of venoms and toxins to be tested. All mixtures were incubated for 30 min at 37 °C and aliquots were assayed for their activities. 2.4. Inhibition of phospholipase activity Indirect hemolytic activity was evaluated using agarose, egg yolk and erythrocyte gels as substrate [27] and a minimum indirect hemolytic dose (MiHD) was defined for each venom or toxin as the amount of enzyme that produces hemolysis zones of 10 mm. LSE-SC, LSE-Pd, SSE-SC and SSE-Pd were tested after incubation with each crude venom at different proportions 1:50, 1:150 and 1:250 (w/w, 1MiHD/SSE) and isolated toxins at 1:50 and 1:150 (w/w, 1MiHD/SSE). Furthermore, SSE-SC was tested after incubation at 1:1, 1:5 and 1:10 (w/w, 1MiHD/SSE). 2.5. Myotoxic activity Swiss male mice (18–22 g) were injected intra-muscularly in the right gastrocnemius muscle with solutions containing doses of 20 µg/50 µl of Cdt and Bjussu venoms or toxins CB, crotoxin, BthTX-I or II. The mixtures of venom/SSE or toxin/SSE (1:50, w/w) were then evaluated. Controls received PBS or SSE solution. Mice were bled from the tail 3 h after injection and blood was collected into heparinized capillary tubes. Plasma creatine kinase activity was determined using the Kit 47-UV (Sigma Chemical Co.). Activity was expressed in units/l, one unit corresponding to the production of 1 µmol of NADH per min at 30 °C. 2.6. Edema-inducing activity Edema was evaluated after sub-plantar injection, in the right footpad of male Swiss mice (18–22 g), of venoms and toxins. Inhibition studies were performed after preincubation of venom or toxin with SSE. Control animals received an injection of PBS under identical conditions. The progression of edema was evaluated with a low-pressure pachymeter (Mitutoyo, Japan) 30 min after injection. The dose administered of crude venoms, crotoxin, CB, BthTX-II and PrTX-III was 10 µg while for BthTX-I, PrTX-I and BthA-I-PLA2 it was 20 µg. The effects of venom/SSE mixtures at 1:5, 1:15 and 1:30 (w/w) and toxin/SSE at 1:0.5, 1:1, 1:5 and 1:10 (w/w) were evaluated. 2.7. Hemorrhagic activity Minimum hemorrhagic dose (MHD) was evaluated only for Bothrops crude venom since it produced a hemorrhagic

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halo of 10 mm [28]. Swiss male mice (18–22 g) were injected intradermically in the back with doses of 1:10, 1:30 and 1:100 (w/w, 1MHDvenom/SSE). Control mice received only PBS. Three hours after injection, mice were killed, and the diameter of the hemorrhage zone in the skin was measured. 2.8. Proteolytic activity on fibrinogen The methodology described by Rodrigues et al. [29], partially modified, was used. Samples of 50 µl of bovine fibrinogen (1 mg/ml) were incubated with venoms (6 µg) at 37 °C for 30 min. The reaction was stopped by addition of 25 µl of 0.05 M Tris–HCl buffer, pH 8.8, containing 10% (v/v) glycerol, 10% (v/v) mercaptoethanol, 2% (w/v) SDS, and 0.05% (w/v) bromophenol blue. Samples were then analyzed by 13.5% (w/v) SDS-PAGE. The effect of SSE on

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the fibrinogenolytic activity was evaluated after preincubation of crude venoms (6 µg) with SSE 1:100 (w/w). 2.9. Proteolytic activity on casein Rodrigues et al. [29] method was carried out using casein as substrate. Briefly, 60 µg of each crude venom was incubated with 1.0 ml of 1% (w/v) casein in 0.1 M Tris–HCl buffer (pH 8.0) for 30 min at 37 °C. The reaction was stopped by addition of 1.0 ml of 5% (v/v) trichloroacetic acid solution and the mixture was left standing for 30 min at room temperature before centrifugation (2000 × g) for 5 min at 25 °C. Proteolytic activity was estimated by measuring the absorbance of the clear supernatant at 280 nm. Proteolytic activity was also assayed in the presence of SSE, after preincubation, for 30 min at 37 °C, of 60 µg of each venom with SS at 1:10 (w/w, venom/SSE). One unit of caseinolytic activity was

Fig. 1. Inhibition of PLA2 activity of snake venoms (A and B) and isolated toxins (C and D) induced by the aqueous extract from subterranean system of M. velutina (SSE) or aqueous extract from leaves and stems (LSE), preincubated with different concentrations of venom extract mixtures for 30 min at 37 °C. Each experiment represents the mean ± S.D. (n = 6).

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defined as the amount of venom that produces an increase in absorbance of 0.001 units/min. 2.10. Coagulant activity The minimum coagulant dose (MCD) was defined as the amount of venom which clots 0.2 ml plasma in 60 s [30]. Aliquots of 0.2 ml plasma were incubated with 50 µl of each venom or venom/SSE 1:30 (w/w, 1MCDvenom/SSE), left standing for 30 min at 37 °C and clotting times determined. Control tubes included plasma incubated with PBS plus calcium or SSE alone. 2.11. Lethality Doses causing 100% lethality (LD100) of male Swiss mice (18–22 g) were determined for Cdt and Bjussu venoms and crotoxin. Groups of eight mice were inject intraperitoneally (i.p.) with doses of mixtures 1:100, 1:250 and 1:500 (w/w, 1LD100venom or toxin/SSE) preincubated for 30 min at 37 °C and survival time within 48 h was measured. PBS and SSE (50 mg) were injected as controls. 2.12. In vitro assay of enzymatic and biological activities displayed by micropropagated plant extracts

potentiating and affecting peptides which membrane permeability play specific roles in the envenomation process acting by different mechanisms [32]. PLA2s constitute by far the most abundant class of proteins in snake venomous gland and are usually present in variant forms. PLA2s from snake venoms are usually Asp49, catalytically active variants, whereas others are Lys49 variants, devoid of catalytic activity [23,33– 35]. A wide variety of pharmacological activities has been described for PLA2s, such as neurotoxic, myotoxic, edematogenic, hypotensive, platelet aggregating, cardiotoxic and anticoagulant [25]. Phospholipase activity was estimated by indirect hemolysis. The minimal effective dose MiHD for all crude venoms, PrTX-III, BthTX-II and BthA-I-PLA2 was approximately 1 µg, while for crotoxin and CB it was 10 µg. Aqueous extract from subterranean system of M. velutina partially inhibited the PLA2 activity of Bpir (38%) and Bmooj (35%) venoms, whereas Crotalus venom was totally inhibited (Fig. 1). This extract was particularly effective against CB, which was totally inhibited, but crotoxin was only 46.5% inhibited. The inhibition effect of toxins from Bothrops venoms was lower. LSE did not induce any significant inhibiting activity. SSE-SC and SSE-Pd showed similar levels of inhibitions upon the assayed snake venoms and toxins. Based on the

Extracts of subterranean systems of 18 month old micropropagated plants were assayed for myotoxic, edemainducing, hemorrhagic and PLA2 activities. The aqueous extract was prepared as described in Section 2.2. The PLA2 activity was evaluated with Cdt venom (1 µg), CB (10 µg) and crotoxin (10 µg). The myotoxic activity was examined using venoms of Cdt (1 µg) and Bjussu (10 µg), BthTX-I (10 µg) and CB (10 µg). Edema-inducing activity was determined with Cdt venom (10 µg), CB (10 µg) and crotoxin (10 µg). Hemorrhagic activity was checked using Bmooj venom (10 µg). All venoms or toxins were incubated at 1:100 (w/w, venom or toxin/SSE) and left standing for 30 min at 37 °C. 2.13. Statistical analysis Data are presented as mean values ± S.D. obtained with recorded number of tested animals. For statistical significance, the data were analyzed by Student’s unpaired t-test at 5% level. 3. Results and discussion In many countries, plant extracts have been traditionally used in the treatment of snakebite envenomations [31], although only in a few cases there has been a scientific validation of such claims. Animal venoms, including snake venoms, are complex mixtures of different classes of proteins and peptides as phospholipases A2, metalloproteases, proteases, bradykinin

Fig. 2. Inhibition of myotoxic activity of crude venoms and toxins after incubation for 30 min at 37 °C with the aqueous extract of subterranean system (SSE), at a ratio of 1:50 (w/w, venom or toxin/SSE). Each denoted value represents the mean ± S.D. (n = 6).

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obtained results, all subsequent studies were carried out with SSE-Pd and PA. Compared to C. sylvestris extract, which neutralizes the phospholipase activity of Cdt (75.7%), Bmooj (76.5%) and Bjussu (62.5%) venoms [14] and to Mimosa pudica root extract which showed 86% inhibition of Naja kaouthia venom PLA2 [17], M. velutina SS extract is more potent to inhibit the phospholipase activity of various venom toxins. Myonecrosis following snake envenomations occurs due to the presence of PLA2 myotoxins in the venom and many types of antivenom are ineffective in antagonizing the myotoxic effects of such toxins [36]. SSE inhibited totally the myotoxic activity of Cdt and CB venoms and only partially Bjussu venom (66.5%), crotoxin (8%), BthTX-I (75%) and BthTX-II (53.3%) (Fig. 2). Regarding CB crotoxin, SSE inhibited it only partially, suggesting a probable interaction among the CB activity sites and SSE.

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Melo et al. [6] reported that extracts of Eclipta prostrata neutralized the myotoxic effects of three crotalid venoms and crotoxin. A purified compound from extracts of T. catharinensis inhibited 40% of Cdt myotoxicity [18]. Edema-inducing activity is a multifactorial pharmacological activity, depending on the combined action of various toxins, suggesting that enzymatic activity is not strictly required to induce this effect [33]. SSE partially inhibited edema-inducing activity of some venoms and toxins, but was ineffective for others. When Cdt and Balt venoms were incubated with SSE 1:30 (w/w) the inhibition was 46% and 30%, respectively. Other venoms did not show any inhibition. CB, crotoxin, BthTX-I and PrTX-I incubated with SSE 1:10 (w/w) was inhibited approximately 60%, 50%, 63% and 65%, respectively (Fig. 3). When venom or toxin/SSE ratio was increased inhibition was not observed.

Fig. 3. Inhibition of edema-inducing activity of crude snake venoms (A) and isolated toxins (B and C) after incubation for 30 min at 37 °C with the aqueous extract of M. velutina subterranean system (SSE) at different concentration. Each denoted value represents the mean ± S.D. (n = 6).

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Previous studies reported the ability of M. velutina extracts to inhibit edema induced by PLA2 from Naja naja venom [16]. Bento et al. [15] showed that inhibition of bradykinin by SSE was correlated to the presence of the triterpene velutinol in the plant extracts. Such biomolecule probably plays an important role in the inhibition of edema induction of all crude venoms and purified toxins assayed. Venoms contain many proteolytic enzymes that degrade a variety of natural substrates such as casein, fibrinogen, collagen and others. Hemorrhagic toxins are among those enzymes and are responsible for the degradation of proteins from the extracellular matrix or alterations in blood coagulation [37] and need a divalent metal ion for their activity. SSE did not inhibit crude venom proteolytic activity on fibrinogen, and these extract did not induce proteolysis in any substrate assayed (results not shown). Casein when incubated with SSE 1:10 (w/w), partially inhibited the proteolytic activity of crude venoms, Bmooj 81%, Bjussu 8%, Balt 85% and Bpir 60% (results not shown). Cdt venom was not significantly inhibited. Thus, M. velutina components do not seem to inhibit the clotting action of venoms by mechanisms of proteolytic degradation, indicating that SSE possesses some unknown inhibitory action which needs further investigation. The MHD evaluated for crude venoms was 10 µg for Balt or Bmooj and 20 µg for Bjussu or Bpir. Aqueous extract 1:100 (w/w) from subterranean system of M. velutina inhibited considerably the hemorrhagic activity of Bjussu venom and totally other venoms (Fig. 4). The total inhibition of hemorrhagic activity induced by Bmooj, Balt and Bpir venoms and 90% inhibition of Bjussu venom suggested interac-

Fig. 4. Inhibition of hemorrhagic activity of crude snake venoms by the aqueous extract of subterranean system of M. velutina (SSE) preincubated at different ratios for 30 min at 37 °C. Each denoted value represents the mean ± S.D. (n = 8).

Table 1 Neutralization of coagulant activity induced by Bothrops crude venom (15 µg) preincubated with the aqueous extract of subterranean system of M. velutina (SSE) in ratio 1:30 (w/w, venom/SSE). Each value represents the mean ± S.D. (n = 6) Venoms Bjussu Bmooj Balt SSE PBS plus Ca ++

Time of coagulation (min) Without SSE 1:30 SSE (w/w) 1'27″ ± 0.06 2'15″ ± 0.09 1'04″ ± 0.12 1'03″ ± 0.01 0'52″ ± 0.06 1'09″ ± 0.01 – No effect 5'00″ ± 0.02 –

tion of SSE with metalloproteases, neutralizing their effects. Anti-hemorrhagic compounds isolated from Curcuma longa roots [38] and wedelolactone Eclipta prostrata leaves [6] were purified. The MCD evaluated for crude venoms was 15 µg. SSE did not inhibit the coagulant effect of crude venoms on plateletpoor plasma, therefore, plasma incubated with SSE did not show anticoagulant effect (Table 1). Regarding a possible action mechanism, our previous studies have shown that no alteration occurs in the electrophoretic pattern of Crotalus and Bothrops venoms and isolated toxins, after incubation with Mandevilla velutina extract (results not shown), excluding proteolytic degradation as a potential mechanism. Our results clearly indicate that M. velutina extract contains substances efficient in neutralizing the hemorrhagic activity induced by crude venoms. Hemorrhagings and phospholipases are enzymes that need a divalent metal ion to promote inhibiting activity. Probably SSE extract contains compounds that bind these ions. On the other hand, components of the extract may occupy sites in toxins, preventing binding of the substrate to the enzymes, and this interaction may be covalent or non-covalent. Lethal activity involves multifactorial actions by compounds of crude venoms and depends on the combined action of various toxins. Lethality caused by snake venoms is due to a systemic action of neurotoxic, myotoxic, hemorrhagic and coagulant compounds. Crotalus venoms are rich in neurotoxins like crotoxin and Bothrops venoms in general are rich in myotoxins. The World Health Organization reported that 30,000–40,000 people die every year due to snakebites [39]. LD100 was determined for Bjussu (100 µg) and Cdt (50 µg) venoms, as well as for crotoxin (30 µg). Bjussu venom preincubated with SSE 1:250 (w/w) resulted in over 5 h survival, while Cdt venom preincubated with SSE 1:250 and 1:500 (w/w) showed a significant increase (14.33 h) in the survival period. The survival period recorded for crotoxin preincubated with SSE 1:500 (w/w) was 11.50 h (Table 2). SSE extended survival duration and inhibited 50% lethality caused by Cdt venom and crotoxin. Crotoxin is a dimeric neurotoxin isolated from Cdt venom and consists of a noncovalent association of a toxic basic subunit CB and a nontoxic and non-enzymatic acid polypeptide called crotapotin [40,41]. Inhibition of crotoxin showed that SSE might be a good source of potentially useful neurotoxin inhibitors.

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Table 2 Survival time of mice injected with crude snake venoms preincubated or not with the aqueous extract of subterranean system of M. velutina (SSE) at different concentrations (w/w, venom/SSE). Each value represents the mean ± S.D. (n = 8) Venoms or toxin

Survival time (h) Without SSE 1.50 a 1.25 a 1.48 a – >48.00 c

Bjussu (100 µg) Cdt (50 µg) Crotoxin (30 µg) SSE (50 mg) PBS

1:100 SSE (w/w) 4.38 6.00 10.18 >48.00 c –

1:250 SSE (w/w) 4.67 10.25 b 11.50 >48.00 c –

1:500 SSE (w/w) 4.08 14.33 b 11.50 b >48.00 c –

a

100% of animal dead. 50% of animal survival. c 100% of animal survival. b

The enzymatic and biological activities of aqueous extract of subterranean system of 18 month old plants cultured in vitro neutralized the effects of crude venoms and toxins (Fig. 5). CB was totally inhibited (Table 3). This is a promising biological method to produce new plants to be used as source of bioactive compounds or for ex situ conservation of genotypes. Extracts from plants collected in Brazilian savanna exhibited higher levels of inhibition than those obtained from in vitro micropropagated plants. The biosynthesis of micromolecules in micropropa-

gated plants is organ specific and physiological state dependent and so the yield of secondary metabolites can be manipulated through biochemical alterations in the culture medium. Fico et al. [42] observed that young plants of Pyracantha coccinea contained pyracanthoside and rutin, while 1 year old plants accumulated hyperoside and isoquercitin. In conclusion, the aqueous extract of Mandevilla velutina is able to inhibit several classes of toxins exhibiting specific interactions with crude venoms and purified toxins from Cdt venom. Investigations of compounds correlated with inhibition of crude venoms and toxins showed that those compounds have high polarity. Furthermore, M. velutina extract also containing velutinol like compounds with polarity even higher than that of velutinol, displayed anti-inflammatory activity.

Acknowledgements

Fig. 5. (A) Subterranean system of adult plant collected in Brazilian savanna and (B) Subterranean system of 18 month old in vitro micropropagated plant.

The authors are indebted to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Universidade de Ribeirão Preto (UNAERP) for financial support and grateful to Flávia Cristina de Jesus (IC), Lucas Blundi Silveira (IC) and Eliandra G. Silva (TT-II) for their helpful technical collaboration. This work was a partial requirement for the Master Degree of Ronaldo Biondo obtained at UNAERP.

Table 3 Inhibition of toxic activities after incubation with the aqueous extract of subterranean system either from in vitro micropropagated (SSi) or native M. velutina plants (SSE). All tests were carried out with 1:100 (w/w, venom or toxin/SSi or SSE) solution. Results are presented as mean values ± S.D. (n = 3) Venom or toxin Cdt CB Crotoxin Bmooj Bjussu BthTX-I a

PLA 2 activity SSi 8.0 100.0 0.0 – – – –

SSE 100.0 100.0 46.5 – – – –

1:30 (w/w, venom/SSE). 1:10 (w/w, venom or toxin/SSE). c 1:50 (w/w, venom or toxin/SSE). b

Inhibition (%) Edema-inducing activity Hemorrhagic activity SSi SSE SSi SSE – – 28.0 46.0 a 37.0 68.0 b – – 0.0 49.0 b – – – – – – – – 28.0 100.0 – – – – – – – –

Myotoxic activity SSi 30.0 0.0 – – – 23.0 51.0

SSE 100.0 c 100.0 c – – – 66.5 75.0

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