Journal of Ethnopharmacology 155 (2014) 1609–1615

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Research paper

Inhibition of the toxic effects of Bothrops asper venom by pinostrobin, a flavanone isolated from Renealmia alpinia (Rottb.) MAAS Isabel Gómez-Betancur a,n, Dora Benjumea a, Arley Patiño a, Nora Jiménez b, Edison Osorio b a Programa de Ofidismo/Escorpionismo, Sede de Investigación Universitaria, Torre 2 Laboratorio 631, Facultad de Química Farmacéutica, Universidad de Antioquia, Calle 70 No. 52-21, Medellin, Colombia b Grupo de Investigación en Sustancias Bioactivas, Facultad de Química Farmacéutica, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia

art ic l e i nf o

a b s t r a c t

Article history: Received 10 June 2014 Received in revised form 5 August 2014 Accepted 7 August 2014 Available online 17 August 2014

Ethnopharmacological relevance: Renealmia alpinia has been traditionally used to treat snakebites by indigenous Embera-Katíos tribes belonging to the regions of Antioquia and Chocó, Colombia, and it has been shown to inhibit the enzymatic and biological activities of Bothrops venoms and their purified phospholipase A2 (PLA2) toxins. In addition to its common local usage against snakebites, Renealmia alpinia is commonly used to treat pain. To evaluate the inhibitory ability of pinostrobin, the main compound in the dichloromethane extract of Renealmia alpinia, on the toxic effects of Bothrops asper venom through in vitro and in vivo models and to evaluate its activity against pain and edema. Materials and methods: Pinostrobin was isolated from the dichloromethane extract of Renealmia alpinia leaves. The protective properties of the extract and of pinostrobin against the indirect hemolytic, coagulant and proteolytic effects of Bothrops asper venom were evaluated in vitro, and the antihemorrhagic and anti-inflammatory activity were evaluated in vivo. Results: Renealmia alpinia extract significantly inhibited the proteolytic activity and indirect hemolytic activity of Bothrops asper venom at a venom:extract ratio of 1:20. Moreover, the present data demonstrate that pinostrobin may mitigate some venom-induced local tissue damage due to hemorrhagic effects, and the compound is also responsible for the analgesic and anti-inflammatory activity of the extract from Renealmia alpinia. This is the first report to describe pinostrobin in the species Renealmia alpinia and its properties in vitro against Bothrops asper venom. Conclusion: Our studies of the activity of Renealmia alpinia against the venom of Bothrops asper have confirmed that this species possesses inhibitory effects against Bothrops asper venom in both in vitro and in vivo models and that these effects may be due to pinostrobin, supporting the traditional usage of the plant. Additionally, pinostrobin may be responsible for the anti-hemorrhagic and analgesic activity (peripheral analgesic activity) of Renealmia alpinia. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Renealmia alpinia Pinostrobin Analgesic Antiinflammatory Bothrops asper Snake venom

1. Introduction Snakebites are a public health concern throughout the world. Annually, between 5.4 and 5.5 million people report being bitten by snakes, resulting in approximately 400,000 amputations and between 20,000 and 125,000 deaths (Williams et al., 2010). In Colombia, Bothrops asper is responsible for 50–80% of the 4526 cases of snakebite reported annually (I.N.S., 2012), with an estimated mortality of 5–8%. In addition, between 6% and 10% of patients suffer consequences as a result of the loss of tissue and/or n

Corresponding author. Tel.: þ 57 4 2196535; fax: þ57 4 2631914. E-mail addresses: [email protected] (I. Gómez-Betancur), [email protected] (D. Benjumea), [email protected] (A. Patiño), [email protected] (N. Jiménez), [email protected] (E. Osorio). http://dx.doi.org/10.1016/j.jep.2014.08.002 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

amputations (Otero-Patiño et al., 2012). The venom from Bothrops spp. induces local (edema, hemorrhage, necrosis) and systemic (coagulation disorders, renal failure) effects, and its function is to immobilize, kill and help digest the prey. For this purpose, the venom includes a variety of toxins with enzymatic activity as well as some inactive toxins, salts and carbohydrates (Chipaux and Goyffon, 1998). The only effective treatment for snakebite is anti-venom, but in most accidents, medical attention is not immediately available, mainly due to geographical factors and the limited availability of anti-venom in remote areas, and thus anti-venoms are usually administered after local effects have already developed to some extent. Furthermore, anti-venom treatment has limited effectiveness in neutralizing local effects, such as hemorrhage, edema and myonecrosis (Oliveira et al., 2005; Otero-Patiño et al., 2012).

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Consequently, there is a need to develop complementary antivenom therapy that can be applied in the field to reduce the effects of snakebites. In this regard, extracts from Renealmia alpinia (Rottb.) Maas, Zingiberaceae family, a plant used by the indigenous people of Antioquia and Choco (Colombia) against snakebites (Otero et al., 2000a, 2000b; Vásquez et al., 2013), has shown neutralizing activity against the toxic effects of Bothrops asper venom (Otero et al., 2000c; Núñez et al., 2004). Moreover, the inhibitory activities of the toxic effects of the whole venom and of some toxins isolated from Bothrops asper, such as PLA2 and metalloproteins, have been explored along with the analgesic activity of the ethanol extract of Renealmia alpinia (Patiño et al., 2012, 2013). In an attempt to continue investigating the biological and chemical aspects of Renealmia alpinia, we screened the extracts obtained from solvents of different polarities for their antihemorrhagic activity. Observing no significant differences in the yield of the principal compound between the dichloromethane and methanolic extracts of Renealmia alpinia leaves, we evaluated the activity of the principal compound isolated from the dichloromethane extract for its capacity to neutralize the toxic effects of Bothrops asper venom by evaluating the indirect hemolytic, coagulant and proteolytic activity in in vitro models and the hemorrhagic, analgesic and anti-inflammatory activity in experimental animals.

2. Materials and methods 2.1. Venom The poison used was collected by manually milked adult specimens of Bothrops asper snakes from different areas of Antioquia and Choco that were held captive in the Serpentarium of the University of Antioquia. The venom was centrifuged, and the supernatant was lyophilized and frozen at 70 1C until use.

2.4. Coagulant activity The coagulant activity of the venom was assessed on citrated human plasma. Samples of 100 μL of various concentrations of venom were added to aliquots of 200 μL plasma previously incubated at 37 1C. The clotting times were recorded using a HumaClot Junior coagulometer (Human; Germany), and the minimum coagulant doses (MCD) for plasma or fibrinogen were determined. The MCD corresponds to the amount of venom that induces clotting in 60 s (Theakston and Reid, 1983). 2.5. Indirect hemolytic activity To establish any possible venom PLA2 activity, indirect hemolysis was determined in agarose erythrocyte–egg yolk gels according to Gutiérrez et al. (2010) using 0.8% agarose dissolved in PBS (0.12 M NaCl, 0.04 M sodium phosphate in distilled water), pH 7.2 and CaCl2. An additional plate without CaCl2 was performed to verify that the hemolytic activity was due to the presence of PLA2. The minimum hemolytic dose (MHeD) was defined as the amount of venom that induced a 20 mm diameter hemolytic halo. The experiments were performed in triplicate. 2.6. Proteolytic activity The proteolytic activity was measured on azocasein (SigmaAldrich, St. Louis, MO) according to Wang et al. (2004) with some modifications. Briefly, 20, 10 or 5 μg of either Renealmia alpinia extract or pinostrobin were dissolved in 20 μL of 25 mM Tris (0.15 M NaCl, 5 mM CaCl2), pH 7.4 (to obtain concentrations of 1.0, 0.5, and 0.25 μg/μL, respectively). These solutions were incubated with 10 mg/mL of azocasein diluted in the same buffer. After an incubation of 90 min at 37 1C, the reaction was stopped by the addition of 200 μL of trichloroacetic acid. The samples were then centrifuged at 360g for 5 min. The supernatant (100 μL) was mixed with an equal volume of 0.5 M NaOH, and the absorbance was measured at 450 nm. The results are shown as units of proteolytic activity, defined as the amount of enzyme that induces a change in absorbance of 0.2.

2.2. Plant specimen and extract preparation 2.7. Neutralization of the hemorrhagic effect of the venom The plant material used in this study was collected in San Rafael (Antioquia) - Colombia, during February 2011. A specimen was deposited at the University of Antioquia Herbarium under voucher number 176395. The dried leaves (1500 g) of Renealmia alpinia were extracted with dichloromethane (3L  3) at room temperature, the dichloromethane was removed using a rotary evaporator, and the extract (74.0 g) was stored at 4 1C until use.

2.3. Isolation and purification Vacuum liquid chromatography (VLC) was performed with the dichloromethane extract (20 g) using different solvent mixtures. The extract was subjected to silica gel column chromatography (CC) and eluted sequentially with hexane:dichloromethane (3:1, 1:1, 1:3), dichloromethane:acetone, and acetone:methanol (3:1, 1:1, 1:3) gradients (each 1 L) to obtain fractions 1–13. Fraction 4 (3.0 g) was applied to a silica gel column and eluted with hexane: dichloromethane (6:4) to obtain fractions 4–1 to 4–4. Fraction 4–3 (1822 mg) afforded pinostrobin (1300 mg). The 1H and 13C NMR spectra were measured on Fourier-300 spectrometers. The definitive assignments of all protons and carbons were obtained using 2D experiments (COSY, HMQC and HMBC). The chemical shifts are shown in d (ppm) with tetramethylsilane (TMS) as an internal reference.

The hemorrhagic activity was determined following described methods (Gutiérrez et al., 1981). Briefly, groups of four mice were injected by an i.d. route in the abdomen with variable doses of extract or pinostrobin along with Bothrops asper venom dissolved in 0.1 mL phosphate buffered saline (PBS) pH 7.2. The control group only received PBS in identical conditions. Two hours later, the mice were sacrificed by the inhalation of ether, and the diameter of the hemorrhagic area was measured. The minimum hemorrhagic dose (MHD) was the lowest dose of venom that induced a hemorrhagic area 10 mm in diameter in 2 h (mean 7SD of three experiments). Three doses (50, 100, 200 mg/kg) of Renealmia alpinia extract and three doses (5, 10, 20 mg/kg) of pinostrobin were pre-incubated at 37 1C for 30 min with 10 mg of Bothrops asper venom (6 MHD) dissolved in 0.1 mL PBS, pH 7.2. The mixture was then injected i.d. to groups of four mice. A control group only received the venom. 2.8. Analgesic activity The analgesic activity was determined using the test of Siegmund et al. (1957). The extract and pinostrobin were diluted in saline solution. Oral doses of 50, 100, 200 and 300 mg/kg of dichloromethane extract or pinostrobin (5, 10, 20 mg/kg) from Renealmia alpinia were administered. The analgesic effect was

I. Gómez-Betancur et al. / Journal of Ethnopharmacology 155 (2014) 1609–1615

determined according to the ability to reduce the number of stretching contractions produced by phenylquinone in the mice. This method is based on the intraperitoneal injection of 2-phenyl1,4-benzoquinone (an analgesic agent), which is capable of eliciting a reflex response or stretching contractions when injected into mice. The mice were randomly divided into homogeneous groups such that, for each dose of the tested product, it was possible to have a group of at least 4 mice. After fasting, ibuprofen was administered to the control group at 1.0 mL per 40 g of body weight as a reference drug. In addition, the treated groups received orally appropriate doses of extract or pinostrobin. After treatment (45 min), all groups of animals received phenylquinone solution i.p. at a dose of 4 mg/kg (prepared from a stock solution of 2 mg/mL in ethanol). After a lag time of 5 min, the contractions were counted individually in each mouse for a period of 10 min. Pain in mice is manifested through contractions, and the mice exhibiting stretching and twisting of the rear legs of the backabdominal musculature were considered positives. The analgesic effects of the extracts of Renealmia alpinia were determined by their ability to reduce the number of painful contractions or stretchings caused by phenylquinone. The inhibition percentage (I%) was calculated by the following formula: Inhibition percentage ðI%Þ ¼

# stretching control group  #stretching treated group  100 #stretching control group

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3. Results 3.1. Isolation and structure elucidation The active component was isolated using a chromatography process different from that used for the dichloromethane extract of the leaves of Renealmia alpinia. The known flavanone pinostrobin was identified. The molecular formula was determined as C16H14O4. The compound was identified by comparison of its spectral data with those reported in the literature (Papotti et al., 2010). 3.2. Inhibition of the coagulant activity induced by the venom of Bothrops asper The dichloromethane extract showed significant in vitro inhibition of the coagulating effect induced by 1.0 mg of venom, corresponding to a clotting time of 15.23 70.14 s. Initially, the coagulant activity was unchanged at low ratios of 1:5 (poison: pinostrobin or poison:extract w/w). However, the extension of the coagulation time became statistically significant when the venom was used at a ratio of 1:20 with the extract or with pinostrobin, with the maximum delay times for clotting onset found to be 24.10 7 1.36 s and 17.30 70.50 s, respectively. From this relationship, the inhibitory effect was shown to depend on the extract concentration. Likewise, after half an hour of incubation, it was possible to validate that the extract relations used for this trial did not generate coagulant activity by themselves (Fig. 1). 3.3. Indirect inhibition of hemolytic activity

2.9. Anti-inflammatory activity The anti-inflammatory activity was determined using the test outlined by Levy (1969). Carrageenan tests in mice divided into groups of 4 animals each were used to determine the antiinflammatory effect of pinostrobin. The treated groups were administered different doses of the extract or of pinostrobin. The control group received a dose of 75 mg/kg ibuprofen as a reference drug. The extract and pinostrobin were diluted in saline solution and administered p.o. 1 h prior to the subplantar injection of carrageenan. The inflammation thickness was quantified by measuring the footpad using a digital electronic caliper, “Caliper Fowler Sylvac”, immediately before carrageenan injection and 2, 3 and 4 h after injection. The difference between the first measurement and the others was taken into account according to the degree of swelling (Δ). The anti-inflammatory effect of pinostrobin was expressed in terms of the percentage reduction of edema, as calculated by the formula: [(ΔC  ΔT)/ΔC]  100, where ΔC represents the control and ΔT represents the treated group. Differences between the treated and control groups with respect to the control at 2, 3 and 4 h were analyzed by Dunnett's test after treatment.

The minimum indirect hemolytic dose (MIHD) of Bothrops asper venom was 2.2 mg. Renealmia alpinia extract and pinostrobin were compared for their ability to neutralize the hemolytic effects of Bothrops asper venom and showed similar neutralizing potencies (Fig. 2). When the venom was pre-incubated with RAE at ratios of 1:20, 1:10 and 1:5, inhibition percentages of 23.35%, 11.65% and 10.00%, respectively, were observed (Fig. 2). Pinostrobin at ratios of 1:20 and 1:10 showed inhibition percentages of 21.65% and 13.35%. These differences were statistically significant. 3.4. Inhibition of the proteolytic activity When the venom was pre-incubated with the dichloromethane extract of Renealmia alpinia at varying ratios, concentrationdependent inhibition of the proteolytic activity of Bothrops asper Pinostrobin 1:1 Pinostrobin 1:5 Pinostrobin1:10 Pinostrobin 1:20

*

extract 1:1

extract 1:10

***

extract 1:20

***

20

30

Venom B . aspe r 10

For the statistical analysis of the neutralizing coagulant effect, analysis of variance (ANOVA) was performed, followed by Dunnett's test to determine significant differences between the positive control group and the different mixtures of neutralization. Assays for activity inhibition and indirect hemolytic and proteolytic activity were performed by one-way ANOVA followed by a Bonferroni test to determine the significant differences between the doses of each extract. The results were expressed as the mean 7SE, and significant differences were considered statistically significant when p o0.05.

extract 1:5

0

2.10. Statistical analysis

time (seg) Fig. 1. Inhibition of the clotting effect induced by the venom of Bothrops asper by the dichloromethane extract and pinostrobin from Renealmia alpinia. Significant differences were determined between the respective control and the compound after performing one way ANOVA followed by Dunnett's test (np o0.05, nnp o 0.01 and nnnp o 0.001).

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25

***

This neutralizing effect was dose-dependent for all doses of the extract and pinostrobin. Although at lower doses hemorrhagic lesions were observed, pinostrobin inhibited the hemorrhagic activity of Bothrops asper venom more effectively than did the Renealmia alpinia extract (Table 1) because effects similar to those observed for the extract were shown at lower concentrations of pinostrobin. The venom controls and pinostrobin and extract assays were carried out in the same manner.

***

Percent inhibition

20 15

**

**

*

10 5

3.6. Analgesic and anti-inflammatory activity

:1 bi Pi n no 1: 20 st ro bi n Pi 1: no 10 st ro bi Pi n 1: no 5 st ro bi n 1: 1

t1

:5

ac

t1

Pi

no

st

ro

Ex

tr

ac tr Ex

Ex

Ex

tr

tr

ac

ac

t1

t1

:1

:2

0

0

0

Doses Venom :Extract/Pinostrobin Fig. 2. Inhibition of the indirect hemolytic effect induced by the venom of Bothrops asper by the dichloromethane extract and pinostrobin from Renealmia alpinia at different doses. Significant differences were determined between the respective control, the compound and the extract after performing one way ANOVA followed by Dunnett's test (np o0.05, nnp o0.01 and nnnp o 0.001).

Percent inhibition

100

*** ***

80

***

60

**

40

* *

20

1 1:

5

in

in

tr os

in

tr os

:P

Table 1 Percentage neutralization induced by the dichloromethane extract (RAE) of Renealmia alpinia or by pinostrobin from Renealmia alpinia on the hemorrhagic activity induced by Bothrops asper venom. The neutralization is expressed as a percentage, where 100% corresponds to total neutralization of the hemorrhagic activity and 0% corresponds to a lack of neutralization (np o 0.05, nnpo 0.01 and nnn p o 0.001) compared with the control group (one-way ANOVA followed by Dunnett's test). Group

Doses (mg/kg)

Number of stretches (SEM 7 SD)

Percentage neutralization (%)

Control  Bothrops asper ( þ) Venom: Renealmia alpinia 1:5 Venom: Renealmia alpinia 1:10 Venom: Renealmia alpinia 1:20 Venom: pinostrobin 1:0.5 Venom: pinostrobin 1:1 Venom: pinostrobin 1:2

4 4 4

– 128.5 7 4.6 55.0 7 11.9nnn

– – 57.2

4

45.8 7 8.6nnn

64.4

4

35.07 8.9nnn

72.8

4

63.0 7 4.5nnn

51.0

4

49.57 9.9

nnn

61.5

4

42.8 7 6.5nnn

66.7

Ve

no

m

in :P

ob

1:

10 1:

ob

in

1:

in

ob

tr

os

in Ve

no

m

:P

:P Ve

no

m

in

os

tr

ob

ra

xt

:E m

no Ve

20

1 1:

5

ct

1:

10

ct m

Ve

no

:E

xt

ra

1: ct

ra xt

m no

Ve

m no

Ve

Ve

no

m

:E

:E

xt

ra

ct

1:

20

0

Fig. 4 shows the percentage pain inhibition in mice following oral administration of the Renealmia alpinia extract or of pinostrobin at different doses. Ibuprofen at a dose of 75 mg/kg showed a percentage pain inhibition of 91.0, while the extract from Renealmia alpinia at doses of 200 and 300 mg/kg showed inhibition rates of 67.8% and 86.6%, respectively. Similarly, pinostrobin, in all doses studied, showed a decrease in the number of stretchings in a dose-

Doses Venom:Extract / Pinostrobin 100

Percent inhibition

*

**

40 20 0

m g/ K g in ia R 50 .a m lp in g ia 10 R 0 .a m lp g in ia 2 R 0 0 .a m lp in g Pi ia no 30 st 0 ro m bi g n Pi 5 m no g/ st K ro g bi Pi n no 10 st m ro g bi n 20 m g

The hemorrhage diameter was measured on the skin, and the hemorrhagic activity was expressed as the percentage of the diameter induced by pure venom. When the dichloromethane extract from Renealmia alpinia or when pinostrobin was independently administered by an oral route before i.d. venom injection (10 mg), there was a significant decrease in the neutralizing ability.

60

***

***

lp

.a

R

3.5. Neutralization of the hemorrhagic effect of the venom

***

***

75

venom was observed (Fig. 3). The venom:extract ratio of 1:20 showed proteolytic activity inhibition of 88.53%. Moreover, pinostrobin was effective at inhibiting the proteolytic effects induced by Bothrops asper venom, although to a lesser degree than was the extract. Pinostrobin at a ratio of 1:20 showed an inhibition percentage of 21.86%. These differences were statistically significant.

***

80

Ib up ro fe n

Fig. 3. Inhibition of the proteolytic effect induced by the venom of Bothrops asper by the dichloromethane extract and pinostrobin from Renealmia alpinia. Significant differences were determined between the respective control, the compound and the extract after performing one way ANOVA followed by Dunnett's test (np o 0.05, nn p o 0.01 and nnnp o0.001).

Doses Fig. 4. Percent inhibition of the pain induced by phenylquinone upon administration of the dichloromethane extract of the leaves or of pinostrobin from Renealmia alpinia. n p o 0.05, nn p o 0.01 and nnnp o 0.001 compared with the control group (one-way ANOVA followed by Dunnett's test, n¼ 4).

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Table 2 Anti-inflammatory activity of the Renealmia alpinia extract, pinostrobin and ibuprofen on carrageenan-induced edema in mice after 2, 3 and 4 h. The differences in the footpad thicknesses of the mice are compared with the first measurement at the start of the experiment (one-way ANOVA followed by Dunnett's test) np o 0.05, nnp o 0.01, n¼ 4. Group

Doses (mg/kg)

Number of stretches (SEM 7 SD) (2 h)

Neutralization (%) (2 h)

Number of stretches (SEM 7 SD) (3 h)

Neutralization (%) (3 h)

Number of stretches (SEM 7SD) (4 h)

Neutralization (%) (4 h)

Control Ibuprofen Renealmia alpinia Renealmia alpinia Renealmia alpinia Pinostrobin Pinostrobin Pinostrobin

— 50 100

3.187 0.08 1.107 0.07nnn 3.007 0.06

— 65.4 5.7

3.337 0.12 1.0 7 0.03nnn 3.05 7 0.21

— 70.0 7.0

3.137 0.13 1.17 0.06nnn 2.707 0.13

— 65.0 13.7

300

2.78 7 0.13

12.6

3.03 7 0,09

9.0

2.65 7 0.08

15.3

500

2.75 7 0.06n

13.5

2.85 7 0,09

14.4

2.63 7 0.15n

16.0

18.0 22.5 22.5

2.687 0.13 2.63 7 0.14n 2.487 0.10nn

14.4 16.0 20.8

5 10 20

2.88 7 0.08 2.687 0.14 2.30 7 0.15n

9.4 15.7 27.7

n

2.737 0.09 2.58 7 0.05n 2.58 7 0.05n

dependent and statistically significant manner compared to the control group, with p o0.001. On the other hand, pinostrobin presented anti-inflammatory action 2, 3 and 4 h post-administration of carrageenan compared with the control (po 0.05) (Fig. 4 and Table 2).

4. Discussion Snake venom is designed to immobilize, kill and help digest prey. It is made up of various proteins and peptides (toxins) that can be grouped into a small number of major families of proteins, including phospholipase A2 (PLA2), zinc-dependent metalloproteinases, serine proteases, C-type lectin proteins, disintegrins, cysteine-rich proteins (CRISPs), bradykinin potentiating peptides and L-aminoacidoxidase, among others (Fernández et al., 2011; Gutiérrez, 2011). Venom also contains non-protein substances such as amines (histamine, bradykinin, serotonin and acetylcholine), which cause edema, hypotension, and severe pain in victims (Oliveira et al., 2005). Therefore, viper snakebites characteristically cause local and systemic pathological changes, such as coagulation disorders, systemic hemorrhage, necrosis, thrombocytopenia, pain and edema, which may vary in intensity depending on the species, age and size of the snake (Otero et al., 2000b, 2000c). The neutralization of all these effects by anti-venoms is a difficult task. However, the plants used traditionally to treat snakebites represent potential sources of inhibitors and a valid alternative for tackling this difficult problem. In this study, similar yields of pinostrobin were isolated from the dichloromethane and methanolic extracts of Renealmia alpinia leaves as the principal compound. Pinostrobin as well as the extract was then evaluated for their ability to neutralize some of the toxic effects of Bothrops asper venom. Bothrops asper venom affects blood coagulation by allowing the formation of fibrin from fibrinogen due to the presence of toxins that activate platelets and factor XII, whereas the molecular factors V and VI found in the venom directly activate factor X. The joint actions of platelets, factor XII and factor X then lead to a hypercoagulable state in the patient. As fibrinogen is converted to fibrin, it becomes more unstable and susceptible to lysis by natural fibrinolytic systems. Therefore fibrinogen is consumed in large quantities, preventing blood from clotting and causing disseminated intravascular coagulation (Gutiérrez, 2011). The components of the Renealmia alpinia extract have the potential to interact with these enzymes and prevent their effect on plasma coagulation. Indeed, our results showed that the delay in coagulation was dependent on the amount of extract used. Pinostrobin, in

contrast, was effective in neutralizing the coagulating effect when administered in a ratio of 1:20. The hemolysis induced by snake venom can occur through two mechanisms: direct hemolysis, which is mediated by the action of a protein component in the venom, the direct lytic factor (FLD); and indirect hemolysis, which requires the action of the PLA2 poison on the exogenous substrate lecithin. PLA2 converts lecithin into lysolecithin (a surfactant), which is in turn responsible for hemolysis. The end result of both of these mechanisms is the disruption of red cell membranes, which leads to the release of hemoglobin into the extracellular medium (Martinez et al., 1991). The abilities of the extract and of pinostrobin to neutralize the indirect hemolytic effect of Bothrops asper venom (an indication of PLA2 activity) were similar when they were administered at the same concentrations (Fig. 2). The inhibition could be explained by the presence of phenolic compounds in the extract. Some compounds of this type have shown the ability to form complexes with metals, such as Ca2 þ , and their hydroxyl groups can form hydrogen bonds with amino acids in the active site or calcium binding loop of PLA2, which can subsequently block the enzymatic action (Leanpolchareanchai et al., 2009). Pinostrobin also inhibited the hemolytic activity of the Bothrops asper venom, although the data presented here suggest the participation of other compounds in addition to pinostrobin, as the effect observed with pinostrobin was in the same range of bioactivity, being its concentration in the extract below. Bothrops asper venom also produces local hemorrhage after intradermal injection. In fact, hemorrhage is one of the most relevant signs of local and systemic envenoming by viper snakes. The toxins responsible for hemorrhage are a group of zincdependent metalloproteinases with catalytic activity. They degrade capillary basal lamina components and are also cytotoxic to endothelial cells, where they create gaps through which erythrocytes can escape (Moreira et al., 1994; Kamiguti et al., 1996). The ability of a drug to inhibit the effects of the minimum hemorrhagic dose (MHD) of venom is considered a good assessment of its anti-snake venom activity (Shirwaikar et al., 2004). Considering that the inhibition of the metalloproteins isolated from Bothrops asper by the ethanol extract of Renealmia alpinia has been previously described (Patiño et al., 2012, 2013), it was not surprising that the Renealmia alpinia extract also displayed significant hemorrhagic inhibition in the present study. We extended these observations to demonstrate that pinostrobin is able to decrease venom-induced local hemorrhage, as the injection of pinostrobin into mice was more effective at reducing the effects of the venom than was the injection of the extract. Most likely, the decrease in the hemorrhagic activity was related to a chelating

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action through of the formation of Zn2 þ complexes (Bottomley et al., 1998). In addition to the anti-hemorrhagic properties of the extract from Renealmia alpinia, an inhibitory effect on the proteolytic activity of the Bothrops asper venom was also observed. In contrast, the administration of pinostrobin did not improve venom neutralization. Although an effect was observed at the maximum concentration of pinostrobin tested (ratio of 1:20), it is likely that other compounds contributed to the extract bioactivity, and therefore the concentration required for pinostrobin to be effective may be quite high. This hypothesis needs to be addressed in further studies, together with the identification of other potential antiproteolytic agents in the extract. The intraperitoneal injection of phenylquinone causes pain. The extract and pinostrobin caused dose-dependent analgesic effects, presumably by affecting the phenylquinone visceral sensory receptors or by inhibiting the production of pain-producing substances such as prostaglandins (Patiño et al., 2012). The present data demonstrate that the extract of Renealmia alpinia significantly inhibited the response of phenylquinoneinduced writhing in a dose-dependent manner. A dose of 300 mg/kg induced an anti-hyperalgesic effect similar to that achieved by ibuprofen at 75 mg/kg. Despite the differences in the dose used, the results obtained explain the potent anti-algesic activity of pinostrobin, which achieved similar effects at 20 mg/kg. The data presented here suggest that pinostrobin is responsible for the biological activity observed in the extract. Previous observations that flavonoids such as rutin, quercetin, luteolin, hesperidin and bioflavonoids produce significant antinociceptive and/or antiinflammatory activities (Calixto et al., 2000) support our findings. The pharmacological modulation of edema was also investigated in this study. Carrageenan has been widely used as a noxious agent to induce experimental inflammation for the screening of compounds possessing anti-inflammatory activity. When injected locally into the mouse paw, this phlogistic agent produces a severe inflammatory reaction within 30 min (Bhandare et al., 2010). In addition, this acute inflammatory model is known to be sensitive to COX inhibitors and has been used to evaluate the effect of nonsteroidal anti-inflammatory drugs (NSAID), which primarily inhibit the COX involved in PG synthesis (Yang et al., 2010). The subcutaneous injection of carrageenan into the rat paw produces inflammation resulting from the release of various inflammatory mediators in a biphasic process. In the first phase, the release of histamine and serotonin begins immediately after the injection of carrageenan and diminishes within 2 h, while in the second phase, the release of prostaglandins, proteases and lysosomes begins and continues for 3–5 h (Eddouks et al., 2012). These results may suggest that pinostrobin has a mechanism of action similar to NSAIDs such as ibuprofen, blocking the synthesis of prostaglandins by inhibiting the enzyme cyclooxygenase, with effects lasting for at least 3 h, encompassing the maximum effect of carrageenan (Crunkhorn and Meacock, 1972). Another mechanism explaining the bioactivity could involve membrane stabilizers, especially on mastocytes, which prevent membrane degradation and the release of inflammatory mediators such as histamine and serotonin. In fact, some flavonoids display anti-inflammatory mechanisms in cancer models, inhibiting COX-2 (Horia and Watkins, 2007; Van Dross et al., 2007). The present data demonstrate that pinostrobin may mitigate some venom-induced local tissue damage, including hemorrhagic effects, and also displays analgesic and anti-inflammatory activity. Metalloproteinases are common in many medically relevant snake venoms and have been shown to participate in the pathogenesis of local tissue damage in two ways: (a) by releasing tumor necrosis factor-α from its membrane-bound precursor, contributing to the generation of a cascade of inflammatory mediators, which may

induce further tissue damage; and (b) by inducing the synthesis of MMPs in affected tissues, causing hemorrhage effects, among others (Escalante et al., 2000; Rucavado et al., 2004). Our findings suggest that pinostrobin could be a metalloproteinase inhibitor. More tangible evidence for this is required, such as the evaluation of pinostrobin and derivative compounds in the development of a selective metalloproteinases inhibitor. Furthermore, the compound showed moderate inhibition of the hemolytic and proteolytic effects of Bothrops asper venom, with the possible participation of other compounds, and therefore it may also possess PLA2 inhibitory activity. 5. Conclusion Extract from Renealmia alpinia was shown to inhibit the indirect hemolytic coagulant effects and proteolytic activity produced by the venom of Bothrops asper in an in vitro model. Furthermore, the extract reduced hemorrhagic effects and exhibited a strong analgesic effect. Pinostrobin was likely responsible for the analgesic and anti-inflammatory activity of the RAE. This is the first report of pinostrobin in the species Renealmia alpinia and of its properties in vitro against Bothrops asper venom. These results shed light on some of the bioactive compounds from Renealmia alpinia, a plant used by indigenous peoples to treat snakebites. Finally, given that metalloproteinases and PLA2 are both present in Bothrops asper venom, the isolation of other bioactive compounds from Renealmia alpinia and their evaluation as inhibitors of these enzymes should be considered in the future.

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