Evaluation of the effect of aqueous extract of Croton urucurana Baillon (Euphorbiaceae) on the hemorrhagic activity induced by the venom of Bothrops jararaca, using new techniques to quantify hemorrhagic activity in rat skin.

Abstract

Aqueous extracts of Croton urucurana (Sangra D'agua), a plant popularly considered a cicatrizant, were analyzed for anti-Bothrops jararaca venom activity. The plant extracts antagonized the hemorrhagic activity of the venom and proanthocyanidins were involved in this activity. Two new methods for the quantification of hemorrhagic activity evoked by bothropic venoms were employed. The first consists of graphic computer analysis of the hemorrhagic halo evoked in rats by dorsal intradermic administration of venom. The second method involves quantification of the hemoglobin present in the hemorrhagic halo. Based on the results, we suggest that these methods, easily implemented in the laboratory routine, allow for quantification of venom-induced hemorrhagic activity. In addition, this study demonstrates that the rich extracts of proanthocyanidins are powerful inhibitors of bothropic venom metalloproteinases.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Bothrops jararaca; Croton urucurana; Hemorrhagic activity; Anti-venom

Introduction

Venoms of snakes in the family Viperidae, which includes the genus Bothrops, induce intense local effects in tissues. These effects include hemorrhage, myonecrosis and edema.

It is believed that the hemorrhagic effect of the venom of Bothrops jararaca (B. jararaca) is caused by metaloenzymes that evoke enzymatic degradation of the basal membrane with loss of vascular wall integrity (Kamiguti et al., 1996). The activity of these enzymes gives rise to the hydrolysis of matrix proteins such as collagen IV, laminin and fibronectin (Baramova et al., 1989).

Normally, homeostasis is maintained by the non-thrombolytic properties of the blood vessel walls. Damage to these walls initiates a response by hemostatic component mechanisms aimed at restructuring damaged vessels. These mechanisms include vasoactive agent release, platelet migration and formation or activation of coagulation factors. When some of these homeostatic components are altered, a hemorrhagic process begins (Kamiguti et al., 1986).

Theakston and Reid (1983) developed standardized assays to quantify hemorrhagic lesions evoked by ophidian venoms. Modifying the methodology proposed by Kondo et al. (1960), the authors measured the hemorrhagic halo present in the internal face of the dorsal skin of the experimental animals. This method of quantification consists of reading the two largest perpendicular diameters of the halo, using the average to reveal the size of the hemorrhagic halo. Ownby et al. (1994) described a semi-quantitative technique based on modifications of these methods. Their technique is based on the standardization of different color intensities by visual comparison of the hemorrhagic lesions, which are quantified on a scale from 1 to 5. To express lesion intensity, the average diameter is multiplied by the number on the scale and the product divided by the amount, in milligrams, of venom administered.

Roodt et al. (2000) reported the difficulties encountered in quantifying hemorrhagic activity when halos evoked by intradermal administration of bothropic venom present similar sizes but different color intensities. The authors compared the results obtained by the determination of average halo diameters and weights with those derived from determination of the amount of hemoglobin extracted from the tissue, measured either directly or by the peroxidase method.

Batina et al. (2000) reported that the root bark of Peschiera fuchsiaefolia (later reclassified as Tabernaemontana catharinensis A.DC.) is used as an anti-venom agent in the rural community of Assis, in the state of Sao Paulo, Brazil. Residents there apply it to snakebites in order to neutralize the effects of the venom. Fresh or stabilized aqueous extracts (AE) of the root of this plant are able to inhibit lethal and myotoxic activities of the venom of the South American Rattlesnake (Crotalus d. terrificus).

Croton urucurana (C. urucurana) also displays medicinal potential and is known popularly in Brazil as "Sangra D'agua". Preparations from this plant are used chiefly as anti-inflammatories, cicatrizants and anti-rheumatics (Peres et al., 1998). In order to evaluate some of these uses, assays of the analgesic and anti-microbial activities of these preparations were carried out, with very encouraging results. Further studies revealed the presence of campesterol, beta-sitosterol (and its glycoside), stigmasterol, acetyl-areulitolic acid, catechin and galocatechin. These compounds are derived from the methanolic extract (Peres et al., 1997, 1998).

The aim of the present study was to report the hemorrhage-inhibiting ability of an extract fraction of C. urucurana. The quantification of hemorrhagic activity was determined using the two new methods.

Materials and methods

Materials

Lyophilized B. jararaca venom was purchased from the Instituto Butantan in Sao Paulo, Brazil. Male Wistar rats averaging 100g body wt. each were maintained in a 24[+ or -]1 [degrees]C environment and provided with commercial rat chow and water ad libitum. C. urucurana Baillon was collected in the city of Ituiutaba, MG, Brazil. Voucher specimens were identified by Dr. Ines Cordeiro of the Herbarium at the University of Sao Paulo, Institute of Botany, and subsequently deposited in the Herbarium (SPFR) of the Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, USP (Reg. # 05660).

Hemorrhage determination

Animals were divided randomly into six groups, composed of six animals per group. To determine hemorrhagic activity, experiments involving six different treatments (one per group) were performed in triplicate. Treatment 1 involved injecting 100 [micro]l of saline solution as a control. For treatments 2-6, animals received incremental doses of venom protein, each increased by a ratio of 1.8 (minimum, 1.54[micro]g/100g body wt.; maximum, 16.20[micro]g/100g body wt.) in a final volume of 100 [micro]l of saline.

The venom was injected intradermically into the shaved dorsal skin under light ethyl ether anesthesia. After 24h, the animals were sacrificed by anesthetic overdose. The dorsal skin was then removed and placed between two glass plates.

Determination of the mean diameter of the hemorrhagic lesion

The hemorrhagic area was drawn onto a transparent sheet and measured with a scale meter. The two largest perpendicular diameters of the lesion were recorded and their arithmetic mean was considered the mean diameter of the lesion (MLD).

Using the values obtained, lesion diameters vs. log doses were plotted with the help of statistical software and utilized for the determination of the minimum hemorrhagic dose (MHD) (Theakston and Reid, 1983).

Lesion intensity analysis of hemorrhagic areas

The skins of the animals were stored in a cold room at 4[degrees]C for 18h. Subsequently, they were scanned and analyzed using ImageTool 1.25 software (University of Texas Health Science Center, San Antonio; http://ddsdx.uthscsa.edu/dig/itdesc.html).

The color intensity of the hemorrhagic area was determined by the following equation: I = A X (N - H), where I is the intensity of the hemorrhagic area, A the hemorrhagic area measured in [mm.sup.2], N the average color intensity in non-hemorrhagic skin and H is the average color intensity in hemorrhagic area. For area analysis, images were transferred to black and white and values of 0 and 255, respectively, were given to the "black" and "white" tonalities.

Determination of hemoglobin concentration

Scanned skin was cut around the limits of halos and the resulting samples were cut into small fragments. The fragments were incubated for 48 h at 4[degrees]C in 3 ml of saline in order to elute the hemoglobin present. Following centrifugation at 800g for 5 min, the concentration of hemoglobin in the supernatant solution was determined using the cyanomethemoglobin method (Drabkin and Austin, 1935; International Committee for Standardization in Haematology, 1978).

Evaluation of the action of C. urucurana aqueous extract on the hemorrhagic activity of B. jararaca venom

Evaluation of the action of C. urucurana AE was based on the determination of hemorrhagic activity. Fractions were dissolved in saline (50 [micro]l), mixed with B. jararaca venom (10.8 [micro]g/50 [micro]l) at room temperature and injected intradermally into the animals. After 24 h, animals were sacrificed by anesthetic overdose, their skins removed and stored in the cold room.

Fractionation of aqueous extract of C. urucurana

After identification, 1 kg of powdered stem bark was extracted with 31 of distilled water, filtered and lyophilized to obtain the AE. The AE (2.1 g) was redissolved in water, applied to an Amberlite XAD-2 column (50 X 3.5 cm) and eluted with water and ethanol. The water-soluble portion was lyophilized (AE1 fraction). The ethanol-soluble portion was evaporated in vacuo (AE2 fraction). The AE2 fraction was dissolved in methanol and cleared by centrifugation. The resulting solution was evaporated in vacuo (AE2M). The AE2M (0.5 g) was then submitted to partition using water (20 ml) and butanol (20 ml), which extracted the fractions AE2MA and AE2MB, respectively. The AE2MB fraction was dissolved in 3 ml of methanol and applied to a Sephadex LH-20 column (30 X 2.5 cm) equilibrated with methanol. The flow rate was 0.5 ml/min and the fraction volume was 10 ml/tube.

Eluates from each tube were analyzed by measurement of absorbance. The fractions dissolved in methanol were measured at 280 and 540 nm. Thin-layer chromatographic analysis on silica gel plate (Sigma) was then performed using a mobile phase of water/butanol/acetic acid (1:4:5). The spray reagent was vanillin (Amarowicz and Shahidi, 1996). Eluates were pooled into seven fractions.

Analysis of condensed tannins

Colorimetric assay was used for analysis of condensed tannins (Dalzell and Kerven, 1998) and an acid-catalyzed oxidative depolymerization of condensed tannins was used to yield red anthocyanidins. This procedure is used to identify polyflavan structures (Schofield et al., 2001).

The fractions obtained were diluted with water (2 mg/ml). Butanol (3 ml) and HCI (0.1 ml) were added to 1 ml of this solution. The mixtures were held in a water bath at 100 [degrees]C for 1 h. Cyanidins in the butanolic phase were evaluated for their absorbance at 540 nm.

Statistical analysis of results

Results were expressed by arithmetic means [+ or -] standard deviation (SD). For linear regression analysis, STATISTIC software (StatSoft) was used. For analysis of differences between two groups, a Student's t-test was employed. For multiple comparisons of groups or protocols, one-way ANOVA, according to Duncan, was employed (Milleken and Johnson, 1984).

Results

Comparison of methods used for determining hemorrhagic activity

Fig. 1 depicts linear regressions of the results of three different measures of hemorrhagic activity. Mean lesion diameter, hemorrhagic halo color intensity (quantified using the ImageTool program) and hemoglobin concentration (eluted from the hemorrhagic halo and plotted as function of the dose of B. jararaca venom applied) are shown. Linear regressions were: 0.97660, 0.99194 and 0.98649, respectively.

Effect of C. urucurana fractions on the hemorrhagic activity of B. jararaca venom

Fig. 2 shows the alterations caused by 2 MHDs (10.8 [micro]g/100 g body wt./100 [micro]l saline) of B. jararaca venom in the presence of the C. urucurana fractions quantified by the lesion intensity method (image analysis). When B. jararaca venom was mixed with SS, AE, AE1, AE2, AE2M, AE2MA and AE2MB, lesion intensities were 11282.05 [+ or -] 1281.48; 8455.26 [+ or -] 1568.47; 9507.54 [+ or -] 961.06; 4182.539 [+ or -] 716.02; 1933.47 [+ or -] 485.02; 1202.19 [+ or -] 353.32 and 319.52 [+ or -] 63.3, respectively.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Hemoglobin concentration

Fig. 3 shows the alterations caused by 2 MHDs (10.8 [micro]g/100 g body wt./100 [micro]l saline) of B. jararaca venom. These alterations were quantified by determination of hemoglobin concentration in the hemorrhagic halos of the same lesions analyzed in Fig. 2.

When B. jararaca venom was mixed with SS, AE, AE1, AE2, AE2M, AE2MA and AE2MB, the concentrations of hemoglobin eluted from the hemorrhagic halos were 0.031 [+ or -] 0.003; 0.02 [+ or -] 0.003; 0.017 [+ or -] 0.002; 0.009 [+ or -] 0.001; 0.006 [+ or -] 0.001; 0.006 [+ or -] 0.001 and 0.005 [+ or -] 0.001, respectively.

Condensed tannins (proanthocyanidins)

Fig. 4 shows the color intensity produced by hydrolysis of condensed tannins at 540 nm. From this, we can see that fractions AE, AE2 and AE2MB had higher absorbance and therefore higher concentrations of condensed tannins. Fractions AE2M, AE2MA and AE1 all had slightly lower absorbance.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Discussion

Numerous studies on hemorrhagic fractions isolated from venoms of various snakes have been undertaken. Inhibitions of this activity by anti-venom serum derived from animal plasma products, crude plant extracts or fractions thereof, have also been described (do Daros et al., 1996; Jurgilas et al., 1999).

Kondo et al. (1960) described a method, later modified by Theakston and Reid (1983), for determining the mean diameter of a lesion evoked on the dorsum of an animal. This method could be employed to determine whether these anti-venom substances alter homeostasis or cause blood vessels to rupture. Ownby et al. (1994) utilized the semi-quantitative measurement of lesion color intensity to verify the inhibitory action of test substances on the hemorrhagic effects of venom. For determination of hemorrhagic halos, Roodt et al. (2000) described a method that consists of elution of the hemoglobin present in hemorrhagic lesions and quantitative estimation by the peroxidase method. But these authors point out that considerable time and work are required for this procedure and do not recommend it for routine laboratory determinations of hemorrhagic activity.

The image analysis method utilized in the present study enables the determination of hemorrhagic areas of any shape, resulting in readings that are more reliable than those obtained by measuring lesion diameters, since lesions are not always circular. Inhibition in the center of the lesion would not be detected.

A method of quantification has been devised in which hemoglobin or red blood cells leaking into the cutaneous tissue are eluted with saline solution and subsequently determined as cyanomethemoglobin (Drabkin and Austin, 1935; International Committee for Standardization in Haematology, 1978). This technique is generally used to study hemorrhagic lesions evoked or inhibited by substances presenting color differentiation or masking of the hemorrhagic halo.

In the present study, image analysis, determination of hemoglobin eluted from hemorrhagic halos and determination of hemorrhagic areas all presented high linear correlation coefficients (r = 0.99194, 0.98649 and 0.97659, respectively).

The image analysis method presented the highest correlation value, but was less sensitive than analyses performed using the halo diameter analysis method, which was in turn more sensitive than hemoglobin concentration measurements.

In order to obtain the values of the hemorrhagic lesions using the MHD method, the arithmetic mean of the greatest perpendicular diameters of the lesions was calculated. This calculation is exact when the area of a perfect circle is measured, but, in this case, the halos contained irregular areas. Therefore, the image analysis method that integrates hemorrhagic areas or the determination of the amounts of hemoglobin eluted from the hemorrhagic halos are more exact ways of determining hemorrhagic activity.

Differences between halos evoked by the plant extract were evident. Halos evoked by B. jararaca venom were reddish in color, in contrast to those evoked by the plant extract, or by the extract and the venom combined.

The software utilized determines the area of the lesion by the color intensity, which displays as a gray scale. Unfortunately, the program does not differentiate the color evoked by the extract from that caused by the venom alone.

When 10.8 [micro]g of the fraction from the extracts were assayed with 2 MHDs of B. jararaca venom, 97% inhibition was obtained. When the lesion intensity was decreased from 11282 U [mm.sup.2] (B. jararaca venom alone) to 319 U [mm.sup.2] (B. jararaca venom pre-mixed with AE2MB), the estimation of hemoglobin from the hemorrhagic halos resulted in 83.87% inhibition. This was paralleled by a decrease in hemoglobin concentration from 0.031 to 0.005 g% (Fig. 3). The AE2MB fraction was found to contain catechin, the basic unit of condensed tannins, as its major component.

In conclusion, the methods reported herein present a high linear correlation coefficient for the determination of hemorrhagic lesions. The image analysis method may be utilized for quantitative determination of a hemorrhagic lesion evoked on the skin of animals. The method for determining the concentration of hemoglobin eluted from the hemorrhagic halo may be utilized to quantify hemorrhagic lesions when the halo is not well defined. These methods are practical and rapid for the quantification of hemorrhagic activity.

The present study suggests that the butanolic fraction of the C. urucurana AE is a promising antidote for the effects of B. jararaca venom.

Acknowledgments

The authors are indebted to Joao Jose Franco and Adelia C.O. Cintra for their technical assistance, to FAPESP and CNPq for financial support and to Capes for the fellowship given.

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L.E. Esmeraldino, A.M. Souza, S.V. Sampaio*

Departamento de Analises Clinicas, Toxicologicas e Bromatologicas da Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, Brazil

Received 2 December 2003; accepted 15 January 2004

*Corresponding author. Tel.: [phone]; fax: [phone].

E-mail address: [e-mail] (S.V. Sampaio).