Merak düzeyi ve sosyal paylaşım sitelerine üyelik durumu arasındaki ilişk

Gabriel Araujo-Silvaa, Naira J.N. Britoa, José B.M. Macedoa, Sibele de O. Tozettob, Alessandra S. Guedesc, Jorge A. Lópezd2, Maria das Graças Almeidaa

a

Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Norte, 59012-57 Natal, RN, Brazil

b

Departamento de Patologia Humana, Escola Bahiana de Medicina, Salvador, BA, Brazil

c

Faculdade de Tecnologia e Ciências, Salvador, BA, Brazil

d

Curso de Biotecnologia, CCBS, Pontifícia Universidade Católica do Paraná, Curitiba - PR, Brazil

Abstract

The study was undertaken to evaluate the effects of a methanolic leaf extract of Spondias sp. (MLES) on CCl4-induced hepatotoxicity in rats. Twenty female Wistar rats

were divided into four groups. Group 1 served as normal control. Group 2, 3 and 4 rats were induced hepatotoxicity by CCl4 administering a single dose. Group 2 was the

toxicant control. Group 3 received MLES (500mg/kg body weight, p.o.) while group 4 was treated with reference drug Legalon (50mg/kg bodyweight, p.o.) by oral administration once daily for 7 days. At the end of the experiment, animals were sacrificed and blood were collected for assaying biochemical parameters. The livers were excised for evaluating peroxidation products and histopahology. The results showed that MLES significantly (p<0.05) lowered alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, total bilirubin (tBIL), reduced the content of the peroxidation products malondialdehyde (TBARS) and elevated the glutathione content (GSH) and the superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) activities, compared to CCl4-treated group. Histopathological

results were also in accordance with the biochemical findings. Therefore, the results of this study suggest that the MLES protective role might be correlated with its antioxidant and free radical scavenger effects.

Keywords: Spondias sp.; methanolic extract; Carbon tetrachloride; Hepatoprotective; antioxidant



Corresponding author. Tel./Fax +55 84 3342 9807

Introduction

Excessive generation and accumulation of free radicals and reactive oxygen species (ROS) have been implicated in a multitude of disease states, including inflammation and some hepatopathies (Halliwell, 2006). Normally, ROS are efficiently neutralised by a cell potent armoury without any untoward effect. However, hampering the balance between ROS production and antioxidant defenses lead to the onset of oxidative stress, which seek stability through electron pairing with biological macromolecules (proteins, lipids and DNA) in healthy human cells and cause protein and DNA damage along with lipid peroxidation (Valko et al. 2007). These changes contribute to numerous manifestations (e.g. cancer, atherosclerosis, cardiovascular diseases, ageing and inflammatory diseases) (Halliwell, 2006), that have been proven to be associated with redox imbalance and oxidative stress (Jones 2006). All cells protect themselves against oxidative damage by enzymes such as catalase (CAT), glutathione peroxidase (GPx) or compounds (e.g. glutathione (GSH), ascorbic acid, tocopherol (Zadák et al. 2007). Therefore, compounds that exhibit antioxidant properties, scavenging of free radicals and inhibition of lipid peroxidation are expected to show protective activity (Handa et al. 1986; Wu et al. 2001; Dhanasekaran et al. 2009.).

Spondias sp (family Anacardiaceae) is a typical fructiferous tree, widely ditributed in

Northeastern Brazil, commonly known as “cajá-umbu” (Lira Júnior et al. 2005). This

species is still in domestication process, possibly the product of natural crossing between Spondias mombim L. and S. tuberosa Arr. Cam. (Almeida et al. 2007). Besides the industrial importance (jelly, juice) (Silva Junior et al. 2004), some pharmaceutical properties have been reported for this plants. Their leaves as infusion are commonly used in folk medicine to produce a wide variety of remedies against ulcers and inflammatory processes (Teixeira and Melo 2006).

Therefore, the aims of this study were to evaluate the antioxidant potential and free radical scavenging activity of a methanol extract of Spondias sp. leaves, employing an in vitro assay and also investigated the protective effect of this extract against liver injury induced by carbon tetrachloride (CCl4) in a murine experimental model.

Materials and methods

AST, ALT, ALP and tBIL kits were supplied by Biosystems (Spain). Legalon was obtained from Nycomed (Brazil). All the other chemicals used were of analytical grade. Spondias sp. leaves, collected from Itaberaba, BA, Brazil, were identified and authenticated by Dr. Maria das Graças Ferreira (Department of Botany, Bahia Company for Agro-Livestock Development (EBDA), Salvador, BA, Brazil). A voucher specimen (Herbarium Nr. 2897/12.09) was deposited at the Herbarium of the Faculty of Technology and Sciences (FTC, Salvador, BA, Brazil). Collected leaves were shade- dried at 40°C and powdered by an expeller. Leaf powder (350 g) was soaked in 3L of methanol for extraction for seven days with periodical shaking. After filtration, the solvent was condensed using a rotary evaporator at 40°C, giving a yield residue of 39.5±2.7 g. The crude extract (MSE) was stored at -20ºC for subsequent experiments. Total phenolics in leaf Spondias sp. extract were determined using Folin-Ciocalteu reagent, based on procedure described by Georgé et al. (2005), and the results were expressed as mg of gallic acid equivalents per g of extract. The determination of radical scavenger activity of the extract was performed by the DPPH test (Brand-Williams et al. 1995), using Carduus marianus extract and BHT as positive controls. The results expressed in % RSC.

Twenty adult female Wistar rats (160 – 210 g) were used. Animals were bred in the Central Animal House (CCS, Federal University of Rio Grande do Norte) at 223ºC in 12 h light/dark cycles with standard diet and water ad libitum. The experiments were performed in accordance with the guidelines provided by the Brazilian College of Animal Experimentation (COBEA) and Institutional Animals Ethics Committee (protocol 100312/09).

Animals were divided into four groups (n = 5). Group 1 served as normal control and received vehicle corn oil. Group 2, 3 and 4 were induced liver toxicity by CCl4 administration by a single dose of CCl4 (2.5 ml/kg CCl4, diluted in corn oil). Groups 2 served as toxicant control, while groups 3 and 4 received MSEL and Legalon® (antioxidant control) at doses of 500 mg/kg and 50 mg/kg, respectively, for seven days. Administration was done orally using the intragastrictubes .Animals were humanly sacrificed 24 h after the last treatment by cervical dislocation, anesthesied with Tiopental (40 mg/kg b.w.). Blood was collected and serum was separated to analyze the

biochemical parameters, while livers were dissected out for analysis and histopathological analysis.

Serum level of ALT, AST, tBIL were estimated according to the manufacturer‟s kit

instructions (Biosystems, Spain). Catalase was determined as described by Beutler (1975). GSH was determined by the method of Beutler et al. (1963), while the levels of TBARS were estimated as describe by Ohkawa et al. (1979) was determined in liver homogenate. For histopathological examination livers sections were fixed in 10% buffered formalin, processed, and embedded in paraffin, cut into 5 - 6 µm thick sections and stained with haematoxylin-eosin (H&E), using a standard protocol and then analyzed by light microscope.

Acute toxicity study was performed according to Organisation for Economic Co- operation and Development guidelines No. 423 (OECD, 1996). Wistar rats of either sex were divided into six groups with six animals each. MESL was administered orally as a single dose to rat at different dose levels of 250, 500, 1 000, 1 500 and 2 000 mg/kg b.w. Animals were observed periodically for the symptoms of toxicity and death within 24 h and then daily for 14 days.

Reverse-phase HPLC analysis were performed by a method development and validation, on the VARIAN ProStar HPLC system (Walnut Creek, USA), consisting of HPLC pump, ProStar 240 quaternary gradient unit, 335 PDA UV/Vis detector. In order of chromatographic separation, we accomplished on a Phenomenex C18 reverse phase column (Phenomenex, 4.6 X 100 mm, 2.6 µm) at room temperature and monitored at 280 nm. The gradient solvent system consisted of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) according the table 01, with a flow rate of 1.3 ml/min. For preparation of stock solutions, extracts and nine phenolic compounds were dissolved in MeOH at concentrations of 5 mg/mL, and 1 mg/mL, respectively. After filtration through a syringe filter (0.22 µm, Allcrom), 9 µl of each sample was injected. The retention times, linear ranges, linear coefficients, correlation coefficients and UV spectrums were showed in table 02. The relative quantity of phenolic compounds in the extract was calculated from each equation, considering the spectrum similarity, and the results were expressed in mg of equivalent standard/ g of the extract.

The experimental results were expressed as meanSD. Analysis of variance was (ANOVA) used for statistical analysis by and post-hos test of Tukey-Kramer, using the Graph PAD Software version 4.0. p <0.05 implied significance.

Results and Discussion

The methanolic crude extract from Spondias sp. leaves produces no mortality at 2 000 mg/kg. Therefore, one-quarter of the maximum no mortality dose of extract, it were selected as therapeutic dose (500 mg/kg) in this study.

In the present study, it was evaluated the methanolic crude extract from Spondias sp. leaves which was tested against the CCl4 induced hepatotoxicity.

Fig. 1 shows the percentage of radical scavenger activity of the extract. It was found that the reduction of DPPH occurred in a concentration-dependent manner, as observed with the decrease in the absorbance of DPPH. This result was in accordance with the concentration of phenolic compounds determined in the extract (12.08±1.20 mg EAG/100 mg of extract).

The hepatoprotective efficacy of the MSEL against CCl4-induced liver injury was

evaluated in a murine model. The administration of a single dose of CCl4 resulted in a significant increase of the serum levels of AST, ALT, ALP and tBIL (p<0.05) due to liver injury. In treated animals with MSEL (group 3) and Legalon (group 4), there was a significant (p<0.05) decrease in these serum hepatic marker levels (Table 1).

The activities of CAT, SOD,GPx and the GSH content were significantly reduced in the CCl4-intoxicated group (p<0.05) when compared to normal control animals. In the group 3 which received CCl4 with MSE and in rats treated with Legalon (group 4), it

was observed a significant (p<0.05) increase in the levels of CAT and GPx activities, as well as in the GSH content, which were near to normal (Group 1) (Table 2).

The administration of CCl4 in rats increased the TBARS level in the liver when compared to normal control animals (p<0.05). However, the TBARS content was significantly reduced by the oral administration of the MSEL or Legalon (p<0.05), when compared with animals from group 2 (Fig.2).

In the histopathological analysis of group 1 (Fig. 3a), animals showed normal architecture of liver. In rats treated with CCl4 alone, the liver normal architecture was

completely lost with the appearance of vacuolated hepatocytes and degenerated nuclei. Vacuolization, fatty changes and necrosis of hepatocytes were severe in the centrilobular region and these changes were also observed in areas other than the centrilobular regions (Fig. 3b). The livers of rats treated with MSEL (Fig. 3c) or Legalon (Fig. 3d), group 3 and 4, respectively, showed a significant recovery from CCl4-induced liver damage as evident from normal hepatocytes with well defined

nuclei, almost comparable to those of the control group. Vacuolization and fatty degeneration were remarkably prevented by the treatment with extract and Legalon. The HPLC method applied to separation of major number of compounds in the Spondias sp. extract, were efficient to identification of the majors compounds and groups in MSEL. In fact, were observed twelve major compounds (Figure 1A), and the group were identified by comparison between their and external standards (Figure 1B) UV spectrums and retention times, which just the compounds 4, 5 and 6 no possible identified the group. The compound 1(RT=1.43) was identified as simple phenolic by shown the RT and UV spectrum similar to Gallic acid, and compound 2 (RT= 1.73) was identified and quantified as 0.69±0.0012 µg Gallic acid/mg of MSEL. The

compound 3 (RT=6.63) was quantified as 1.34 ±0.0065 µg de Chlorogenic acid /mg de

MSEL. The compounds 7, 8 and 9 (RT=9.23, 10.05 and 12.65) corresponding at glycosylated flavonoids, and the compound 7 was identified and quantified as 21.63 ± 0.0039 µg of rutin/mg de MSEL. Flavonoids aglicon, compounds 10(15.99), 11(18.92)

and 12(19.30), were found, and the compound 10 corresponding at 2.14 ± 0.02 µg of

quercetin/mg MSEL. The compound 11 were quantified as 4.44 ± 0.0046 µg of kaempferol/mg de MSEL.

The results indicate that the methanolic crude extract from Spondias sp. leaves was found to be rich in phenolics and flavonoids, a flavonol-3-O-glycosilate (compound 7) with near retention time and similar UV spectrum of rutin, and were identified gallic acid, chlorogenic acid, quercetin and kaempferol.

Acknowledgements

The authors thank to FAPESB, CNPq (proc. 478652/2010-0), Faculdade de Tecnologia e Ciências, Salvador – BA for the preliminary assay and Centro de Ciências da Saúde, Universidade Federal do Rio Grande do Norte, Natal for Grant and financial support.

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Table 1. Scheme of the elution gradient established for the separation of phenolic compounds

Time (min) ACN (%) Remarks

0-3 5 Isocratic range 3-7 520 Linear increase 7-9 20 Isocratic range 9-10 2023 Linear increase 10-15 23 Isocratic range 15-19 2350 Linear increase

Table 2. Parameters to quantification of nine phenolic compounds used a external standard method.

Compound Retention time

(min) Linear range (mg/mL) a R2 UV (nm) Gallic acid 1.71 1.5-50 9.2054 0.9945 271 Chlorogenic acid 6.47 1.5-50 9.7922 0.9973 326 Catechin 7.19 1.5-50 5.3171 0.9993 278 (-)-Epigallocatechin gallate 8.18 1.5-50 8.7029 0.9989 275 Rutin 9.20 1.81-100 5.3938 0.9976 257 - 354 Hiperin 9.40 1.81-100 7.2496 0.9995 256 - 354 Quercetin 16.03 1.5-50 10.379 0.9991 255 - 371 Apigenin 18.51 1.5-50 10.022 0.9989 266 - 337 Kaempferol 18.85 1.5-50 5.443 0.9984 263 - 367

Table 3. Effect of methanolic leaf extract of Spondias sp. on serum aminotransferases and total bilirubin in control and treated animals groups.

Parameters Group 1 Group 2 Group 3 Group 4

AST (IU/L) 140.7±30.6 871.3±19.5 169.3±45.0 160.7±13.6 ALT (IU/L) 070.7±18.1 438.0±21.2 076.7± 7,6 069.3± 8.3 ALP (IU/L) 142.0± 7,0 213.0± 2.8 97.2± 5.2 137.0±28.3 tBIL (mg/dL) 0.29 ± 0,01 0.56 ± 0.2 0.35± 0.01 0.30 ± 0.01

Values are expressed as means±S.D.

Values not sharing a common superscript differ significantly at p<0.05

Table 4. Effect of methanolic leaf extract of Spondias sp. on antioxidant parameter (CAT, GPx, SOD and GSH) in control and treated animals groups.

Parameters Group 1 Group 2 Group 3 Group 4

CAT (Umol/min.L) 223.01±79.12 84.25±43.10 242.08±53.33 271.71±78.39 GPx (U/mg protein) 18.21±4.54 11.42±0.95 23.18±2.78 20.93±3.61 SOD(U/mg protein) 74.58±3.55 24.27±6.55 105.90±4.25 98.52±7.01

GSH (nmol/mL) 5.51±0.40 2.89±0.29 6.74±0.76 5.45±0.93

Values are expressed as means±S.D.

Fig. 1. HPLC–UV chromatogram of phenolic compounds in MESL. Detection was at 280 nm (A), HPLC–UV chromatogram of standard phenolic compounds(B).