PHYTOTHERAPY RESEARCH Phytother. Res. 26: 10881091 (2012) Published online 30 December 2011 in Wiley Online Library (wileyonlinelibrary.

com) DOI: 10.1002/ptr.3703

Hepatoprotective Effect of Filipendula hexapetala Gilib. (Rosaceae) in Carbon Tetrachloride-induced Hepatotoxicity in Rats
Tatjana ebovi1* and Zoran Maksimovi2
1 2

Institute of Biochemistry, School of Medicine, University of Novi Sad, Novi Sad, Serbia Institute of Pharmacognosy, School of Pharmacy, University of Belgrade, Belgrade, Serbia

The inuence of methanol extract produced from the owers of Filipendula hexapetala Gilib. (Rosaceae) on some liver biochemical parameters in rats intoxicated with carbon tetrachloride (CTC) was evaluated in this study. The results Obtained revealed that pretreatment with the extract investigated inhibited CTC-induced liver injury by decreasing lipid peroxidation and increasing the content of reduced glutathione in a dosage dependent manner, bringing the levels of all antioxidant enzymes close to control values. The administration of CTC diminished hepatic antioxidant defense mechanisms by signicant reduction of peroxidase and catalase activities. The catalase activity was signicantly recovered in groups treated with the extract investigated and intoxicated with a single CTC dose. A similar impact on hepatic peroxidase activity has also been observed, indicating a partial detoxication of hydrogen peroxide by both catalase and peroxidase. Copyright 2011 John Wiley & Sons, Ltd.
Keywords: Filipendula hexapetala; owers; methanol extract; hepatoprotective activity.

INTRODUCTION In the ora of Serbia, the genus Filipendula L., Rosaceae is represented by two species: Filipendula ulmaria (L.) Maxim. and Filipendula hexapetala Gilib. (Gaji, 1972). Filipendula ulmaria is a medicinal and melliferous plant that owers, and the aerial parts are often used in both traditional and ofcial medicine. Pharmacopoeial herbal drugs Spiraeae os and Filipendulae ulmariae herba (dried owers or owering summits of F. ulmaria, respectively) are used for their antidiaphoretic, diuretic, astringent, tonic, antirheumatic and antiinammatory activities (Wichtl, 2002). These herbal drugs contain phenolic glycosides (primverosides of salicylaldehyde and methylsalicylate), which undergo hydrolytic cleavage during drying and storage, yielding a small quantity of the essential oil with salicyl aldehyde and methyl salicylate as major constituents. Flavonoids spiraeoside (quercetin4-O-b-D-glucopyranoside) and kaempferol-4-O-b-Dglucopyranoside, elagitannins and the other minor polyphenolic constituents are also identied in the owers of this plant (Wichtl, 2002). Specic use of F. hexapetala both in traditional and ofcial medicine is not recognized so far. In the available literature, it is indicated that its inorescence is not used in medical treatments analogous to the herbal drugs Spiraeae os and/or Filipendulae ulmariae herba, but is considered as their adulterant (Wichtl, 2002). However, other authors report that the owers of this plant have a long record of regular use in traditional medicine (Serbian, in particular), in cases of rheumatism

as a diuretic, or as an astringent in haemorrhoids, bleedings, etc. (Tucakov, 1990). At present, knowledge about the chemical inventory and pharmacological properties of this herbal drug is not supported by available reference data; hence, the plant species came into the focus of our scientic interest. As one of the rst results of a broad study on various chemical and pharmacological aspects of F. hexapetala owers, the chemical composition of the essential oil of F. hexapetala owers, rich in salicylic acid derivatives (such as salicylaldehyde, benzyl- and methyl salicylate), has been reported recently (Pavlovi et al., 2007). The starting point for joint investigation on F. hexapetala owers was the evaluation of antioxidant properties of the methanolic extract by three commonly used in vitro procedures, which provided the basis for an activity-guided fractionation assay towards the isolation and chemical characterization of a avonoid (spiraeoside) responsible for the observed activity (Maksimovi et al., 2007). The main objective of the present study was, therefore, to estimate the hepatoprotective potential of methanol extract F. hexapetala owers in a model of carbon tetrachloride (CTC)-induced liver toxicity in rats.

MATERIALS AND METHODS Plant material and extraction procedure. Plant material (owers of F. hexapetala) was collected from a meadow at Mount Zlatibor (western Serbia), during the full-owering period in June 2008 and dried naturally at room temperature. Identication was conrmed by Professor Dr Radia Jani, Head of the Department of Botany, School of Pharmacy, University of Belgrade, Serbia. A voucher specimen has been deposited there.
Received 22 September 2011 Revised 06 October 2011 Accepted 07 October 2011

* Correspondence to: Tatjana ebovi, Institute of Biochemistry, School of Medicine, University of Novi Sad, Novi Sad, Serbia. E-mail: cebovictatjana@gmail.com

Copyright 2011 John Wiley & Sons, Ltd.

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Values are expressed as mean standard deviation of six rats at p < 0.001. Intensity of lipid peroxidation (LPx) is expressed in nmolMDA/mg of protein; MDA, malonyldialdehyde. Content of hepatic reduced glutathione (GSH) is expressed in nmolGSH/mg of protein. Activities of xanthine oxidase (XOD), catalase (CAT), peroxidase (Px), glutathione peroxidase (GSHPx) and glutathione reductase (GR) are expressed in nmol/mg of protein min1. a Significantly different from the control group at p < 0.001. b Significantly different from the control + CTC group at p < 0.001.

Table 1. Effect of MeOH extract of Filipendula hexapetala owers and CTC on the biochemical parameters in the rat liver homogenate

Biochemical assays. Activities of several antioxidant enzymes were determined in liver homogenate. Activity
Copyright 2011 John Wiley & Sons, Ltd.

Control Control + CTC E1 E2 E3 E1 + CTC E2 + CTC E3 + CTC

control group the animals received 1 mL/kg distilled water for 7 days; control + CTC the animals were treated with 1 mL/kg distilled water for 7 days and 2 mL/kg of CTC (day 7) 24 hours before killing; E1, E2 and E3 (FHME-treated groups) the animals were treated with the investigated extract, receiving 1, 2 or 3 mL/kg for 7 days, respectively; E1 + CTC, E2 + CTC and E3 + CTC the animals were treated with the same dosage of FHME, followed by 2 mL/kg of CTC (day 7) 24 h before killing. At the end of experiment (day 8), the animals were anaesthetized with isouorane, decapitated and exsanguinated. The liver weight was taken upon removal of the gall bladder. Samples weighing 1 g were homogenized with TrisHCl:saccharose solution (50 mmol/L; 0.25 mol/L; pH 7.40), 1:3, 4  C, using a glass PotterElvehjem homogenizer set. The resulting homogenate was centrifuged afterwards at 3000 rev/min for 10 min and protein concentrations were determined by the biuret reaction using bovine serum albumine as the standard (Wood, 1989).

Group

2.951 0.049 9.832 0.138a 2.903 0.014b 2.917 0.229b 3.898 0.181a,b 7.827 0.403a,b 3.611 0.653b 3.081 0.123b

LPx

8.504 0.011 21.270 0.222a 8.728 4.264b 15.430 1.347a,b 11.470 0.822a,b 14.690 0.608a,b 11.760 1.120a,b 16.660 0.602a,b

GSH-Px

4.984 0.008 0.730 0.002a 4.942 0.021b 4.980 0.006b 4.509 0.343a,b 2.836 0.166a,b 3.975 0.023a,b 3.025 0.034a,b

In vivo antioxidant activity. Animals and treatment. An aliquot of FHME was dissolved under sonication in a volume of water sufcient to make 5% (w/v) solution, and ltered through 0.45 mm membrane lter. The resulting solution was kept refrigerated (4  C) until applied. Animal care and all experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animal Resources edited by the Commission of Life Sciences, National Research Council. Seven- to eight-week-old Albino Wistar rats of both sexes (obtained from Biochemical Laboratory, School of Medicine, Clinical Centre Novi Sad, University of Novi Sad, Serbia), weighing 200250 g, were housed individually in temperature (25  C) and humidity controlled (3050%) cages and left for 46 weeks to adapt to a reversed lightdark cycle. The animals were maintained on standard pellet diet (LM2, Veterinarski zavod, Subotica, Serbia) and allowed access to tap water ad libitum. The FHME solutions were administered intraperitoneally (1, 2 and 3 mL/kg body weight, corresponding to 50, 100 and 150 mg/kg FHME) for 7 days. The CTC was administered intraperitoneally (2 mL/kg) 24 h before killing. The animals were randomly allocated to eight groups of six, under the following conditions and treatments:

Biochemical parameters

GSH

6.109 0.007 1.250 0.199a 6.032 0.109b 6.620 0.594b 6.109 0.214b 4.785 0.744a,b 4.336 0.214a,b 5.919 0.075b

GR

10.82 0.002 4.937 0.018a 9.289 0.764a,b 9.595 0.946b 8.376 0.443a,b 9.136 0.185a,b 7.541 0.506a,b 8.742 0.727a,b

Px

1.931 0.002 1.899 0.035 2.180 0.146 3.673 0.281a,b 2.692 0.003a,b 2.393 0.279a,b 1.719 0.224 2.215 0.131

Dried plant material (100 g) was reduced to a coarse powder and defatted in a Soxhlet-type all-glass apparatus with light petroleum and chloroform. Residual plant material was extracted by percolation with methanol at a moderate ow rate, and the extract was evaporated to dryness under reduced pressure at 50  C. Dry methanol extract (40 g of brownish gumous mass) was suspended in 500 mL of ethyl acetate and stirred with a magnetic stirrer overnight, at room temperature. The insoluble part was separated by vacuum-ltration over a sintered glass funnel, washed with cold ethyl acetate and dried in a vacuum desiccator (yield: 35 g of goldyellowish, non-hygroscopic ne powder; denoted hereon as FHME).

XOD

9.560 0.002 3.332 0.051a 9.256 0.271b 10.510 0.909b 8.820 0.266b 5.976 0.137a,b 8.234 0.384a,b 7.546 0.447a,b

CAT

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of xanthine oxidase (XOD) was determined following the Bergmayer (1970) method, catalase (CAT) according to Beers and Sizer (1950), peroxidase (Px) according to Simon et al. (1974), glutathione peroxidase (GSH-Px) according to Beuthler (1984) and glutathione reductase (GR) according to Goldberg and Spooner (1983). Amounts of reduced glutathione (GSH; Beuthler et al., 1983) and non-protein SH (Higashi, 1988) were also determined as well as the intensity of lipid peroxidation (LPx) using the Buege and Aust (1978) protocol. Statistical analysis. All the measurements were performed in triplicate, and the results were expressed as mean SD. Mean values between the groups in biochemical analyses were considered signicantly different at the p < 0.05 condence level, after performing a one-way single factor ANOVA, followed by Dunnetts multiple comparison post hoc test.

RESULTS AND DISCUSSION As presented in Table 1, a single 2 mL/kg dose of CTC resulted in a strong induction of LPx intensity, which is caused by free radical generation by CTC. Applied along with a single CTC dose, FHME expressed a clear and dose-dependent suppressive effect against lipid peroxidation in liver homogenate. The relative effect of FHME administration was particularly strong in cases when the extract was applied in doses of 100 and 150 mg/kg, lowering the LPx intensity in liver homogenate by 63 and 69%, respectively (Fig. 1). On the other hand, the administration of FHME during the experiment had insignicant inuence on LPx intensity in the liver homogenate, with the exception of E3 (150 mg/kg), when a notable and statistically signicant pro-oxidant activity was observed, compared with control values. The treatment of rats with 100 and 150 mg/kg doses of FHME produced a statistically signicant increase in activity of GSH-Px in liver homogenate. As expected, a dose of CTC strongly induced the same enzyme.

However, simultaneous application of FHME and CTC led to a signicant decrease in GSH-Px activity in animals that received the combined treatment, compared with those treated with a single dose of CTC. Apparently, although it induced the GSH-Px activity in the rst place, FHME was able to diminish the harmful effect of excessive production of free radicals generated by CTC. The strongest relative effect of FHME to GSH-Px activity was observed when the investigated extract was applied at a dose of 100 mg/kg, which lowered the GSH-Px activity by 45%, compared with the average value recorded in the group of animals treated with a single dose of CTC. The treatment of experimental animals with FHME has not inuenced signicantly the GSH content in the liver homogenate. Intraperitoneal administration of a 2 mL/kg CTC dose caused a substantial drop in GSH levels. As presented in Table 1, the application of FHME reduced signicantly the impact of CTC on the animals, keeping the GSH content in liver homogenate close to the basal level recorded in the control group. A similar inuence on GR activity has been observed in liver of the animals that received combined treatment with FHME and CTC. However, although corresponding values remained lower when compared with basal GR activity (control group), the protective effect of FHME was unambiguous. The results obtained revealed that administration of CTC diminished hepatic antioxidant defense mechanisms by the signicant reduction of Px and CAT activities. The catalase activity was signicantly decreased in the CTC group, but to a much less extent in groups treated with FHME and intoxicated with a single CTC dose. A similar impact on hepatic Px activity has been also observed, indicating the presence of an increased production of hydrogen peroxide (H2O2), partially detoxicated by both CAT and Px. Some of the present results conrmed earlier ndings that CTC intoxication does not alter signicantly the activity of hepatic XOD (ebovi et al., 2006). However, the opposite situation has been observed in groups of animals treated with different doses of FHME: a signicant and dose-dependent

Figure 1. Relative effects of treatment, compared with the group of animals that received a single 2 mL/kg dose of CTC (control + CTC = 100%). Negative values indicate a lowering effect of F. hexapetala methanol extract to increased enzyme activity caused by CTC and vice versa.
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increase of XOD activity has been recorded (the highest value for the 150 mg/kg dose). Reported changes in various antioxidant enzyme activities may also point to an indirect antioxidant effect due to the involvement of both CTC and extract components in complex metabolic pathways, for which there are insufcient experimental data. One of the possible explanations for the detected changes in linear parameters could be related to hormesis (induction of stress response; here due to 7-day extract pre-treatment). In conclusion, in addition to an indirect antioxidant effect, which is expressed via stimulation or supression of certain enzymatic systems involved in free radical metabolism, a possible mechanism of F. hexapetala extract as a hepatoprotective agent may include direct antioxidant activity, which impairs the activation of carbon tetrachloride into the reactive form. The antioxidant activity of spiraeoside observed in previous studies correlates well to actual knowledge about the chemical features critical for in vitro antioxidant activity of avonoids in general, and to antioxidant properties of this

avonoid previously reported (Lamaison et al., 1991; Suh et al., 1999; Yeilada et al., 2000; Lachman et al., 2003; Oh et al., 2004; Maksimovi et al., 2007; Jin et al., 2011; Kim et al., 2011). The results of the present study suggest that, under the experimental conditions described, methanol extract of F. hexapetala owers exerts a protective potential in carbon tetrachloride induced liver injury in vivo, which might substantiate its prospective use in medicine.

Acknowledgements
Supported by Grant No. 173021 of the Ministry of Education and Science, Republic of Serbia.

Con ict of Interest

The authors have declared that there is no conict of interest.

REFERENCES
Beers RFJ, Sizer JW. 1950. Spectrophotometric method for measuring of breakdown of hydrogen peroxide by catalase. J Biol Chem 195: 133140. Bergmayer UH. 1970. Methoden der enzymatischen Analyse. Verlag Chemies: Weinheim; 483484. Beuthler E. 1984. Red Cell Metabolism. A Manual of Biochemical Methods, 3rd edn. University Press: London; 234236. Beuthler E, Duron O, Kelly B. 1983. Improved methods for the determination of blood glutathione. J Lab Clin Med 61: 882889. Buege AL, Aust DS. 1978. Microsomal lipid peroxidation. In Methods in Enzymology, Fleisher S, Parker L (eds). Academic Press: New York; 306310. ebovi T, Spasi S, Popovi M, Borota J, Leposavi G. 2006. The European mistletoe (Viscum album L.) grown on plum extract inhibits CCl4-induced liver damage in rats. Fres Environ Bull 15(5): 393400. Gaji M. 1972. Filipendula L. In Flora of Serbia, Josifovi M (ed.). Vol. IV. Serbian Academy of Sciences and Arts, Department of Natural and Mathematical Sciences: Belgrade; 1214. Goldberg DM, Spooner RJ. 1983. Glutathione reductase. In Methods of Enzymatic Analysis Volume III, 3rd edn., Bergmayer HU (ed.). Weinheim: Basel; 258265. Higashi T. 1988. Critical review on the determination of glutathione in biological preparations. Prot Nucleic Enz 33: 13701382. Jin XF, Qian J, Lu YH. 2011. The role of hepatoprotective effect of a flavonoid-rich extract of Salvia plebeia R. Br. on carbon tetrachloride-induced acute hepatic injury in mice. J Med Plants Res 5(9): 15581563. Kim SM, Kang K, Jho EH, et al. 2011. Hepatoprotective effect of flavonoid glycosides from Lespedeza cuneata against oxidative stress induced by tert-butyl hyperoxide. Phytother Res 25(7): 10111017. Lachman J, Pronk D, Hejtmnkov A, Dudjak J, Pivec V, Faitov K. 2003. Total polyphenol and main flavonoid antioxidants in different onion (Allium cepa L.) varieties. Hort Sci (Prague) 30(4): 142147. Lamaison JL, Carnat A, Petitjean-Freytet C. 1991. Main flavonoid contents of commercial samples of Filipendula ulmaria (L.) Maxim. Plant Med Phytother 25:15. Maksimovi Z, Petrovi S, Pavlovi M, Kovaevi N, Kuki J. 2007. Antioxidant activity of Filipendula hexapetala flowers. Fitoterapia 78: 265267. Oh H, Kim DH, Cho JH, Kim YC. 2004. Hepatoprotective and free radical scavenging activities of phenolic petrosins and flavonoids isolated from Equisetum arvense. J Ethnopharmacol 95(23): 421424. Pavlovi M, Petrovi S, Risti M, Maksimovi Z, Kovaevi N. 2007. Essential oil of Filipendula hexapetala. Chem Nat Comp 43: 228229. Simon LM, Fatrai Z, Jonas DE, Matkovics B. 1974. Study of metabolism enzymes during the development of Phaseous vulgaris. Biochem Physiol Pflanz 166: 389393. Suh HJ, Lee JM, Cho JS, Kim YS, Chung SH. 1999. Radical scavenging compounds in onion skin. Food Res Int 32: 659664. Tucakov J. 1990. Leenje biljem. Rad: Beograd. Wichtl M. 2002. Teedrogen und Phytopharmaka. Wissenschaftliche Verlagsgesellschaft mbH: Stuttgart; 587589. Wood EJ. 1989. Practical Biochemistry for Colleges. Pergamon Press: Oxford. Yeilada E, Tsuchiya K, Takaishi Y, Kawazoe K. 2000. Isolation and characterization of free radical scavenging flavonoid glycosides from the flowers of Spartium junceum by activity-guided fractionation. J Ethnopharmacol 73: 471478.

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