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Protective effects of dried flower extracts of Hibiscus sabdariffa L. against oxidative stress in rat primary hepatocytes
Pergamon
Food and Chemical Toxicology 35 (1997) 1159-1164
Protective Effects of Dried Flower Extracts of Hibiscus sabdariffa L. against Oxidative ',Stress in Rat Primary Hepatocytes T.-H. T S E N G , E.-S. K A O , C.-Y. C H U , F.-P. C H O U , H.-W. L I N W U a n d C.-J. W A N G * Institute of Biochemistry, Chung Shah Medical and Dental College, Taiwan, ROC (Accepted 22 April 1997) Abstract--Dried flower extracts of Hibiscus sabdarrifa L., a local soft drink material and medical herb, was found to possess antioxidant activity in the present study. In the preliminary studies, antioxidant potential of three fractions of the ethanol crude extract (HS-C: chloroform-soluble fraction; HS-E: ethyl acetate soluble fraction; HS-R: residual fraction) obtained from the dried flowers of Hibiscus sabdarrifa L. were evaluated by their capacity of quenching l, 1-diphenyl-2-picrylhydrazyl(DPPH) free radical and inhibiting xanthine oxidase (XO) activity. HS-E showed the greatest capacity of scavenging free radical (ECso=0.017mg/ml), and HS-C showed the strongest inhibitory effect on XO activity (ECs0 = 0.742 mg/ml). Furthermore, antioxidant bioactivities of these crude extracts were investigated using a model of tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat primary hepatocytes. All fractions were found to inhibit significantly the unscheduled DNA synthesis (UDS) induced by t-BHP at a concentration of 0.20 mg/ml. HS-C and HS-E also decreased the leakage of lactate dehydrogenase (LDH) and the formation of malondialdehyde (MDA) induced by t-BHP (1.5 m i ) considerably at a concentration of 0.10 and 0.20 mg/ml in the rat primary hepatocyte cultures. These results indicated that the dried flower extracts (HS-C and HS-E) of H. sabdarrifa L. protect rat hepatocytes from t-BHP-induced cytotoxicity and genotoxicity by different mechanisms. © 1997 Elsevier Science Ltd. All righi's reserved.
Abbreviations:t-BHP = tert-butyl hydroperoxide; DMSO = dimethyl sulfoxide; DPPH = 1,1-diphenyl2-picrylhydrazyl radical; LDH = lactate dehydrogenase; MDA = malondialdehyde; PBS = phosphate buffered saline; ROS = reactive oxygen species; UDS = unscheduled DNA synthesis; XO = xanthine oxidase.
I:NTRODUCTION Reactive oxygen species (ROS) can be originated from several internal and external sources, such as metabolic reactions, dietary intake or cigarette smoking. Excessiw: formation of oxidants in biological systems and the consequent oxidative damage are topics of growing interest in recent years, since these processes probably contribute to several inflammatc,ry diseases and cancer (Morse and Stoner, 1993). Studies have shown that ROS play an important role in carcinogenesis, especially in the promotion stage (Breimer, 1990; Cerutti, 1985). Although the body possesses defence mechanisms to reduce the oxidative damage, such as using enzymes and antioxidant nutrients to arrest the damaging properties of excited oxygen species, *Author for correspondence at: Institute of Biochemistry, Chung Shan Medical and Dental College, No. l l0, Section 1, Chien Kuo N. Road, Taichung 402, Taiwan, ROC.
continuous exposure to chemicals and contaminants may lead to an increase in the amount of free radicals in the body beyond control, and cause irreversible oxidative damage. Therefore, antioxidants are considered effective inhibitors of carcinogenesis and also in other conditions which are pathogenetically associated with oxidative mechanisms. Many studies have supported that antioxidant nutrients or/and medicines play a protective role in human health (Ames, 1983; Weisburger, 1991). The study of numerous compounds that could be useful as antioxidants, ranging from ct-tocopherol and fl-carotene to plant antioxidants such as flavones and tannins, has generated increasing interest in the field of food and/or medicine. The dried flowers of H. sabdariffa L. (Malvaceae) have been used effectively in folk medicines against hypertension, pyrexia and liver disorders. Recently, it has gained importance as a soft drink material in local regions. However, little information is available about the pharmacological and biological effects of H. sabdarzffa L., even though the polysaccharide constituents
0278-6915/97/$19.00 + 0.00 © 1997 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PH S0278-6915(97)00{)96-3
1160
T.-H. Tseng et al.
have been assayed for their possible immunomodulating effects (Mtiller and Franz, 1992). To understand the antioxidant action of the dried flowers of H. sabdariffa L. in intact cells, we used the model of t-BHP-induced oxidative damage in the primary cultures of rat hepatocytes for the study, t-BHP is widely applied to investigate the mechanism of cell injury initiated by oxidative stress (Rush et al., 1985). It can be metabolized to free radical intermediates by cytochrome P-450 (hepatocyte) or haemoglobin (erythrocytes), which in turn can initiate lipid peroxidation, affect the cell integrity and mediate DNA damage (Davies, 1989; Thornalley et al., 1983). Polyunsaturated fatty acids, present in cell membranes, are easily oxidized by enzymatic and autooxidative peroxidation via free radical chain reactions. Initiation of lipid peroxidation can be prevented at the early stage by free radical scavengers or XO inhibitors (Chang et al., 1993; Husain et al., 1987; Reiners et al., 1987). XO is a flavoprotein that catalyses purine to form uric acid and also yield superoxide radical (07) which is then converted into hydrogen peroxide by superoxide dismutase. In this study we evaluated the antioxidant activity of the crude extracts of the dried flowers of H. sabdariffa L. by analysing their free radical-scavenging capacities and inhibitory effect on XO activity. We further investigated the effect of these crude extracts on the t-BHP-induced hepatic cytotoxicity including LDH leakage and lipid peroxidation, and DNA damage.
MATERIALS
AND METHODS
Plant materials and chemicals The dried flowers of H. sabdariffa L. were purchased from a traditional herbal store in Taichung, Taiwan. Solvents and chemicals for chromatography were procured from E. Merck Co. (Darmstadt, Germany). Cell culture medium and reagents were obtained from GIBCO BRL (Grand Island, NY, USA). Dishes for cell culture were obtained from Nunc (Denmark). All other chemicals (e.g. t-BHP, kit for LDH) were procured from Sigma Chemical Co. (St Louis, M e , USA).
Preparation o f crude extracts The dried flowers of H. sabdariffa L. were extracted twice with ethanol for 2 wk at room tem-
Table 2. Effectof the crude extracts of H. sabdariffa L. flowerso n the inhibitionof XO activity Dose (mg/ml)
HS-C
0.10 0.20 0.50 1.00
8.5 17.9 34.5 66.3
% of inhibition* HS-E HS-R 13.5 20.0 26.9 43.9
1.8 3.5 10.9 22.4
*% of inhibition= (absorbanceof control group- absorbaneeof crude extract added group)/absorbanceof controlx 100%. perature. The ethanol extract was designated as HSI and mixed with chloroform to obtain a soluble fraction (HS-C) and an insoluble fraction (HS-II). HS-II was then extracted further with ethyl acetate to obtain a soluble fraction (HS-E) and an insoluble fraction (HS-R). After the organic solvents were evaporated, the dried fractions of HS-C, HS-E and HS-R were dissolved in dimethyl sulfoxide (DMSO) (final concentration not more than 0.2%) and phosphate buffered saline (PBS, pH = 7.0) for following studies.
Determination o f free radical-scavenging capacity The free radical-scavenging capacity of HS-C, HS-E and HS-R was tested by bleaching (at 517 nm) the stable DPPH according to the method of Ursini et al. (1994). A reaction mixture containing methanol (3 ml), DPPH (10 mM, 30 #1) and the crude extract (HS-C, HS-E or HS-R at a final concentration of 0.01, 0.05, 0.10 or 0.5 mg/ml) was left to stand at room temperature for 30min before being mixed with redistilled water (1 ml) and toluene (3 ml). The solution was then centrifuged, and the absorbance of the upper phase was read at 517 nm against a blank without crude extract, and processed as above. The fraction of DPPH bleaching was calculated with the absorbance of the DPPH solution alone as 0%.
Assay for XO activity The XO activity using xanthine as substrate was assayed spectrophotometrically at 295 nm by the method described in our previous work (Tseng et al., 1995). HS-C, HS-E or HS-R (final concentration: 0.1, 0.2, 0.5, 1.0 mg/ml) was added before the enzyme was instilled and its inhibition effect on the XO activity was compared according to the absorbance. The fraction of XO inhibition was calculated and expressed.
Preparation o f rat hepatocytes Table 1. Effectof the crude extracts of H. sabdariffa L. flowerson DPPH bleaching Dose (mg/ml)
% of DPPH bleaching* HS-C HS-E HS-R
0.01 0.05 0.10 0.50
42.6 44.2 47.7 65.9
46.5 55.8 60.5 93.8
43.6 45.7 47.1 58.7
*% of DPPH bleaching= (absorbance of control group-absorbance of the crude extract added group)/absorbanceof control x 100%.
Male Sprague-Dawley rats (Taichung Veterans General Hospital Animal Center) (250-300 g) were used for these experiments. Hepatocytes were prepared by the two-stage coUagenase perfusion (Bonney et al., 1974) and cultured in Williams' E medium supplemented with PSN (1%; penicillin, streptomycin and neomycin; GIBCO) antibiotic mixture, glutamine (1%; GIBCO) and calf bovine serum (10%; GIBCO) under O2/CO2 (95/5%) gas. The cells were plated onto a petri dish and treated
Antioxidant activity of Hibiscus sabdariffa L. extracts in rat hepatocytes
1161
cultured at a density of l x 106 cells/60 mm dish, were treated with the crude extract (HS-C, HS-E or HS-R at concentrations of 0.10 and 0.20 mg/ml) for 10 min, followed by t-BHP (1.5 mM) exposure for 30 min. The cells were then incubated in a fresh medium containing [methyl-3H]thymidine (1 ttCi/ml) for an additional 18 hr. Finally, the cells were harvested and lysed to measure the radioactivity with a liquid scintillation counter, and D N A was quantified in lystate according to the DABA (3,5-diaminobenzoic acid) method (Vytask, 1982).
with chemicals as indicated in the following tests 3 hr after their attachment.
Hepatic cytotoxicity and lipid peroxidation assay After pretreatment with the non-toxic concentrations (data not shown) of the crude extract (HSC, HS-E or HS-R; 0.10 and 0.20 mg/ml) for 10 min, hepatocytes were iLncubated with t-BHP (1.5mM) for 30 min (Joyeux et al., 1990). The cells were centrifuged (600 g), a~Ld the medium was removed for the determination of LDH leakage by a commercial kit (Sigma). Hepatic cytotoxicity was expressed in terms of the activity of LDH released from the treated cells. For the assay of lipid peroxidation, the pellet was dissolved and transferred to a tube containing 1.5 ml 0.67'% thiobarbituric acid. The mixture was boiled for 30min followed by rapid cooling to room temperature, and then extracted with butanol. The formation of M D A was assayed by fluorometric method at 553 nm with excitation at 515nm using 1,1,3,3-tetramethoxypropane as standard (Yagi, 1987).
Statistical analysis The results were reported as means (standard deviations from individual magnitudes). Statistical differences were analysed according to Student's ttest wherein the differences were considered to be significant at P < 0.05. RESULTS
Measurement of DNA repair synthesis
Free radical-scavenging capacity of HS-C, HS-E and HS-R
The extent of D N A damage was determined by the method of UDS (Hsia et al., 1983). After pretreatment with hydroxyurea for I hr, hepatocytes,
To determine the free radical-scavenging capacity of the crude extracts, DPPH was used to provide stable free radicals. Although a decrease in absor-
250
200
=o 150 E v,
m 100 _¢ ,.1a .--I
50
t-BHP
+
HS-C
HS-E
0,20 0,10 0,20
0.20 0.10 0.20
+
+
+
HS-R
+
0.20 0.10 0.20 mg/ml
+
+
Fig. 1. Effect of the crude extracts of H. sabdariffa flowers on the t-BHP-induced leakage of LDH in primary hepatocytes. Primary hepatocyte cultures were pretreated respectively with (a) HS-C, (b) HS-E and (c) HS-R for 10 min, then t-BHP was added for 30 min. *P < 0.05; **P < 0.01, compared with t-BHP treated alone (n = 3).
T.-H. Tseng et al.
1162
bance, in other words loss of DPPH free radical, indicates the capability of the reagents to capture free radicals, it is not a clear-cut definition of antioxidant effect. However, in the present study, HS-E showed the greatest capacity for scavenging free radicals (ECs0=0.017mg/ml; Table 1). This result might be the contribution of the phenolic constituents in the fraction of HS-E (unpublished data).
concentrations of the three crude extracts were assessed. After the addition of the crude extracts at concentrations of 0.10 and 0.20mg/ml to the primary cultured hepatocytes, it was observed that only HS-C and HS-E significantly suppressed the hepatic cytotoxicity, as seen by the t-BHP-induced leakage of LDH (Fig. 1). Lipid peroxidation has been recognized as being a potential mechanism for cell injury. The concentration of MDA, an index of lipid peroxidation, was found to be increased in the hepatocytes exposed to 1.5mM t-BHP alone. Pretreatment of HS-C and HS-E (0.10 and 0.20 mg/ ml) to the cells decreased the formation of M D A (P < 0.05 and P<0.01) significantly as compared with that of the non-pretreated ones (Fig. 2).
Inhibitory effect of HS-C, HS-E and HS-R on XO activity XO, a flavoprotein, catalyses the oxidation of hypoxanthine to xanthine and of xanthine to uric acid showing maximal absorbance at 295nm. Therefore, XO activity was evaluated by spectrophotometric measurement of the formation of uric acid from xanthine. The tests showed that HS-C displayed the strongest inhibitory effect on XO activity (EC50=0.742 mg/ml; Table 2). According to our analysis, the major constituents of HS-C are steroid glycosides and flavonoids (unpublished data).
Effects of HS-C, HS-E and HS-R on t-BHP-induced DNA damage UDS is usually used as an indicator of D N A damage (genotoxicity). The present experiment was performed to study the effect of the crude extracts on D N A damage induced by 30 min of t-BHP treatment in the rat hepatocytes. The results showed that all the crude extracts, at a concentration of 0.20 mg/ml, significantly inhibited the t-BHPinduced D N A repair synthesis (P < 0.05; Table 3).
Effects of HS-C, HS-E and HS-R on t-BHP-induced hepatic cytotoxicity and lipid peroxidation In the preliminary experiments of hepatic cytotoxicity and lipid peroxidation, non-hepatotoxic
1.t 14
12
8 =o __.
10
,'-
8
o E c o
6 < Q
HS-C 0.20 0.t0 0.20 FBHP
+
+
4-
HS-E 0.20 0.t0 0.20 ÷
+
HS-R 0.20 0.t0 0.20 mg/ml 4-
÷
Fig. 2. Effect of the crude extracts of H. sabdariffa flowers on the t-BHP-induced lipid peroxidation in primary hepatocytes. Primary hepatocyte cultures were pretreated respectively with (a) HS-C, (b) HS-E and (c) HS-R for l0 rain, then t-BHP was added for 30 min. *P < 0.05; **P < 0.01, compared with t-BHP treated alone (n = 3).
Antioxidant activity of Hibiscus sabdariffa L. extracts in rat hepatocytes Table 3. Effect of the crude extracts of H. sabdariffa L. on the t-
BHP-inducedDNA damage in rat primary hepatocyte cultures Treatment* Control (DMSO 0.2%) t-BHP (1.5 mM) t-BHP (1.5 mM) plus HS-C (0.10 mg/ml) HS-C (0.20 mg/ml) HS-E (0.10 mg/ml) HS-E (0.20 mg/ml) HS-R (0.10 mg/ml) HS-R (0.20 mg/ml)
dpm//~g DNA
% of inhibition:l:
58 + 11 179 _+18
-0
136-t- 12t 107 __.10t 130 _ 1It 102 + 12t 172 ___20 125 + 24t
35 60 40 64 6 45
*See Materials and Methods for details. Values were obtained as the averages of triplicate determinations. tP < 0.05, compared with the treatment with t-BHP alone. :1:% of inhibition = difference between t-BHP with and without HS/difference betweent-BHP alone and control x 100%. DISCUSSION Recently, there ]aas been an explosion of interest in studying the in~1olvement of free radicals in carcinogenesis (Troll and Weisner, 1985). Much of the emphasis of these studies, linking free radicals and cancer, has been on the intermediates, such as hydroxyl radicals, that are formed during oxygen reduction. XO catalyses the hydroxyl action of many purine substrates and converts hypoxanthine into xanthine and then into uric acid in the presence of molecular oxyger, to yield superoxide anion. Inhibition of the enzyme leads to a decrease in the ROS generation ill target cells. XO inhibitors are suggested to be scavengers of hydroxyl radicals and, therefore, be the antitumour promotion agents (Husain et al., 198"7; Reiners et al., 1987). The results of the present study revealed that chloroform and ethyl acetate soluble fractions (HSC and HS-E) of the ethanol crude extract of the dried flowers of H. sabdariffa L. presented strong antioxidant potential. HS-C displayed greatest inhibition of XO activity (Table 2) and HS-E scavenged D P P H free radicals most effectively (Table 1), although all tested crude extracts showed effects on these two parameters to some extent. To expose further the protective action of the crude extracts, t-BHP-induced oxidative damage model was applied in the rat hepatocytes. Hepatocytes metabolize t-BHP by two distinct pathways. One pathway involves the cytochrome P-450 and leads to the formation of toxic peroxyl and alkoxyl radicals (Rush et al., 1985). The second is a detoxification reaction involving glutathione peroxidase which gives rise to tert-butyl alcohol and oxidized glutathione. These metabolic pathways will increase cell free radicals which may attack phospholipids, proteins and nucleic acid. L D H leakage is a general index of hepatic cytotoxicity. M D A which is the major oxidative degradation of unsaturated fatty acid membrane has been shown to be biologically active with cytotoxic and genotoxic properties (Esterb~Luer et al., 1988; Vaca et al., 1992). Oxidative D N A damage is thought to be potentially carcinogenic. The occurrence of D N A damage can be measured, either directly or in-
1163
directly, through the determination of D N A repair synthesis (Hsia et al., 1983). In the present investigation, HS-C and HS-E crude extracts were observed to inhibit the leakage of LDH, formation of M D A (Figs 1 and 2) and the unscheduled D N A repair synthesis (Table 3) induced by t-BHP in the rat hepatocyte cultures whereas HS-R (0.20 mg/ml) could only inhibit the genotoxicity. It was interesting that HS-R exhibited an inhibition effect on the t-BHP-induced genotoxicity but not on the hepatic cytotoxicty and lipid peroxidation. These results might represent different biological mechanisms for the different constituents of HS-C, HS-E and HS-R. Presently, we are conducting experiments to analyse the compositions of the crude extracts. The preliminary data indicated that HS-E contains phenolic constituents (such as protocatechnic acid) and the major components of HS-C are steroid glycosides (such as fl-sitosteroid glycoside) and flavonoids. We will be able to provide more detailed explanation on the protective effects of the crude extracts on t-BHP-induced damage when the analysis is complete. In conclusion, HS-C and HS-E of H. sabdariffa L. flower extracts showed efficiently protective action against t-BHP-induced hepatic cytotoxicity and genotoxicity, possibly by different mechanisms associated with different antioxidants existing in the crude extracts. The exact mechanisms and constituents need to be investigated further. Acknowledgements--The authors acknowledge the ChungShan Medical and Dental College Research Fund (CSMC 83-NS-A-002) and National Science Council Grant (NSC 84-2331-B-040-003), ROC for their support.
REFERENCES
Ames B. N. (1983) Dietary carcinogens and anticarcinogens: oxygen radicals and degenerative diseases. Science 221, 1256-1264. Bonney V. R., Becker J. E., Walker P. R. and Potter V. R. (1974) Primary monolayer cultures of adult rat liver parenchymal cells suitable for study of regulation of enzyme synthesis. In Vitro 9, 399~,13. Breimer L. H. (1990) Molecular mechanism of oxygen radical carcinogenesis and mutagenesis: the role of DNA base damage. Molecular Carcinogenesis 3, 188-197. Cerutti P. A. (1985) Proxident states and tumor promotion. Science 227, 375-380. Chang W. S., Lee Y. J., Lu F. J. and Chiang H. C. (1993) Inhibitory effects of flavonoids on xanthine oxidase. Anticancer Research 13, 2165-2170. Davies M. J. (1989) Detection of peroxyl and alkoxyl radicals produced by reaction of hydroperoxides with rat liver microsomal fractions. Biochemical Journal 257, 603-606. Esterbauer H., Zollner H. and Schaur R. J. (1988) Hydroxyalkenals: cytotoxic products of lipid peroxidation. Science and Biochemistry 1, 31 i-317. Hsia M. T. S., Kreamerand B. L. and Dolara P. (1983) A rapid and simple method to quantitate chemically induced unscheduled DNA synthesis in freshly isolated rat hepatocytes facilitated by DNA retention of membrane filters. Mutation Research 122, 177-185. Husain S. R., Cillard J. and Cillard P. (1987) Hydroxyl radical scavenging activity of flavonoids. Phytochemistry 26, 2489-2491.
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Joyeux M., Rolland A., Fleurentin J., Mortier F. and Dorfman P. (1990) tert-Butylhydroperoxide-induced injury in isolated rat hepatocytes: a model for studying antihepatotoxic crude drugs. Planta Medica 56, 171174. Morse M. A. and Stoner G. D. (1993) Cancer chemoprevention principles and prospects. Carcinogenesis 14, 1743-1746. Mfiller B. M. and Franz G. (1992) Chemical structure and biological activity of polysaccharides from Hibiscus sabdariffa. Planta Medica 58, 60-67. Reiners J. J., Pence B. C., Barcus M. C. S. and Cantu A. R. (1987) 12-O-Tetra-decanoylphorbol-13-acetatedependent induction of xanthine dehydrogenase and conversion to xanthine oxidase in murine epidermis. Cancer Research 47, 1775-1779. Rush G., Gorski J. R., Ripple M. G,, Sowinski J., Bugelski P. and Hewitt W. R. (1985) Organic hydroperoxide-induced lipid peroxidation and cell death in isolated hepatocytes. Toxicology and Applied Pharmacology 78, 473-483. Thornalley P., Trotta R. J. and Stern A. (1983) Free radical involvement in the oxidative phenomena induced by tert-butyl hydroperoxide in erythrocytes. Biochimica et Biophysica Acta 759, 16-22.
Troll W. and Weisner R. (1985) The role oxygen radicals as possible mechanism of tumor promotion. Annual Review of Pharmacology and Toxicology 25, 509-528. Tseng T. H., Chu C. Y., Huang J. M., Shiow S. J. and Wang C. J. (1995) Crocetin protects against oxidative damage in rat primary hepatocytes. Cancer Letters 97, 61-67. Ursini F., Maiorino M., Morazzoni P., Roveri A. and Pifferi G. (1994) A novel antioxidant flavonoid (IdB 1031) affecting molecular mechanisms of cellular activation. Free Radical Biology and Medicine 16, 547-553. Vaca C. E., Vodicka P. and Hemminki K. (1992) Determination of malonaldehyde-modified 2(-deoxyguanosine-3)-monophosphate and DNA by 32P-postlabelling. Carcinogenesis 13, 593-599. Vytask R. (1982) A sensitive fluorometric assay for the determination of DNA. Analytical Biochemistry 120, 243248. Weisburger J. H. (1991) Nutritional approach to cancer prevention with emphasis on vitamins, antioxidants ahd carotenoids. Journal of Environmental Health C8, 339351. Yagi K. (1987) Lipid peroxide and human disease. Chemistry and Physics of Lipids 45, 337-351.
Food and Chemical Toxicology 35 (1997) 1159-1164
Protective Effects of Dried Flower Extracts of Hibiscus sabdariffa L. against Oxidative ',Stress in Rat Primary Hepatocytes T.-H. T S E N G , E.-S. K A O , C.-Y. C H U , F.-P. C H O U , H.-W. L I N W U a n d C.-J. W A N G * Institute of Biochemistry, Chung Shah Medical and Dental College, Taiwan, ROC (Accepted 22 April 1997) Abstract--Dried flower extracts of Hibiscus sabdarrifa L., a local soft drink material and medical herb, was found to possess antioxidant activity in the present study. In the preliminary studies, antioxidant potential of three fractions of the ethanol crude extract (HS-C: chloroform-soluble fraction; HS-E: ethyl acetate soluble fraction; HS-R: residual fraction) obtained from the dried flowers of Hibiscus sabdarrifa L. were evaluated by their capacity of quenching l, 1-diphenyl-2-picrylhydrazyl(DPPH) free radical and inhibiting xanthine oxidase (XO) activity. HS-E showed the greatest capacity of scavenging free radical (ECso=0.017mg/ml), and HS-C showed the strongest inhibitory effect on XO activity (ECs0 = 0.742 mg/ml). Furthermore, antioxidant bioactivities of these crude extracts were investigated using a model of tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat primary hepatocytes. All fractions were found to inhibit significantly the unscheduled DNA synthesis (UDS) induced by t-BHP at a concentration of 0.20 mg/ml. HS-C and HS-E also decreased the leakage of lactate dehydrogenase (LDH) and the formation of malondialdehyde (MDA) induced by t-BHP (1.5 m i ) considerably at a concentration of 0.10 and 0.20 mg/ml in the rat primary hepatocyte cultures. These results indicated that the dried flower extracts (HS-C and HS-E) of H. sabdarrifa L. protect rat hepatocytes from t-BHP-induced cytotoxicity and genotoxicity by different mechanisms. © 1997 Elsevier Science Ltd. All righi's reserved.
Abbreviations:t-BHP = tert-butyl hydroperoxide; DMSO = dimethyl sulfoxide; DPPH = 1,1-diphenyl2-picrylhydrazyl radical; LDH = lactate dehydrogenase; MDA = malondialdehyde; PBS = phosphate buffered saline; ROS = reactive oxygen species; UDS = unscheduled DNA synthesis; XO = xanthine oxidase.
I:NTRODUCTION Reactive oxygen species (ROS) can be originated from several internal and external sources, such as metabolic reactions, dietary intake or cigarette smoking. Excessiw: formation of oxidants in biological systems and the consequent oxidative damage are topics of growing interest in recent years, since these processes probably contribute to several inflammatc,ry diseases and cancer (Morse and Stoner, 1993). Studies have shown that ROS play an important role in carcinogenesis, especially in the promotion stage (Breimer, 1990; Cerutti, 1985). Although the body possesses defence mechanisms to reduce the oxidative damage, such as using enzymes and antioxidant nutrients to arrest the damaging properties of excited oxygen species, *Author for correspondence at: Institute of Biochemistry, Chung Shan Medical and Dental College, No. l l0, Section 1, Chien Kuo N. Road, Taichung 402, Taiwan, ROC.
continuous exposure to chemicals and contaminants may lead to an increase in the amount of free radicals in the body beyond control, and cause irreversible oxidative damage. Therefore, antioxidants are considered effective inhibitors of carcinogenesis and also in other conditions which are pathogenetically associated with oxidative mechanisms. Many studies have supported that antioxidant nutrients or/and medicines play a protective role in human health (Ames, 1983; Weisburger, 1991). The study of numerous compounds that could be useful as antioxidants, ranging from ct-tocopherol and fl-carotene to plant antioxidants such as flavones and tannins, has generated increasing interest in the field of food and/or medicine. The dried flowers of H. sabdariffa L. (Malvaceae) have been used effectively in folk medicines against hypertension, pyrexia and liver disorders. Recently, it has gained importance as a soft drink material in local regions. However, little information is available about the pharmacological and biological effects of H. sabdarzffa L., even though the polysaccharide constituents
0278-6915/97/$19.00 + 0.00 © 1997 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PH S0278-6915(97)00{)96-3
1160
T.-H. Tseng et al.
have been assayed for their possible immunomodulating effects (Mtiller and Franz, 1992). To understand the antioxidant action of the dried flowers of H. sabdariffa L. in intact cells, we used the model of t-BHP-induced oxidative damage in the primary cultures of rat hepatocytes for the study, t-BHP is widely applied to investigate the mechanism of cell injury initiated by oxidative stress (Rush et al., 1985). It can be metabolized to free radical intermediates by cytochrome P-450 (hepatocyte) or haemoglobin (erythrocytes), which in turn can initiate lipid peroxidation, affect the cell integrity and mediate DNA damage (Davies, 1989; Thornalley et al., 1983). Polyunsaturated fatty acids, present in cell membranes, are easily oxidized by enzymatic and autooxidative peroxidation via free radical chain reactions. Initiation of lipid peroxidation can be prevented at the early stage by free radical scavengers or XO inhibitors (Chang et al., 1993; Husain et al., 1987; Reiners et al., 1987). XO is a flavoprotein that catalyses purine to form uric acid and also yield superoxide radical (07) which is then converted into hydrogen peroxide by superoxide dismutase. In this study we evaluated the antioxidant activity of the crude extracts of the dried flowers of H. sabdariffa L. by analysing their free radical-scavenging capacities and inhibitory effect on XO activity. We further investigated the effect of these crude extracts on the t-BHP-induced hepatic cytotoxicity including LDH leakage and lipid peroxidation, and DNA damage.
MATERIALS
AND METHODS
Plant materials and chemicals The dried flowers of H. sabdariffa L. were purchased from a traditional herbal store in Taichung, Taiwan. Solvents and chemicals for chromatography were procured from E. Merck Co. (Darmstadt, Germany). Cell culture medium and reagents were obtained from GIBCO BRL (Grand Island, NY, USA). Dishes for cell culture were obtained from Nunc (Denmark). All other chemicals (e.g. t-BHP, kit for LDH) were procured from Sigma Chemical Co. (St Louis, M e , USA).
Preparation o f crude extracts The dried flowers of H. sabdariffa L. were extracted twice with ethanol for 2 wk at room tem-
Table 2. Effectof the crude extracts of H. sabdariffa L. flowerso n the inhibitionof XO activity Dose (mg/ml)
HS-C
0.10 0.20 0.50 1.00
8.5 17.9 34.5 66.3
% of inhibition* HS-E HS-R 13.5 20.0 26.9 43.9
1.8 3.5 10.9 22.4
*% of inhibition= (absorbanceof control group- absorbaneeof crude extract added group)/absorbanceof controlx 100%. perature. The ethanol extract was designated as HSI and mixed with chloroform to obtain a soluble fraction (HS-C) and an insoluble fraction (HS-II). HS-II was then extracted further with ethyl acetate to obtain a soluble fraction (HS-E) and an insoluble fraction (HS-R). After the organic solvents were evaporated, the dried fractions of HS-C, HS-E and HS-R were dissolved in dimethyl sulfoxide (DMSO) (final concentration not more than 0.2%) and phosphate buffered saline (PBS, pH = 7.0) for following studies.
Determination o f free radical-scavenging capacity The free radical-scavenging capacity of HS-C, HS-E and HS-R was tested by bleaching (at 517 nm) the stable DPPH according to the method of Ursini et al. (1994). A reaction mixture containing methanol (3 ml), DPPH (10 mM, 30 #1) and the crude extract (HS-C, HS-E or HS-R at a final concentration of 0.01, 0.05, 0.10 or 0.5 mg/ml) was left to stand at room temperature for 30min before being mixed with redistilled water (1 ml) and toluene (3 ml). The solution was then centrifuged, and the absorbance of the upper phase was read at 517 nm against a blank without crude extract, and processed as above. The fraction of DPPH bleaching was calculated with the absorbance of the DPPH solution alone as 0%.
Assay for XO activity The XO activity using xanthine as substrate was assayed spectrophotometrically at 295 nm by the method described in our previous work (Tseng et al., 1995). HS-C, HS-E or HS-R (final concentration: 0.1, 0.2, 0.5, 1.0 mg/ml) was added before the enzyme was instilled and its inhibition effect on the XO activity was compared according to the absorbance. The fraction of XO inhibition was calculated and expressed.
Preparation o f rat hepatocytes Table 1. Effectof the crude extracts of H. sabdariffa L. flowerson DPPH bleaching Dose (mg/ml)
% of DPPH bleaching* HS-C HS-E HS-R
0.01 0.05 0.10 0.50
42.6 44.2 47.7 65.9
46.5 55.8 60.5 93.8
43.6 45.7 47.1 58.7
*% of DPPH bleaching= (absorbance of control group-absorbance of the crude extract added group)/absorbanceof control x 100%.
Male Sprague-Dawley rats (Taichung Veterans General Hospital Animal Center) (250-300 g) were used for these experiments. Hepatocytes were prepared by the two-stage coUagenase perfusion (Bonney et al., 1974) and cultured in Williams' E medium supplemented with PSN (1%; penicillin, streptomycin and neomycin; GIBCO) antibiotic mixture, glutamine (1%; GIBCO) and calf bovine serum (10%; GIBCO) under O2/CO2 (95/5%) gas. The cells were plated onto a petri dish and treated
Antioxidant activity of Hibiscus sabdariffa L. extracts in rat hepatocytes
1161
cultured at a density of l x 106 cells/60 mm dish, were treated with the crude extract (HS-C, HS-E or HS-R at concentrations of 0.10 and 0.20 mg/ml) for 10 min, followed by t-BHP (1.5 mM) exposure for 30 min. The cells were then incubated in a fresh medium containing [methyl-3H]thymidine (1 ttCi/ml) for an additional 18 hr. Finally, the cells were harvested and lysed to measure the radioactivity with a liquid scintillation counter, and D N A was quantified in lystate according to the DABA (3,5-diaminobenzoic acid) method (Vytask, 1982).
with chemicals as indicated in the following tests 3 hr after their attachment.
Hepatic cytotoxicity and lipid peroxidation assay After pretreatment with the non-toxic concentrations (data not shown) of the crude extract (HSC, HS-E or HS-R; 0.10 and 0.20 mg/ml) for 10 min, hepatocytes were iLncubated with t-BHP (1.5mM) for 30 min (Joyeux et al., 1990). The cells were centrifuged (600 g), a~Ld the medium was removed for the determination of LDH leakage by a commercial kit (Sigma). Hepatic cytotoxicity was expressed in terms of the activity of LDH released from the treated cells. For the assay of lipid peroxidation, the pellet was dissolved and transferred to a tube containing 1.5 ml 0.67'% thiobarbituric acid. The mixture was boiled for 30min followed by rapid cooling to room temperature, and then extracted with butanol. The formation of M D A was assayed by fluorometric method at 553 nm with excitation at 515nm using 1,1,3,3-tetramethoxypropane as standard (Yagi, 1987).
Statistical analysis The results were reported as means (standard deviations from individual magnitudes). Statistical differences were analysed according to Student's ttest wherein the differences were considered to be significant at P < 0.05. RESULTS
Measurement of DNA repair synthesis
Free radical-scavenging capacity of HS-C, HS-E and HS-R
The extent of D N A damage was determined by the method of UDS (Hsia et al., 1983). After pretreatment with hydroxyurea for I hr, hepatocytes,
To determine the free radical-scavenging capacity of the crude extracts, DPPH was used to provide stable free radicals. Although a decrease in absor-
250
200
=o 150 E v,
m 100 _¢ ,.1a .--I
50
t-BHP
+
HS-C
HS-E
0,20 0,10 0,20
0.20 0.10 0.20
+
+
+
HS-R
+
0.20 0.10 0.20 mg/ml
+
+
Fig. 1. Effect of the crude extracts of H. sabdariffa flowers on the t-BHP-induced leakage of LDH in primary hepatocytes. Primary hepatocyte cultures were pretreated respectively with (a) HS-C, (b) HS-E and (c) HS-R for 10 min, then t-BHP was added for 30 min. *P < 0.05; **P < 0.01, compared with t-BHP treated alone (n = 3).
T.-H. Tseng et al.
1162
bance, in other words loss of DPPH free radical, indicates the capability of the reagents to capture free radicals, it is not a clear-cut definition of antioxidant effect. However, in the present study, HS-E showed the greatest capacity for scavenging free radicals (ECs0=0.017mg/ml; Table 1). This result might be the contribution of the phenolic constituents in the fraction of HS-E (unpublished data).
concentrations of the three crude extracts were assessed. After the addition of the crude extracts at concentrations of 0.10 and 0.20mg/ml to the primary cultured hepatocytes, it was observed that only HS-C and HS-E significantly suppressed the hepatic cytotoxicity, as seen by the t-BHP-induced leakage of LDH (Fig. 1). Lipid peroxidation has been recognized as being a potential mechanism for cell injury. The concentration of MDA, an index of lipid peroxidation, was found to be increased in the hepatocytes exposed to 1.5mM t-BHP alone. Pretreatment of HS-C and HS-E (0.10 and 0.20 mg/ ml) to the cells decreased the formation of M D A (P < 0.05 and P<0.01) significantly as compared with that of the non-pretreated ones (Fig. 2).
Inhibitory effect of HS-C, HS-E and HS-R on XO activity XO, a flavoprotein, catalyses the oxidation of hypoxanthine to xanthine and of xanthine to uric acid showing maximal absorbance at 295nm. Therefore, XO activity was evaluated by spectrophotometric measurement of the formation of uric acid from xanthine. The tests showed that HS-C displayed the strongest inhibitory effect on XO activity (EC50=0.742 mg/ml; Table 2). According to our analysis, the major constituents of HS-C are steroid glycosides and flavonoids (unpublished data).
Effects of HS-C, HS-E and HS-R on t-BHP-induced DNA damage UDS is usually used as an indicator of D N A damage (genotoxicity). The present experiment was performed to study the effect of the crude extracts on D N A damage induced by 30 min of t-BHP treatment in the rat hepatocytes. The results showed that all the crude extracts, at a concentration of 0.20 mg/ml, significantly inhibited the t-BHPinduced D N A repair synthesis (P < 0.05; Table 3).
Effects of HS-C, HS-E and HS-R on t-BHP-induced hepatic cytotoxicity and lipid peroxidation In the preliminary experiments of hepatic cytotoxicity and lipid peroxidation, non-hepatotoxic
1.t 14
12
8 =o __.
10
,'-
8
o E c o
6 < Q
HS-C 0.20 0.t0 0.20 FBHP
+
+
4-
HS-E 0.20 0.t0 0.20 ÷
+
HS-R 0.20 0.t0 0.20 mg/ml 4-
÷
Fig. 2. Effect of the crude extracts of H. sabdariffa flowers on the t-BHP-induced lipid peroxidation in primary hepatocytes. Primary hepatocyte cultures were pretreated respectively with (a) HS-C, (b) HS-E and (c) HS-R for l0 rain, then t-BHP was added for 30 min. *P < 0.05; **P < 0.01, compared with t-BHP treated alone (n = 3).
Antioxidant activity of Hibiscus sabdariffa L. extracts in rat hepatocytes Table 3. Effect of the crude extracts of H. sabdariffa L. on the t-
BHP-inducedDNA damage in rat primary hepatocyte cultures Treatment* Control (DMSO 0.2%) t-BHP (1.5 mM) t-BHP (1.5 mM) plus HS-C (0.10 mg/ml) HS-C (0.20 mg/ml) HS-E (0.10 mg/ml) HS-E (0.20 mg/ml) HS-R (0.10 mg/ml) HS-R (0.20 mg/ml)
dpm//~g DNA
% of inhibition:l:
58 + 11 179 _+18
-0
136-t- 12t 107 __.10t 130 _ 1It 102 + 12t 172 ___20 125 + 24t
35 60 40 64 6 45
*See Materials and Methods for details. Values were obtained as the averages of triplicate determinations. tP < 0.05, compared with the treatment with t-BHP alone. :1:% of inhibition = difference between t-BHP with and without HS/difference betweent-BHP alone and control x 100%. DISCUSSION Recently, there ]aas been an explosion of interest in studying the in~1olvement of free radicals in carcinogenesis (Troll and Weisner, 1985). Much of the emphasis of these studies, linking free radicals and cancer, has been on the intermediates, such as hydroxyl radicals, that are formed during oxygen reduction. XO catalyses the hydroxyl action of many purine substrates and converts hypoxanthine into xanthine and then into uric acid in the presence of molecular oxyger, to yield superoxide anion. Inhibition of the enzyme leads to a decrease in the ROS generation ill target cells. XO inhibitors are suggested to be scavengers of hydroxyl radicals and, therefore, be the antitumour promotion agents (Husain et al., 198"7; Reiners et al., 1987). The results of the present study revealed that chloroform and ethyl acetate soluble fractions (HSC and HS-E) of the ethanol crude extract of the dried flowers of H. sabdariffa L. presented strong antioxidant potential. HS-C displayed greatest inhibition of XO activity (Table 2) and HS-E scavenged D P P H free radicals most effectively (Table 1), although all tested crude extracts showed effects on these two parameters to some extent. To expose further the protective action of the crude extracts, t-BHP-induced oxidative damage model was applied in the rat hepatocytes. Hepatocytes metabolize t-BHP by two distinct pathways. One pathway involves the cytochrome P-450 and leads to the formation of toxic peroxyl and alkoxyl radicals (Rush et al., 1985). The second is a detoxification reaction involving glutathione peroxidase which gives rise to tert-butyl alcohol and oxidized glutathione. These metabolic pathways will increase cell free radicals which may attack phospholipids, proteins and nucleic acid. L D H leakage is a general index of hepatic cytotoxicity. M D A which is the major oxidative degradation of unsaturated fatty acid membrane has been shown to be biologically active with cytotoxic and genotoxic properties (Esterb~Luer et al., 1988; Vaca et al., 1992). Oxidative D N A damage is thought to be potentially carcinogenic. The occurrence of D N A damage can be measured, either directly or in-
1163
directly, through the determination of D N A repair synthesis (Hsia et al., 1983). In the present investigation, HS-C and HS-E crude extracts were observed to inhibit the leakage of LDH, formation of M D A (Figs 1 and 2) and the unscheduled D N A repair synthesis (Table 3) induced by t-BHP in the rat hepatocyte cultures whereas HS-R (0.20 mg/ml) could only inhibit the genotoxicity. It was interesting that HS-R exhibited an inhibition effect on the t-BHP-induced genotoxicity but not on the hepatic cytotoxicty and lipid peroxidation. These results might represent different biological mechanisms for the different constituents of HS-C, HS-E and HS-R. Presently, we are conducting experiments to analyse the compositions of the crude extracts. The preliminary data indicated that HS-E contains phenolic constituents (such as protocatechnic acid) and the major components of HS-C are steroid glycosides (such as fl-sitosteroid glycoside) and flavonoids. We will be able to provide more detailed explanation on the protective effects of the crude extracts on t-BHP-induced damage when the analysis is complete. In conclusion, HS-C and HS-E of H. sabdariffa L. flower extracts showed efficiently protective action against t-BHP-induced hepatic cytotoxicity and genotoxicity, possibly by different mechanisms associated with different antioxidants existing in the crude extracts. The exact mechanisms and constituents need to be investigated further. Acknowledgements--The authors acknowledge the ChungShan Medical and Dental College Research Fund (CSMC 83-NS-A-002) and National Science Council Grant (NSC 84-2331-B-040-003), ROC for their support.
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