Evaluation of the hepatoprotective and antioxidant activity of Boehmeria nivea var. nivea and B. nivea var. tenacissima

Journal of Ethnopharmacology 60 (1998) 9 – 17

Evaluation of the hepatoprotective and antioxidant activity of Boehmeria ni6ea var. ni6ea and B. ni6ea var. tenacissima Chun-Ching Lin a,*, Ming-Hong Yen a, Tsae-shiuan Lo a, Jer-Min Lin b a

Graduate Institute of Natural Products, Kaohsiung Medical College, Kaohsiung, Taiwan, ROC b Ziel Enterprise, 14F-3, No. 247, Min Sheng RD., Kaohsiung, Taiwan, ROC Accepted 13 October 1997

Abstract In this study, the relationship between liver protective effects and antioxidant activity of Boehmeria ni6ea var. ni6ea ( =B. ni6ea) and B. ni6ea var. tenacissima (= B. frutescens) was investigated. The water extracts of both plants exhibited a hepatoprotective activity against CCl4-induced liver injury. B. ni6ea var. ni6ea and B. ni6ea var. tenacissima, also showed anti-oxidant effects in FeCl2-ascorbate induced lipid peroxidation in rat liver homogenate. Moreover, the active oxygen species scavenging potencies were evaluated by an electron spin resonance (ESR) spin-trapping technique. B. ni6ea var. tenacissima displayed better superoxide radical scavenging activity than B. ni6ea. Based on these findings, we suggest that in the liver protective and antioxidative effects of B. ni6ea var. ni6ea and B. ni6ea var. tenacissima, possibly involve mechanisms related to free radical scavenging effects. © 1998 Elsevier Science Ireland Ltd. Keywords: Boehmeria ni6ea var. ni6ea; B. ni6ea var. tenacissima; Hepatoprotective; Antioxidative; Electron spin resonance (ESR)

1. Introduction Boehmeria ni6ea (L.) Gaudish. var. ni6ea ( =B. ni6ea) and B. ni6ea (L.) Gaudish. var. tenacissima (Koidz.) Kitam. (=B. frutescens), two plants of the Urticaceae family, are widely distributed in * Corresponding author.

China and Taiwan. B. ni6ea var. ni6ea has been used traditionally for diuretic and antipyretic purposes. B. ni6ea var. tenacissima has been used to eliminate inflammation, neutralize poison and to dissipate heat. Recently in Taiwan, B. ni6ea var. tenacissima has also been used locally as a remedy for hepatitis (Kan, 1986). However, the pharmacological effects are obscure.

0378-8741/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 7 8 - 8 7 4 1 ( 9 7 ) 0 0 1 2 2 - 0

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Several components of B. ni6ea var. tenacissima have been isolated and identified (Matsuura et al., 1973). These components are boehnic acid, palmatic acid, stearic acid, ursolic acid, 19a-hydroxyursolic acid, b-sitosterol, b-sitosteryl-b-Dglucoside, etc. All the plants of the Boehmeria family have the same components. In addition, the pollen of B. ni6ea var. ni6ea are reported to cause asthma (Miura, 1993). Therefore, the people who use the leaves of B. ni6ea var. ni6ea must be warned against possible allergic reactions to the compounds mentioned above. In addition, chlorogenic acid and rhoifolin were also isolated from the leaves of B. ni6ea var. ni6ea. Chlorogenic acid reduced LDL, antilipid oxidation (Laranjinha et al., 1992) and inhibited tongue cancer induced by 4-nitroguindine-1-oxide (Tanaka et al., 1993). Serious attention is now paid to the cytotoxicity of active oxygen/free radicals as the cause of various pathological conditions. Lipid peroxides, produced from unsaturated fatty acids via radicals, cause histotoxicity and promote the formation of additional free radicals in a chain reaction-type manner. It is thought that, if the in vivo activity of enzymes or scavengers is not high enough to inhibit these radicals, various diseases such as arteriosclerosis, liver disease, diabetes, inflammation, renal failure or accelerated aging may result (Niki, 1995). In this study, we evaluated the relationship between pharmacological and antioxidant effect of Boehmeria ni6ea var. ni6ea and B. ni6ea var. tenacissima. The CCl4 induced hepatotoxicity in rats was used to study the hepatoprotective effect. Liver damage was assessed by biochemical studies (sGOT and sGPT) and by histopathological examinations. Furthermore, the antioxidant effect of the crude drug extracts were evaluated by two experiments: FeCl2-ascorbic acid induced lipid peroxidation in rat liver homogenate and active oxygen scavenging activity by electron spin resonance (ESR) spectrometry, using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap (Mitsuta et al., 1990; Kohno et al., 1991).

2. Materials and methods

2.1. Test animals Male Wistar albino rats were purchased from the National Laboratory Animal Breeding and Research Center, National Science Council and were kept for 1 week on a commercial diet under environmentally controlled conditions (room temperature 2293°C, relative humidity 559 5%) with free access to food and water. A controlled 12 h light/dark cycle was maintained. Rats weighing 180–230 g were used for CCl4-induced hepatotoxicity.

2.2. Plant resources and preparation of crude drug extracts The roots of B. ni6ea var. ni6ea (specimen No. BNN-R, KMC Herbarium) and B. ni6ea var. tenacissima (specimen No. BNT-R, KMC Herbarium) were collected from Chaiyi Hsien, 8 June 1995 and authenticated by Dr. Yang, YuenPo, Department of Biology, National Sun YatSen (Chunshan) University, Kaohsiung, Taiwan, ROC. All samples were chopped and dried at 60°C for 24 h. A total of 200 g of each sample was decocted with 1 l boiling water for 1 h, three times. The decoction was filtered, mixed, concentrated and lyophilized. The lyophilized powder of each sample was dissolved in normal saline (100, 300, 500 mg/ml/kg in rats) prior to oral administration to the experimental animals.

2.3. CCl4 -induced hepatotoxicity in rats The hepatoprotective effect was induced by CCl4 according to methods described previously (Shibayama, 1989; Suzuki et al., 1990; Yoshitake et al., 1991). Liver damage was induced in rats with a 1:1 (v/v) mixture of carbon tetrachloride and olive oil, administered s.c. at a dose of 3 ml/kg body weight. Animals were divided into nine groups of six rats each. Group 1 received normal saline (10 ml/kg, p.o.) as normal control. Group 2 was injected with CCl4/olive oil alone (3 ml/kg). Groups 3–9 were administered orally, solutions of tested drugs and silymarin, once be-

C.-C. Lin et al. / Journal of Ethnopharmacology 60 (1998) 9–17

fore the injection of CCl4/olive oil and twice at 24 and 48 h after the time of injection, respectively.

2.4. Assay of serum GOT and GPT acti6ities All animals were anaesthetized with ether and then blood was withdrawn from the carotid artery 72 h after CCl4 intoxication. The blood was centrifuged with 3000× rpm at 4°C for 10 min to separate the serum. Glutamic oxaloacetate transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities were measured according to the method previously described (Reitman and Frankel, 1957).

2.5. Histopathological obser6ation After the blood was collected, sections were taken from each lobe of the liver immediately. The tissues were fixed in 10% neutral formalin for a period of at least 24 h, dehydrated in graded (50 100%) alcohol and embedded in paraffin, cut into 45 mm thick sections and stained with haematoxylin-eosin for photomicroscopic assessment.

2.6. Chemicals Thiobarbituric acid (TBA), sodium dodecyl sulfate (SDS), L-(−)-ascorbic acid (AA) were purchased from Sigma (St. Louis, MO). Carbon tetrachloride (CCl4) was obtained from Merck. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was supplied by Labotec (Tokyo, Japan). Xanthine oxidase (XOD) was purchased from Boehringer Mannheim. Ferreous sulfate, ferreous chloride and hydrogen peroxides were obtained from Wako (Osaka, Japan). The other chemicals used were of reagent grade.

2.7. FeCl2 -ascorbic acid stimulated lipid peroxidation in rat li6er homogenate In rat liver homogenate, the young male Wistar albino rats weighing 150 g were used, following the method of Masao et al. (1993). Rats were killed by decapitation and their liver tissues were quickly removed. A 2 g portion of liver tissue was

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sliced and then homogenized with 10 ml of 150 mM KCl-Tris–HCl buffer (pH 7.2). The protein content was determined by the method of Lowry et al. (1951). According to the method of Yoshiyuki et al. (1981), the reaction mixture was composed of 0.25 ml of liver homogenate, 0.1 ml of Tris–HCl buffer (pH 7.2), 0.05 ml of 0.1 mM ascorbic acid, 0.05 ml of 4 mM FeCl2 and 0.05 ml of various tests crude drug extract. The mixture was incubated at 37°C for 1 h in a capped tube, then 0.5 ml of HCl (0.1 N), 0.2 ml of SDS (9.8%), 0.9 ml of distilled water and 2 ml of TBA (0.6%) were added to each tube and vigorously shaken. The tubes were placed in a boiling water bath (100°C) for 30 min. After cooling, the flocculent precipitate was removed by adding 5 ml of nBuOH and centrifugated at 3000× rpm for 25 min, then the absorbance of the supernatant was measured at 532 nm.

2.8. Assay of superoxide anion sca6enging acti6ity Using an ESR spectrometer, we analyzed the superoxide anion radical ( · O2− ) from the spin adduct of · O2− (DMPO–OOH) (Mitsuta et al., 1990; Kohno et al., 1991; Lin et al., 1995a,b,c). Superoxide radical · O2− was generated from a hypoxanthine (HPX)-xanthine oxidase reaction system. The mixture contained 50 ml of 2.0 mM hypoxanthine, 35 ml 5.5 mM enetriaminepentaacetic acid (DETAPAC), 50 ml of various concentrations of crude drug extract and SOD (superoxide dismutase) and 15 ml of 9.2 mM DMPO (5,5-dimethyl-1-pyrroline-oxide) were put into a test tube. Then 50 ml of 0.4 units/ml XOD was added to the mixed solution. The reaction was initiated by addition of XOD. A period of 40 s after the addition of XOD, the spin adduct DMPO–OOH was determined by ESR spectrometer (JEOL-JES-FR80, JEOL, Tokyo). ESR spectra were recorded at 37°C with a field set 335.49 5.0 mT for superoxide radicals, modulation frequency 100 KHz, modulation amplitude 0.79×0.1 mT, response time 0.1 s, sweep time 2 min, microwave power 8.0 mM (9.423 GHz), receiver gains 2×100 when superoxide radicals were trapped.

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2.9. Statistical analysis All experimental data were expressed as mean 9S.E. The Student’s t-test was applied for detecting the significance of difference between different groups. PB0.05 was regarded as significant.

3. Results

3.1. CCl4 -induced hepatotoxicity The activities of sGOT and sGPT after administration of CCl4/olive oil were summarized in Table 1, the protective effects were compared with silymarin. The sGOT and sGPT levels were elevated significantly after CCl4/olive oil injection. In contrast, treatment with B. ni6ea var. ni6ea (500 mg/kg), B. ni6ea var. tenacissima (100, 300, 500 mg/kg) and silymarin reduced enzyme activities caused by CCl4/olive oil administration (P B0.01, P B 0.05). According to the results, B. ni6ea var. tenacissima showed a better hepatoprotective effect than B. ni6ea var. ni6ea. The histological observation includes the amount of necrosis, ballooning degeneration, fatty changes and broad infiltration of lymphocytes and Kupffer cell around the central vein. In the CCl4-induced Table 1 The hepatoprotective effect of the aqueous extracts of B. ni6ea var. ni6ea (BN), B. ni6ea var. tenacissima (BT) and silymarin on carbon tetrachloride (CCL4)-induced hepatitis in rats Groups Control CCl4/Olive oil BN

BT

Silymarin

Dose (mg/kg) sGOT (IU/l)

sGPT (IU/l)

121.9 9 4.2 728.89 32.5

55.5 91.0 243.5913.4

675.1 974.9 644.49 55.3 594.29 46.8b 608.8 9 40.8b 515.2 9 29.5a 468.6 9 57.3a 590.0 9 34.8b

209.69 26.7 241.99 14.5 157.899.2a 168.3 9 8.5a 162.19 13.5a 158.19 11.9a 137.59 14.5a

— 3 ml/kg 100 300 500 100 300 500 25

Values represent the mean 9S.E. of six animals each group. a PB0.01. b PB0.05 (Student’s t-test), significantly different from CCl4treated group.

group, more severe lesions were noted than in the other drug-treated groups. This result correlated with the changes of serum enzymatic alternation as shown in Fig. 1.

3.2. FeCl2 -ascorbic acid induced lipid peroxidation in rat li6er homogenate The rat liver homogenate was induced with ascorbic acid/Fe2 + (FeCl2-AA) to cause nonenzymatic lipid peroxidation and the action of crude extracts on this system were determined. For the purpose of investigating the effects of B. ni6ea var. ni6ea and B. ni6ea var. tenacissima on the in vitro lipid peroxidation, these drugs were incubated with a rat liver homogenate in the presence of FeCl2-AA. The lipid peroxide concentration was determined by the absorbance of a MDA– TBA adduct (complexion of malondialdehyde with thiobarbituric acid) at 532 nm (Mihara et al., 1980; Wong et al., 1987). On incubation for 1 h at 37°C, most of the water extract drugs inhibited the formation of TBA-RS at concentrations of 1–10 mg/ml (Table 2). There was significant increase of MDA level compared to the normal control without FeCl2AA (PB 0.01). The water extracts 10 mg/ml of B. ni6ea var. ni6ea and B. ni6ea var. tenacissima showed significant anti-lipid peroxidation activities (PB 0.01) and the inhibition rates were 76.0 and 66.7%, respectively.

3.3. Superoxide radical sca6enger acti6ity Fig. 2 shows the spin adduct DMPO–OOH which formed by the HPX–XOD reaction system and the manganese oxide (Mn2 + ) signal as the internal standard. Moreover, the same results were obtained after the addition of the water extracts of Boehmeria ni6ea var. ni6ea and B. ni6ea var. tenacissima (Fig. 2). However, the signal intensities of DMPO–OOH decreased with increasing superoxide dismutase (SOD) concentration and a linear calibration curve (Fig. 3) was obtained by determining the relationship of SOD concentration and [I0/I–1]. In the present case, I indicates the averaged relative peak height at various concentration of SOD (Table 3).

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Fig. 1. The photomicrographs of liver section taken from rats. (A) control group; (B) CCl4/olive oil (1:1, 3 ml/kg, s.c.); (C) CCl4 +BN (100 mg/kg, p.o.); (D) CCl4 + BN (300 mg/kg, p.o.); (E) CCl4 +BN (500 mg/kg, p.o.); (F) CCl4 +BT (100 mg/kg, p.o.); (G) CCl4 +BT (300mg/kg, p.o.); (H) CCl4 + BT (500 mg/kg, p.o.); (I) CCl4 +silymarin (25 mg/kg, p.o.).

After measuring the signal intensities of the crude drugs groups (Fig. 2) and transforming them to the calibration curve (Fig. 3), the · O2− scavenging activity (SOD-like activity) of B. ni6ea var. ni6ea (1.5×104 unit/g) and B. ni6ea var. tenacissima (1.7×104 unit/g) were obtained, as

shown in Table 4. The IC50, which caused approximately the same effect as 4.58 unit/ml of SOD with respect to inhibiting averaged relative peak height by 50%, is 0.61 mg/ml to B. ni6ea var. ni6ea and 0.54 mg/ml to B. ni6ea var. tenacissima, respectively (Table 4).

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Table 2 The inhibitory effects of aqueous extracts of B. ni6ea var. ni6ea (BN), B. ni6ea var. tenacissima (BT) on FeCl2-Ascorbic acid induced lipid peroxidation in a rat liver homogenate in vitro Groups

Concentration (mg/ml)

MDA (nmole/mg protein)

Inhibition (%)

Normal FeCl2-AA FeCl2-AA+BN FeCl2-AA+BN FeCl2-AA+BN FeCl2-AA+BT FeCl2-AA+BT FeCl2-AA+BT FeCl2-AA+Vit E

— — 1 3 10 1 3 10 0.3

1.35 90.07 2.31 90.08a 2.15 90.07 2.11 90.06 1.58 90.03b 2.159 0.01 2.00 90.12 1.67 9 0.05b 1.42 90.05a

— — 16.7 20.8 76.0 16.7 32.3 66.7 90.9

Values represent the mean 9S.E. (n= 5). PB0.01. b PB0.05 (Student’s t-test), significantly different from normal control group. a

4. Discussion and conclusions Studies on the hepatoprotective experimental model indicated that CCl4 is first metabolized by cytochrome P450 in the liver endoplasmic reticulum to the highly reactive · CCl3 radical (Recknagel, 1967; Noguchi et al., 1982a,b).The free radical, in the presence of oxygen, leads to autooxidation of the fatty acids present in the cytoplasmic membrane phospholipids (Hruszkewycz et al., 1978; Recknagel, 1983) and causes functional and morphological changes in the cell membrane. Furthermore, influx of extracellular Ca-ions into cell is claimed to be an important step leading to cell death. Therefore, the examination of the preventive action in liver damage, caused by CCl4, may give an indication of the

liver-protective action of drugs in general. The results of this study show that the water extracts of B. ni6ea var. ni6ea (500 mg/kg) and B. ni6ea var. tenacissima (100, 300, 500 mg/kg) have preventive action on CCl4-induced hepatotoxicity. This phenomenon was also confirmed by histological observation. It has been hypothesized that one of the principal causes of CCl4-induced liver injury is lipid peroxidation by free radical derivatives of CCl4. Thus, the antioxidant activity or the inhibition of the generation of free radicals is important in the protection against CCl4-induced liver lession (Castro et al., 1974; Maling et al., 1974). The in vitro lipid peroxidation in a liver homogenate proceeds in an enzymatic and a nonenzymatic processes. The former process is

Fig. 2. ESR signals of the standard manganese oxide (Mn2 + ) and superoxide radical (DMPO – OOH) peak without SOD and the inhibitory effect of the crude drugs on ESR signals of the superoxide radical. (A) Boehmeria ni6ea var. ni6ea (BN); (B) B. ni6ea var. tenacissima (BT).

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Fig. 3. The relationship between (SOD) and (I0/I–1). Calibration curve: y =0.237598x– 0.088816, where y =(I0/I – 1), x =SOD (unit/ml).

NADPH-dependent, but the latter process is induced by ascorbate in the presence of Fe2 + /Fe3 + , even with the boiled liver homogenate. It was reported that Fe2 + and ascorbic acid stimulated lipid peroxidation in rat liver microsomes and mitochondria. In order to clarify the mechanism of action of these drugs, in vitro experiments were

undertaken. According to the results, all of the drugs inhibited the FeCl2-ascorbic acid-stimulated lipid peroxidation in rat liver homogenate. The water extract of 10 mg/ml B. ni6ea var. ni6ea and B. ni6ea var. tenacissima exhibited 76.0 and 66.7% inhibitory action against the lipid peroxidation, respectively.

Table 3 ESR signal activity of Mn2+ and superoxide radical in various concentrations of SOD SOD (Unit/ml)

ESR Signal Mn2+

Peak height radical

Averaged relative peak height

0.0000 1.6320 4.0800 8.1600 12.2400 16.3200

85.400 84.800 86.000 84.200 85.600 87.800

247.000 180.200 134.400 86.800 65.600 52.400

2.892 2.125 1.563 1.031 0.766 0.597

(Regression coefficient) I0/I–1= 0.237598×SOD−0.088816; correlation coefficient = 0.999248; I indicates the relative peak height when various concentration of SOD was added; calibration curve: y = 0.237598×−0.088816, where y =(I0/I – 1), x =SOD (unit/ml).

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Table 4 SOD-like (superoxide radical scavenger) activity assay Sample

Concentration (g/ml)

Signal peak Mn2+

Height radical

SOD activitya

SOD-like activity (unit/g)

BN BT

1.25×10−4 1.00×10−3

84.80 85.80

77.60 84.60

9.476 8.511

1.5×104 1.7×104

IC50 (mg/ml)

0.61 0.54

a Calibration curve: y = 0.237598x−0.088816, where y= [I0/I– 1], x= SOD (unit/ml). I0, 2.892 (from Table 3, without SOD or crude drug); I, the averaged relative peak height at various concentration of crude drugs extracts.

Active oxygen species and free radicals are involved in a variety of pathological events, cancer and the aging process. Any compound, natural or synthetic, with antioxidant properties that might contribute towards the partial or total alleviation of this damage, may have a significant role in maintaining health when continuously taken as components of dietary foods, spices and drugs. Therefore, removing · O2− is probably one of the most effective defences of a living body against diseases. Table 4 show the scavenging activity of B. ni6ea var. ni6ea and B. ni6ea var. tenacissima. The scavenging activity, shown by the extract of B. ni6ea var. ni6ea and B. ni6ea var. tenacissima, demonstrated that both crude drugs inhibited the DMPO–OOH signal intensity generated from the HPX –XOD system. The results suggest that B. ni6ea var. ni6ea and B. ni6ea var. tenacissima contain a free radical scavenging activity, which could exert a beneficial action against pathological alterations caused by the presence of CCl4, which appears to be metabolized to a free radical during intoxication (Hruszkewycz et al., 1978; Noguchi et al., 1982a; Recknagel, 1983). Moreover, we also investigated the antilipid peroxidation in rat liver homogenate and the active oxygen scavenging activity of crude drugs, which at least in part, can explain the mechanism of hepatoprotective effect of B. ni6ea var. ni6ea and B. ni6ea var. tenacissima. In conclusion, the aqueous extracts of B. ni6ea var. ni6ea and B. ni6ea var. tenacissima exhibited a liver protective effect against CCl4 induced hepatotoxicity and possessed anti-lipid peroxidative and free radical scavenging activities. The hepato-

protective effect, anti-lipid peroxidation, superoxide radical scavenging activity of compounds of B. ni6ea var. ni6ea and B. ni6ea var. tenacissima require further study.

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