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Antioxidant and hepatoprotective activity of aqueous extract of Solanum fastigiatum (false “Jurubeba”) against paracetamol-induced liver damage in mice
Journal of Ethnopharmacology 120 (2008) 226–232
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Antioxidant and hepatoprotective activity of aqueous extract of Solanum fastigiatum (false “Jurubeba”) against paracetamol-induced liver damage in mice S.M. Sabir a,b,∗ , J.B.T. Rocha b a b
University of Azad Jammu and Kashmir, Faculty of Agriculture Rawalakot, 12350 A.K., Pakistan Departmento de química, Bioquímica Toxicologia (CCNE), Universidade Federal de Santa Maria, Brazil
a r t i c l e
i n f o
Article history: Received 14 January 2008 Received in revised form 28 July 2008 Accepted 15 August 2008 Available online 23 August 2008 Keywords: Solanum fastigiatum Lipid peroxidation Pro-oxidants Serum enzymes Antioxidant Paracetamol
a b s t r a c t Aim of the study: Solanum fastigiatum is a medicinal plant widely distributed in the south of Brazil and has been used mainly to treat hepatitis, spleen disorders, uterine tumors, irritable bowel syndrome and chronic gastritis. The present research was aimed to evaluate the potential antioxidant and hepatoprotective activity of aqueous extracts of leaves using in vitro and in vivo models to validate the folkloric use of the plant. Materials and methods: Antioxidant activity was evaluated by different assays, including thiobarbituric acid reactive species (TBARS), total antioxidant, 2,2-diphenlyl-1-picrylhydrazyl (DPPH) radical and metal ion-chelating activities. The hepatoprotective activity of the aqueous extracts was studied on mice liver damage induced by paracetamol (250 mg/kg) by monitoring biochemical parameters. Results: The extract showed inhibition against TBARS, induced by 10 M FeSO4 and 5 M sodium nitroprusside in rat liver, brain and phospholipid homogenates from egg yolk. The plant exhibited strong antioxidant activity in the DPPH (IC50 , 68.96 ± 1.25 g/ml) assay. The aqueous extract also showed significant hepatoprotective activity that was evident by enzymatic examination and brought back the altered levels of TBARS, non-protein thiol and ascorbic acid to near the normal levels in a dose dependent manner. Acute toxicity studies revealed that the LD50 value of the extract is more than the dose 4 g/kg body weight of mice. Conclusions: The results indicate that this plant possesses potential antioxidant and hepatoprotective properties and has therapeutic potential for the treatment of liver diseases. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Solanum fastigiatum Willd. (Solanaceae) commonly know as “jurubeba” is widely distributed in the southern states of Brazil (Claudia et al., 1988). The leaves and roots of the plant are used in Brazilian medicine as a tonic for fevers, anemia, erysipelas, liver diseases, hepatitis, spleen disorders (Costa, 1940; Penna, 1964), uterine tumors, irritable bowel syndrome, chronic gastritis and other digestive problems such as sluggish digestion, bloating and flatulence (Cruz, 1965). Jurubeba leaf tea is a very common household remedy throughout Brazil for hangovers. Phytochemical analysis has shown the presence of flavonoids and glycosides in the leaves of the plant (Higa et al., 2007). Many species of Solanum
are known by the Brazilian population as “jurubeba” but the Brazilian Pharmacopoeia described the species Solanum paniculatum L. as the true “jurubeba” (Corrêa, 1984). Solanum paniculatum has been extensively studied mainly because of its protective effects on the liver and anti-secretory gastric properties (Comissão Geográfica e Geologia, 1972, p. 74). However, scientific literature data supporting the folkloric use of the Solanum fastigiatum in liver diseases are not available and its tentative mechanism(s) are still unknown. The present study was therefore, aimed to evaluate the potential in vitro and in vivo antioxidant properties of aqueous extracts of Solanum fastigiatum and its in vivo hepatoprotective activity against paracetamol-induced hepatotoxicity.
2. Material and methods ∗ Corresponding author at: Departmento de química, Bioquímica Toxicologia (CCNE), Universidade Federal de Santa Maria, Brazil. Tel.: +55 55 81345365; fax: +55 55 32208978. E-mail addresses: [email protected], [email protected] (S.M. Sabir), [email protected] (J.B.T. Rocha). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.08.017
2.1. Analytical materials Thiobarbituric acid (TBA), malonaldehyde-bis-dimethyl acetal (MDA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), quercetin, rutin,
S.M. Sabir, J.B.T. Rocha / Journal of Ethnopharmacology 120 (2008) 226–232
epinephrine and phenanthroline were purchased from Sigma (St. Louis, MO, USA). Sodium nitroprusside (SNP) was obtained from Merck (Darmstadt, Germany) and iron(II) sulphate from Reagen (Rio de Janeiro, RJ, Brazil). 2.2. Preparation of plant extract The whole plant was collected close to the campus of Santa Maria University (RS, Brazil) during October–November, 2007 and identified by a botanist Renato Aquino Záchia. A voucher specimen (SMDB 11.115) was deposited at the Herbarium of University Federal Santa Maria, Department of Pharmacy. Dried leaves of the plant (25 g) were ground and soaked in boiling water (250 ml) for 15 min, allowed to cool and filtered using whatman filter paper. The obtained residue was further extracted twice and finally the whole extract was concentrated using a rotary evaporator. The extract weight and percentage yield were found to be 2.8 g (11.2%). Serial dilutions were made to obtain the desired concentration of plant for the experiments. 2.3. Test animals All animal procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by University Federal Santa Maria Ethical Council (UFSM 10067). Wistar male rats (200–250 g) from our own breeding colony were used for in vitro studies. For in vivo studies male albino mice weighing 24–30 g were used. The animals were kept in separate cages with access to water and food ad libitum, in a room with controlled temperature (22 ± 3 ◦ C) and in 12 h light/dark cycle with lights turn on at 7:00 a.m.
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2.4.3. DPPH radical scavenging Scavenging of the stable DPPH radical (ethanolic solution of 0.25 mM) was assayed in vitro (Hatano et al., 1988) and the absorbance was measured at 517 nm. Percentage inhibition was calculated from the control. Ascorbic acid was used as a standard compound in DPPH assay. 2.4.4. Antioxidant potential assay The total antioxidant potential of the extracts was estimated using the phosphomolybdenum reduction assay according to Prieto et al. (1999). The reducing capacity of the extracts was expressed as the ascorbic acid equivalent. 2.4.5. Metal chelating activity The ability of the aqueous extract to chelate Fe2+ was determined using a modified method of Puntel et al. (2005) at 510 nm. 2.5. Acute toxicity Twenty-five male albino mice (35–45 g) were divided into five groups comprising five animals in each group. Mice were orally treated with graded doses of extract ranging up to 4 g/kg body weight for two consecutive days and were observed for signs of toxicity and mortality for 7 days (Silva et al., 2007). The activities of enzymes, glutamate pyruvate transaminase (GPT) and gulatamate oxaloacetate transaminase (GOT) were determined in serum using assay kits (Lab Test, MG, Brazil) according to the supplier specifications. 2.6. In vivo hepatoprotective activity
2.4. In vitro assays 2.4.1. Production of thiobarbituric acid reactive species (TBARS) from animal tissues Production of TBARS was determined using a modified method of Ohkawa et al. (1979). The rats were anesthetized with ether and then sacrificed by decapitation. Liver and brain were quickly removed and placed on ice. Tissues were homogenized in cold 100 mM Tris buffer pH 7.4 (1:10 w/v) and centrifuged at 1000 × g for 10 min. The homogenates (100 l) were incubated with or without 50 l of the various freshly prepared oxidants (iron and sodium nitroprusside) and different concentrations of the plant extracts together with an appropriate volume of deionized water to give a total volume of 300 l at 37 ◦ C for 1 h. The color reaction was carried out by adding 200, 500 and 500 l each of the 8.1% sodium dodecyl sulphate (SDS), acetic acid (pH 3.4) and 0.6% TBA, respectively. The reaction mixtures, including those of serial dilutions of 0.03 mM standard MDA, were incubated at 97 ◦ C for 1 h. The absorbance was read after cooling the tubes at a wavelength of 532 nm in a spectrophotometer. TBARS unit was expressed as nmol/g tissue. 2.4.2. Production of TBARS from phospholipid Production of TBARS from phospholipid was determined using the method of Ohkawa et al. (1979) with slight modifications. One gram of egg yolk was extracted with 100 ml of hexane–isopropanol (3:2) and filtered. The solution was dried in a rotary evaporator at 60 ◦ C until it is pastured. The remaining procedure was the same as mentioned above except that the color reaction was carried out without adding SDS. The tubes were cooled and 2 ml of n-butanol was finally added and centrifuged at 1000 × g. The organic layer (supernatant) was collected and the absorbance was read at 532 nm in a spectrophotometer.
The 30 animals were divided into six groups comprising five mice in each group. Group I (control) received only distilled water. Group II (plant group) received aqueous extract at a dose of 100 mg/kg body weight of animals. Group III (plant group) received aqueous extract at a dose of 200 mg/kg. Group IV (paracetamol control) received 250 mg/kg paracetamol only. Group V received single dose of paracetamol (250 mg/kg) + (100 mg/kg) of dose 1 of extract. Whereas, Group VI received single dose of paracetamol (250 mg/kg) + (200 mg/kg) of dose 2 of extract. Plant extract was administered 3 h after the administration of paracetamol. All the treatments were instilled orally by means of a gastric tube. The treatments were continued for 7 days and on the eighth day of the experiment all animals were sacrificed under light ether anesthesia. Liver was quickly removed, placed on ice and homogenized in seven volumes of NaCl (0.9%). The homogenates were centrifuged at 4000 × g for 10 min to yield a clear supernatant fraction that was used for the biochemical analysis. 2.6.1. Enzyme assays Serum was obtained from clotted blood samples by centrifugation at 1500 × g for 10 min and the activities of enzymes, GPT and GOT were determined. Catalase (CAT) activity was estimated by following the breakdown of hydrogen peroxide according to the method of Aebi (1983). Superoxide dismutase (SOD) was assayed according to Misra and Fridevich (1972) based on the inhibition of epinephrine auto-oxidation by the enzyme. Ascorbic acid was measured by the method of Natelson (1963). Lipid peroxidation was measured in terms of TBARS following the thiobarbituric acid method of Ohkawa et al. (1979). Non-protein thiol (NPSH) content in the liver homogenate was determined as described by Jollow et al. (1974).
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Table 1 The inhibitory effect of aqueous extracts of Solanum fastigiatum (SF) on iron sulphate induced and sodium nitroprusside (SNP)-induced lipid peroxidation in a rat liver homogenate
Table 2 The inhibitory effect of aqueous extracts of Solanum fastigiatum (SF) on iron sulphate induced and sodium nitroprusside (SNP)-induced lipid peroxidation in a rat brain homogenate
Treatments
SF concentration (g/ml)
TBARS (×100)
TBARS inhibition (%)
Treatments
SF concentration (g/ml)
TBARS (×100)
Normal Control Iron + SF Iron + SF Iron + SF Iron + SF Iron + SF
– – 20 40 60 80 100
0.83 4.31 4.16 3.02 0.92 0.33 0.13
± ± ± ± ± ± ±
0.11 0.1b 0.1b 0.12 0.3 0.04c 0.03c
– – 3.36 30 78.6 92.3 97
Normal Control Iron + SF Iron + SF Iron + SF Iron + SF Iron + SF
– – 20 40 60 80 100
0.72 5.44 3.01 1.18 0.47 0.30 0.174
± ± ± ± ± ± ±
0.11 0.16 0.42 0.1 0.05ab 0.04ab 0.1
– – 44.6 78.2 81.3 91.3 97
Normal Control SNP + SF SNP + SF SNP + SF SNP + SF SNP + SF
– – 20 40 60 80 100
0.83 4.1 3.62 3.4 3.17 2.16 1.72
± ± ± ± ± ± ±
0.11 0.12 0.03a 0.062a 0.09b 0.09b 0.07
– – 11 16.5 22 46.6 57.6
Normal Control SNP + SF SNP + SF SNP + SF SNP + SF SNP + SF
– – 20 40 60 80 100
0.72 5.65 4.9 3.19 2.12 1.7 1.05
± ± ± ± ± ± ±
0.1 0.36 0.12 0.1 0.16 0.11 0.12
– – 13.5 43.6 62.5 70 81.4
TBARS inhibition (%)
Values represent the means ± S.D. (n = 6), TBARS expressed as nmol/g tissue. p < 0.05 by DMRT is significantly different from control group. Values in columns followed by same letters are non-significantly different from each other.
Values represent the means ± S.D. (n = 6), TBARS expressed as nmol/g tissue. p < 0.05 by DMRT is significantly different from control group. Values in columns followed by same letters are non-significantly different from each other.
2.7. Phenolics content
more effective against Fe2+ than SNP-induced TBARS. Similar to the results obtained with tissues, Fe2+ and SNP stimulated TBARS production in phospholipid and aqueous extract reduced (p < 0.05) TBARS production in a concentration dependent manner (Table 3) The extract also exhibited strong antioxidant activity in the DPPH and iron chelation assays (Fig. 1). The IC50 value obtained for DPPH scavenging was 68.96 ± 1.25 g/ml which is comparable to the reference standard ascorbic acid (IC50 = 28.4 g/ml). For iron chelation assay the extract showed maximum inhibition of 48.7% at the lowest concentration. Indeed, the chelation was not found to be concentration dependent (Fig. 1). The total antioxidant capacity of the extract (equivalent to ascorbic acid) was found to be 50.22 ± 0.33 g/ml at 100 g/ml and was increased with increasing concentrations of extract (Fig. 2). Phytochemical analysis of the leaves showed high content of total phenolics (110.67 ± 0.003 mg/g) and flavonoids (2.24 ± 0.004 mg/g; Table 4). The results of TLC analysis also confirmed the presence of two important flavonoids, rutin (Rf = 0.59) and quercetin (Rf = 0.91) in the methanolic extracts of leaves.
The total phenol content was determined by following the method of Singleton et al. (1999). The mean of three readings was used and the total phenol content was expressed in milligram of gallic acid equivalents/g extract. 2.8. Determination of total flavonoids The total flavonoid content as quercetin equivalents/g extract was based on the method reported by Kosalec et al. (2004). 2.9. Thin layer chromatography (TLC) of plant extracts Methanolic extract of leaves was characterized by thin layer chromatography (Silica gel coated TLC plates, Merck; mobile phase, n-butanol:ethyl acetate:water 2:4:4). Chromatograms were evaluated under UV light at 254 and 365 nm to detect the presence of flavonoids. The presence of flavonoids was further confirmed by spraying the plates with 5% AlCl3 in ethanol. Rutin and quercetin were used as standard flavonoids. 2.10. Statistical analysis The results were expressed as mean ± S.D. The data were analyzed statistically by one-way ANOVA and different group means were compared by Duncan’s multiple range test (DMRT); p < 0.05 was considered significant in all cases. The software package Statistica was used for analysis of data. 3. Results 3.1. In vitro assays Ferrous sulphate and SNP caused a significant increase in TBARS production in rat liver homogenates (Table 1) and the aqueous extract of Solanum fastigiatum significantly reduced (p < 0.05) Fe2+ and SNP-induced TBARS production in a concentration dependent manner. Cerebral TBARS production was also stimulated by Fe2+ and SNP and plant extract caused a concentration dependent inhibition of TBARS production (p < 0.05, Table 2). However, Solanum fastigiatum was
Table 3 The inhibitory effect of aqueous extracts of Solanum fastigiatum (SF) on iron sulphate induced and sodium nitroprusside (SNP)-induced lipid peroxidation in phospholipid homogenate Treatments
SF concentration (g/ml)
TBARS (×100)
Normal Control Iron + SF Iron + SF Iron + SF Iron + SF Iron + SF
– – 20 40 60 80 100
1.96 12.21 8.34 7.13 6.74 6.06 6.86
± ± ± ± ± ± ±
0.34 0.03 0.12 0.02a 0.03 0.02 0.14a
TBARS inhibition (%) – – 31.7 41.6 44.8 50.4 43.8
Normal Control SNP + SF SNP + SF SNP + SF SNP + SF SNP + SF
– – 20 40 60 80 100
1.96 7.40 4.48 4.10 3.74 3.38 2.44
± ± ± ± ± ± ±
0.34 0.08 0.12 0.03 0.07 0.04 0.03
– – 29.8 39.4 49.4 54.3 67
Values represent the means ± S.D. (n = 6), TBARS expressed as nmol/g tissue. p < 0.05 by DMRT is significantly different from control group. Values in columns followed by same letters are non-significantly different from each other.
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Fig. 1. DPPH radical scavenging and iron chelation capacity of the extract of Solanum fastigiatum in vitro. Values are mean ± S.D. (n = 3).
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Fig. 3. Hepatoprotective action of Solanum fastigiatum in mice: serum enzyme activity of GPT and GOT among different treatment groups. Values are mean ± S.D. (n = 5); b p < 0.05 versus normal control group and c p < 0.05 versus paracetamol (PM) control group; PM = 250 mg/kg, D1 = 100 mg/kg, and D2 = 200 mg/kg.
Fig. 2. Total antioxidant activity of Solanum fastigiatum leaves measured by phosphomolybdenum assay. Values are mean ± S.D. (n = 3).
3.2. In vivo assays Administration of paracetamol (250 mg/kg) caused significant liver damage as evidenced by the altered serum and liver biochemical parameters (Figs. 3–8). However, the administration of aqueous extract of Solanum fastigiatum at a dose of 100 mg/kg and 200 mg/kg exhibited marked protection against paracetamol-induced liver damage (Figs. 3–8). The aqueous leaf extract of Solanum fastigiatum was found to be practically non-toxic when administered orally to mice and we could not calculate its LD50 because the treatment did not cause any deaths and consequently its LD50 is higher than 4 g/kg body weight. The animals treated with extract did not manifest any overt signs of toxicity and there was no significant difference in the weight of the treated animals compared to the negative control (data not shown). However, safety margin of the drug can be considered below 4 g/kg because this dose caused a small increase (p < 0.05) in the level of GPT and GOT, which indicates a potential hepatotoxicity for very high doses of plant (Fig. 9).
Fig. 4. Hepatoprotective action of Solanum fastigiatum in mice: catalase activity among different treatment groups. Values are mean ± S.D. (n = 5); a p < 0.05 versus normal control group, b p < 0.05, and c p < 0.05 versus paracetamol (PM) control group. PM = 250 mg/kg, D1 = 100 mg/kg, and D2 = 200 mg/kg.
Table 4 Content of phenolics and flavonoids in water and methanolic extracts of Solanum fastigiatum Extracts
Total phenolics (mg/g)
Aqueous Methanol
128.6 ± 0.03 149.1 ± 0.05
Total flavonoids (mg/g) 0.173 ± 0.03 2.24 ± 0.043
Fig. 5. Hepatoprotective action of Solanum fastigiatum in mice: superoxide dismutase activity among different treatment groups. Values are mean ± S.D. (n = 5); a p < 0.05 versus normal control group, b p < 0.05, and c p < 0.05 versus paracetamol (PM) control group. PM = 250 mg/kg, D1 = 100 mg/kg, and D2 = 200 mg/kg.
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Fig. 6. Hepatoprotective action of Solanum fastigiatum in mice: TBARS (nmol/g.tissue) among different treatment groups. Values are mean ± S.D. (n = 5); a p < 0.05 versus normal control group, b p < 0.05 versus normal control group, and c p < 0.05 versus paracetamol (PM) control group. PM = 250 mg/kg, D1 = 100 mg/kg, and D2 = 200 mg/kg.
Fig. 7. Hepatoprotective action of Solanum fastigiatum in mice: non-protein thiol (NPSH) content among different treatment groups. Values are mean ± S.D. (n = 5); b p < 0.05 versus normal control group and c p < 0.05 versus paracetamol (PM) control group. PM = 250 mg/kg, D1 = 100 mg/kg, and D2 = 200 mg/kg.
4. Discussion Oxidative stress is now recognized to be associated with more than 100 diseases, as well as with the normal aging process
Fig. 8. Hepatoprotective action of Solanum fastigiatum in mice: ascorbic acid content among different treatment groups. Values are mean ± S.D. (n = 5); a p < 0.05 versus normal control group, b p < 0.05 versus normal control group, and c p < 0.05 versus paracetamol (PM) control group. PM = 250 mg/kg, D1 = 100 mg/kg, and D2 = 200 mg/kg.
Fig. 9. Effect of different doses of Solanum fastigiatum aqueous extracts on serum parameters (GPT and GOT) in mice. Values are mean ± S.D. (n = 5); *p < 0.05 versus normal control group.
(Ghasanfari et al., 2006). Here Fe2+ and SNP were used as a tool to induce lipid peroxidation. The aqueous extracts of Solanum fastigiatum exhibit good antioxidant activity against two pro-oxidants in all tissues. However, in brain and liver it was more effective against Fe2+ inhibition compared to SNP-induced TBARS. While, in phospholipid homogenate the extract showed high antioxidant activity against SNP as compared to Fe2+ . Increases in the formation of TBARS in Fe2+ (10 M)-induced oxidative stress as compared to the basal suggest possible damage of tissues with an overload of iron. Rats overloaded with iron showed toxic effects such as hepatocellular hypertrophy, cardiomyopathy, pancreatic atrophy, splenic white pulp atrophy, and hemosiderosis in the liver, heart, pancreas and endocrine glands, respectively (Whittaker et al., 1997). The protections offered by the Solanum fastigiatum suggest that the aqueous extract may protect the liver and brain against toxicities resulting from potential overload of iron. Sodium nitroprusside is an anti-hypertensive drug that acts by relaxation of vascular smooth muscle and consequently dilates peripheral arteries and veins. However, SNP has been reported to cause cytotoxicity through the release of cyanide and or nitric oxide (Bates et al., 1990). The protection offered by Solanum fastigiatum extract on tissues (brain and liver) and phospholipids confirms the antioxidant activity of extract and indicate its use in accidental intoxications resulting from the overload with SNP. The present results revealed that the extract of Solanum fastigiatum protects the tissues against the lipid peroxidation at low concentration (less than 100 g/ml) and in most of the cases have the ability to reduce the TBARS production to the basal level. The DPPH radical scavenging activity of the extracts also revealed high antioxidant activity considering the fact that quenching properties were obtained only from the crude extracts with the IC50 value 68.96 ± 1.25 g/ml. Such high free radical scavenging properties of the crude extracts are shown by few other plants (Sabir and Rocha, 2008). The use of iron chelation is a popular therapy for the management of Fe2+ associated oxidative stress in brain. The iron chelating ability of the Solanum fastigiatum is expected to potentially enhance the neuroprotective properties of the plant particularly in view of the fact that iron overload is involved in the pathogenesis of brain diseases such as Alzheimer (Elise and James, 2002). In the phosphomolybdenum assay, which is a quantitative method to evaluate water-soluble and fat-soluble antioxidant capacity (total antioxidant capacity), the extract demonstrated electron-donating capacity showing its ability to act as chain terminators, transforming relative free radi-
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cal species into more stable non-reactive products (Dorman et al., 2003). Here we have demonstrated for the first time that Solanum fastigiatum aqueous extract has and important hepatoprotective effect against paracetamol. This is particularly important in view of the fact that the treatments of acute human intoxications with paracetamol are limited and frequently not effective and relies basically on the use of N-acetylcysteine (Groote and Steenbergen, 1995; Angela et al., 2005). Paracetamol is a well-known antipyretic and analgesic agent, which is safe in therapeutic doses, but can produce fatal hepatic necrosis in experimental animals and humans (Schidt et al., 1997; Amar and Schiff, 2007) and is employed as an experimental hepatotoxic agent. An obvious sign of hepatic injury is the leaking of cellular enzymes into the plasma due to the disturbance caused in the transport functions of hepatocytes (Zimmerman and Seeff, 1970). The estimation of enzymes in the serum is a useful quantitative marker of the extent and type of hepatocellular damage. The mice treated with an overdose of paracetamol developed significant hepatic damage, which was observed by a substantial increase in the concentration of serum hepatic enzymes (GPT and GOT). Administration of Solanum extract (100 mg/kg and 200 mg/kg p.o.) after paracetamol treatment resulted in a significant reduction (p < 0.05) of paracetamol-induced elevation of GPT and GOT and appears to be protective in reducing the injurious effect of paracetamol. We also observed significant increase (p < 0.05) in the levels of TBARS in liver, which was decreased by the administration of the plant extract in a dose dependent manner. The administration of the paracetamol (250 mg/kg, p.o., 7 days) significantly decreased (p < 0.05) the activity of CAT and SOD enzymes, which were restored to normal levels by the subsequent treatment with the plant. In fact, extract treatment alone caused a significant increase (p < 0.05) in the activity of CAT and SOD, which can explain at least in part its hepatoprotective effect. It is a well-known fact that paracetamol toxicity is associated with sharp decrease in glutathione (Gerber et al., 1977) and ascorbic acid content (Dahlin et al., 1984). Here we observed that treatment with extracts of Solanum fastigiatum restored the levels of NPSH and ascorbic acid that were depleted by paracetamol intoxication. In fact, treatment with plant extracts increased the levels of ascorbic acid, indicating an additional mechanism for its hepatoprotective action. The protective action of aqueous extracts Solanum fastigiatum may be attributed to the presence of high content of phenolics and flavonoids (rutin and quercetin), which are well known hepatoprotective agents (Khalid et al., 2002). The data obtained on safety studies suggest that the aqueous extract of Solanum fastigiatum was relatively non-toxic when administered to mice for two consecutive days as the calculated LD50 value was found to be higher than 4 g/kg body weight and since to be non-toxic in humans a single oral dose of 4 g/kg should cause no acute symptoms or death of animals (Zbinden and FluryRoversi, 1981). However, the small increase in the level of serum enzymes (GPT and GOT) at very high dose of the extract suggests that the use of the plant in humans (or phytotherapics) should be accompanied by a detailed and carefully conducted dose response curves in healthy volunteers. Infact, this plant is commonly sold in Brazil by non-informed persons and people use it without having any indication of safe daily or chronic doses. In conclusions, the aqueous extracts of Solanum fastigiatum exhibited protective effect against paracetamol-induced hepatotoxicity and possess anti-lipid peroxidative and free radical scavenging activities. The result supports the use of the plant as described in folk medicine, that the aerial parts of plant can be used to treat liver and gastric disorders, even though it is considered to be the false “jurubeba”. Further studies are required to isolate the active constituents involved in the antioxidant and hepatoprotective activity of the plant.
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Acknowledgments We gratefully acknowledge the financial support and the offer of doctoral fellowship to Syed Mubashar Sabir by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil and the Academic of Science for Developing Countries (TWAS), Italy. J.B.T.R is the beneficiary of CNPq research fellowship. FINEP research grant “Rede Instituto Brasileiro de Neurociência (IBN-Net)” # 01.06.084200 is also acknowledged. References Aebi, H.E., 1983. In: Bergmeyer, H.O. (Ed.), Catalase, methods enzymology, vol. 3. Academic Press, New York, p. 2. Amar, P.J., Schiff, E.R., 2007. Acetaminophen safety and hepatotoxicity. Where do we go from here? Expert Opinion on Drug Safety 6, 341–355. Angela, B.R., Richard, C.K., Sandra, S.M., Robert, W.B., Jack, A.H., 2005. Mechanism of acetaminophen-induced hepatotoxicity: role of oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes. 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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Antioxidant and hepatoprotective activity of aqueous extract of Solanum fastigiatum (false “Jurubeba”) against paracetamol-induced liver damage in mice S.M. Sabir a,b,∗ , J.B.T. Rocha b a b
University of Azad Jammu and Kashmir, Faculty of Agriculture Rawalakot, 12350 A.K., Pakistan Departmento de química, Bioquímica Toxicologia (CCNE), Universidade Federal de Santa Maria, Brazil
a r t i c l e
i n f o
Article history: Received 14 January 2008 Received in revised form 28 July 2008 Accepted 15 August 2008 Available online 23 August 2008 Keywords: Solanum fastigiatum Lipid peroxidation Pro-oxidants Serum enzymes Antioxidant Paracetamol
a b s t r a c t Aim of the study: Solanum fastigiatum is a medicinal plant widely distributed in the south of Brazil and has been used mainly to treat hepatitis, spleen disorders, uterine tumors, irritable bowel syndrome and chronic gastritis. The present research was aimed to evaluate the potential antioxidant and hepatoprotective activity of aqueous extracts of leaves using in vitro and in vivo models to validate the folkloric use of the plant. Materials and methods: Antioxidant activity was evaluated by different assays, including thiobarbituric acid reactive species (TBARS), total antioxidant, 2,2-diphenlyl-1-picrylhydrazyl (DPPH) radical and metal ion-chelating activities. The hepatoprotective activity of the aqueous extracts was studied on mice liver damage induced by paracetamol (250 mg/kg) by monitoring biochemical parameters. Results: The extract showed inhibition against TBARS, induced by 10 M FeSO4 and 5 M sodium nitroprusside in rat liver, brain and phospholipid homogenates from egg yolk. The plant exhibited strong antioxidant activity in the DPPH (IC50 , 68.96 ± 1.25 g/ml) assay. The aqueous extract also showed significant hepatoprotective activity that was evident by enzymatic examination and brought back the altered levels of TBARS, non-protein thiol and ascorbic acid to near the normal levels in a dose dependent manner. Acute toxicity studies revealed that the LD50 value of the extract is more than the dose 4 g/kg body weight of mice. Conclusions: The results indicate that this plant possesses potential antioxidant and hepatoprotective properties and has therapeutic potential for the treatment of liver diseases. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Solanum fastigiatum Willd. (Solanaceae) commonly know as “jurubeba” is widely distributed in the southern states of Brazil (Claudia et al., 1988). The leaves and roots of the plant are used in Brazilian medicine as a tonic for fevers, anemia, erysipelas, liver diseases, hepatitis, spleen disorders (Costa, 1940; Penna, 1964), uterine tumors, irritable bowel syndrome, chronic gastritis and other digestive problems such as sluggish digestion, bloating and flatulence (Cruz, 1965). Jurubeba leaf tea is a very common household remedy throughout Brazil for hangovers. Phytochemical analysis has shown the presence of flavonoids and glycosides in the leaves of the plant (Higa et al., 2007). Many species of Solanum
are known by the Brazilian population as “jurubeba” but the Brazilian Pharmacopoeia described the species Solanum paniculatum L. as the true “jurubeba” (Corrêa, 1984). Solanum paniculatum has been extensively studied mainly because of its protective effects on the liver and anti-secretory gastric properties (Comissão Geográfica e Geologia, 1972, p. 74). However, scientific literature data supporting the folkloric use of the Solanum fastigiatum in liver diseases are not available and its tentative mechanism(s) are still unknown. The present study was therefore, aimed to evaluate the potential in vitro and in vivo antioxidant properties of aqueous extracts of Solanum fastigiatum and its in vivo hepatoprotective activity against paracetamol-induced hepatotoxicity.
2. Material and methods ∗ Corresponding author at: Departmento de química, Bioquímica Toxicologia (CCNE), Universidade Federal de Santa Maria, Brazil. Tel.: +55 55 81345365; fax: +55 55 32208978. E-mail addresses: [email protected], [email protected] (S.M. Sabir), [email protected] (J.B.T. Rocha). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.08.017
2.1. Analytical materials Thiobarbituric acid (TBA), malonaldehyde-bis-dimethyl acetal (MDA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), quercetin, rutin,
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epinephrine and phenanthroline were purchased from Sigma (St. Louis, MO, USA). Sodium nitroprusside (SNP) was obtained from Merck (Darmstadt, Germany) and iron(II) sulphate from Reagen (Rio de Janeiro, RJ, Brazil). 2.2. Preparation of plant extract The whole plant was collected close to the campus of Santa Maria University (RS, Brazil) during October–November, 2007 and identified by a botanist Renato Aquino Záchia. A voucher specimen (SMDB 11.115) was deposited at the Herbarium of University Federal Santa Maria, Department of Pharmacy. Dried leaves of the plant (25 g) were ground and soaked in boiling water (250 ml) for 15 min, allowed to cool and filtered using whatman filter paper. The obtained residue was further extracted twice and finally the whole extract was concentrated using a rotary evaporator. The extract weight and percentage yield were found to be 2.8 g (11.2%). Serial dilutions were made to obtain the desired concentration of plant for the experiments. 2.3. Test animals All animal procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by University Federal Santa Maria Ethical Council (UFSM 10067). Wistar male rats (200–250 g) from our own breeding colony were used for in vitro studies. For in vivo studies male albino mice weighing 24–30 g were used. The animals were kept in separate cages with access to water and food ad libitum, in a room with controlled temperature (22 ± 3 ◦ C) and in 12 h light/dark cycle with lights turn on at 7:00 a.m.
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2.4.3. DPPH radical scavenging Scavenging of the stable DPPH radical (ethanolic solution of 0.25 mM) was assayed in vitro (Hatano et al., 1988) and the absorbance was measured at 517 nm. Percentage inhibition was calculated from the control. Ascorbic acid was used as a standard compound in DPPH assay. 2.4.4. Antioxidant potential assay The total antioxidant potential of the extracts was estimated using the phosphomolybdenum reduction assay according to Prieto et al. (1999). The reducing capacity of the extracts was expressed as the ascorbic acid equivalent. 2.4.5. Metal chelating activity The ability of the aqueous extract to chelate Fe2+ was determined using a modified method of Puntel et al. (2005) at 510 nm. 2.5. Acute toxicity Twenty-five male albino mice (35–45 g) were divided into five groups comprising five animals in each group. Mice were orally treated with graded doses of extract ranging up to 4 g/kg body weight for two consecutive days and were observed for signs of toxicity and mortality for 7 days (Silva et al., 2007). The activities of enzymes, glutamate pyruvate transaminase (GPT) and gulatamate oxaloacetate transaminase (GOT) were determined in serum using assay kits (Lab Test, MG, Brazil) according to the supplier specifications. 2.6. In vivo hepatoprotective activity
2.4. In vitro assays 2.4.1. Production of thiobarbituric acid reactive species (TBARS) from animal tissues Production of TBARS was determined using a modified method of Ohkawa et al. (1979). The rats were anesthetized with ether and then sacrificed by decapitation. Liver and brain were quickly removed and placed on ice. Tissues were homogenized in cold 100 mM Tris buffer pH 7.4 (1:10 w/v) and centrifuged at 1000 × g for 10 min. The homogenates (100 l) were incubated with or without 50 l of the various freshly prepared oxidants (iron and sodium nitroprusside) and different concentrations of the plant extracts together with an appropriate volume of deionized water to give a total volume of 300 l at 37 ◦ C for 1 h. The color reaction was carried out by adding 200, 500 and 500 l each of the 8.1% sodium dodecyl sulphate (SDS), acetic acid (pH 3.4) and 0.6% TBA, respectively. The reaction mixtures, including those of serial dilutions of 0.03 mM standard MDA, were incubated at 97 ◦ C for 1 h. The absorbance was read after cooling the tubes at a wavelength of 532 nm in a spectrophotometer. TBARS unit was expressed as nmol/g tissue. 2.4.2. Production of TBARS from phospholipid Production of TBARS from phospholipid was determined using the method of Ohkawa et al. (1979) with slight modifications. One gram of egg yolk was extracted with 100 ml of hexane–isopropanol (3:2) and filtered. The solution was dried in a rotary evaporator at 60 ◦ C until it is pastured. The remaining procedure was the same as mentioned above except that the color reaction was carried out without adding SDS. The tubes were cooled and 2 ml of n-butanol was finally added and centrifuged at 1000 × g. The organic layer (supernatant) was collected and the absorbance was read at 532 nm in a spectrophotometer.
The 30 animals were divided into six groups comprising five mice in each group. Group I (control) received only distilled water. Group II (plant group) received aqueous extract at a dose of 100 mg/kg body weight of animals. Group III (plant group) received aqueous extract at a dose of 200 mg/kg. Group IV (paracetamol control) received 250 mg/kg paracetamol only. Group V received single dose of paracetamol (250 mg/kg) + (100 mg/kg) of dose 1 of extract. Whereas, Group VI received single dose of paracetamol (250 mg/kg) + (200 mg/kg) of dose 2 of extract. Plant extract was administered 3 h after the administration of paracetamol. All the treatments were instilled orally by means of a gastric tube. The treatments were continued for 7 days and on the eighth day of the experiment all animals were sacrificed under light ether anesthesia. Liver was quickly removed, placed on ice and homogenized in seven volumes of NaCl (0.9%). The homogenates were centrifuged at 4000 × g for 10 min to yield a clear supernatant fraction that was used for the biochemical analysis. 2.6.1. Enzyme assays Serum was obtained from clotted blood samples by centrifugation at 1500 × g for 10 min and the activities of enzymes, GPT and GOT were determined. Catalase (CAT) activity was estimated by following the breakdown of hydrogen peroxide according to the method of Aebi (1983). Superoxide dismutase (SOD) was assayed according to Misra and Fridevich (1972) based on the inhibition of epinephrine auto-oxidation by the enzyme. Ascorbic acid was measured by the method of Natelson (1963). Lipid peroxidation was measured in terms of TBARS following the thiobarbituric acid method of Ohkawa et al. (1979). Non-protein thiol (NPSH) content in the liver homogenate was determined as described by Jollow et al. (1974).
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Table 1 The inhibitory effect of aqueous extracts of Solanum fastigiatum (SF) on iron sulphate induced and sodium nitroprusside (SNP)-induced lipid peroxidation in a rat liver homogenate
Table 2 The inhibitory effect of aqueous extracts of Solanum fastigiatum (SF) on iron sulphate induced and sodium nitroprusside (SNP)-induced lipid peroxidation in a rat brain homogenate
Treatments
SF concentration (g/ml)
TBARS (×100)
TBARS inhibition (%)
Treatments
SF concentration (g/ml)
TBARS (×100)
Normal Control Iron + SF Iron + SF Iron + SF Iron + SF Iron + SF
– – 20 40 60 80 100
0.83 4.31 4.16 3.02 0.92 0.33 0.13
± ± ± ± ± ± ±
0.11 0.1b 0.1b 0.12 0.3 0.04c 0.03c
– – 3.36 30 78.6 92.3 97
Normal Control Iron + SF Iron + SF Iron + SF Iron + SF Iron + SF
– – 20 40 60 80 100
0.72 5.44 3.01 1.18 0.47 0.30 0.174
± ± ± ± ± ± ±
0.11 0.16 0.42 0.1 0.05ab 0.04ab 0.1
– – 44.6 78.2 81.3 91.3 97
Normal Control SNP + SF SNP + SF SNP + SF SNP + SF SNP + SF
– – 20 40 60 80 100
0.83 4.1 3.62 3.4 3.17 2.16 1.72
± ± ± ± ± ± ±
0.11 0.12 0.03a 0.062a 0.09b 0.09b 0.07
– – 11 16.5 22 46.6 57.6
Normal Control SNP + SF SNP + SF SNP + SF SNP + SF SNP + SF
– – 20 40 60 80 100
0.72 5.65 4.9 3.19 2.12 1.7 1.05
± ± ± ± ± ± ±
0.1 0.36 0.12 0.1 0.16 0.11 0.12
– – 13.5 43.6 62.5 70 81.4
TBARS inhibition (%)
Values represent the means ± S.D. (n = 6), TBARS expressed as nmol/g tissue. p < 0.05 by DMRT is significantly different from control group. Values in columns followed by same letters are non-significantly different from each other.
Values represent the means ± S.D. (n = 6), TBARS expressed as nmol/g tissue. p < 0.05 by DMRT is significantly different from control group. Values in columns followed by same letters are non-significantly different from each other.
2.7. Phenolics content
more effective against Fe2+ than SNP-induced TBARS. Similar to the results obtained with tissues, Fe2+ and SNP stimulated TBARS production in phospholipid and aqueous extract reduced (p < 0.05) TBARS production in a concentration dependent manner (Table 3) The extract also exhibited strong antioxidant activity in the DPPH and iron chelation assays (Fig. 1). The IC50 value obtained for DPPH scavenging was 68.96 ± 1.25 g/ml which is comparable to the reference standard ascorbic acid (IC50 = 28.4 g/ml). For iron chelation assay the extract showed maximum inhibition of 48.7% at the lowest concentration. Indeed, the chelation was not found to be concentration dependent (Fig. 1). The total antioxidant capacity of the extract (equivalent to ascorbic acid) was found to be 50.22 ± 0.33 g/ml at 100 g/ml and was increased with increasing concentrations of extract (Fig. 2). Phytochemical analysis of the leaves showed high content of total phenolics (110.67 ± 0.003 mg/g) and flavonoids (2.24 ± 0.004 mg/g; Table 4). The results of TLC analysis also confirmed the presence of two important flavonoids, rutin (Rf = 0.59) and quercetin (Rf = 0.91) in the methanolic extracts of leaves.
The total phenol content was determined by following the method of Singleton et al. (1999). The mean of three readings was used and the total phenol content was expressed in milligram of gallic acid equivalents/g extract. 2.8. Determination of total flavonoids The total flavonoid content as quercetin equivalents/g extract was based on the method reported by Kosalec et al. (2004). 2.9. Thin layer chromatography (TLC) of plant extracts Methanolic extract of leaves was characterized by thin layer chromatography (Silica gel coated TLC plates, Merck; mobile phase, n-butanol:ethyl acetate:water 2:4:4). Chromatograms were evaluated under UV light at 254 and 365 nm to detect the presence of flavonoids. The presence of flavonoids was further confirmed by spraying the plates with 5% AlCl3 in ethanol. Rutin and quercetin were used as standard flavonoids. 2.10. Statistical analysis The results were expressed as mean ± S.D. The data were analyzed statistically by one-way ANOVA and different group means were compared by Duncan’s multiple range test (DMRT); p < 0.05 was considered significant in all cases. The software package Statistica was used for analysis of data. 3. Results 3.1. In vitro assays Ferrous sulphate and SNP caused a significant increase in TBARS production in rat liver homogenates (Table 1) and the aqueous extract of Solanum fastigiatum significantly reduced (p < 0.05) Fe2+ and SNP-induced TBARS production in a concentration dependent manner. Cerebral TBARS production was also stimulated by Fe2+ and SNP and plant extract caused a concentration dependent inhibition of TBARS production (p < 0.05, Table 2). However, Solanum fastigiatum was
Table 3 The inhibitory effect of aqueous extracts of Solanum fastigiatum (SF) on iron sulphate induced and sodium nitroprusside (SNP)-induced lipid peroxidation in phospholipid homogenate Treatments
SF concentration (g/ml)
TBARS (×100)
Normal Control Iron + SF Iron + SF Iron + SF Iron + SF Iron + SF
– – 20 40 60 80 100
1.96 12.21 8.34 7.13 6.74 6.06 6.86
± ± ± ± ± ± ±
0.34 0.03 0.12 0.02a 0.03 0.02 0.14a
TBARS inhibition (%) – – 31.7 41.6 44.8 50.4 43.8
Normal Control SNP + SF SNP + SF SNP + SF SNP + SF SNP + SF
– – 20 40 60 80 100
1.96 7.40 4.48 4.10 3.74 3.38 2.44
± ± ± ± ± ± ±
0.34 0.08 0.12 0.03 0.07 0.04 0.03
– – 29.8 39.4 49.4 54.3 67
Values represent the means ± S.D. (n = 6), TBARS expressed as nmol/g tissue. p < 0.05 by DMRT is significantly different from control group. Values in columns followed by same letters are non-significantly different from each other.
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Fig. 1. DPPH radical scavenging and iron chelation capacity of the extract of Solanum fastigiatum in vitro. Values are mean ± S.D. (n = 3).
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Fig. 3. Hepatoprotective action of Solanum fastigiatum in mice: serum enzyme activity of GPT and GOT among different treatment groups. Values are mean ± S.D. (n = 5); b p < 0.05 versus normal control group and c p < 0.05 versus paracetamol (PM) control group; PM = 250 mg/kg, D1 = 100 mg/kg, and D2 = 200 mg/kg.
Fig. 2. Total antioxidant activity of Solanum fastigiatum leaves measured by phosphomolybdenum assay. Values are mean ± S.D. (n = 3).
3.2. In vivo assays Administration of paracetamol (250 mg/kg) caused significant liver damage as evidenced by the altered serum and liver biochemical parameters (Figs. 3–8). However, the administration of aqueous extract of Solanum fastigiatum at a dose of 100 mg/kg and 200 mg/kg exhibited marked protection against paracetamol-induced liver damage (Figs. 3–8). The aqueous leaf extract of Solanum fastigiatum was found to be practically non-toxic when administered orally to mice and we could not calculate its LD50 because the treatment did not cause any deaths and consequently its LD50 is higher than 4 g/kg body weight. The animals treated with extract did not manifest any overt signs of toxicity and there was no significant difference in the weight of the treated animals compared to the negative control (data not shown). However, safety margin of the drug can be considered below 4 g/kg because this dose caused a small increase (p < 0.05) in the level of GPT and GOT, which indicates a potential hepatotoxicity for very high doses of plant (Fig. 9).
Fig. 4. Hepatoprotective action of Solanum fastigiatum in mice: catalase activity among different treatment groups. Values are mean ± S.D. (n = 5); a p < 0.05 versus normal control group, b p < 0.05, and c p < 0.05 versus paracetamol (PM) control group. PM = 250 mg/kg, D1 = 100 mg/kg, and D2 = 200 mg/kg.
Table 4 Content of phenolics and flavonoids in water and methanolic extracts of Solanum fastigiatum Extracts
Total phenolics (mg/g)
Aqueous Methanol
128.6 ± 0.03 149.1 ± 0.05
Total flavonoids (mg/g) 0.173 ± 0.03 2.24 ± 0.043
Fig. 5. Hepatoprotective action of Solanum fastigiatum in mice: superoxide dismutase activity among different treatment groups. Values are mean ± S.D. (n = 5); a p < 0.05 versus normal control group, b p < 0.05, and c p < 0.05 versus paracetamol (PM) control group. PM = 250 mg/kg, D1 = 100 mg/kg, and D2 = 200 mg/kg.
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Fig. 6. Hepatoprotective action of Solanum fastigiatum in mice: TBARS (nmol/g.tissue) among different treatment groups. Values are mean ± S.D. (n = 5); a p < 0.05 versus normal control group, b p < 0.05 versus normal control group, and c p < 0.05 versus paracetamol (PM) control group. PM = 250 mg/kg, D1 = 100 mg/kg, and D2 = 200 mg/kg.
Fig. 7. Hepatoprotective action of Solanum fastigiatum in mice: non-protein thiol (NPSH) content among different treatment groups. Values are mean ± S.D. (n = 5); b p < 0.05 versus normal control group and c p < 0.05 versus paracetamol (PM) control group. PM = 250 mg/kg, D1 = 100 mg/kg, and D2 = 200 mg/kg.
4. Discussion Oxidative stress is now recognized to be associated with more than 100 diseases, as well as with the normal aging process
Fig. 8. Hepatoprotective action of Solanum fastigiatum in mice: ascorbic acid content among different treatment groups. Values are mean ± S.D. (n = 5); a p < 0.05 versus normal control group, b p < 0.05 versus normal control group, and c p < 0.05 versus paracetamol (PM) control group. PM = 250 mg/kg, D1 = 100 mg/kg, and D2 = 200 mg/kg.
Fig. 9. Effect of different doses of Solanum fastigiatum aqueous extracts on serum parameters (GPT and GOT) in mice. Values are mean ± S.D. (n = 5); *p < 0.05 versus normal control group.
(Ghasanfari et al., 2006). Here Fe2+ and SNP were used as a tool to induce lipid peroxidation. The aqueous extracts of Solanum fastigiatum exhibit good antioxidant activity against two pro-oxidants in all tissues. However, in brain and liver it was more effective against Fe2+ inhibition compared to SNP-induced TBARS. While, in phospholipid homogenate the extract showed high antioxidant activity against SNP as compared to Fe2+ . Increases in the formation of TBARS in Fe2+ (10 M)-induced oxidative stress as compared to the basal suggest possible damage of tissues with an overload of iron. Rats overloaded with iron showed toxic effects such as hepatocellular hypertrophy, cardiomyopathy, pancreatic atrophy, splenic white pulp atrophy, and hemosiderosis in the liver, heart, pancreas and endocrine glands, respectively (Whittaker et al., 1997). The protections offered by the Solanum fastigiatum suggest that the aqueous extract may protect the liver and brain against toxicities resulting from potential overload of iron. Sodium nitroprusside is an anti-hypertensive drug that acts by relaxation of vascular smooth muscle and consequently dilates peripheral arteries and veins. However, SNP has been reported to cause cytotoxicity through the release of cyanide and or nitric oxide (Bates et al., 1990). The protection offered by Solanum fastigiatum extract on tissues (brain and liver) and phospholipids confirms the antioxidant activity of extract and indicate its use in accidental intoxications resulting from the overload with SNP. The present results revealed that the extract of Solanum fastigiatum protects the tissues against the lipid peroxidation at low concentration (less than 100 g/ml) and in most of the cases have the ability to reduce the TBARS production to the basal level. The DPPH radical scavenging activity of the extracts also revealed high antioxidant activity considering the fact that quenching properties were obtained only from the crude extracts with the IC50 value 68.96 ± 1.25 g/ml. Such high free radical scavenging properties of the crude extracts are shown by few other plants (Sabir and Rocha, 2008). The use of iron chelation is a popular therapy for the management of Fe2+ associated oxidative stress in brain. The iron chelating ability of the Solanum fastigiatum is expected to potentially enhance the neuroprotective properties of the plant particularly in view of the fact that iron overload is involved in the pathogenesis of brain diseases such as Alzheimer (Elise and James, 2002). In the phosphomolybdenum assay, which is a quantitative method to evaluate water-soluble and fat-soluble antioxidant capacity (total antioxidant capacity), the extract demonstrated electron-donating capacity showing its ability to act as chain terminators, transforming relative free radi-
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cal species into more stable non-reactive products (Dorman et al., 2003). Here we have demonstrated for the first time that Solanum fastigiatum aqueous extract has and important hepatoprotective effect against paracetamol. This is particularly important in view of the fact that the treatments of acute human intoxications with paracetamol are limited and frequently not effective and relies basically on the use of N-acetylcysteine (Groote and Steenbergen, 1995; Angela et al., 2005). Paracetamol is a well-known antipyretic and analgesic agent, which is safe in therapeutic doses, but can produce fatal hepatic necrosis in experimental animals and humans (Schidt et al., 1997; Amar and Schiff, 2007) and is employed as an experimental hepatotoxic agent. An obvious sign of hepatic injury is the leaking of cellular enzymes into the plasma due to the disturbance caused in the transport functions of hepatocytes (Zimmerman and Seeff, 1970). The estimation of enzymes in the serum is a useful quantitative marker of the extent and type of hepatocellular damage. The mice treated with an overdose of paracetamol developed significant hepatic damage, which was observed by a substantial increase in the concentration of serum hepatic enzymes (GPT and GOT). Administration of Solanum extract (100 mg/kg and 200 mg/kg p.o.) after paracetamol treatment resulted in a significant reduction (p < 0.05) of paracetamol-induced elevation of GPT and GOT and appears to be protective in reducing the injurious effect of paracetamol. We also observed significant increase (p < 0.05) in the levels of TBARS in liver, which was decreased by the administration of the plant extract in a dose dependent manner. The administration of the paracetamol (250 mg/kg, p.o., 7 days) significantly decreased (p < 0.05) the activity of CAT and SOD enzymes, which were restored to normal levels by the subsequent treatment with the plant. In fact, extract treatment alone caused a significant increase (p < 0.05) in the activity of CAT and SOD, which can explain at least in part its hepatoprotective effect. It is a well-known fact that paracetamol toxicity is associated with sharp decrease in glutathione (Gerber et al., 1977) and ascorbic acid content (Dahlin et al., 1984). Here we observed that treatment with extracts of Solanum fastigiatum restored the levels of NPSH and ascorbic acid that were depleted by paracetamol intoxication. In fact, treatment with plant extracts increased the levels of ascorbic acid, indicating an additional mechanism for its hepatoprotective action. The protective action of aqueous extracts Solanum fastigiatum may be attributed to the presence of high content of phenolics and flavonoids (rutin and quercetin), which are well known hepatoprotective agents (Khalid et al., 2002). The data obtained on safety studies suggest that the aqueous extract of Solanum fastigiatum was relatively non-toxic when administered to mice for two consecutive days as the calculated LD50 value was found to be higher than 4 g/kg body weight and since to be non-toxic in humans a single oral dose of 4 g/kg should cause no acute symptoms or death of animals (Zbinden and FluryRoversi, 1981). However, the small increase in the level of serum enzymes (GPT and GOT) at very high dose of the extract suggests that the use of the plant in humans (or phytotherapics) should be accompanied by a detailed and carefully conducted dose response curves in healthy volunteers. Infact, this plant is commonly sold in Brazil by non-informed persons and people use it without having any indication of safe daily or chronic doses. In conclusions, the aqueous extracts of Solanum fastigiatum exhibited protective effect against paracetamol-induced hepatotoxicity and possess anti-lipid peroxidative and free radical scavenging activities. The result supports the use of the plant as described in folk medicine, that the aerial parts of plant can be used to treat liver and gastric disorders, even though it is considered to be the false “jurubeba”. Further studies are required to isolate the active constituents involved in the antioxidant and hepatoprotective activity of the plant.
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