ANTIOXIDANT ACTIVITIES IN VITRO AND HEPATOPROTECTIVE EFFECTS OF NELUMBO NUCIFERA LEAVES IN VIVO

Background: Herbal medicines played a major role in the treatment of hepatic disorders, and a number of medicinal plants and their compounds were widely used for the treatment of these disorders, and oxidant stress injury was one of the mechanism of liver injury. Materials and Methods: Antioxidant activity of Nelumbo nucifera leaves (NU) extracts was assayed by the methods of scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2 ́-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) radical and ferric reducing antioxidant power (FRAP) in vitro. By intraperitoneal injection carbon tetrachloride (CCl4) to establish acute liver injury model in mice, the levels of Glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), superoxide dismutase (SOD) and the content of and maleicdialdehyde (MDA) were detected to evaluate hepatoprotective effect of NU using corresponding test kit. Results: EtOAC (NUEA) and n-BuOH extracts (NUBU) of N. nucifera leaves had good scavenging DPPH and ABTS radical activity and ferric reducing antioxidant power in vitro. DPPH radical scavenging activity and ferric reducing antioxidant power of NUEA (IC50= 6.68±0.29 μg/mL, RACT50=1749.82±67.03 μmol/g) and NUBU (IC50= 4.61±0.01 μg/mL, RACT50=1995.27±135.71 μmol/g ) were higher than that of BHT (IC50=8.76±0.20 μg/mL, RACT50=1581.68±97.41 μmol/g) and Dangfeiliganning (IC50=28.06±0.17 μg/mL, RACT50=1028.55±3.28 μmol/g). ABTS radical scavenging activity of NUEA (IC50= 5.32±0.12 μg/mL) and NUBU (IC50= 8.16±0.27 μg/mL) were higher than that of Dangfeiliganning (IC50= 9.76±0.16 μg/mL). Thus, hepatoprotective effect of NUEA and NUBU was evaluated on CCl4-induced acute liver injury mice. The results showed that the levels of GOT and GPT in each treatment group significantly decreased (p<0.001 and p<0.01, p<0.05, respectively) except for the group of NUEA (130.8 mg/kg) (p>0.05). The contents of malondialdehyde (MDA) in liver in groups of NUEA (523 mg/kg), NUBU (840.5 and 420.5 mg/kg, repectively) had significant decrease (p<0.001 and p<0.05, respectively), and the level of SOD in liver for each treatment group could significantly decrease (p<0.001, p<0.05, respectively). Conclusion: NUEA and NUBU had significantly hepatoprotective effect for Calcium tetrachloride CCl4-induced liver injury, which might be attributable to its antioxidant activity.


Introduction
Liver injury can be induced by various factors, such as CCl 4 , ethanol and acetaminophen, which are metabolized by Cytochrome P450 2E1 (CYP2E1) to generate unstable free radicals and reactive oxygen species (ROS), these free radicals and ROS can induce liver cell apoptosis and necrosis (Sun et al., 2001;Kuzu et al., 2007), and up-regulation of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in necrotic hepatocytes that accelerate the progression of liver cell injury.
Carbon tetrachloride (CCl 4 ) is one of the oldest and most widely used toxins for experimental induction of liver injury in laboratory animals.At present, the mechanism of CCl 4 -induced liver injury is accepted widely, CCl 4 is metabolized to a highly reactive trichloromethyl free radical (CCl 3 -) and chlorine free radical (Cl -) by cytochrome P450 in liver microsome.These free radicals may be to bind to macromolecular in liver cells covalently, and also attack saturated lipids, which can induce lipid peroxidation and lead to hepatocellular membrane damage (Koch et al., 1974).Herbal medicines play a major role in the treatment of hepatic disorders.A number of medicinal plants and their compounds are widely used for the treatment of these disorders (Park et al., 2000;Gong et al., 2012;Wei et al., 2012;Praveen et al., 2009;).Berberine is an isoquinoline alkaloid of the protoberberine type and silymarin is an antioxidant flavonoid complex, derived from the herb used for treatment of hepatic disorders (Domitrovi´c et al., 2011).
Nelumbo nucifera leaves (NU) is a well known Chinese herbal medicine.It distributed throughout china, and all parts of N. nucifera have been used as foodstuffs and Chinese traditional medicines.Moreover, many biological and pharmacological studies have been performed on each part of the plant.Phytochemical researches have shown that flavonoids and alkaloids were the main active components of N. nucifera leaves, for example astragalin, queretin, hyperin, armepavine and coclaurine (Yang et al., 2007;Xiao et al., 2006).
In this study, antioxidant activity of N. nucifera leaves extracts was assayed by methods of DPPH, ABTS and FRAP.By intraperitoneal injection CCl 4 to establish acute liver injury model in mice, the levels of GOT, GPT, SOD and the content of MDA were detected to evaluate hepatoprotective effect of NU.

Animals
Male Kunming normal mice weighing 20 ± 2 g were obtained from the Experimental Animal Center of Henan Province.(Zhengzhou, Hennan, China), (12 h light/dark cycle, 25°C and humidity 45 to 65%) and were fed with standard rodent diet and water ad libitum.All animal procedures were approved by the ethical committee in accordance with the 'Institute Ethical Committee Guidelines' for animal experimentation and care (HNPR-2009-05003).Animals were housed in polycarbonate cages.The air dried Nelumbo nucifera leaves (3.25 kg) were extracted three times with acetone water solution (acetone:distilled water=7:3) for 7 days at room temperature.After evaporation of solvent in a vacuum, the concentrated extract was suspended in water and extract with petroleum ether, EtOAC and n-BuOH, respectively.The solution was concentrated under reduced pressure to yield petroleum ether extract (55 g, NUPE), EtOAC extract (81.5 g, NUEA) and n-BuOH extract (175 g, NUBU), respectively.

Antioxidant activity using DPPH assay
DPPH radical scavenging activity was assayed according to the method of Kang (Kang and Wang, 2010).0.1 mL different extracts of NU in methanol had been mixed with 3.5 mL DPPH methanol solution (0.06 mmol/L).The solution was measured at 515 nm after 30 min at room temperature with PG, BHA and BHT as positive control.The antioxidant activity was expressed as an IC 50 value, that is the concentration in μg/mL that scavenging DPPH• absorption by 50%, and was calculated from the concentration-effect linear regression curve.

Antioxidant activity using ABTS assay
Scavenging activity on ABTS radical of different extract of NU was evaluated in accordance with the literature (Li et al., 2011;Yue and Kang et al., 2011).The different extracts (0.15 mL) were mixed with ABTS radical stock solution (2.85 mL).The absorbance was observed at 734 nm after incubated 10 min at 37 0 C with PG, BHA and BHT as positive control.The Scavenging rate of ABTS •+ was calculated using the formula: Scavenging rate (%) = [(A 0 -A 1 )/A 0 ]×100, where A 0 was the absorbance of the control and A 1 was the absorbance of the sample and the standard compound.

Hepatoprotective effect in vivo
Ninety KM male mice (20±2 g) were randomly divided into nine groups of ten each.Group 1 (normal control) was treated with distilled water.Group 2 (liver injury model control) was treated with distilled water.Group 3 was fed Dangfeiliganning capsule (1400 mg/kg) as positive control.Group 4, 5 and 6 were given 523, 261.5 and 130.8 mg/kg of NUEA, respectively.Group 7, 8 and 9 were received 840.5, 420.5 and 210 mg/kg of NUBU, respectively.Administration adopted gastric lavage daily for 8 days.On the eighth day, except for the normal group, other group animals were given a single dose of 0.1 ml/10 g b.w.CCl 4 that was diluted with corn oil by intraperitoneally injection after 2 h of the last administration.All groups were added to the drinking water but not feeding for 16 h.Blood was collected from mice eyeball for the levels of GPT and GOT in serum.The liver was removed to prepare liver homogenate solution of 10% and 1% for determining the content of MDA and the level of SOD by tissue homogenizer, respectively.The content of protein was determined by the method of Comassie brilliant blue G-250.The levels of GPT, GOT and SOD and the content of MDA were determined by the requirements of kits.

Statistical analysis
All data were statistically evaluated with SPSS 17.0 software.Statistical comparisons were compared by one-way analysis of variance (ANOVA).The results were considered statistically significant if the p values were 0.05 or less.All results are expressed as mean ± standard deviation (SD) for ten mice in each group.

Effect of NUEA and NUBU on the levels of GPT and GOT in serum
As shown in Table 2 and Fig. 1, compared with blank of group, the levels of GPT and GOT were significantly increased (p<0.001) in CCl 4 group, which indicated CCl 4 -induced liver injury mice model was established successfully.Compared with CCl 4 group, the levels of GPT and GOT (p<0.01 and p<0.05, respectively) in high and middle doses of NUEA groups (523 mg/kg and 261.5 mg/kg) were significantly decreased.
The low dose of NUEA group (130.8 mg/kg) had no significantly increased (p>0.05).The levels of GPT and GOT (p<0.001 and p<0.01, p<0.05, respectively) in three doses of NUBU groups were significantly decreased.As shown in Table 3, Fig. 2 and Fig. 3, compared with blank of group, the content of MDA in liver homogenate solution was significantly increased in (p<0.001) and the level of SOD in liver homogenate solution was significantly decreased (p<0.001)liver injury control mice, which indicated that CCl 4 could cause oxidative stress in mice.Compared with CCl 4 group, the content of MDA was significantly decreased (p<0.01 and p<0.05, respectively) in high dose of NUEA group (523 mg/kg) and high and middle doses of NUBU groups (840.5 and 420.5 mg/kg, respectively), and the other treatment groups had no significantly increased (p>0.05).The levels of SOD in each treatment group were significantly increased (p<0.001,p<0.01 and p<0.05, respectively).Cell membrane was protected and prevent GPT and GOT in the endochylema to plunge into the serum, so the levels of GPT and GOT was decreased in serum.In addition, a significant increase in the level of SOD as a defense against the presence of oxygen free radicals, and reduced the damage of cells.So, antioxidant was one of hepatoprotective mechanism to decrease lipid peroxidation and oxidant stress for NU hepatoprotective effects.
According to the literature(Li et al., 2011;Yue and Kang et al., 2011), the NU extracts (0.2 mL) and fresh prepared TPTZ stock solution (3.8 mL) were mixed and incubated at 37 0 C for 30 min.The absorbance was measured at 593 nm.Trolox was used as a reference standard.The standard curve was linear between 25 and 400 μmol/L Trolox.Results were expressed in μmol Trolox equivalents (TEAC) (TE)/g sample.In this study, RACT 50 was used to express Trolox equivalent (RACT 50 = the concentration of Trolox cleared 50% free radical/ the concentration of compound or condensate cleared 50% free radical).

Figure 2 :
Figure 2: Effect of NUEA and NUBU on the levels of MDA in liver tissue

Figure 3 :
Figure 3: Effect of NUEA and NUBU on the contents of SOD in liver tissue

Table 1 :
Antioxidant activity of NU extracts by DPPH, ABTS and FRAP assay Dangfeiliganning capsule is one of the Chinese patent drugs, not include adjuvant and can been detected in antioxidant capability.
B: Three positive Controls: Propyl gallate (PG), Butylated hydroxyanisole(BHA) and Butylated hydroxytoluene(BHT)are finest antioxidants, have strong toxicity and often use in antioxidant experiments in vitro.

Table 2 :
Effect of NUEA and NUBU on GPT and GOT in acute liver injury mice

Figure 1 :
Effect of NUEA and NUBU on the level of GPT and GOT in serum.