ANTIOXIDANT ACTIVITY IN VITRO AND HEPATOPROTECTIVE EFFECT OF PHLOMIS MAXIMOWICZII IN VIVO

Background: A number of medicinal plants and there compounds played a major role in the treatment of hepatic disorders. They were widely used for the treatment of these disorders, and oxidant stress injury was one of the liver injury mechanisms. The present study evaluated the antioxidant activity and the hepatoprotective effect of each extracts of Phlomis maximowiczii. Materials and Methods: The antioxidant activity was assayed by the methods of ABTS, FRAP and DPPH in vitro. Hepatoprotective effect of P. maximowiczii extracts was examined using carbon tetrachloride-induced acute liver injury in mice. Results: P. maximowiczii n-butanol (PMBU) extract, ABTS (IC50=18.96 μg/mL), DPPH (IC50=25.15 μg/mL), and FRAP (RACT50=2775.6±144.18 μmol/g), showed higher scavenging capacity than that of P. maximowiczii ethyl acetate (PMEA). The n-butanol extract could significantly reduce the level of GPT, GOT and MDA (P<0.05, P<0.001 and P<0.001, respectively) and increase the level of SOD (P<0.001), respectively. Conclusion: The antioxidant activity of n-butanol extract in vitro was related with the level of MDA and SOD in vivo, and hepatoprotective effect of n-butanol extract also had relationship with its antioxidant activity in vivo.


Introduction
As a metabolic organ, the liver is vulnerable to injury by a variety of xenobiotics, such as CCl 4 , ethanol and acetaminophen which are metabolized by cytochrome P450 2E1 (CYP 2E1), (Sun et al., 2001).Carbon tetrachloride (CCl 4 ), as a well-known hepatotoxin used as chemical inducer of experimental liver injury in a range of laboratory animals (Recknagel et al., 1989).The mechanism of CCl 4 -induced acute liver injury is widely accepted as metabolized to a highly reactive trichloromethyl radical (CCl 3 • ), by cytochrome P450 in liver.CCl 3 • in liver, and can leads to lipid peroxidation, as well as to hepatocellular membrane damage (Ohta et al., 1998;Drill, 1952).Natural antioxidants have been accepted widely as preventing the deleterious effects of toxic agents by scavenging free radicals and other reactive oxygen species (Domitrović et al., 2011).
Phlomis maximowiczii, belongs to Labiatae family, is widely distributed in Asia.It has been used in various folk medications for the treatment of inflammatory cold, haemorrhage, and fever (Xie et al., 1996).Fat-soluble ingredients of P. maximowiczii in our previous studies were reported (Gu and Chen, et al., 2012).There is no research of P. maximowiczii on chemical research, hepatoprotective and antioxidant effects.In order to investigate the hepatoprotective and antioxidant effects of P. maximowiczii, P. maximowiczii extracts were assayed using the CCl 4 -induced liver injury mice in vivo, and the methods of ABTS, FRAP and DPPH in vitro.

Scavenging of 2, 2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid)(ABTS)
The different extracts of P. maximowiczii (0.15 mL) were mixed with ABTS (Fluka; USA), radical stock solution (2.85 mL), and incubated at 37 °C.The absorbance was observed at 734 nm after 10 min with PG, BHA and BHT as positive control (Kang and Wang et al., 2010).The percentage inhibition of ABTS •+ was calculated using the formula: % Inhibition = [(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.

Ferric reducing antioxidant power (FRAP) reducing activity assay
The P. maximowiczii (0.2 mL), and fresh prepared TPTZ (Acros organics; USA) stock solution (3.8 mL) were mixed and incubated at 37°C for 30 min.The absorbance was measured at 593 nm (Thipong et al., 2006).Trolox (Aldrich; USA), was used as a reference standard.The standard curve was linear between 25 and 400 mol/L Trolox (R=0.999).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).

Materials and animals in experiments in vivo
Male KM normal rats weighting 20±2 g were obtained from the Experimental Animal Center of Henan Province (Zhengzhou, Hennan, China), (

Biochemical analyses (Lowry et al., 1951)
The protein content in homogenates was assayed by the method of using bovine plasma albumin as a standard.The levels of GOT, GPT, SOD and MDA were measured following the commercial kit's instructions.

Statistical analysis
Statistical analyses were carried out using SPSS 17.0 software.The overall significance of the results was examined using one-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons test.P<0.05 was considered statistically significant.All values were expressed as mean values ± standard deviation (SD).

Assay for free radical scavenging activity in vitro
The antioxidant activity of P. maximowiczii with half inhibitory concentration (IC 50 ), and Trolox equivalent (RACT 50 ), is shown in Table 1.

Effect of PMBU and PMEA on GPT and GOT in serum
The level of GPT and GOT in normal and acute liver injury mice is shown in Table 2.The level of hepatic GPT and GOT in CCl 4 -treated mice increased significantly compared with the normal control (P<0.001), and indicated that the acute liver injury mice model was established.
Compared with model group, the level of GPT and GOT of PMBU group decreased significantly (P<0.05 and P<0.001, respectively), showed that had a therapeutic effect.Compared with bifendate (75 mg/kg), as positive control, intra-gastric administration of PMBU had no significantly difference (P>0.05) and presented dose-dependent manner.(Table 2 and figure 1).

Effect of PMBU and PMEA on MDA and SOD in liver
The effects of different doses of PMBU and PMEA on the level of SOD, MDA in normal and CCl 4 -induced liver injury mice are shown in Table 3.The level of MDA in liver significantly increased in liver injury control mice (P<0.001), and the level of SOD in liver significantly decreased (P<0.001), when compared with normal control group.The level of MDA in liver only in administration of PMEA (400 mg/kg), had no significant reduction (P>0.05), the other treatment groups had significantly reduced (P<0.001).The level of SOD of each treatment groups had significant increase (P<0.001 and P<0.01, respectively), when compared with model group.Compared with bifendate (75 mg/kg), intra-gastric administration of PMBU showed no significant difference (P>0.05) and presented dose-dependent manner.(Table 3 and Figure 2 and 3).

Discussion
Acute and chronic liver diseases is mostly induced by viral hepatitis, alcoholism, iron overload or drug toxicity.Among these types of liver injuries, there is consistent evidence of enhanced production of free radicals and/or a significant decrease in antioxidant defense mechanisms

Figure 1 :
Figure 1: Effect of PMBU and PMEA on GPT and GOT in serum

Figure 2 :
Figure 2: Effect of Phlomis maximowiczii on the level of SOD in acute liver injury in mice.

(
Hoek and Pastorino, 2002).Carbon tetrachloride (CCl 4 ) is a well-established model for screening hepato-protective drugs, with a marked elevation in the serum levels of the aminotransferases enzymes GOT and GPT.Hepatic cells damaged by free radicals and released these enzymes into the blood.Results showed that a significant in the level of GOT and GPT in CCl 4 -treated mice (P<0.001).Levels of GPT and GOT, MDA decreased significantly (P<0.05 and P<0.01 and P<0.001, respectively), and SOD in administration of each dose group of PMBU.Results of scavenging activity of P. maximowiczii showed that n-butanol extract showed higher antioxidant activity than that of ethyl acetate extracts in vitro.

Figure 3 :
Figure 3: Effect of Phlomis maximowiczii on the level of MDA in acute liver injury in mice

Assays for antioxidant properties of extracts in vitro Scavenging of 2, 2-Diphenyl-1-Picrylhydrazyl (DPPH)
ExtractionDried whole plants of about (2.0 kg), was extracted three times with methanol at room temperature for three days.The total methanol extract was filtered and concentrated in-vacuo, and extracted with petroleum ether, ethyl acetate and n-butanol respectively.Solution was concentrated under reduced pressure to yield petroleum ether of P. maximowiczii (PMPE), ethyl acetate of P. maximowiczii (PMEA), and n-butanol extracts (Voucher No: 20090723) whole plant was collected in July 2009, from Tianchi Mountain in Henan Province and identified by Professor Changqin Li (Institute of Natural Products, Henan University).Voucher specimen was deposited at the Institute of Natural Products, Henan University, Kaifeng, China.
(Gong et al., 2012)le, 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 Institute ethical committee guidelines' for Animal Experimentation and Care.Animals were housed in polycarbonate cages.Mice were randomly divided into nine groups with 10 mice in each group; and normal control group, CCl 4 model group, bifendate (75 mg/kg), group, PMBU (600, 300 and 150 mg/kg, respectively), groups and the PMEA (800, 400 and 200 mg/kg, respectively), groups.Mice were administered orally by gastric gavage with different doses of PMBU, PMBU and bifendate at a volume of 10 mL/kg once a day for 8 days.The normal control group and the CCl 4 model group were administered with an equivalent volume of distilled water.On the eighth day, at 2 h after the final administration, except of normal control group, the mice in other groups were intra-peritoneally injected with CCl 4 diluted in olive oil at the dose of 0.05 mL/kg body weight, and the normal control group was injected with an equivalent volume of olive oil alone(Chen et al., 2004).At 16 hr after the CCl 4 injection, each mouse was weighed and then killed under light ether anesthesia for blood collection via puncture of the retro-orbital venous plexus.Serum was obtained from the collected blood by centrifugation immediately after death.Liver homogenate was homogenized with physiological saline.The homogenates were centrifuged at 3000 rpm for 10 min at 4 °C and clear supernatants were used immediately for assessment of MDA and SOD(Gong et al., 2012).

Table 1
Antioxidation in vitro of Phlomis maximowiczii NT unavailable, because of low activity.BHT, BHA and PG were used as positive control.

Table 2
Effect of Phlomis maximowiczii on the level of GOT and GPT in acute liver injury in mice Data expressed as means ± SD (n=10).Bifendate was as the positive control drug.P<0.05, ∆∆ P<0.01 and ∆∆∆ P<0.001, normal group compared with CCl 4 -induced acute liver injury.* P<0.05, ** P<0.01, and *** P<0.001, treated group compared with CCl 4 -induced acute liver injury.