Department of Medical Biochemistry, Delta State University, Abraka, Nigeria

Article Publishing History

Introduction

Herbs   have   been   investigated   for   their   antioxidant properties (Gazzaneo et al., 2005) and medicinal plants containing  active  chemical  constituents  with  high  anti- oxidant  activity  play  important  role  in  prevention  of various  degenerative  diseases  (Lukmanul  et  al.,  2008). Antioxidants can abstract lone electron from free radi- cal molecules such as reactive oxygen species, ROS and help humans to control these harmful substances. ROS, are usually produced in the body by chemical reactions which occur during normal or pathological cellular proc- esses (Lakenbrink et al., 2000 and Onyesom et al., 2015). Excess formation of these ROS can overwhelm body defense and cause oxidative stress. Oxidative stress plays significant roles in processes of ageing and pathogenesis of numerous diseases like diabetes, cancer, neurodegen- erative and respiratory tract disorders (Anderson et al., 2000). Halliwell (1996), opined that the sum of endog- enous and food derived antioxidants represents thetotal antioxidant capacity of a system. The role of antioxidant is to detoxify reactive oxygen intermediates in the body (Delay, 1993). Therefore,improved antioxidant status can minimize oxidative stress and associated damages. This delays or decreases the risk of developing free radical induced diseases.

Protective antioxidants bestowed by many plant extracts and products make these agents promising therapeutic drugs for free radicals induced pathologies. Phyllanthus amarus (P. amarus) is a medicinal plant of the family Euphorbiacae. It has about 800 species which are found in tropical and subtropical regions of the world including Nigeria (Mazumderetal., 2006). Phyllan- thus amarus is used as a chemoprotective agent (Kumar and Kulta, 2005), and it has been observed to exhibit hypoglycaemic property (Kussuya et al., 2003). Recently we have demonstrated that crude ethanolic leaf extract of P. amarusrestored renal dysfunction associated with P. berghei malarial parasite- induced oxidative stress in experimental mice (Onyesom et al., 2015). P.amarus has been reported to contain antioxidant phytocompounds, but this study, however, investigated the bioavailability and impact of these substances in graded crude ethanolic leaf extract on serum total antioxidant capacity and asso- ciated levels of oxidative stress in experimental mice.

Materials And Methods

Harvesting And Preparation Of Plant Extract

Fresh whole plants of wild type Phyllantusamarus grow- ing in uncultivated farmland in Abraka, Ethiope East Local Government Area of Delta State, Nigeria were obtained in May, 2015 and authenticated (No: FHI: 109728) in the Herbarium Unit, Forest Research Institute of Nigeria, Ibadan. Crude ethanolic leaf extract of the harvested plant was prepared as earlier described (Onye- som et al., 2015).

Animal Grouping And Extract Administration

Forty (40) adult Swiss albino mice of mixed sexes weighing between 20-30g were assigned into four (4) groups (n=10/group). Group 1: Control (was given pla- cebo — normal saline), Groups 2, 3 and 4: Experimental (were administered placebo — 150, 300 and 450mg/kg/d of P. amarus ethanolic leaf extract, respectively). The graded doses of extract were prepared and administered as already documented (Onyesom et al., 2015).

Animal Sacrifice and Collection Of Specimen

After 7 days of extract administration and another 14 days of observation, the mice were fasted overnight and sacrificed (n=5 mice per each time) the next day under chloroform anaesthesia.Whole blood was collected by heart puncture and centrifuged (Cent 80D, Serico, China) to obtain serum which was used for the biochemical analyses of total antioxidant capacity, TAC and malond- ialdehyde, MDA levels in blood.

Serum TAC was determined by the Trolox Equivalent Antioxidant Capacity (TEAC) method described by Miller et al., (1993) and MDA level was estimated by assess- ing amount of Thio Barbituric Acid Reacting Substances (TBARS) (Ohkawa et al., 1979).

Statistical  Analysis

Data were assessed by the one way analysis of variance (ANOVA) and Dunnett’s post hoc test using SPSS soft- ware package version 20. Level of significant difference was established at p<0.05.

Results and Discussion

The results obtained from the investigation into  the total antioxidant capacity and malondialdehyde levels in serum of mice administered with graded crude eth- anolic leaf extract of Phyllanthus amarusare presented in Table 1.

Table 1 showed the data on total antioxidant capacity, TAC as well as malondialdehyde, MDA levels in experi- ment mice administered graded doses of crude ethanolic leaf extract of Phylanthus amarus.

Table 1: Total antioxidant capacity, TAC and levels of malondialdehyde, MAD in serum of experimental mice administered varying doses of crude ethanolic leaf extract of Phyllanthus amarus
TAC(mM)  MDA(μM)
Group	7 Days	21 Days	7 Days	21 Days
1.	0.13±0.06a	0.09±0.02a	38.33±4.73a	40.33±1.53a
2.	0.29±0.05b	0.19±0.05b	26.33±3.51b	30.67±6.66b
3.	0.30±0.04b	0.23±0.03b	23.33±2.50b	27.67±3.72b
4.	0.29±0.01b	0.25±0.06b	28.67±6.66b	31.67±3.51b
Values are expressed as Mean±SD for n=5 mice. Values that bear another superscript on a column differ significantly (p<0.05). Group 1 = Control (given placebo— normal saline) Group 2 = Experimental (treated with 150mg/kg P. amarus) Group 3 = Experimental (treated with 300mg/kg P. amarus) Group 4 = Experimental (treated with 450mg/kg P. amarus) TAC = Total antioxidant capacity MDA = Malondialdehyde

The administration of varying doses (150, 300 and 450mg/kg/d) of Phyllanthus amarus crude ethanolic leaf extract to experimental mice significantly (p<0.05) increased serum total antioxidant capacity, TAC, but reduced (p<0.05) levels of malondialdehyde, MDA (an oxidative stress  / lipid peroxidation  biomarker) after 7days of administration and another 14 days of obser- vation when compared with control values at the 5% probability level.

Herbs have been investigated for their antioxidant properties (Gazzaneo et al., 2005). Medicinal plants hav- ing active chemical constituents with good antioxidant property play significant role in prevention of various (degenerative) diseases (Lukmanul et  al.,  2008).  Natu- ral antioxidants from plant sources are potent, safe and harmless because of their low toxicity reports (Calixtoe- tal., 1998; Ogbonon etal., 2008; Onocha and Ali, 2010).

This study assessed total antioxidant capacity and lev- els of oxidative damage in serum of mice administered crude ethanolic leaf extract of Phyllanthus amarus. The estimation of malondialdehyde, MDA levels was used to ascertain the levels of oxidative damage because MDA is one of the final products of polyunsaturated fatty acids (PUFAs) peroxidation in cells. An increase in free radicals causes overproduction of MDA. Hence, malon- dialdehyde level is commonly used as oxidative stress biomarker.

The results (Table 1) indicate that Phyllathus amarus crude ethanolic leaf extract administered to experimental mice in apparent good health for seven days and obser- vation for another fourteen days thereafter induced an increase in total antioxidant capacity resulting in decreased levels of malondialadehyde. These changes were found to be significant (p<0.05) when compared with control values.

Therefore, oral administration of the P. amarus leaf extract served as a factor that improved antioxidant defense and significantly reduce oxidative stress.

The phytochemicals identified in theleaf of Phyllan- thus amarus include flavonoids, tannins, saponins, alka- loids, terpenoids, gycosides and anthroquinones(Faremi et al., 2008; Onyesom et al., 2015). Flavonoids from this plant have been shown to possess several pharmaco- logical properties such as anti-inflammatory (Joy and Kuttan, 1998; Kassuya etal., 2003; Adeneye, 2006) and antioxidant activities (Wampa et al., 2012).

Total antioxidant capacity, TAC of systems include the summation of both endogenous and food – derived anti- oxidants which involve some enzymes such as superox- ide dismutase, catalase and glutathione peroxidase, and arrays of small macromolecules like ascorbic acid, toco- pherol, carotene, reduced glutathione (Halliwell, 1996).

Antioxidants interact with free radicals including reactive oxygen species ROS — which are usually pro- duced by the body as a result of chemical reactions dur- ing normal cellular processes (Lakenbrink et al., 2000) in order to terminate their cell damaging activities. So, increased activities of ROS can lead to oxidative stress are known to play significant role in the process of age- ing and pathogenesis of numerous diseases like diabetes, cancer, neurodegenerative diseases and respiratory tract disorders (Anderson et al., 2000).Improved antioxidant status therefore, helps to minimize oxidative  damage and this could delay or decrease the risk of developing many age related free – radical induced diseases.

Evidence indicates that Phyllanthus amarus crude ethanolic leaf extract contributes to the improvement of antioxidant defense which invigorated the blood and provide health benefits at low, moderate and high doses.

Conclusion

On the whole, crude ethanolic leaf extract of P. amarus was observed to improve antioxidant defense, reduced oxidative stress and invigorated the blood in experi- mental mice. This ability could be due to the bioactivi- ties of identified phytochemicals especially phenolics (flavonoids and tannins) which have been observed to display significant antioxidant activity (Ettebong et al., 2012; Sen and Batra, 2013). As a corollary, this study hereby confirms the bioavailability and bioactivity of P. amarus antioxidant phytochemicals. The antioxidant phytochemicals (flavonids and tannins) should be puri- fied and further studied in order to identify the chemical compounds that possess the invigorating property.

Acknowledgements

We gratefully acknowledge the services of Affamefune Biomedical Consult, Abraka, Delta State.

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