Phytochemical screening and antioxidant activity of Terminalia muelleri benth. leaf extract

In the present investigation, leaves of Terminalia muelleri were assayed for their phytochemical constituents, as well as its free radical scavenging activity. The antioxidant effect was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) radical scavenging assay, the phytochemical constituents were screened to assess their corresponding effect on antioxidant activity. The results showed that the ethanol soluble fraction of T. muelleri possesses a potent radicalscavenging activity using the DPPH • (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assay (IC50 value = 2.7 μg/mL, while the standard ascorbic acid has an IC50 value = 10.5 μg/mL).


INTRODUCTION
Free radicals including superoxide anions, hydrogen peroxide, nitric oxide, and hydroxyl radical are chemically unstable atoms; they are generated during normal cellular function and are part of the natural physiological process of all living beings.The imbalance between the generation of reactive oxygen species (ROS) and the antioxidant enzymes, results in damage to lipid cells, proteins, and DNA [1].ROS have a crucial role in the development of oxidative stress, which is considered as the major cause of numerous diseases, such as cancer, diabetes, liver and kidney impairment, cardiovascular diseases and aging.
Natural and synthetic antioxidants can scavenge these free radicals.Synthetic antioxidants have been reported to be toxic.Therefore, natural antioxidants have gained much attention nowadays because they are considered safer.The high antioxidant capacity of plants may be due to certain phytochemical constituents, including flavonoids, tannins, and other phenolic compounds.These natural antioxidants act as reducing agents, hydrogendonating antioxidants, and singlet oxygen quenchers owing to the presence of conjugated aromatic rings [2, 3].The main objective of this study was to determine the phytochemical constituents of powdered leaves of T. muelleri, as well as investigate the free radical scavenging activity of T. muelleri leaf extract.

Plant material
Fresh leaves of Terminalia muelleri Benth.(Combretaceae) were collected in November 2012 from trees grown in the Zoo Garden, Giza, Egypt.The plant was kindly authenticated by Eng.Therese Labib, the consultant at Orman Botanical Garden, Giza, and National Gene Bank at the Ministry of Agriculture, Egypt.A voucher specimen of T. muelleri Benth was deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt (ASU TMC2012).

Extract preparation
The air-dried powdered leaves of T. muelleri were extracted with 80% aqueous MeOH.The total extract was concentrated and freeze-dried to obtain a dry powder, then defatted with petroleum ether.The remaining part was concentrated and freeze-dried to obtain a dry powder, which was dissolved in absolute EtOH.
The EtOH-soluble portion was then concentrated and freeze-dried to obtain the total extract dry powder abbreviated as (TMEF).

Methods for phytochemical screening
Powdered leaves of T. muelleri were screened for the following phytoconstituents: carbohydrates and/ or glycosides, flavonoids, sterols and/ or triterpenes, saponins, tannins, alkaloids, and anthraquinones according to the standard procedures [11, 12].

Methods for DPPH
• radical scavenging assay The antioxidant effect of the TMEF and pet.ether-fraction was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) radical scavenging assay, according to the method described by Bourgou, Ksouri [13] with slight modification.Tested samples and L-ascorbic acid (standard), both prepared at different concentrations or ethanol in case of control, were added to 0.25 mM freshly prepared DPPH • ethanolic solution.
The mixture was slightly shaken and kept in dark for 30 min at room temperature, the absorbance was determined against a blank at 517 nm using a UV Spectrophotometer.All assays were conducted in triplicates.

Percentage inhibition of free radical DPPH
• was calculated as follow: Where: A Control is the absorbance of the control reaction.
A Sample is the absorbance in the presence of the tested samples.
To calculate the IC50 [14] the concentration of the substrate that causes 50% loss of the DPPH • activity (color), different concentrations of the tested samples where used and the percentage inhibition was calculated.The IC 50 values were calculated according to the equation for Boltzmann sigmoidal concentration-response curve using the nonlinear regression fitting models (Graph Pad, Prism Version 6, La Jolla, CA, USA).From the table, it can be concluded that the phytochemical constituents of T. muelleri include tannins, flavonoids, sterols and /or triterpenes and carbohydrates and/or glycosides.Also, results revealed that this species most probably doesn't contain alkaloids, anthraquinones, and saponins.

Results of the antioxidant activity of ethanol-soluble fraction (TMEF)
The IC 50 (the concentration that inhibits 50% of the absorbance of DPPH • ), was determined from the graph plotted for the % inhibition against the concentration (Fig. 1).TMEF IC 50 was 2.7 μg/mL compared to 60 μM ≈ 10.5 μg/mL for ascorbic acid.
It could be concluded from the obtained values that the ethanol-soluble fraction (TMEF) of T. muelleri leaf extract showed a more potent antioxidant activity when compared to the standard ascorbic acid.

CONCLUSION
This study demonstrated the potent antioxidant activity of T. muelleri leaf extract which might be attributed to its high tannins and flavonoids content.The wide use of genus terminalia in traditional medicine for the treatment of various diseases may be in part due to their antioxidant potency.

Conflict of Interest
We declare that we have no conflict of interest.

Table 1
Results of phytochemical screening