International Journal of Drug Development and Research

Key words

Aphanamixis polystachya, Free-radical scavenging, Ferric Reducing Ability.

Introduction

Aphanamixis polystachya (Wall) Parker also familiar as Amoora Rohituka, belonging to the family Meliaceae is the large handsome tree continues to holding green leaves throughout the year, with an umbrella shaped or dense spreading crown. It has a straight cylindrical bole up to 15m in height and 1.5-1.8m in girth. It is distributed in the sub-Himalayan tract from Gonda (Uttar Pradesh) eastwards to Bengal, Sikkim, Assam up to 6000ft and in Western ghats, chota Nagpur, Konkarn, Andamans and adjoining hill ranges from the Poona district southwards to Tinnevelly up to 3500ft [1].

The stem barks of Aphanamixis polystachya is used traditionally in treatment of tumors, cancer, skin and spleen diseases, leprosy, diabetes, opthalmopathy, intestinal worm, jaundice and rheumatism [2]. The hepatoprotective activity [3], antimicrobial, antiviral and antibacterial activity of isolated limonoid rohitukin [4,5] and cytotoxicity of amoorastatin were also established [6]. Aphanamixis polystachya has disclosed the presence of Aphanmixis a triterpenes [7], tetranortriterpenoid i.e. aphanamixinin, sterol, saponins [8], flavanone and anthraquinone glycosides [9].

Material and Methods

Plant Material

The stem bark of Aphanmixis polystachya family meliaceae was collected from Forest Research Institute, Dehradun (Uttarakhand) and authenticated by Dr. Arvind Bhardwaj, who is a Botanist. The stem barks were also authenticated by comparison of macroscopic and microscopic characteristics with monograph of Pharmacopoeia [10].

Prepration of Extract

About 500gm of stem barks were washed, air dried and ground into course powder. It was extracted in soxhlet apparatus with alcoholic (95.0%), aqueous methanol (methanol: water, 50:50) and petroleum ether solvent for 36 hours. The solvents were then removed under pressure to yield brownish residue (yields sequentially: 12.51%, 05.82% and 02.41% w/w with respect to dried stem barks) [11]. Now the alcoholic, aqueous methanol and petroleum ether extracts were studied for in vitro antioxidant activity.

DPPH free-radical scavenging activity: DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical-scavenging activity was measured by the method of Szabo et al. [12]. The reaction mixture contained 1.5×10-7M Methanolic solution of DPPH and various concentrations of the test substances and were kept in dark for 50min. Optical Density (OD) of the samples was measured at 517nm against a blank and IC50 values were calculated using linear regression analysis.

FRAP assay: The Ferric Reducing Antioxidant Power (FRAP) of various extracts of A. polystachya was performed based on the method of Benzie and Strain [13]. The assay mixture contained 2.5mL of 300mM acetate buffer at pH 3.6, 0.25mL of 10mM TPTZ solution in 40mM HCl, 0.25mL of 20mM FeCl3 and test substances in 0.1mL water or methanol. The absorbance was measured after 30min incubation at 593nm.

Standard graphs were constructed using known concentrations of ferrous salt in water/methanol to replace FeCl3. All tests were run in triplicate and mean values were used to calculate EC1 values. EC1 is defined as concentration of an antioxidant having a ferric reducing ability equivalent to that of mM ferrous salt.

Result and Discussion

DPPH free radical scavenging activity: The results of the DPPH scavenging activity of A. polystachya bark extracts are shown in [Fig. 1]. The IC50 values exhibited that the scavenging ability of alcohol (>25μg mL-1), aqueous methanol (5.33μg mL- 1) and petroleum ether (>25μg mL-1) extracts was comparable to Vitamin C (4.50μg mL-1) [Table 01].

FRAP assay: A. polystachya bark extracts exhibited superior ferric reducing antioxidant power as depicted in [Fig. 2], compared to that of vitamin C. The EC1 values indicated that the ferric reducing antioxidant potential of alcoholic extract was about 2 fold higher compared to vitamin C. The FRAP EC1 values were found to be 13.8, 18.6 and >25μg mL-1 for alcoholic, aqueous methanolic extracts of A. polystachya and Vitamin C respectively whereas petroleum ether extract did not exhibited ferric reducing antioxidant power [Table 01].

The DPPH test provided information on the reactivity of test compounds with a stable free radical. Because of its odd electron, 2, 2-Diphenyl- Picryl Hydrazyl radical (DPPH) gives a strong absorption band at 517 nm in visible spectroscopy (deep violet colour). The efficacies of anti-oxidants are often associated with their ability to scavenge stable free radicals. In the present study, alcoholic, aqueous methanol and petroleum ether extracts exhibited >25μg mL-1, 5.33μg mL-1 and >25μg mL-1 DPPH radical scavenging activity with IC50 values respectively in comparison to vitamin C (IC50 4.50μg mL-1). FRAP assay measures the reducing ability of antioxidants against oxidative effects of reactive oxygen species. Electron donating anti-oxidants can be described as reductants and inactivation of oxidants by reductants can be described as redox reactions. Total antioxidant power may be referred analogously to total reducing power. In the current study methanol and aqueous methanol extracts of A. polystachya exhibited 13.8 and 18.6μg mL-1 antioxidant power with EC1 values respectively whereas Vitamin C having EC1 >25μg mL-1.

CONCLUSION

In the present investigation, we evaluated comparative antioxidant activity of various A. polystachya extracts in comparison to Vitamin C. Alcohol and aqueous methanol extracts exhibited up to 3 fold better efficacy in various in vitro antioxidant assays. These antioxidant studies proved superior efficacy of A. polystachya extracts. It is a safe and effective intervention for free radical mediated diseases.

Tables at a glance

Table 1

Figures at a glance

Figure 1 Figure 2

References

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