1. ABTS radical scavenging activity
1.1 Principle
The colorless ABTS molecule is converted into the blue-green colored radical, ABTS·+, by oxidation of one electron. Addition of antioxidants to the preformed radical cation reduces it to ABTS, to an extent and on a time-scale depending on the antioxidant activity, the concentration of the antioxidant & the duration of the reaction. Decrease of the absorption after addition of antioxidants is directly proportional to the number of ABTS·+ radicals (Tirzitis, G. and Bartosz, G. 2010).
1.2 Procedure
The method described by Zhang et al. (2011) was used. Briefly, 7 mM ABTS solution and 2.45 mM potassium persulphate solution were prepared in distilled water respectively. Equal volumes of both stocks were mixed and kept at room temperature in dark for 12 to 16 hours. 700 µl of ABTS radical was diluted with 6 ml of methanol to obtain an absorbance of 0.7 ± 0.02 at 734 nm.1000 ppm stock solution of MPGL in methanol was prepared. Dilutions were made to obtain concentration of 100 to 900 µg/ml. 10 µl of extract was added to 1.99 ml of the blue-green ABTS radical solution. The mixture was shaken vigorously and incubated in darkness for 30 minutes. The control consisted of 10 µl of methanol and 1.99 ml of ABTS radical solution. The decrease in absorbance was measured at 734 nm.
Percent inhibition was calculated from the following formula
Percent inhibition = Abs. (Control) – Abs (Test)
1.3 Results:
ABTS radical scavenging activity of MPGL:
MPGL showed scavenging effect on ABTS radical in a dose dependent manner (Fig 1). MPGL showed 73.703% inhibition at the concentration of 1000 µg/ml. The IC50 value is tabulated below.
Fig. 1 Effect of MPGL on ABTS radical scavenging activity
Table 1. IC50 value of MPGL
Extract / IC50 value (µg/ml)(Mean ± S.E.M.)
MPGL / 724.323 ± 16.451
2. In vitro inhibition of advanced glycation end-products
Evaluation of methanolic extract of Punica granatum leaves and fruits on in vitro glycation of protein
2.1 Principle:
When reducing sugars such as glucose react non-enzymatically with amino groups in proteins, lipids and nucleic acids, a series of reactions (known as Maillard reaction) takes place leading to the formation of Schiff bases, Amadori products followed by formation of cross-linked fluorescent and non-fluorescent adducts called advanced glycation end products (AGEs) (Jariyapamornkoon, N. et al., 2013) (Singh, R. et al., 2001). The fluorescent AGEs formation can be determined using a spectrofluorometer.
2.2 Procedure:
In vitro glycation of protein was carried out in accordance to a previously published method (Jariyapamornkoon, N. et al., 2013). Bovine serum albumin (10 mg/ml) was incubated with glucose (500 mM) in 0.1 M phosphate buffered saline (PBS), pH 7.4 containing 0.02% sodium azide, MPGL of different concentration (10-1000 µg/ml) and PBS was added to make up the total volume of 5 ml. Aminoguanidine was used as a positive control for this study. Each solution was incubated in dark at 37°C for 21 days for formation of AGEs. After incubation 200 µl of samples were added from each solution to 96 well plate and fluorescent AGEs were determined by measuring fluorescence intensity (excitation wavelength of 370 nm and emission wavelength of 440 nm) using spectrofluorometer (Synergy, epoch well plate reader). The study was carried out in triplicates.
Percent Inhibition was calculated as follows:
% Inhibition = [(Absorbance of control- Absorbance of sample)/ Absorbance of Control] X 100
2.3 Results
Effect of MPGL on in vitro glycation of protein
MPGL at dose of 40 – 1000 µg/ml and aminoguanidine showed significant decrease in relative fluorescence intensity as compared to negative control group (Fig. 2).
Figure 2: Effect of MPGL on in vitro glycation of protein
Each value is given as Mean ± S.E.M. (n= 3)
* p<0.05; ** p<0.01; *** p<0.001 treatment group compared with negative control; using one-way ANOVA with Dunnett’s test
References:
1. Tirzitis, G. and Bartosz, G. Determination of antiradical and antioxidant activity: basic principles and new insights. Acta Biochimica Polonica Journal. 2010, 57(1), 139-142.
2. Zhang, L.; Fu, Q.; Zhang, Y. Composition of anthocyanins in pomegranate flowers and their antioxidant activity. Food Chemistry. 2011, 127, 1444-1449.
3. Singh, R.; Barden, A.; Mori, T.; Beilin, L. Advanced glycation end-products: review. Diabetologia. 2001, 44, 129-146.
4. Jariyapamornkoon, N.; Yib-chok-anun, S.; Adisakwattana, S. Inhibition of advanced glycation end products by red grape skin extract and its antioxidant activity. BMC Complementary and Alternative Medicine. 2013, 13, 171-179.