# Garlic Extract: Inhibition of Biochemical and Biophysical Changes in Glycated HSA
## 3 Results
### Biophysical Analyses
#### 3.2.2 UV and Tryptophan Spectral Analyses  
Overall spectral analysis was completed for glycated and non-glycated protein molecules by measuring all the samples at 280 nm. Significant differences in the UV intensities were observed between non-glycated (0.43 OD) vs. G-HSA-10W (0.16 OD; \(p<0.001\)) samples  
Figure 4: Freshly isolated PBMCs were incubated with different concentrations of garlic extract (0.781–100 \(\upmu\)g/mL) and durations (0–48 h) of incubations.  
Figure 5: AGE molecule pentosidine specific fluorescence was recorded for non-glycated and glycated HSA samples (**a**). Effect of garlic extract on the formation of AGEs (pentosidine) during glycation of HSA (10 weeks) with different concentrations (0.78–100 \(\upmu\)g/mL) of garlic (**b**). Data presented as mean \(\pm\) SD values. \(t\) test was adopted for comparison between non-glycated vs. glycated HSA samples and significance was calculated.  
followed by G-HSA-5W (0.32 OD; \(p\) < 0.05) (Figure 6a). However, G-HSA-1W did not show a significant change compared to N-HSA. The hypochromicity in UV intensities after 5 and 10 weeks of incubation of the glycation reaction indicated protein folding.  
The UV intensity of G-HSA-10W increased to near UV intensity of N-HSA, when garlic extracts were introduced into the reaction mixture. However, significant increase in the UV intensity was observed mainly at 12.5 \(\upmu\)g/mL of garlic extract. This shift further increased with the increase in extract concentrations (25, 50, and 100 \(\upmu\)g/mL) (Figure 6b). Hence, garlic extract (12.5 \(\upmu\)g/mL or more) reduces the protein folding of HSA molecule and helps in retaining the normal protein structure, which was altered by the glycation.  
HSA protein molecule has a single tryptophan residue. Fluorescence analysis of tryptophan residue indicates site-specific changes in the molecule due to glycation. All the samples were analyzed for fluorescence emission spectra over 290-430 nm, with an excitation wavelength of 285 nm. Emission spectra showed maximum intensity at 330 nm for N-HSA (25.3 AU), G-HSA-1W (23.0 AU), and G-HSA-5W (20.1 AU). However, the G-HSA-10W exhibited 8.7 AU intensity at 320 nm (Figure 7a). This change in the emission spectra, showing a 10 nm blue shift, and a significant fluorescence intensity (N-HSA vs. G-HSA-10W), was due to glycation. Glycation may induce tryptophan microenvironment protein folding, leading the tryptophan residue away from polar to a more nonpolar environment.  
Figure 6: UV-spectra were analyzed for N-HSA, G-HSA-1W, G-HSA-5W, and G-HSA-10W (**a**). Effect of garlic extract on the overall protein structure changes during glycation of HSA (10 weeks) with different concentrations (0.78–100 \(\upmu\)g/mL) (**b**). All the sample readings were recorded in the range of 260–360 nm. Data is presented as mean \(\pm\) SD values. \(t\) test was adopted for the comparison between N-HSA vs. G-HSA-1W, G-HSA-5W, and G-HSA-10W.  
The above result showed significant tryptophan microenvironment alterations in HSA molecules due to glycation. In this result, we examined the inhibition in tryptophan microenvironment changes exerted by the different concentrations of the extract. Extract concentration of 12.5 \(\upmu\)g/mL showed a respectable increase in tryptophan fluorescence (14.0 AU) in the glycation mixture vs. glycation reaction without extract (8.7 AU) (Figure 7b). Moreover, the 25 \(\upmu\)g/mL concentration of extract exhibited both significant increase in the fluorescence intensity (20.1 AU) towards the native protein and blueshift of \(\sim\)10 nm occurs. This result shows higher concentrations (12.5 \(\upmu\)g/mL or more) of garlic extract inhibit changes in the tryptophan microenvironment and induce the exposure of tryptophan residue to a polar environment.