2.3.5. Spectral Studies

**Figure 6.** Percentage amyloid structure vs. concentration of garlic extract. Native HSA and G-HSA were incubated for 10 weeks as negative and positive controls, respectively. G-HSA was incubated with various concentrations (0–100 µg/mL) of garlic extract (green). The results are presented as means ± SEM (*n* = 3). All the percentage amyloid structure inhibition results (0.78–100 µg/mL garlic extract) were compared with G-HSA sample. Comparison between two groups was performed based on *t* test, and significance was defined as \* *p* < 0.05*.* UV spectral studies were conducted for all the protein samples. HSA incubated with glucose has been reported to have significant decrease in absorption peak at 280 nm as compared with the native HSA, indicating considerable hypochromicity. The observed hypochromicity of glycated sample at 280 nm might be attributable to an alteration in the protein microenvironment and the change in aromatic amino acids. Glycated HSA samples incubated with the extract (50 µg/mL) showed significantly increased UV absorption (Figure 7).

2.3.5. Spectral Studies

(Figure 7).

**Figure 7.** Absorbance of G-HSA vs. concentration of garlic extract. Native HSA and G-HSA were incubated for 10 weeks as negative and positive controls, respectively. G-HSA was incubated with various concentrations (0–100 µg/mL) of garlic extract (green). The results are presented as means ± SEM (*n* = 3). Change in absorbance results (0.78–100 µg/mL garlic extract) were compared with G-HSA sample. Comparison between two groups was performed based on *t* test, and significance was defined as \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. **Figure 7.** Absorbance of G-HSA vs. concentration of garlic extract. Native HSA and G-HSA were incubated for 10 weeks as negative and positive controls, respectively. G-HSA was incubated with various concentrations (0–100 µg/mL) of garlic extract (green). The results are presented as means ± SEM (*n* = 3). Change in absorbance results (0.78–100 µg/mL garlic extract) were compared with G-HSA sample. Comparison between two groups was performed based on *t* test, and significance was defined as \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

The formation of AGEs in the glycated samples was detected using AGE pentosidine-

UV spectral studies were conducted for all the protein samples. HSA incubated with glucose has been reported to have significant decrease in absorption peak at 280 nm as compared with the native HSA, indicating considerable hypochromicity. The observed hypochromicity of glycated sample at 280 nm might be attributable to an alteration in the protein microenvironment and the change in aromatic amino acids. Glycated HSA samples incubated with the extract (50 µg/mL) showed significantly increased UV absorption

specific autofluorescence. For each sample, the specific fluorescence of AGEs was observed to be in the range of 400–480 nm. The fluorescence intensity vs. wavelength (400– 480 nm) spectra were found to be rather wide (data not shown), which might be attributable to the variety of fluorescent molecules created during the glycation process. Extract containing samples showed a steady reduction in AGE-specific fluorescence at 450 nm as the extract concentration increased in the incubated samples (Figure 8). The fluorescence intensity of HSA treated with glucose was maximum at 450 nm. Our data show that garlic extract exhibited protection against AGEs synthesis by glycation. Protection level increased as the concentration of garlic extract increased. The formation of AGEs in the glycated samples was detected using AGE pentosidinespecific autofluorescence. For each sample, the specific fluorescence of AGEs was observed to be in the range of 400–480 nm. The fluorescence intensity vs. wavelength (400–480 nm) spectra were found to be rather wide (data not shown), which might be attributable to the variety of fluorescent molecules created during the glycation process. Extract containing samples showed a steady reduction in AGE-specific fluorescence at 450 nm as the extract concentration increased in the incubated samples (Figure 8). The fluorescence intensity of HSA treated with glucose was maximum at 450 nm. Our data show that garlic extract exhibited protection against AGEs synthesis by glycation. Protection level increased as the concentration of garlic extract increased. *Molecules* **2022**, *27*, x FOR PEER REVIEW 8 of 19

Garlic extract (µg/mL)

gether with garlic extract with varying concentrations (0–100 µg/mL) was investigated using a Jasco J 810 spectropolarimeter (Table 2). The presence of secondary structural elements was estimated as relative percentages by using the Chen and Yang equation [19] via a computer data processor. CD spectra were recorded for all the samples at a similar wavelength range (200 to 280 nm). As we have shown previously, there is a significant change in α-helix (−10.5%) and β-sheet (+14.9%) structures upon glycation of HSA [1]. Inhibition in the change of both α-helix (−2.8%) and β-sheet (+4.6%) structures of glycated samples were observed when incubated with garlic extract of 100 (µg/mL). Significant decreases in all the secondary structure (α-helix (*p* < 0.05), β-sheet (*p* < 0.01), β-turns (*p* < 0.01), and random coils (*p* < 0.05)) changes in glycated HSA samples were observed at 25 µg/mL concentration of garlic extract. Furthermore, the highest reduction in all the secondary structures was achieved at 100 µg/mL concentration of extract used (Table 2).

**Table 2.** Secondary structure composition of N-HSA, G-HSA, and G-HSA incubated with different

39.8 ± 0.3 \* (−6.8%)

28.8 ± 0.2 \* (+9.9%)

19.1 ± 0.3 \* (+3.2%)

12.3 ± 0.4 (−2.4%)

The values are in percentage. Each sample was read in triplicate. Data are mean ± standard deviation. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 vs. control (N-HSA). Values in parentheses represent the percentage change in the secondary structure from N-HSA. Percentage decrease and increase

40.5 ± 0.3 \* (−5.2%)

28.1 ± 0.2 \*\* (+7.3%)

19.0 ± 0.3 \*\* (+2.7%)

12.4 ± 0.4 \* (−1.6%)

40.9 ± 0.3 \*\* (−4.2%)

27.9 ± 0.2 \*\* (+6.5%)

18.8 ± 0.3 \*\* (+1.6%)

12.4 ± 0.4 \* (−1.6%)

41.5 ± 0.3 \*\*\* (−2.8%)

27.4 ± 0.2 \*\*\* (+4.6%)

18.6 ± 0.3 \*\* (+0.5%)

12.5 ± 0.4 \*\*\* (−0.8%)

40.8 ± 0.3 \*\* (−4.4%)

27.8 ± 0.2 \*\* (+6.1%)

19.0 ± 0.3 \*\* (+2.7%)

12.4 ± 0.4 \* (−1.6%)

**- 0.78 1.56 3.12 6.25 12.5 25 50 100 AG** 

38.8 ± 0.3 (−9.1%)

29.6 ± 0.2 (+13.0%)

19.2 ± 0.3 (+4.3%)

12.3 ± 0.4 (−2.4%)

**Figure 8.** AGE-specific fluorescence intensity at 450 nm vs. concentration of garlic extract. Native HSA and G-HSA were incubated for 10 weeks as negative and positive controls, respectively. G-HSA was incubated with various concentrations (0–100 µg/mL) of garlic extract (green). The results are presented as means ± SEM (*n* = 3). Reduction in AGE fluorescence results (0.78–100 µg/mL garlic extract) were compared with G-HSA sample. Comparison between two groups was performed based on *t* test, and significance was defined as \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. **Figure 8.** AGE-specific fluorescence intensity at 450 nm vs. concentration of garlic extract. Native HSA and G-HSA were incubated for 10 weeks as negative and positive controls, respectively. G-HSA was incubated with various concentrations (0–100 µg/mL) of garlic extract (green). The results are presented as means ± SEM (*n* = 3). Reduction in AGE fluorescence results (0.78–100 µg/mL garlic extract) were compared with G-HSA sample. Comparison between two groups was performed based on *t* test, and significance was defined as \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

2.3.6. Circular Dichroism

concentrations of garlic extract (0–100 µg/mL).

**N-HSA G-HSA G-HSA with Garlic Extracts (μg/mL)** 

38.4 ± 0.3 (−10.0%)

29.9 ± 0.2 (+14.1%)

19.4 ± 0.3 (+4.9%)

12.3 ± 0.4 (−2.4%)

**Conformation** 

<sup>α</sup>-helix 42.7 ± 0.6 38.2 ± 0.3

<sup>β</sup>-sheet 26.2 ± 0.5 30.1 ± 0.2

<sup>β</sup>-turn 18.5 ± 0.2 19.4 ± 0.3

Random coil 12.6 ± 0.5 12.3 ± 0.4

(−10.5%)

(+14.9%)

(+4.9%)

(−2.4%)

38.2 ± 0.3 (−10.5%)

30.1 ± 0.2 (+14.9%)

19.4 ± 0.3 (+4.9%)

12.3 ± 0.4 (−2.4%)

38.3 ± 0.3 (−10.3%)

30.0 ± 0.2 (+14.5%)

19.4 ± 0.3 (+4.9%)

12.3 ± 0.4 (−2.4%)
