Phytochemical Screening

The chemical and biological properties detected in preliminary screening of aqueous solution of garlic extract are shown in Table 1. The presence of flavonoids was determined using an alkaline reagent test. FeCl<sup>3</sup> test was used to estimate phenolic content. The total phenolic compounds in garlic extract were determined to be 21.45 ± 0.02 mg gallic acid equivalent/g dry weight of the extract. Phenolic content serves as a means of protection against both infection and oxidative stress. Calculation of total flavonoid content of the extract was performed by AlCl<sup>3</sup> test, with quercetin as a reference. Thus, total flavonoid content was found to be 16.58 ± 0.03 mg quercetin equivalents (QE)/g dry weight of the extract.

extract.


against both infection and oxidative stress. Calculation of total flavonoid content of the extract was performed by AlCl3 test, with quercetin as a reference. Thus, total flavonoid content was found to be 16.58 ± 0.03 mg quercetin equivalents (QE)/g dry weight of the

**Table 1.** Chemical and biological studies of aqueous garlic extract. **Preliminary Screening Garlic Extract** 

**Table 1.** Chemical and biological studies of aqueous garlic extract.

*Molecules* **2022**, *27*, x FOR PEER REVIEW 3 of 19

+ sign indicates presence of flavonoids and polyphenolic compounds.

### *2.2. Antioxidant and Free Radical Scavenging Activities of Garlic Extract* 2.2.1. Assay for Ferric Reducing Antioxidant Power (FRAP) *2.2. Antioxidant and Free Radical Scavenging Activities of Garlic Extract*  2.2.1. Assay for Ferric Reducing Antioxidant Power (FRAP)

Using the FRAP test and ascorbic acid as a standard reference, we evaluated the reducing capability of garlic extract. The reduction of Fe+3 to Fe+2 by the extract is the basis for this test. At 700 nm, the solution of ascorbic acid (0–100 µg/mL) followed Beer's Law with a regression coefficient (R<sup>2</sup> ) of 0.9973 and a slope (m) of 0.004. For this figure, the intercept was 0.0216. The standard curve's equation is y = 0.004x + 0.0216 (not given). Garlic extract had FRAP values of 32.41 ± 0.86 g ascorbic acid/100 mg dry weight of extract. Garlic extract was found to have improved ferric reducing power in a dosage-dependent manner (Figure 1). Using the FRAP test and ascorbic acid as a standard reference, we evaluated the reducing capability of garlic extract. The reduction of Fe+3 to Fe+2 by the extract is the basis for this test. At 700 nm, the solution of ascorbic acid (0–100 µg/mL) followed Beer's Law with a regression coefficient (R2) of 0.9973 and a slope (m) of 0.004. For this figure, the intercept was 0.0216. The standard curve's equation is y = 0.004x + 0.0216 (not given). Garlic extract had FRAP values of 32.41 ± 0.86 g ascorbic acid/100 mg dry weight of extract. Garlic extract was found to have improved ferric reducing power in a dosage-dependent manner (Figure 1).

**Figure 1.** Percentage reducing power of ascorbic acid (red) and aqueous garlic extract (green). Samples in the histogram showed varying concentrations of ascorbic acid and garlic extract (0–100 µg/mL). The *y* axis shows the corresponding percentage reducing power. The results are presented as means ± SEM (*n* = 3). All the results (0.78–100 µg/mL garlic extract) were statistically significant compared with the sample without extract (0 µg/mL). Comparison between two groups was performed based on *t* test, and significance was defined as *p* < 0.05*.* **Figure 1.** Percentage reducing power of ascorbic acid (red) and aqueous garlic extract (green). Samples in the histogram showed varying concentrations of ascorbic acid and garlic extract (0–100 µg/mL). The *y* axis shows the corresponding percentage reducing power. The results are presented as means ± SEM (*n* = 3). All the results (0.78–100 µg/mL garlic extract) were statistically significant compared with the sample without extract (0 µg/mL). Comparison between two groups was performed based on *t* test, and significance was defined as *p* < 0.05.

### 2.2.2. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Assay 2.2.2. 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Assay

The DPPH free radical scavenging method is a widely used method for determining antioxidant capacity of various compounds. Figure 2 depicts the DPPH radical scavenging The DPPH free radical scavenging method is a widely used method for determining antioxidant capacity of various compounds. Figure 2 depicts the DPPH radical scavenging capabilities of aqueous garlic extract. Garlic extract exhibits a substantial DPPH scavenging activity that was observed to rise as extract concentrations were raised from 0–100 µg/mL. The DPPH scavenging activity of 100 µg/mL extract was 66.18%. However, ascorbic acid showed 39.59% at 100 µg/mL.

bic acid showed 39.59% at 100 µg/mL.

bic acid showed 39.59% at 100 µg/mL.

*Molecules* **2022**, *27*, x FOR PEER REVIEW 4 of 19

**Figure 2.** Percentage of free radical reduced vs. aqueous garlic extract concentrations. Various concentrations (0–100 µg/mL) of garlic extracts (green) and 100 µg/mL of ascorbic acid (red). The results are presented as means ± SEM (*n* = 3). All the results (0.78–100 µg/mL garlic extract) were statistically significant compared with the sample without extract (0 µg/mL). Comparison between two groups was performed based on *t* test, and significance was defined as *p* < 0.05*.* **Figure 2.** Percentage of free radical reduced vs. aqueous garlic extract concentrations. Various concentrations (0–100 µg/mL) of garlic extracts (green) and 100 µg/mL of ascorbic acid (red). The results are presented as means ± SEM (*n* = 3). All the results (0.78–100 µg/mL garlic extract) were statistically significant compared with the sample without extract (0 µg/mL). Comparison between two groups was performed based on *t* test, and significance was defined as *p* < 0.05. **Figure 2.** Percentage of free radical reduced vs. aqueous garlic extract concentrations. Various concentrations (0–100 µg/mL) of garlic extracts (green) and 100 µg/mL of ascorbic acid (red). The results are presented as means ± SEM (*n* = 3). All the results (0.78–100 µg/mL garlic extract) were statistically significant compared with the sample without extract (0 µg/mL). Comparison between two groups was performed based on *t* test, and significance was defined as *p* < 0.05*.*

capabilities of aqueous garlic extract. Garlic extract exhibits a substantial DPPH scavenging activity that was observed to rise as extract concentrations were raised from 0–100 µg/mL. The DPPH scavenging activity of 100 µg/mL extract was 66.18%. However, ascor-

capabilities of aqueous garlic extract. Garlic extract exhibits a substantial DPPH scavenging activity that was observed to rise as extract concentrations were raised from 0–100 µg/mL. The DPPH scavenging activity of 100 µg/mL extract was 66.18%. However, ascor-

### *2.3. Inhibition of Structural Changes by Garlic Extract 2.3. Inhibition of Structural Changes by Garlic Extract 2.3. Inhibition of Structural Changes by Garlic Extract*

### 2.3.1. Protein Denaturation Inhibition 2.3.1. Protein Denaturation Inhibition 2.3.1. Protein Denaturation Inhibition

Inhibition in in vitro HSA protein denaturation was examined using garlic extracts. Tissue proteins have been reported to be denatured by inflammatory and oxidative reactions. Natural products were used to evaluate their potential to protect proteins from denaturation and would be employed as an anti-inflammatory dietary source. Garlic extract (50 µg/mL) inhibited heat-induced albumin denaturation at a higher percentage of 50.66% (Figure 3). Inhibition in in vitro HSA protein denaturation was examined using garlic extracts. Tissue proteins have been reported to be denatured by inflammatory and oxidative reactions. Natural products were used to evaluate their potential to protect proteins from denaturation and would be employed as an anti-inflammatory dietary source. Garlic extract (50 µg/mL) inhibited heat-induced albumin denaturation at a higher percentage of 50.66% (Figure 3). Inhibition in in vitro HSA protein denaturation was examined using garlic extracts. Tissue proteins have been reported to be denatured by inflammatory and oxidative reactions. Natural products were used to evaluate their potential to protect proteins from denaturation and would be employed as an anti-inflammatory dietary source. Garlic extract (50 µg/mL) inhibited heat-induced albumin denaturation at a higher percentage of 50.66% (Figure 3).

**Figure 3.** Percentage protection from denaturation induced by heat vs. garlic extract concentration (0–100 µg/mL). The results are presented as means ± SEM (*n* = 3). All the percentage denaturation **Figure 3.** Percentage protection from denaturation induced by heat vs. garlic extract concentration (0–100 µg/mL). The results are presented as means ± SEM (*n* = 3). All the percentage denaturation **Figure 3.** Percentage protection from denaturation induced by heat vs. garlic extract concentration (0–100 µg/mL). The results are presented as means ± SEM (*n* = 3). All the percentage denaturation inhibition results (0.78–100 µg/mL garlic extract) were statistically significant compared with the sample without extract (0 µg/mL). Comparison between two groups was performed based on *t* test, and significance was defined as *p* < 0.05.
