Garlic cubes were purchased from the local market of Rasht (Iran). The garlic was washed and peeled. Before extraction, the garlic was ground in a blender and the resulting particles were immediately used for extraction.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical, Folin-Ciocalteu reagent, and the standard substances including gallic acid were purchased from Sigma (Sigma Aldrich GmbH, Sternheim, Germany), and methanol and ethanol were prepared from Merck KGaA (Darmstadt, Germany). An ultrasonic bath process (purchased from Elma, Germany) was used to perform the ultrasonic-assisted extraction.

The method proposed by Sharifi et al. (2003): water was selected as the solvent to prepare the extract. Small (500g) pieces of garlic were mixed with 1 liter of distilled water (ratio 1:2) using a blender. The mixture was put in a shaking incubator at 40°C for 72 h. Finally, the resulting solution was filtered and dried in a freeze dryer 15.

The method proposed by Pinilla et al. (2017), with some modifications: garlic cloves were washed, crushed and mixed with distilled water (ratio 1:2 w/v). The mixture was put in an ultrasonic bath at 40 kHz for 15 min and then centrifuged at 10,000 rpm and 4°C for 10 min. The resulting supernatant was collected, passed through a 0.22-µm membrane and stored in sterile glass containers at 4°C for the tests 16.

The method proposed by Pinilla et al. (2017), with some modifications: garlic cloves were washed, crushed and mixed with 50% ethanol (ratio 1:2 w/v). The mixture was placed in an ultrasonic bath at 40 kHz for 15 min and then centrifuged at 10,000 rpm and 4°C for 10 min. The resulting supernatant was collected, filtered using a 0.22-µm membrane, and stored in sterile glass containers at 4°C for the tests 16.

The Nazari method with some modifications: garlic cloves were peeled, washed, crushed and then mixed with distilled water (ratio 1:10 w/v). The mixture was boiled over a flame for half an hour. The resulting solution was filtered using Whatman Filter Paper No. 1 and dried 17.

The method proposed by Swami et al. (2008), with some modifications: cloves of garlic were peeled, washed, crushed and then mixed with distilled water (ratio 1:10 w/v). The mixture was put in an oven at 35°C for 24 h. The resulting solution was filtered using Whatman Filter Paper No. 1 and dried 18.

The method proposed by Swami et al. (2008), with some modifications: garlic cloves were peeled, washed and crushed and then mixed with 50% ethanol (ratio1:10 w/v). The mixture was put in an oven at 35°C for 24 h. The resulting solution was filtered using Whatman Filter Paper No. 1 and dried 18.

The content of total phenolic compounds in the garlic extracts was determined using the Folin–Ciocalteu reagent 19. Absorbance was measured at 765 nm using a UV-VIS spectrophotometer (Shimadzu, Japan). The content of phenolic compounds was expressed as gallic acid equivalents (GAE) per dry weight of extract. All experiments were performed in three replicates.

The free-radical scavenging capacity of garlic extracts was determined as described by Fenfang et al. (2017). Different amounts of the extract was mixed with methanol (95%) and DPPH in order to obtain different final concentrations of the extract. After 60 min at room temperature and in darkness, the absorbance was measured at 517 nm using a UV-VIS spectrophotometer (Shimadzu, Japan). All experiments were performed in threereplicates. Radical scavenging capacity (%RSC) was calculated from the following equation 20:

….(Eq. 1)

IC50 is a parameter commonly used to compare the free radical scavenging activity of different extracts. IC50 refers to the half-maximal inhibitory concentration of the extract against the free radical in the reaction medium 21. The amount of IC50 Obtained using Graph Pad Prism8 software.

Quantitative Determination of Allicin in the Optimum extract

The quantity of allicin in the optimum extract was measured by high-performance liquid chromatography (HPLC) (Agilent, 1200) based on the method described by British Pharmacopoeia (2015). The internal standard was used of butyl parahydroxy benzoate and column size was 0.25 m and 4 mm diameter. The mobile phase consisted of mixing 60% methanol and 40% anhydrous formic acid solution (v/ v) at a flow rate of 0.7 ml/ min and detection done with Spectrophotometer at 254 nm 22.

….(Eq. 2)

S1: area of the peak corresponding to allicin

S2: area of the peak corresponding to butylparahydroxybenzoate

C1: sample concentration

C2: internal standard concentration

Vis: internal standard volume

Vs: sample volume

Vt: total volume

This was done based on the criteria in chromatography (retention time) and spectrometry (mass spectral interpretation, comparison with library information and standard compounds). Chromatographic and spectroscopic data were collected using a gas chromatograph (Thermo Scientific Trace OQ301) attached to a DSQ mass spectrometer (electron impact ionization, eV 70; Thermo Scientific). The length of DB-5MS (J & W Scientific) column was 30 m, 0.52 mm internal diameter, with 5% phenyl polyimethylsiloxane static phase, or 0.25 μm thickness, was designed as follows:Temperature of 50 °C (1 min), temperature rise at 10 °C/ min to 280 °C and stop for 15 min. Temperature of injection area and transmission line were set to 250 and 280 °C, respectively. Helium (99.99%) as carrier gas with a pressure of 155 KPa and a rate of 27 cm/ min (constant current 1 ml/ min). Mass spectrometry and chromatograms were adjusted by mass scanning method in the range of 45-500 mlz at 5.1 scan/ sec 23.

SPSS 22 software was used for data analysis and Excel 2007 software was used for drawing charts. IC50 values were obtained using Graph Pad Prism8 software. All samples were tested in three replications.

(Table 1) shows the results related to total phenolic compounds based on mg gallic acid equivalent/ g dry weight of each extract. The results showed that phenolic compounds retention was affected by the extraction method and the type of solvent. The largest amounts of phenolic compounds were observed in the ultrasonic aqueous extract followed by the aqueous extract prepared after 72 h in a shaking incubator. There was a significant difference between these two extracts and the other extracts in this regard (p<0.05).

The results regarding the inhibition rate of the free radical (DPPH) by the prepared extracts are shown in Figure 1. They indicated that the inhibition rate was affected by the extraction method and the type of solvent. The highest inhibition rate (50% at 5000 ppm) was that of the ultrasonic aqueous extract and the shaken aqueous extract. Other extracts achieved an inhibition rate of 50% at 8000 ppm (p<0.05).

The high rate of DPPH inhibition in this study is due to the higher quantities of phenolic and tocopherol compounds. The inhibitory potency of the different extracts largely depends on the numbers and positions of the hydroxyl groups and on the molecular weights of the phenolic compounds 24. Like previous research, the results of this study showed that the extracts containing more phenolic compounds exhibited greater inhibitory effects 25. It is noteworthy that phenolic compounds act more efficiently as hydrogen donors and, therefore, serve as effective antioxidants. In addition, the inhibitory effects of phenolic compounds on DPPH increase with increases in their concentrations or hydroxylation degrees 26.

IC₅₀ is a parameter commonly used to compare the free radical scavenging activity of different extracts. IC₅₀ refers to the half-maximal inhibitory concentration of the extract against the free radical (DPPH in the present research) in the reaction medium. Therefore, lower IC₅₀ values indicate higher free radical scavenging activity. Table 2 presents the IC₅₀ values of the extracts prepared in this study. The results demonstrated that the highest and the lowest IC₅₀ were those of the ultrasonic aqueous extract (4,376 µg/ml) and the boiled aqueous extract (8,540 µg/ml), respectively. The amounts of phenolic compounds are associated with antioxidant properties: the antioxidant property of extracts improves with increases in their content of phenolic compounds. This can justify the lower IC₅₀ values of all boiled extracts that Table 2 shows 21.

The results showed that the aqueous extract outperformed the others in free radical inhibition. Moreover, there were significant differences between aqueous and ethanol extracts at all concentrations, and increased concentrations of phenolic compounds directly increased the ability of the different extracts to inhibit the activity of the free radical. Due to the increasing number of hydroxyl groups existing in the reaction medium, higher concentrations of phenolic compounds are more likely to increase the possibility of donating hydrogen to the free radicals, following which the inhibitory effects of extracts improve 27. The inhibitory potency of different extracts largely depends on the number and position of hydroxyl groups and the molecular weight of phenolic compounds. Hydroxyl groups are more available in phenolic compounds with lower molecular weights 24.

Zou et al. (2008) found that the ultrasonic extraction of ginseng saponins was about three times faster than the traditional extraction method 28. Ultrasound is considered an appropriate method for extracting biologically active compounds from Salvia officinalis and Hibiscus tiliaceus, antioxidants from rosemary 29. Compared to traditional methods in water or ethanol, the ultrasound method can increase the efficiency of extracting fennel, hops, parsley, and mint by 34%, 12%, 3%, and 7%, respectively 12. Variations in extraction efficiency are mainly due to the specific structures of the products. Albu et al. (2004) investigated the effects of different solvents and the ultrasound method on the extraction of carnosic acid from rosemary and reported that the traditional extraction method using ethanol was significantly less efficient than extraction with ethyl acetate and butanone. In addition, the application of ultrasound improved the relative efficiency of ethanol compared to the other two solvents. Therefore, the use of ultrasound may reduce the dependence on a specific solvent and makes it possible to apply alternative solvents that are more appropriate with respect to economic, environmental, health, and safety considerations 30.

HPLC chromatograms show the results of regarding the allicin contents of the garlic extracts prepared in this study. Allicin content was calculated based on the area under the peak and Equation 2.

As shown in Table 3, the highest allicin content (0.086%) was observed in the ultrasonic aqueous extract. The allicin content significantly decreased in garlic extracts prepared in the oven, probably due to the temperature conditions. The lowest allicin content (0.009%) was that of the boiled aqueous extract. This can corroborate the decline in allicin content at high temperatures or in long exposure to heat. In fact, heat reduces the active ingredients or the enzymes effective in their production. In addition, processes like cooking soften the cell wall, facilitating the extraction of carotenoids, cause their release into the water and reduce their content in tissues 3132. Ilic et al. (2011) stated that allicin was derived from its precursor alliin through the activity of alliinase. Although allicin is a molecule of low polarity, it is often extracted by using polar solvents such as water at room pressure (0.1 MPa) because allicin is very unstable in non-polar organic solvents. This confirms the results of the present study 33.

The quantities of compounds present in the ultrasonic aqueous extract, measured by GC-MS according to the retention time (chromatogram in Figure 2), are listed in Table 4. The two main compounds in this garlic extract were diallyl disulfide (34.87%) and dipropyl trisulfide (25.88%). Pyrogallol (13.38%) and methyl propyl trisulfide (11.36%) were among the other compounds found in the ultrasonic aqueous extract (Table 4, Figure 2). The amounts of volatile compounds in garlic extract depend on various factors including genetic factors (type and species of garlic) and agricultural factors (planting, caring for the crop and harvesting). For example, the diallyl disulfide contents of Cameroonian, Chinese, Moroccan, French and Mexican garlic are 37.3, 35, 23.2, 21.8 and 17.2%, respectively 34 ⁴⁵.