Comparative study on the composition of four different varieties of garlic

Materials and preparation

Four different varieties of garlic were purchased from the local agricultural market; the garlic bulbs and peeled garlic cloves are shown in Fig. 1. ‘Taicangbaipi’ and ‘Ershuizao’ were purchased in supermarket of Chengdu, and ‘Hongqixing’ and ‘Single-clove’ were bought in local market of Wenjiang. The garlic bulbs were peeled and crushed.

Determination of soluble sugar and starch

Garlic juices (0.3 g) were extracted in 15 mL of 80% ethanol, and then centrifuged at 7,000 rpm for 10 min; the supernatant was retained for soluble sugar determination, whereas the precipitate was kept for starch extraction (Irigoyen, Einerich & Sanchez-Diaz, 1992). The precipitate was dissolved in 12 mL of 1.1% HCl and thoroughly mixed. The solution was heated in a water bath at 100 °C for 30 min to extract the starch, and after cooling, it was centrifuged at 7,000 rpm for 10 min; the supernatant was retained and diluted to 50% solutions.

The total soluble sugar and starch were estimated using anthrone reagent (Sadasivam & Manickam, 1997). One millilitre of the diluted solutions were added to five mL of freshly prepared anthrone reagent (0.2 g anthrone was dissolved in 100 mL of 72% sulphuric acid), and the mixtures were heated in a boiling water bath for 10 min. The tubes were removed and cooled, and then the absorbance of the content was measured at 625 nm in a spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). The amount of total sugar present in the sample was calculated from a standard curve drawn from variable amounts of glucose and total starch obtained from a standard curve for potato starch.

Determination of protein content

Garlic juices (one g) were extracted in 3.5 mL protein extraction buffer containing 15% (v/v) 1M Tris–HCl (pH 8), 25% (v/v) glycerol, and 2% (w/v) polyvinylpyrrolidone. The mixture was kept on ice for 3 h, and then centrifuged at 10,000 rpm for 20 min at 4 °C. Samples of each supernatant (200 μL) were treated with 800 μL Coomassie Brilliant Blue G-250. After 10 min, the mixtures were subjected to colorimetric analysis in a T6-spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China) at 595 nm. A standard curve was drawn using bovine serum albumin.

GC–MS analysis

Gas chromatography analyses were performed using a Shimadzu GC/MS-QP2010 system (Kyoto, Japan). A Rtx-Wax capillary column (30 m × 0.25 mm, 0.25 μm) was used, with helium carrier gas flowing at 1 mL/min; the column temperature was first kept at 60 °C for 5 min, then increased by 10 °C/min up to 280 °C. The injection port temperature was maintained at 220 °C. The MS data libraries NIST05.LIB and NIST05s.LIB were used for the spectrum analyses and compound identification.

Microbial strains, culture, and antimicrobial test

Xanthomonas campestris pv. malvacearum (BNCC138498), Pseudomonas syringae (BNCC134219), Fusarium proliferatum (BNCC143058), and Alternaria brassicicola (CICC264) were obtained from Beina Chuanglian Biological Research Institute (Beijing, China). The garlic juices were mixed with deionized water at pH 3.0 at a ratio of 1:2 g/mL (Chen et al., 2017), for 2 h at 4 °C.

Oxford cup assays were used in the antimicrobial experiments, with some modifications (Shang et al., 2014). Bacteria (X. campestris pv. malvacearum, P. syringae) were grown to an OD₆₀₀ of 0.6 in nutrition broth (NB) at 28 °C in a shaker, then 100 mL NB agar was mixed with one mL of the bacterial solutions and added to the plates. Fungi (F. proliferatum and A. brassicicola) were cultivated on potato dextrose agar (PDA) plates at 28 °C. The spores were washed with 10 mL of sterile water and centrifuged at 6,000 rpm for 5 min to remove the supernatant. The spores were then mixed with sterile water to a concentration of approximately 2 × 10⁷/mL as assessed by a haemocytometer. Then, 100 mL PDA agar was mixed with 0.5 mL spore suspension and added to the plates. Four Oxford cups (six-mm diameter) were placed above the agar surface, and 20 μL garlic extract (‘Taicangbaipi’, ‘Ershuizao’, ‘Hongqixing’, and ‘Single-clove’) was added to each cup, respectively. The bacterial inhibition zones were observed after 24 h at 28 °C, and the fungal inhibition zones were observed after 2–4 days at 28 °C. Three replicates were performed for each treatment.

Cell membrane permeability

After 24 h of incubation at 28 °C, X. campestris pv. malvacearum cells were treated with the extracts of the four garlic varieties. The culture samples (five mL) were treated with 500 μL of the garlic extracts, and the mixtures were incubated at 28 °C for 1–12 h, respectively. Conductance was recorded using a conductivity meter (DDS-307; SPSIC-Rex Instrument Factory, Shanghai, China) after incubation, and was expressed as the bacterial membrane permeability relative conductivity and measured according to the method of Diao et al. (2014).

Integrity of cell membrane

Bacterial cultures containing 500 μL of the extracts of the four garlic varieties were centrifuged for 10 min at 6,000 rpm, and the supernatants were obtained. The supernatants (200 μL) were treated with 800 μL Coomassie Brilliant Blue G-250, and the mixtures were analysed at 595 nm on a T6-spectrophotometer (Beijing Purkinje General In-strument Co., Ltd, Beijing, China). The breakdown of bacterial cell membrane integrity manifested as protein leakage.

Cytotoxic effects (antitumor) of the extracts of different garlic varieties on cells

Cell Culture: Colorectal cancer (CRC) cells (SW480 and HCT116) were obtained from the American Type Culture Collection (Rockville, MD, USA). The extracts of the four garlic varieties were dissolved in phosphate-buffered saline. The dilution ratios were 0, 1/32, 1/16, 1/8, 1/4, and 1/2. SW480 and HCT116 cells were seeded in 96-well plates. Various concentrations of the garlic extracts were applied to the cells, and cell viability was analysed after 72 h of incubation.

Soluble sugar, total starch, and protein content in different varieties of garlic

As shown in Table 1, different varieties of garlic resulted in significantly different soluble sugar, total starch, and protein content. The highest soluble sugar content was observed in ‘Single-clove’, at a mean of 186.91 mg/g. The mean starch levels of ‘Hongqixing’ and ‘Ershuizao’ were 287.71 and 263.98 mg/g, respectively, which were higher than that of both ‘Taicangbaipi’ and ‘Single-clove’. It was also observed that ‘Hongqixing’ and ‘Ershuizao’ had higher protein contents, with mean levels of 45.23 and 51.04 mg/g, respectively. However, the overall content of these parameters in the ‘Taicangbaipi’ extract was relatively lower than those in the other three extracts.

Constituents by GC–MS analysis

The identities of the compounds in the four varieties of garlic are listed in Table 2, and the GC–MS spectra of the constituents is shown in Figs. 2 and 3. The most abundant constituent in the ‘Taicangbaipi’ extract was Β-D-fructofuranosyl α-D-glucopyranoside (27.34%), followed successively by ethylic acid (22.40%) and 2-amino-5-methylbenzoic acid (17.05%). The main constituents of the ‘Hongqixing’ extract were Β-D-fructofuranosyl α-D-glucopyranoside (44.18%) and 5-hydroxymethylfurfural (26.78%). The primary compounds in the ‘Ershuizao’ extract were 5-hydroxymethylfurfural (47.10%), and Β-D-fructofuranosyl α-D-glucopyranoside (31.00%). Finally, the primary constituents of the ‘Single-clove’ extract were Β-D-fructofuranosyl α-D-glucopyranoside (61.07%), 2-amino-5-methylbenzoic acid (9.07%), and 5-hydroxymethylfurfural (4.88%), in descending order.

Activity of the extracts from different garlic varieties against plant pathogenic bacteria and fungi

In order to determine the antimicrobial effects of the extracts of the different garlic varieties, two types of bacteria (X. campestris pv. malvacearum, P. syringae) and two types of fungi (F. proliferatum and A. brassicicola) were treated with the four garlic extracts. After incubation, the zones of inhibition showed that the ‘Hongqixing’ extract had the highest activity against the microorganisms, followed by ‘Ershuizao’, ‘Taicangbaipi’, and ‘Single-clove’, in descending order of activity. The experimental results are shown in Fig. 4.

Changes in cell membrane permeability

Membrane permeability experiments were performed to study the effect of the garlic extracts on bacterial cell membranes. Figure 5A shows the effects of the extracts from the different garlic varieties on the membrane permeability of X. campestris pv. malvacearum. During the first 8 h, there was almost no change in the relative conductivity of the control; however, an increase in the conductivity was observed at 12 h, which may have been due to bacterial death. Similar to the control, the relative conductivity of the samples for ‘Single-clove’ and ‘Taicangbaipi’ were only slightly higher. However, compared to the control, with increasing treatment time and concentration of the ‘Hongqixing’ and ‘Ershuizao’ extracts, the conductivity increased rapidly. This meant that the permeability of the bacterial membrane would have increased correspondingly, resulting in the loss of intracellular components, especially K⁺, Na⁺, and other electrolytes.

Leakage of cellular contents

Similar to the membrane permeability test, the extracts from the different garlic varieties elevated protein leakage through the plasma membrane of X. campestris pv. malvacearum (Fig. 5B). ‘Single-clove’ and ‘Taicangbaipi’ caused slight protein leakage, whereas ‘Hongqixing’ and ‘Ershuizao’ caused extensive protein leakage. These results indicate that the ‘Hongqixing’ and ‘Ershuizao’ extracts may cause irreversible damage to the bacterial plasma membrane, resulting in the loss of cellular components such as proteins and some essential molecules, leading to cell death.

Analysis of cytotoxic (antitumor) effects

After 72 h of incubation with extracts from different garlic varieties at different concentrations (dilution ratios of 0, 1/32, 1/16, 1/8, 1/4, and 1/2), we analysed the viability of SW480 (Fig. 6A) and HCT116 (Fig. 6B) cells. The cell viability for the control (no added garlic extract) was 100%. After incubation with the extracts from the different varieties of garlic at different concentrations, the cell viability decreased by varying degrees. At 1/2 concentration, after 72 h, the SW480 cell viability was 21.18% when incubated with ‘Ershuizao’, 29.14% with ‘Hongqixing’, 38.46% with ‘Single-clove’, and 39.07% with ‘Taicangbaipi’. The extracts of ‘Ershuizao’ and ‘Hongqixing’ had strong cytotoxic effects on HCT116, with cell viabilities of 5.80% and 7.13%, respectively. Conversely, the cell viability for HCT116 with ‘Single-clove’ was 40.76% and with ‘Taicangbaipi’ was 40.88%.