Biochemical Characterization and Antimicrobial and Antifungal Activity of Two Endemic Varieties of Garlic ( Allium sativum L.) of the Campania Region,Southern Italy

Abstract

Extracts of the bulbs of the two endemic varieties “Rosato” and “Caposele” of Allium sativum of the Campania region, Southern Italy, were analyzed. The phenolic content, ascorbic acid, allicin content, and in vitro antimicrobial and antifungal activity were determined. Ultra performance liquid chromatography with diode array detector performed polyphenol profile. The polyphenolic extracts showed antioxidant activity (EC₅₀) lower than 120 mg. The amount of ascorbic acid and allicin in the two extracts was similar. Polyphenol extract exhibited antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and (only by the extract of Rosato) against Bacillus cereus. The extract of Caposele was more effective in inhibiting the growth of Aspergillus versicolor and Penicillum citrinum. On the other hand, the extract of Rosato was effective against Penicillium expansum.

Introduction

Polyphenols received certain attention due to their potent antioxidant, antimicrobial, antifungal, and antiviral activities; indeed, they are capable of inhibiting proliferation of cancer cells.¹ For these reasons, they have been the matter of numerous investigations, and crops received special attention to identify those species/varieties capable to act as or contribute functional ingredients, providing beneficial effects on human health.

The recovery of biodiversity in agriculture has long been the focus of public opinion and of community policy. Biodiversity is essential to the sustainable development of agricultural production in rural areas. Garlic (Allium sativum L.) is a plant belonging to the family Liliaceae. It is a fundamental component in many dishes, principally of Asia, the Middle East, Africa, South and Central America, and Mediterranean area. In Italy, garlic is cultivated mainly in Campania, Sicily, in Emilia-Romagna and Veneto. In the Campania region are present different varieties, such as “Bianco locale,” “Salomone,” “Caposele,” “Tondo di Torella,” “Schiacciato,” and “Rosato” locale.² The extracts obtained from bulb or from other not edible parts of the plant also exhibit antioxidant, antibacterial, antifungal, antiviral, immunostimulating, and cholesterol-lowering properties.^3
–5 Due to their beneficial effects on health, the phytochemical content and biological properties of garlic have been investigated. These studies identified several bioactive constituents, such as organo-sulfur compounds, volatile sulfur compounds (e.g., diallyl sulfide and diallyl disulfide), and polyphenols.⁵ Recently, Fratianni et al. ⁶ focused their attention on the biochemical study of some varieties of garlic, present in the Campania region for over 60 years and therefore defined as “native.” This study placed further emphasis on some biological properties of these varieties, starting with the ability of the extracts to exhibit antimicrobial and antiproliferative activity, evaluated by in vitro tests.

Therefore, the aim of this study was to evaluate the biochemistry and biological properties of the bulbous portion of two different endemic varieties of garlic cultivated in the Campania region in southern Italy, and to assess the antimicrobial and antifungal activities of their polyphenols extracts against different bacteria and molds pathogenic strains using the inhibition halo test.

Materials and Methods

The variety “Caposele” of A. sativum (Fig. 1a) is typical of the Irpinian province, in particular of the village of Caposele. The variety “Rosato” (Fig. 1b) is typical of the Acerra-Marigliano area (Naples, Campania, Italy). Both varieties were grown in the same experimental area of “F. Marsocci” located in Acerra (Naples, Campania, Italy).

FIG. 1.

The two endemic varieties of Allium sativum of the Campania region of southern Italy: variety “Caposele” (a), and “Rosato” (b). Color images available online at www.liebertpub.com/jmf

Standards, reagents, and solvents

Caffeic, ferulic, p-coumaric, gallic, chlorogenic, and ascorbic acids; epicatechin, rutin, quercetin, and allicin standards; 2,2-diphenyl-1-picrylhydrazyl (DPPH), H₂SO₄, butyl-parahydroxybenzoate; high performance liquid chromatography (HPLC)-grade methanol; sulfuric, metaphosphoric, acetic, and formic acids; acetonitrile; ethanol; and acetone were purchased from Sigma-Aldrich. Apigenin, luteolin, and hyperoside were purchased by Extrasynthese.

Extraction procedures

The extracts were prepared following the method of Fratianni et al. ⁶ Briefly, in the first step samples were maintained (1:1 w/v) at room temperature without exposure to light, in an acetone:ethanol:ultrapure water mixture (70:15:15) for 24 h. The supernatants were recovered by centrifugation (11,600×g, 15 min; Beckman Italia). Pellets were treated for 24 h at 4°C with ultrapure water (1:1 w/v), without exposure to light, to enhance the extraction of acidic polyphenols; the supernatants were recovered by centrifugation. The two supernatants were pooled and concentrated before analysis.

Colorimetric analysis of total polyphenols

The total phenolic content was determined using Folin–Ciocalteu reagent.⁷ The absorbance at λ = 760 nm was determined at room temperature using a Cary 73 UV/Vis spectrophotometer (Varian). Quantification was based on a standard curve generated using gallic acid at concentration ranging from 0.5 to 5 mM (y = 0.5488x + 0.0148 R ² = 0.9919). The results were expressed as μg of gallic acid equivalent (GAE)/g of the fresh product ± standard deviation (SD).

Free radical scavenging capacity

The free radical scavenging activity was determined using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.⁸ The analysis was performed in microplates by adding 7.5 μL of an extract to 303 μL of a methanolic DPPH solution (153 mM). Next, the absorbance at λ = 517 nm was measured (Cary 50 MPR; Varian). The absorbance of DPPH in the absence of antioxidants (control sample) was used to determine the baseline value. The EC₅₀ value defined the sample concentration (in mg) necessary to inhibit the DPPH radical activity by 50% during a 60-min incubation.

Determination of the ascorbic acid content

Ascorbic acid was extracted following the method of Nazzaro et al. ⁹ The samples were homogenized with 10 volumes of metaphosphoric acid (4%) and were maintained for 1 h at 4°C without direct exposure to light. After centrifugation (at 11,600×g for 10 min at 4°C; Beckman), the supernatants were collected and filtered (0.45-μm; Waters). The ascorbic acid content was determined using reverse-phase (RP)-HPLC, using a Gold System chromatographic device equipped with an UV detector (Beckman). A Khromasil KR 100-5 C₁₈ column (25 cm ×4.6 mm) was used at room temperature. The mobile phase consisted of 0.005 M H₂SO₄ in HPLC-grade water. The injection volume was 20 μL, and the detection wavelength was set at 245 nm. The ascorbic acid concentration was determined based on a standard curve generated using an ascorbic acid standard solution.

Extraction and evaluation of allicin

Allicin was extracted and quantified following the method of Fratianni et al. ⁶ The samples were dried at 60°C to a constant weight and were subsequently ground; 0.8 g of the ground samples were homogenized by ultrasound for 5 min in 20 mL ultrapure water. The homogenates were maintained at room temperature for 60 min and subsequently centrifuged for 30 min at 4600 × g (Beckman). The supernatants were collected, filtered, and stored at −26°C until analysis. A 10-mL aliquot of each supernatant was diluted with 25 mL formic acid: methanol (40:60 v/v) and centrifuged for 5 min at 4600 × g. A 0.5-mL aliquot of a butyl-parahydroxybenzoate internal standard (prepared by dissolving 20 mg of butyl-parahydroxybenzoate in 10 mL of 50% methanol) was added to 10 mL of each sample. The content of allicin was determined using RP-HPLC, using a Gold System chromatographic device equipped with an UV detector (Beckman). Samples were analyzed on a Khromasil KR 100-5 C₁₈ column (25 cm ×4.6 mm) at room temperature at a flow rate of 0.7 mL/min. The mobile phase consisted of 50% methanol. The injection volume was 20 μL, and the detection wavelength was set at 254 nm. The concentration of allicin was determined based on a standard curve generated using an allicin standard solution and expressed as mg/g of product.

All experiments were performed in triplicate. Results were expressed as the mean values ± standard deviation.

Chromatographic analysis

An ACQUITY Ultra Performance LC™ system (Waters) linked to a PDA 2996 photodiode array detector (Waters) was used for ultra high-performance liquid chromatography analyses. The Empower software controlled the instruments, and acquired and processed the data. The extracts and standards (previously dissolved in methanol) were filtered (0.45-μm; Waters) before analysis. The analyses were carried at 30°C using a reversed phase column (BEH C₁₈, 1.7 μm, 2.1 × 100 mm; Waters) following the method of Fratianni et al. ¹⁰ The mobile phase consisted of solvent A (7.5 mM acetic acid) and solvent B (acetonitrile) at a flow rate of 250 μL·min⁻¹. Gradient elution was employed, starting with 5% B for 0.8 min; then 5–20% B over 5.2 min; isocratic 20% B for 0.5 min; 20–30% B for 1 min; isocratic 30% B for 0.2 min; 30–50% B over 2.3 min; 50–100% B over 1 min; isocratic 100% B for 1 min; 100–5% B over 0.5 min. At the end of this process, the system equilibrated the column under the initial conditions for 2.5 min. The pressure ranged from 6000 to 8000 psi during the chromatographic run. The effluent was introduced into an LC detector (scanning range: 210–400 nm, resolution: 1.2 nm). The injection volume was 5 μL. The samples were tested in triplicate, and the results were expressed as the mean values ± standard deviations.

Antimicrobial activity

The inhibition halo test on agar plates was employed to investigate the antibacterial activity of the different varieties of garlic,¹¹ using the following pathogens: the Gram-positive strains Bacillus cereus DSM 4313 and Staphylococcus aureus DSM 25923 and the Gram-negative strains Escherichia coli DSM 8579 and Pseudomonas aeruginosa ATCC 50071. All strains were provided by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). Each strain was incubated at 37°C for 18 h in nutrient broth (Sigma-Aldrich). The microbial suspensions at 1 × 10⁸ colony forming units (cfu)/mL were uniformly spread on the solid media plates (Ø = 90-mm dishes). Sterile Whatman No. 1 paper filter discs (Ø = 5 mm) were individually placed on the inoculated plates (Ø = 90 mm dishes) and impregnated with different amounts of the extracts previously dried and resuspended in sterile ultrapure water (final amount ranging from 1 to 4 μg/paper disc, corresponding to 4 to 16 μL). After 30 min under sterile conditions at room temperature, the plates were incubated at 37°C for 24–48 h, depending on the strain. The diameter of the clear zone on the plates was measured and the antibacterial activity was expressed in mm. Sterile ultrapure water was used as the negative control. Tetracycline (7 μg; Sigma-Aldrich) and gentamycin (8 μg; Sigma-Aldrich) were used as the reference samples. The samples were tested in triplicate, and the results were expressed as the mean values ± standard deviations.

Antifungal activity

Some fungal strains of agro-food interest, Penicillium citrinum DSM 1997, Penicillium expansum DSM 1994,¹² and Aspergillus versicolor DSM1943 were used. The strains were purchased from DSMZ. Sterile Whatman No. 1 paper filter discs (Ø = 5 mm) were individually placed on the inoculated plates (Ø = 90 mm dishes) and impregnated with different amounts of the extracts (final amount ranging from 1 to 4 μg/paper disc, corresponding to 4 to 16 μL), were used. A cell suspension of fungi was prepared in sterile distilled water, adjusted to contain ∼10⁶ CFU/mL, and 50 μL were plated onto Potato Dextrose Agar (PDA) (Oxoid). After 20 min under sterile conditions at room temperature, plates were incubated at 28°C until the mycelium of fungi reached the edges of the control plate (negative control without the sample added extracts); the resulting clear zones of inhibition were measured in mm, expressing the antifungal activity. DMSO (10 μL) was used as negative control. Samples were tested in triplicate and the results are expressed as mean ± standard deviation. Data were subjected to analysis of variance (ANOVA) and to Tukey's test using the SPSS statistical software package version 13.0 (2004) to detect the significance of the antimicrobial and antifungal activity of the two polyphenolic extracts.

Data were subjected to the statistical analysis and Tukey's test using the SPSS statistical software package version 13.0 (2004).

Results

Biochemical analysis

The antioxidant activities of the polyphenol extracts of Rosato and Caposele endemic varieties of garlic showed an EC₅₀ value, defined as the concentration necessary to inhibit the activity of the stable radical DPPH by 50%, of 114.96 and 81.34 mg, corresponding to 21.74% and 30.73%, respectively (Table 1). Such values were very similar to those reported for other endemic varieties of garlic present in the Campania region⁶ and different from those reported by Eghdami et al. ¹³ Our values were also higher than those reported in the compositional tables of the Italian National Centre of Agriculture.¹⁴ Nencini et al. ¹⁵ observed that some Italian Allium wild species exhibited an antioxidant activity no higher than 25.02%, and that those ecotypes with a red papery coat showed an antioxidant activity no higher than 16.54% indeed. Caposele and Rosato showed similar level of ascorbic acid (Table 1), greater than that reported by Montano et al. ¹⁶ and Park et al. ¹⁷ Such data confirm that the content of such metabolites is variable depending on the cultivar. However, our results were essentially related to cultivar, unlike what was reported by Beato et al. ¹⁸ The content of total polyphenols and of allicin found in two varieties was practically the same (Table 1). This later was lower respect to other endemic Italian varieties of A. sativum ¹⁹ We did not observe a strict correlation between the antioxidant contents of the extracts with the ascorbic acid content and the total polyphenols present. Therefore, other endemic varieties of the Campania, even if exhibited different amount of ascorbic acid, or of allicin, showed similar inhibitory capacities, that is, 50% inhibition of the stable radical.⁶

Table 1.

Antioxidant Activity, Total Polyphenols, Ascorbic Acid, and Allicin Amount Present in the Two Endemic Garlic Varieties Caposele and Rosato of the Campania Region of Southern Italy

	Antioxidant activity EC₅₀ (mg)	Antioxidant activity (% of inhibition)	Total polyphenols μgGAE/g	Allicin (mg/g)	Ascorbic acid (mg/g)
Caposele	81.34 ± 9.28^a	30.73 ± 1.25^a	87.35 ± 5.06^a	3.1 ± 0.1^a	0.645 ± 0.02^a
Rosato	114.96 ± 11.41^b	21.74 ± 1.09^b	90.41 ± 1.56^a	3.4 ± 0.2^a	0.683 ± 0.03^a

Antioxidant activity is expressed both as the EC₅₀/mL of DPPH and as percentage of inhibition; total polyphenols were calculated using gallic acid as standard (used at concentration ranging between 0.5 and 5 mM, and following the equation y = 0.5488x + 0.0148 R ² = 0.9919), and expressed as μg GAE/g of fresh product. Ascorbic acid and allicin are expressed as mg/g of fresh product (n = 3). Means followed by the same letter in superscripts within the same column are not significantly different by Tukey test (P < .05).

GAE, gallic acid equivalent.

Chromatographic analysis of the polyphenol portion

The most abundant phenolic compounds in the two varieties of garlic (Table 2) were gallic acid, hyperoside, and allyl isothiocyanate (AITC). The last in particular was most abundant in the extract of Rosato (150 μg/g) than Caposele. On the contrary, hyperoside resulted in double quantity in the extract of Caposele than Rosato (89 and 42 μg/g, respectively). Hyperoside is a major active constituent in many medicinal plants, which folk medicines use for their neuroprotective, anti-inflammatory, and antioxidative effects. Intestinal bacteria would metabolize this metabolite after oral administration. Furthermore, it can efficiently prevent the in vitro shrinkage and apoptosis of PC12 cells induced by hydrogen peroxide and tert-butyl hydroperoxide, affecting thus their proliferative capability.^20
–22 The level of gallic acid were similar in the two extracts. Gallic acid is a key element in the metabolic pathways of vegetables because both acids are substrates for the synthesis/catabolism of various secondary metabolites.²³ Compositional peculiarities were, in the extract of Caposele, the presence of chlorogenic acid and an amount of ferulic acid, which, although very low, turned about 26 times higher than that found in the extract of Rosato. This last contained epicatechin (39.18 μg/g), which turned out completely absent in the extract of Caposele. Starting from the similarity as regards the amount of total polyphenols, we could say that the increased antioxidant activity exhibited by the polyphenol extract of Caposele could be attributable to the greater amount of hyperoside than the variety Rosato. This last failed to have the same capacity to inhibit the activity of the DPPH radical, despite the presence of epicatechin and of a greater amount of AITC.

Table 2.

Polyphenol Profiles of the Extracts of the Endemic Garlic Varieties Caposele and Rosato of the Campania Region of Southern Italy

Polyphenols	Rosato, μg GAE/g ( ± SD)	Caposele, μg GAE/g ( ± SD)
Gallic acid	39.66 ± 1.86	34.58 ± 1.24
Chlorogenic acid	—	1.81 ± 0.01
Epicatechin	39.18 ± 1.65	—
Hyperoside	42.23 ± 2.01	89.24 ± 3.24
Ferulic acid	0.06 ± 0	1.55 ± 0.02
Allysiothiocianate	150.22 ± 9.02	124.90 ± 2.31

The polyphenols were identified and quantified based on the retention times of the corresponding standards. The data are mean values (expressed as μg ± standard deviations) of compounds present per g product (n = 3).

Antimicrobial and antifungal activity

In Table 3 we report the widths (mm) of the inhibition halos of the polyphenolic extracts obtained tested at 1–4 μg, using different bacteria and molds. Table 4 shows the widths of the inhibition halos of two antibiotics, tetracycline and gentamycin, positive control, and DMSO, used as negative control. Polyphenolic extract of the variety Rosato was active against all four microorganisms used as tester strains, producing halos ranging from 5 to 16.33 mm. The extract of the variety Caposele was inactive against B. cereus but it was more effective against the entheropathogenic strain of E. coli with respect to the extract of variety Rosato. The antimicrobial activity exhibited by the two extracts against S. aureus and E. coli might be used for food and pharmaceutical purposes, taking also into consideration the resistance of these microbial species toward the synthetic antibiotic.²⁴ Taking into account the results of Fratianni et al.,⁶ and Dziri et al.,²⁵ we could say that the level of antimicrobial activity might be dependent not only on the garlic variety but also on polyphenol composition. On the contrary, it was not linked to the antioxidant activity that was essentially related to the presence of specific metabolites. In fact, the presence of epicatechin in the polyphenolic extract of Rosato did not affect the antioxidant capability of such extract, but, on the contrary, probably gave it the capability to exhibit antimicrobial activity against B. cereus. Therefore, the extract of variety Caposele, which did not contain this metabolite, did not show such capability. Indeed, the presence of the higher level of hyperoside, strictly linked to the antioxidant activity, did not meliorate the antimicrobial activity of this extract against that specific microorganism.

Table 3.

Inhibition of Bacterial and Fungal Growth by the Garlic Endemic Varieties Caposele and Rosato of the Campania Region of Southern Italy

	μg	Escherichia coli	Bacillus cereus	Pseudomonas aeruginosa	Staphylococcus Aureus	Penicillum citrinum	Penicillium expansum	Aspergillus versicolor
Rosato	4	10.66 ± 0.57^a	10.66^a ± 0.57	11.33 ± 0.57^a	16.33 ± 1.52^a	—	11.33 ± 0.57^a	—
Rosato	2	7.33 ± 0.57^b	7.33^b ± 2.51^b	10.66 ± 0.57^a	10.66 ± 0.57^b	—	10.66 ± 0.57^a	—
Rosato	1	5.33 ± 0.57	5 ± 0^c	5 ± 0	5 ± 0^d	—	5 ± 0^c	9.33 ± 0.57^b
Caposele	4	25.33 ± 0.57^a	—	16.66^a ± 0.57^a	16.33 ± 1.52^a	13.33 ± 2.88^a	—	15.33 ± 2.88^a
Caposele	2	19.66 ± 0.57^b	—	10 ± 0^c	10.66 ± 0.57^c	9.33 ± 0.57^b	—	10.66 ± 1.15^b
Caposele	1	15.33 ± 0.57^c	—	5 ± 0^d	5 ± 0^d	6.33 ± 0.57^c	—	6.33 ± 0.57^c

The results shown (expressed in mm) are the mean values ± standard deviations of the widths of the inhibition zones (n = 3). Means followed by the same letter in superscripts within the same column are not significantly different by the Tukey test (P < .05).

Table 4.

Inhibition of Bacterial Growth by Gentamycin, Tetracycline, and DMSO

	Gentamycin 8 μg	Tetracycline 7 μg	DMSO
E. coli	16.0 ± 1.3	12.0 ± 0.4	—
B. cereus	17.7 ± 1.3	10.0 ± 0.5	—
P. aeruginosa	15.4 ± 0.4	10 ± 0	—
S. aureus	6.0 ± 0	6.5 ± 0.5	—

The results shown (expressed in mm) are the mean values ± standard deviations of the widths of the inhibition zones (n = 3).

The polyphenolic extracts showed different antifungal activity (Table 4). The extract of variety Rosato was effective against P. expansum, a mycotoxin-producer strain, producing halos ranging from 5 to 11.33 mm. On the other hand, the polyphenols of the variety Caposele were effective against P. citrinum and A. versicolor. The presence of epicatechin in the extract Rosato probably determined the ability of such extract to inhibit the growth of P. expansum. Moreover, the extract Caposele (in which this metabolite was absent) did not exhibit such capability. On the contrary, the activity carried out from the extract Caposele versus A. versicolor could be related to its content of hyperoside (twice abundant with respect to the extract Rosato), thus, in a certain sense, also to the antioxidant activity. We could make the same consideration also with regard to the antifungal activity exhibited by the extract Caposele and not by Rosato versus P. citrinum. Our work confirmed previous reports of polyphenols present in garlic, which respond to the antibacterial, antifungal, and antioxidant activity.²⁶ The different capability to inhibit various microorganisms, exhibited by the two extracts, could be exploited for food purposes, in developing systems of preservation that use extracts as such or entrapped in packaging systems. In this way, the quality of food could be preserved from oxidation and its contamination by citrinin-producing molds (P. citrinum and P. expansum) could be limited. The effectiveness in inhibiting the growth of A. versicolor, shown mainly by the extract Caposele, may also be used for pharmaceutical purposes: this fungus is responsible for different pathologies ranging from onychomycosis to pulmonary aspergillosis. In addition, toxins of A. versicolor can be extremely dangerous: they give hepatotoxicity and probably carcinogenicity and teratogenicity.^27,28 Glycotoxin inhibits phagocytosis by macrophages and the activation of B-lymphocytes.²⁹

In conclusion, these two endemic varieties of A. sativum L. of the Campania region may be considered promising sources of natural metabolites with antioxidant and antibacterial activities. More studies of the phytochemical contents and biological properties of the areal portions of these garlic varieties may stimulate new bioactive uses, particularly in food technology and in the pharmaceutical industry instead of the synthetic molecules.

Footnotes

Acknowledgment

The study was supported by the Project “SALVE” (Safeguard of the endemic vegetal genetic resources of the Campania Region”), PSR 2007–2013, Campania region, mis. 214 az f2.

Author Disclosure Statement

No competing financial interests exist.

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