Physicochemical Characterization and Biological
Activities of Black and White Garlic: In Vivo and
In Vitro Assays †
MaríaÁngeles Toledano Medina 1, Tania Merinas-Amo 2, Zahira Fernández-Bedmar 2,* ,
Rafael Font 3, Mercedes del Río-Celestino 3, Jesús Pérez-Aparicio 1,
Alicia Moreno-Ortega 4,5 ,Ángeles Alonso-Moraga 2and Rafael Moreno-Rojas 4
1Department of Food Science and Health, IFAPA-Palma del Río, Avda. Rodríguez de la Fuente, s/n,
14700 Palma del Río, Córdoba, Spain; email@example.com (M.A.T.M.);
2Department of Genetics, University of Córdoba, Gregor Mendel Building, Campus Rabanales,
14071 Córdoba, Spain; firstname.lastname@example.org (T.M.-A.); email@example.com (A.A.-M.)
3Agrifood Laboratory, CAPDER Avda. Menéndez Pidal s/n, 14004 Córdoba, Spain;
firstname.lastname@example.org (R.F.); email@example.com (M.d.R.-C.)
4Department of Bromatology and Food Technology, University of Córdoba, Darwin Building, Campus
Rabanales, 14071 Córdoba, Spain; firstname.lastname@example.org (A.M.-O.);
5Department of Food Science and Health, IFAPA-Alameda del Obispo, Avda. Menéndez-Pidal, s/n,
14004 Córdoba, Spain
*Correspondence: email@example.com or firstname.lastname@example.org; Tel.: +34-957-218-674
†The work was a part of MaríaÁngeles Toledano Medina’s doctoral thesis.
Received: 11 May 2019; Accepted: 12 June 2019; Published: 21 June 2019
White and three types of black garlic (13, 32, and 45 days of aging, named 0C1, 1C2, and
2C1, respectively) were selected to study possible diﬀerences in their nutraceutic potential. For this
purpose, garlic were physicochemically characterized (Brix, pH, aW, L, polyphenol, and antioxidant
capacity), and both
assays were carried out. Black garlic samples showed higher
polyphenol content and antioxidant capacity than the white ones. The biological assays showed
that none of the samples (neither raw nor black garlic) produced toxic eﬀects in the Drosophila
melanogaster animal genetic model, nor exerted protective eﬀects against H
, with the exception of
the 0C1 black garlic. Moreover, only white garlic was genotoxic at the highest concentration. On the
other hand, 0C1 black garlic was the most antigenotoxic substance. The
showed signiﬁcant extension of lifespan at some concentrations of white and 0C1and 1C2 black
experiments showed that all of the garlic samples induced a decrease in leukemia
cell growth. However, no type of garlic was able to induce proapoptotic internucleosomal DNA
fragmentation. Taking into account the physicochemical and biological data, black garlic could be
considered a potential functional food and used in the preventive treatment of age-related diseases.
In addition, our ﬁndings could be relevant for black-garlic-processing agrifood companies, as the
economical and timing costs can signiﬁcantly be shortened from 45 to 13 days of aging.
Keywords: black garlic; physicochemical proﬁle; polyphenol content; HL-60 cell line
Garlic (Allium sativum) is probably one of the oldest known medicinal plants, used since ancient
times to cure diﬀerent human diseases. Garlic started taking part in humans’ daily diet in Ancient
Foods 2019,8, 220; doi:10.3390/foods8060220 www.mdpi.com/journal/foods
Foods 2019,8, 220 2 of 18
]. Several scientiﬁc researches and clinical trials have been conducted during the last decade
to determine the eﬀects of garlic consumption on human health. Garlic’s principal medicinal uses
have focused on prevention and treatment of cardiovascular disease by lowering blood pressure and
cholesterol, and, more recently, on its antimicrobial properties and as a preventive agent for cancer [
The physiological eﬀects of garlic are due mainly to the presence of volatile sulfur compounds
like thiosulfates, which give it its characteristic pungent aroma. Several recent studies have shown that
these organosulfur compounds show anti-cancer, anti-cardiovascular, anti-neurological, and anti-liver
disease eﬀects, as well as eﬀects for the prevention of allergies and arthritis [
]. This group of
compounds, originating from the allicin decomposition, are associated with Allium species’ pungent
aroma and taste as well as their antioxidant activity [
]. However, rats fed with fresh garlic at high
doses (0.5 g/kg of body weight/day) showed toxicity in the liver .
Even though the health beneﬁts of garlic are known, its global consumption is declining. In general,
people are reluctant to eat raw garlic due to its pungent taste, smell, and gastrointestinal discomfort.
Because of this, researchers are interested in developing aged garlic products to decrease these negative
With a growing awareness of the health beneﬁts of garlic, black garlic, an aged garlic product,
has emerged as one of the fastest-growing health-oriented food products in world markets [
Black garlic is produced through the natural aging of whole ordinary garlic under controlled high
C) and humidity (90%) conditions for several days, without any artiﬁcial treatments or
]. Thermal processes are commonly used in food manufacturing to enhance the sensorial
quality of foods, their palatability, and to extend the range of colors, tastes, aromas, and textures in
]. In addition, heating processes have led to the formation of biological compounds that are
not originally present in food [
]. However, the inﬂuence of thermal processes on the concentration of
single ﬂavonoids and phenolic acids in garlic still remains unknown.
During aging, the cloves of normal garlic change their color from white to brown and ﬁnally
become black due to the Maillard reaction. At the same time, unstable compounds in raw garlic are
transformed into stable soluble compounds with a high antioxidant power [
]; the organoleptic
characteristics in black garlic are improved due to the conversion of unstable and odorous compounds
to stable and odorless compounds such as S-allyl-L-cysteine (SAC), or decomposed to organosulfur
compounds such as diallyl sulphide (DAS), diallyl disulphide (DADS), diallyl trisulphide (DATS),
dithiins, and ajoene [
]. Previous studies on black garlic have reported that the increase in its
antioxidant capacity could be due to the increase in polyphenols and S-allyl-cysteine, a compound
derived from alliin, during the heat processing .
Compared with fresh garlic, black garlic contains a polyphenol content that is three times higher in
whole black garlic bulbs and six times higher in peeled black garlic cloves [
], which is directly related
to the increase in the antioxidant activity. The amino acids, carbohydrates, and the S-allyl-L-cysteine
contents are increased 2.5 times, 28.7%–47.0%, and eight times, respectively [16,17].
Diﬀerent beneﬁcial health properties of black garlic have been described previously:
(i) antioxidant eﬀects using diﬀerent indicators such as super-oxide dismutase (SOD),
-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), and hydroxy radical scavenging,
as well as Fe
-chelating activities; (ii)
chemopreventive eﬀects in diﬀerent
cancers—including ethanol extracts of aged black garlic, which reduce the viability of several human
cancer cell lines (i.e., AGS, A549 lung, HepG2 liver, and MCF-7 breast cancer cells), and hexane extracts,
which induce caspase-dependent apoptosis in leukemic cells; (iii) anti-inﬂammatory eﬀects have been
shown by inactivation of NF-
B, upregulation of heme oxygenase-1, and inhibition of the COX-2 and
5-lipooxygenase activities, among other eﬀects .
The aim of the present study is to perform a qualitative and quantitative evaluation of the
health-beneﬁcial activities of white and three types of black garlic using a multi-assay experimental
design at the individual, cell, and DNA levels. We assessed the genotoxic, antigenotoxic, and lifespan
eﬀects using an
animal model of the common fruit ﬂy (Drosophila melanogaster) and their
Foods 2019,8, 220 3 of 18
proapoptotic capacities against cancer processes, including cytotoxicity and clastogenic DNA activity,
using an in vitro human cancer model (HL-60 cell line).
2. Materials and Methods
2.1. Preparation of Samples
White and black garlic were used in this study. Raw white garlic was purchased in a local market.
Black garlic was manufactured at 60
C and 90% relative humidity (RH). Samples at 0 (White), 13 (0C1),
32 (1C2), and 45 (2C1) days aging were taken during the manufacturing process. After peeling bulbs,
samples were crushed and divided into three subsets (to be physicochemically analyzed at speciﬁed
times). Garlic samples were lyophilized (
C, less than 1% water) before the biological assays and
then dissolved in distilled water to obtain the diﬀerent concentrations tested. The lyophilized extracts
were stored at room temperature in a dark and dry atmosphere until use.
The concentrations of garlic for the diﬀerent bioassays were established taking into account the
average daily food intake of D. melanogaster (1 mg/day) and the average body weight (1 mg) [
The concentration range for all tested substances was calculated in order to make it comparable to
the recommended garlic daily intake for humans. Although there is no standard intake for garlic, the
German Kommission E monograph proposed that a daily intake of approximately 1–2 garlic cloves
(about 4 g) of intact garlic may have health beneﬁts [
]. Unfortunately, this recommendation is not
substantiated by any scientiﬁc reference.
2.2. Measurement of Soluble Solid Content, pH, aw, and Browning Intensity
Total soluble solid content (
Brix), pH, water activity (a
), and browning intensity (Lvalue)
values were determined in triplicate for all samples following the method previously described by
Toledano–Medina et al. [
]. Garlic soluble solids (
Brix) were measured with an Abbe Refractometer
ORT-1 of KERN (Kern & Sohn GmbH, Balingen-Frommern, Germany). Garlic pH was measured with
a pH meter Crison Basic 20 (Crison Instruments, Barcelona, Spain). Garlic water activity (a
measured with an Aqualab Series 3/3TE meter with a temperature stabilizer (MeterGroup, München,
Germany). Garlic browning intensity was determined with a Konica Minolta CR-410 Croma Meter
colorimeter (Konica Minolta, Inc., Tokyo, Japan) as an Lvalue (L=100, white; L=0, black), following
the method described previously by Toledano–Medina et al. .
2.3. Total Polyphenol Content and Antioxidant Capacity
A Perkin Elmer Lambda 20 UV VIS spectrophotometer (Perkin Elmer, Waltham, MA, USA)
was used to determine total polyphenol content and antioxidant capacity in raw and heated garlic.
A previous extract was prepared to analyze antioxidant properties. Brieﬂy, samples were lyophilized
C, less than 1% water) and spliced into ﬁve extracts per sample. Garlic extract was prepared
dissolving 0.3 g of the lyophilized sample in 10 mL of a mixture of 50% (v/v) ethanol and distilled water.
Next, samples were stirred for one hour and then ﬁltered using a Buchner funnel with Whatman paper
(Whatman PLC, Maidstone, UK) into a vacuum ﬂask connected to a vacuum pump ﬁlter. The ﬁltered
extract was levelled at 25 mL with a 50% (v/v) hydroalcoholic solution.
The polyphenol concentration of garlic samples was determined by the Folin–Ciocalteu
]. To a volumetric 25 mL ﬂask, 0.5 mL of extract, 10 mL of distilled water, 1 mL
of Folin–Ciocalteu reagent, and 3 mL of sodium carbonate 20% (w/v) were added and diluted to
volume (25 mL) with distilled water. The mixture was heated to 50
C for 5 min to accelerate the
coloration reaction. Subsequently, it was cooled with water, and the reading was carried out in the
spectrophotometer (Perkin Elmer) at 765 nm. The reading was compared with a calibration curve
prepared with diﬀerent gallic acid solutions: 75, 100, 200, 250, 300 ppm. Polyphenol content results
were expressed considering the dilution of the sample (0.3 g in 25 mL) in grams of gallic acid equivalents
per kilogram of lyophilized sample.
Foods 2019,8, 220 4 of 18
Raw and heated garlic antioxidant capacity was determined by the ABTS method [
]. A mix of
2.557 mL of a solution of 7 mM ABTS reagent (Sigma, St. Louis, MI, USA) and 0.333 mL of a solution of
2.25 mM potassium persulfate in distilled water was made. This solution was stored in darkness for 16
h, enough time for radical cation (ABTS
) formation. Then, 0.15 mL of the ABTS
solution was diluted
in 15 mL of ethanol. The absorbance value at 734 nm was adjusted near 0.7 (A
). Next, 0.980 mL of
solution and 0.02 mL of garlic extract were added. After stirring, the absorbance was read at
734 nm after 7 minutes (A1). The inhibition percentage was calculated by the following expression:
% inhibition =(A0−A1)×100/A0. (1)
A calibration curve was built with the following Trolox (6-hydroxy-2, 5, 7,
8-tetramethylchroman-2-carboxylic acid) concentrations: 0.1, 0.5, 1, and 1.5 mM. Considering the
sample dilution, results were expressed in mmol Trolox-equivalents per kilogram of lyophilized sample.
2.4. In Vivo Assays
2.4.1. D. melanogaster Strains
The following Drosophila strains, each carrying a third chromosome hair marker, were used: (i)
mwh/mwh are homozygous for the recessive multiple wing hairs (mwh) mutation that produces multiple
tricomas per cell instead of one [
], and (ii) ﬂr
/In (3LR) TM3, rip
, where the ﬂr
marker is a homozygous recessive lethal mutation that produces deformed tricomas but is viable in
homozygous somatic cells once larvae start the development [
]. For detailed information on the
mutations, see Lindsley and Zimm .
2.4.2. Toxicity and Antitoxicity Assays
Five concentrations (4, 2, 1, 0.5, and 0.25 mg/mL) for each tested garlic, along with negative (H
and positive (0.12 M H
) controls were assayed after toxicity screening experiments. The toxicity
index was calculated as the percentage of individuals born in each treatment with respect to the negative
control. The antitoxicity tests consisted of combined treatments using the same concentrations as in
the toxicity assays, with the exception of the highest one (4 mg/mL), by adding the toxicant hydrogen
peroxide at 0.12 M . The percentage of emerging adults was compared with the positive control.
2.4.3. Genotoxicity and Antigenotoxicity Assays
The genotoxicity assays were carried out following the method described by Graf et al. [
trans-heterozygous larvae for mwh and ﬂr
gene markers were obtained by crossing four-day-old
females with mwh males in a 2:1 ratio. Four days after fertilization, females were allowed
to lay eggs in fresh yeast medium (25 g yeast and 4 mL sterile distilled water) during 8 h to obtain
synchronized larvae. After 72
4 h, the larvae were collected, washed with distilled water to remove
the remaining medium, and transferred, in groups of 100 individuals, to the treatment tubes where
they were chronically fed with the diﬀerent compounds. Treatment tubes contained 0.85 g of Drosophila
Instant Medium (Formula 4-24, Carolina Biological Supply, Burlington, NC, USA) and 4 mL of solutions
with diﬀerent concentrations of garlic (2 mg/mL and 0.25 mg/mL).
The antigenotoxicity trials were carried out following the method described by Graf et al. [
which consists of combined treatments of genotoxin (0.12 M H
) (Sigma, cat. number H-1009)
and the same concentrations used in genotoxicity assays of lyophilized garlic. For the evaluation
of the inhibition potency, negative (H
O) and positive (0.12 M H
) (Sigma, cat. number H-1009)
concurrent controls were included. After emergence, adult ﬂies were stored in 70% ethanol until the
removal and mounting of wings on slides using Faure’s solution (30 g Arabic gum, 20 mL glycerol,
50 g chloral hydrate, and 50 mL distilled water) for mutation screening under a photonic microscope
(Leica, Wetzlar, Germany) at 400×magniﬁcation.
Foods 2019,8, 220 5 of 18
Similar numbers of male and female wings for each treatment and concentration were mounted,
and wing hair mutations were scored among a total of 24,400 monotricoma wild-type cells per wing [
Wing hair spots were grouped into three diﬀerent categories: S, a small single spot corresponding
to one or two cell clones exhibiting the mwh phenotype that occurs in the latest stages of the mitotic
division; L, a large single spot with three or more cell clones showing mwh or ﬂr
occur in the early stages of larval development; or T, a twin spot corresponding to two juxtapositioned
clones, one showing the mwh phenotype and other the ﬂr
phenotype. Small and large spots are caused
by somatic point mutations, chromosome aberrations, and somatic recombinations, while twin spots
are produced exclusively by somatic recombinations between the ﬂr3locus and the centromere.
The total number of clones was also counted and a multiple-decision procedure was applied to
determine whether a result was positive, inconclusive, or negative [
]. The inhibition percentages
(IPs) for the combined treatments were calculated from the total spots per wing statistics with the
following formula :
IP =((single genotoxin −combined treatment)/single genotoxin) ×100. (2)
2.4.4. Lifespan Assays
In order to compare the genotoxicity and longevity results, ﬂies that underwent the lifespan
trials carried the same genotype as in genotoxicity assays. Hence, the F1 progeny from mwh and
parental strains produced by a 24 h egg-laying in fresh yeast medium was used in the longevity
experiments. All experiments were carried out at 25
C and according to the procedure described
ndez–Bedmar et al. [
]. Brieﬂy, synchronized 72
12 hour-old trans-heterozygous larvae
were washed, collected, and transferred in groups of 100 individuals to test vials containing 0.85 g of
Drosophila Instant Medium (formula 4-24, Carolina BiologicalSupply, Burlington NC, USA) and 4 mL
of the diﬀerent concentrations of the selected compounds.
Sets of 25 emerged individuals of the same sex were selected and placed into sterile vials
containing 0.21 g of Drosophila Instant Medium (formula 4-24, Carolina BiologicalSupply, Burlington
NC, USA) and 1 mL of the diﬀerent concentrations of solution of the compounds (4 mg/mL–0.25 mg/mL
range). Two replicates were followed during the complete life extension for each control and for the
concentrations established. Alive animals were counted, and the media was renewed twice a week.
2.5. In Vitro Assays
2.5.1. HL-60 Cell Line Culture Conditions
Cells were grown in RPMI-1640 medium (Sigma, R5886, St. Louis, MI, USA) supplemented
with 50 mL heat-inactivated fetal bovine serum (Linus, S01805, Madrid, Spain), L-glutamine at 200
mM (Sigma, G7513), and antibiotic-antimycotic solution with 10,000 units of penicillin, 10 mg of
streptomycin, and 25
g amphotericin B per mL (Sigma, A5955). Cells were incubated at 37
C in a
humidiﬁed atmosphere of 5% CO
(Shel Lab, Cornelius, OR, USA) [
]. The cultures were plated at
2.5 ×104cells/mL density in 10 mL culture bottles and passed every 2 days.
2.5.2. Cytotoxicity Assay
HL-60 cells were placed in 96-well culture plates (2
cells/mL) and treated for 72 h with
the lyophilized white and black garlic at diﬀerent concentrations (4 mg/mL, 2 mg/mL, 1 mg/mL,
0.5 mg/mL, 0.25 mg/mL, 0.12 mg/mL, 0.06 mg/mL, 0.03 mg/mL, and 0.015 mg/mL for white garlic and
4 mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL, and 0.25 mg/mL for black garlic samples). This wide range
of concentrations was intended to estimate the inhibitory concentration 50 (IC50).
Cell viability was determined by the trypan blue dye (Sigma, T8154) exclusion test. Trypan
blue solution was added to the cell cultures at a 1:1 volume ratio and 20
L of cell suspension were
immediately loaded into a Neubauer chamber. Cells were counted with an inverted microscope at
Foods 2019,8, 220 6 of 18
magniﬁcation (AE30/31, Motic, Wetzlar, Germany). Curves were plotted as survival percentages
with respect to the control growing at 72 h. At least three independent repetitions were carried out.
2.5.3. Determination of DNA Fragmentation
DNA fragmentation is a hallmark of apoptosis and has been regarded as a critical step in apoptosis.
Brieﬂy, HL-60 cells (1
cells/mL) were treated with diﬀerent concentrations of lyophilized garlic
(4 mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL, and 0.25 mg/mL, respectively) for 5 h. Treated cells were
collected and centrifuged at 3000 rpm for 5 min, and DNA was extracted with lysis, precipitation,
and wash steps according to Merinas–Amo et al. [
]. The total extracted DNA was quantiﬁed
in a spectrophotometer (Nanodrop
ND-1000, Thermo Fisher Scientiﬁc, Waltham, MA, USA), and
1200 ng of DNA was loaded into a 2% agarose gel electrophoresis, stained with ethidium bromide,
and visualized under UV light.
2.6. Statistical Analysis
The statistical analysis of the solid content, pH, a
, browning intensity, polyphenol content,
antioxidant capacity, and total polyphenol index for each type of garlic was evaluated with the SPSS
Statistics 17.0 software SPSS (IBM, Armonk, NY, USA) using one-way ANOVA and Tukey’s test
(homogeneous subsets) to assess the signiﬁcance of the subsets.
Signiﬁcant diﬀerences with respect to the concurrent control in toxicity assays were determined
using the Chi-square method, and a concentration was considered as a toxic when the Chi-square
value was higher than 5.02.
The frequency of each type of mutant clone/wing in the anti/genotoxicity assays was compared
with the negative concurrent control, and signiﬁcance was given at the 5% error level. Inconclusive and
positive results were further analyzed with the Mann–Whitney–Wilcoxon (
U-test using the SPSS Statistics 17.0 software SPSS.
The statistical treatment of life- and health-span data for each control and concentration was
assessed with the SPSS Statistics 17.0 software, using the Kaplan–Meier method. The signiﬁcance of
the curves was determined using the Log-Rank method (Mantel-Cox).
To obtain the tumor growth inhibition curves, the mean of three independent assays of the
alive-treated cells for each compound and concentration was used. The standard errors of the three
repetitions were calculated, and the Excel-given curve was added. Finally, the inhibitory concentration
50 (IC50) was estimated.
3. Results and Discussion
3.1. Soluble Solids Content, pH, Water Activity, and Browning Intensity
A weight reduction was observed during the garlic manufacturing procedure, with the 0C1
black garlic being the sample with the nearest weight to the white garlic (Table 1). According to
similar studies, changes in garlic weight during processing are mainly caused by a reduction of the
amount of water [
]. The main organosulfur in black garlic is considered to be the water-soluble
S-allyl-L-cysteine (SAC) [
]. Hence, after aging, SAC increased in the processed black garlic matrix,
and its precursor garlic
-glutamyl-S-allyl-L-cysteine decreased [
]. The manufacturing of black garlic
in this manner is not a microbe-associated fermentation but a Maillard and Browning reaction because
the processing temperature of garlic does not allow bacterial growth to elicit fermentation .
Soluble solids content (
Brix), pH, water activity (a
), and browning intensity (L) are shown in
Table 1. During heat treatment, soluble solids content increased in garlic, whereas pH, a
, and browning
intensity decreased. Similar Tukey’s test values were obtained in
Brix readings for white and 0C1
black garlic (40.47), meanwhile, signiﬁcant soluble solids content diﬀerences were observed in the 1C2
and 2C1 black garlic (43.17 and 45.67, respectively). The sugar content (
Brix) of black garlic increased
with respect to white garlic. This result is in agreement with the data of Choi et al., which show that
Foods 2019,8, 220 7 of 18
sugar content (e.g., glucose, fructose, sucrose, and maltose) increased in black garlic compared to
fresh and steamed garlic [
]. Furthermore, this increment might be related to its sweeter taste [
pH signiﬁcantly decreased during the manufacturing process. White garlic pH was the highest with a
value of 5.94, whereas black garlic pH decreased rapidly starting at 3.69 and reaching 3.49 at 45 days
of aging. These results are in agreement with the report by Shin et al., who showed that black garlic
pH decreased from 6.40 to 5.29 after 6 days of aging [
]. The same observation has recently been
]. Water activity (a
) decreased with aging to a lesser extent than other parameters
because the black garlic was manufactured maintaining a high relative humidity. According to Kaanane
and Labuza and Labuza and Saltmarch, the rate of the browning reaction is known to reach a maximum
values in the range of 0.5–0.7 [
]. However, signiﬁcant diﬀerences between white and black
are found (Table 1). The high RH and time required for producing black garlic in the present
study might have created a balanced situation between the a
of the heated garlic sample and the RH
inside the chamber where black garlic was produced. This a
condition is thought to facilitate the
browning reaction in heated garlic samples. As Table 1shows, browning intensity (L) in white and
black garlic was signiﬁcantly diﬀerent, with more than 28 units of diﬀerence between them, although
the 1C2 and 2C1 black garlic showed similar luminescence (17.85 and 17.58, respectively). Browning
intensity happened earlier at higher temperatures. Several studies have shown a positive relationship
between temperature increasing and browning product formation; however, at the initial induction
period a decrease is observed [
]. The garlic’s color eventually changed to dark brown/black,
mainly due to the formation of numerous compounds resulting from the non-enzymatic browning
reaction (Maillard reaction).
Table 1. Physicochemical characterization of four types of garlic according to the days of aging.
Type of Garlic White 0C1 Black 1C2 Black 2C1 Black
Aging process (days) 0 13 32 45
Weight of 10 garlic cloves (g) 49.69 ±0.25 a,1 45.83 ±0.32 b,c 37.57 ±0.38 b19.67 ±0.19 d
Soluble solid content (ºBrix) 40.47 ±0.29 c40.47 ±0.34 c43.17 ±0.48 b45.67 ±0.42 a
pH 5.94 ±0.01 a3.69 ±0.03 b3.60 ±0.04 c3.49 ±0.06 d
Water activity (aw) 0.97 ±0a0.93 ±0c0.93 ±0c0.93 ±0c
Browning intensity (L) 47.16 ±0.15 a18.73 ±0.21 c17.85 ±0.24 b17.58 ±0.25 b
Polyphenol content (g GAE/kg) 4.30 ±0.04 d10.94 ±0.28 c14.67 ±0.19 b16.17 ±0.29 a
Antioxidant activity (TROLOX equivalents/kg)
10.20 ±0.27 d67.65 ±1.26 b57.35 ±1.74 c78.61 ±2.41 a
Values are means
standard error (SE) (n=3).
Diﬀerent letters (a, b, c, d) in the same row show signiﬁcant values
in a one-way ANOVA using the post hoc Tukey’s test. GAE, gallic acid equivalents.
3.2. Total Polyphenol Content and Antioxidant Capacity
Total polyphenol (g/kg in Gallic) content and antioxidant capacity (inhibition percentage) are
shown in Table 1. During heat treatment, unstable compounds of raw garlic are transformed into stable
soluble compounds with a high antioxidant power [
]. Previous studies on black garlic reported
that this enhancement of the antioxidant capacity could be due to the increase in polyphenols and
S-allyl-cysteine, the compound derived from alliin [
]. The antioxidant power of polyphenols has
been demonstrated, so it seems logical to state that an increase in polyphenol content in black garlic
is responsible for the antioxidant properties in this product [
]. It is well known that the higher
antioxidant eﬀect of black garlic is due to the presence of S-allyl-cysteine, a compound derived from
alliin during heat processing .
Signiﬁcant diﬀerences among all the samples were found for the total polyphenol content and
the antioxidant capacity. Both parameters increased signiﬁcantly as heat increased. The highest
concentration of polyphenol content was obtained in 2C1 black garlic, although all black garlic samples
showed increases between 6 and 12 times in relation to the heat treatment (Table 1). Previous studies
carried out with whole bulbs of black garlic at 70, 72, 75, and 78
C have described an increase
of 2–3 times in polyphenol content compared to raw garlic [
]. Our results on the increase of
polyphenol content after heating agree in part with those obtained by other authors who found a
threefold increase in content .
Foods 2019,8, 220 8 of 18
To clarify the antioxidant properties of black garlic during aging, we focused on the analysis of
total polyphenol content. At the end of the heating process, an increase in antioxidant capacity was
observed in garlic. Black garlic samples showed an increase rank of 5.7–7.8 times with respect to white
garlic (Table 1). Several studies described that aged black garlic exerts stronger antioxidant activity
than white garlic, both
]. The total polyphenol content of black garlic
was not only signiﬁcantly higher than that of raw garlic, but also increased signiﬁcantly at the 13th
day of aging. Similar results were obtained by Sasaki et al., who showed an antioxidant potency
increase in aged black garlic extracts reaching 25-fold compared with fresh garlic [
]. According to Xu
and Chang, heat treatment of the phenolic compounds increased the free fraction of phenolic acids,
whereas it decreased the ester, glycoside, and ester-bound fractions, leading to an increase in free
phenol forms [
]. Gorinstein et al. showed that the garlic processing conditions lead to changes in the
content of its bioactive compounds (polyphenols such as ﬂavonoids and anthocyanins), and this is
related to the type and duration of treatment [
]. From the results regarding total polyphenols and
antioxidant capacity, it is possible to state that the optimum aging period for black garlic in order to
maximize antioxidant content may be 13 days.
The toxicity and antitoxicity of the four samples of tested garlic was assessed in the D. melanogaster
model. Figure 1A shows the relative percentage of emerging adults after treating larvae with
diﬀerent concentrations of these substances, showing that none of the garlic samples were toxic at the
assayed doses in D. melanogaster. These results agree with those by which the safety of garlic extracts
was well established through general, chronic, acute, and subacute toxicity, teratogenicity, and toxicity
tests conducted by the U.S. Food and Drug Administration, and clinical studies as well [47–52].
Figure 1B shows the results of the antitoxicity assays using hydrogen peroxide as a toxicant.
The individuals treated with 0.12 M of the oxidative toxin reached an average survival rate of 63.4%
with respect to the water negative control. In addition, 0C1 black garlic was the only preventive
substance against H
at two of the assayed concentrations (0.5 and 1 mg/mL). On the other hand,
white and 1C2 and 2C2 black garlic did not exhibit protective eﬀects against the genotoxin at any
tested concentrations (0.25–0.5 and 0.5–1 mg/mL, respectively). Lei et al. studied the eﬀects that black
10–15 days-aged garlic extracts had in D. melanogaster. The results from this study demonstrated that
black garlic extracts possess strong antioxidant capacities in vitro and in vivo .
To assess the genotoxicity/antigenotoxicity of the studied compounds, we used the SMART
(Somatic Mutation and Recombination Test) Test in D. melanogaster [
]. Increasing concentrations of
tested compounds, a negative control corresponding to water used as a solvent, and a positive control
) for periodic validation of the assay were concurrently assayed. Furthermore, antigenotoxicity
experiments were carried out using combined treatments consisting of repeating every concentration
tested and by adding the same concentration of hydrogen peroxide, which we have demonstrated to
be a potent mutagen in the SMART system .
Table 2shows the results of genotoxicity assays in the SMART test for white and the three black
garlics. Negative controls showed a frequency of mutations per wing equal to 0.195, which falls into
the historical range for the wing spot test [
]. The ﬁnal concentration of H
used (0.12 M) has
been demonstrated to exert a potent genotoxic eﬀect capable of inducing somatic mutations and mitotic
recombination in D. melanogaster [
]. The average frequency of total mutations per wing obtained in
the treatment with H
was 0.425. For each concentration and compound, single small, single large,
twin, and total clones were analyzed in the wings of chronically treated animals. The results showed
that all garlic showed non-genotoxic activity except for the white one, which signiﬁcantly increased the
frequency of mutations to 0.425 at the highest concentration tested. Similar results were obtained by
Abraham and Kesavan and Shukla and Taneja, who demonstrated that aqueous garlic extracts (5% v/v)
Foods 2019,8, 220 9 of 18
and ﬁne garlic powder (7.5, 5 and 2.5 g/kg body weight) supplementation do not induce chromosomal
aberrations nor DNA damage in mouse bone marrow cells [
]. Similar results were obtained by
Sowjanya et al. at 3, 6, and 12 mg/culture in human lymphocytes [
] and by Chughtai et al. using
extracts of fresh garlic bulbs in a yeast model .
) and antitoxicity (
) levels of black and white garlic studied in D. melanogaster.
) Percentage of viability of Drosophila treated with diﬀerent concentrations of the assayed garlic.
) Viability of Drosophila tested with diﬀerent concentrations of the tested garlic combined with the
genotoxicant hydrogen peroxide at 0.12 M. Values represent the mean
SE from three independent
experiments. *: signiﬁcant (p
0.05), with respect to their concurrent controls. 0C1: black garlic with
13 days aging, 1C2: black garlic with 32 days aging, and 2C1: black garlic with 45 days aging.
Vegetables contain polyphenols and oligoelements with antimutagenic activity [
]. The 0C1 black
garlic was the only one able to inhibit the genotoxic activity of hydrogen peroxide in a dose-dependent
manner (Table 2). The highest concentration tested for 0C1 black garlic in the combined treatments
partially counteracted part of the genotoxic eﬀect of H
, showing a decrease in the total mutation
frequency to 0.266 spots/wing and inhibiting around 37% of the genotoxicity induced by H
control correction). The rest of the compounds tested did not show signiﬁcant protective results against
DNA damage at the highest concentration and a just a slight inhibition percentage of mutations induced
by the genotoxin were observed (24% for white, 18.6% for 1C2 black, and 7.5% for 2C1 black garlic).
In general, garlic has signiﬁcant antioxidant activity and protective eﬀects against oxidative
DNA damage regardless of the processing method [
]. Our antitoxicity and antigenotoxicity results
showed that 0C1 black garlic (aged for 13 days) is able to protect from the genomic damage of
this genotoxin in a dose-dependent manner. This eﬀect could probably be due to the antioxidative
and free-radical scavenging capacity of their respective organosulfur compounds, which agree with
previous reports [
]. Besides the antioxidant activity, our results about the stronger antioxidant
Foods 2019,8, 220 10 of 18
activity shown by black garlic, compared with fresh garlic, are in agreement with previous
in vitro garlic assays [65,66].
Genotoxicity and antigenotoxicity of white (0 days aging), 0C1 black (13 days aging), 1C2
black (32 days aging), and 2C1 black (45 days aging) garlic in the Drosophila wing spot test.
Clones per Wing (n◦spots) 1
Percentage (%) 2
Small Single Clones
Large Simple Clones
(More Than 2 Cells)
H2O 41 0.146 (6) 0.049 (2) 0 0.195 (8)
H2O240 0.350 (14) 0.075 (3) 0 0.425 (17) +
White garlic (mg/mL)
0.25 40 0.225 (9) 0.025 (1) 0.025 (1) 0.275 (11) −
2 40 0.375 (15) 0.050 (2) 0.000 0.425 (17) +
0C1 Black garlic (mg/mL)
0.25 40 0.175 (7) 0.025 (1) 0.000 0.200 (8) −
2 41 0.122 (5) 0.000 0.000 0.122 (5) −
1C2 Black garlic (mg/mL)
0.25 40 0.200 (8) 0.025 (1) 0.025 (1) 0.250 (10) -
2 40 0.175 (7) 0.025 (1) 0.000 0.200 (8) -
2C1 Black garlic (mg/mL)
0.25 40 0.175 (7) 0.05 (2) 0.000 0.225 (9) −
2 40 0.250 (10) 0.000 0.000 0.250 (10) −
Combined Treatment With H2O2(0.12 M)
White garlic (mg/mL)
0.25 34 0.235 (8) 0.088 (3) 0.000 0.323 (11) −24
2 34 0.265 (10) 0.206 (7) 0.000 0.500 (17) +−17
0C1 Black garlic (mg/mL)
0.25 30 0.5 (15) 0.033 (1) 0.000 0.533 (16) +−25.4
2 30 0.233 (7) 0.033 (1) 0.000 0.266 (8) −37.4
1C2 Black garlic (mg/mL)
0.25 26 0.307 (8) 0.038 (1) 0.000 0.346 (9) −18.6
2 38 0.368 (14) 0.053 (2) 0.000 0.421 (16) −0.17
2C1 Black garlic (mg/mL)
0.25 28 0.357 (10) 0.036 (1) 0.000 0.393 (11) −7.5
2 28 0.357 (10) 0.250 (7) 0.000 0.607 (17) +−42.8
Statistical diagnosis according to Frei and Wurgler [
]. +, positive (p<0.05);
, negative. m, multiplication
factor. Levels of signiﬁcance
=0.05, tail test without Bonferroni correction. Inconclusive results were resolved
by Mann–Whitney–Wilcoxon U-test.
The inhibition percentages for the combined treatments were calculated from
total spots per wing according to Abraham .
3.5. Longevity Assays
Drosophila melanogaster is a choice model organism in the study of aging due to its relatively
short life expectancy. Moreover, a large number of individuals can be reared in controlled laboratory
conditions, and adults show many aspects of the observed cellular senescence events in mammals.
Thus, ﬂies have been frequently used to study physiological and pathological processes that aﬀect
life expectancy and can help to understand the relationship between nutrient metabolism and the
mechanisms of aging .
The entire lifespan curves and signiﬁcances obtained by the Kaplan–Meier method for each
substance and concentration are shown in Figure 2and Table 3, respectively. Drosophila had an average
lifespan expansion of 60 days in the control treatment. White and 1C2 black garlic signiﬁcantly
increased Drosophila’s lifespan at the lowest and the two moderated concentrations tested (0.25, 1,
and 2 mg/mL), with an extension with respect to the concurrent control of 10.1, 11.1, and 18.5 days
for white garlic and 9.4, 10.1, and 9.8 days for black garlic, respectively (Table 3). Furthermore, every
concentration assayed of 0C1 black garlic, except the highest one, induced a lifespan extension of
10 days in D. melanogaster compared to the control. On the other hand, 2C1 black garlic did not
inﬂuence the lifespan extension of D. melanogaster at any tested concentration. No previous
studies on longevity activities of black garlic as a food have been reported. However, several authors
have reported beneﬁcial eﬀects on animal lifespans using white garlic extracts in D. melanogaster at 37.5
Foods 2019,8, 220 11 of 18
and 75 mg/mL, Caenorhabditis elegans at 0.05 mg/mL, and senescence-accelerated mice (SAMP8) at 2%
Survival curves of D. melanogaster fed with diﬀerent concentrations of garlic (black and white)
) White garlic,
) 0C1: black garlic with 13 days aging,
) 1C2: black garlic with 32 days
aging and D) 2C1: black garlic with 45 days aging.
Mean and signiﬁcances of lifespan and healthspan curves for the diﬀerent garlic treatments
assayed in D. melanogaster.
Compound Title Treatment (mg/mL) Mean Lifespan (Days) Mean Healthspan (Days)
Negative Control 0 60.31 32.46
0.25 70.47 * 38.40 ns
0.5 68.72 ns 29.40 ns
1 71.43 ** 40.39 *
2 78.89 *** 40.44 *
4 58.21 ns 31.91 ns
0.25 70.15 * 37.67 ns
0.5 71.72 ** 38.18 ns
1 70.73 * 30.85 ns
2 71.80 * 43.90 *
4 62.60 ns 33.10 ns
0.25 69.73 * 29.36 ns
0.5 65.19 ns 25.15 *
1 70.49 ** 31.10 ns
2 70.18 * 28.57 ns
4 60.26 ns 26.20 ns
0.25 59.06 ns 24.07 ns
0.5 64.75 ns 41.29 ns
1 59.82 ns 28.50 ns
2 57.30 ns 23.46 **
4 66.38 ns 40.50 ns
Results were calculated by the Kaplan–Meier method, and the signiﬁcance of the curves was determined by the
Log-Rank method (Mantel–Cox). ns: non-signiﬁcant (p>0.05), *: signiﬁcant (p <0.05), **: signiﬁcant (p<0.01),
***: signiﬁcant (p<0.001). WG: white garlic (0 days aging); 0C1: black garlic (13 days aging); 1C2: black garlic (32
days aging); 2C1: black garlic (45 days aging).
Foods 2019,8, 220 12 of 18
We suggest that the diﬀerences found between these results and ours could be due to the diﬀerent
types of sample presentation. We used crude entire garlic material, and all data available elsewhere on
lifespan trials come from extracts. In this sense, Prowse et al. demonstrated that garlic juice exerted
insecticidal activity across life stages of ﬂies at a wide range of concentrations (0.25–5%) in two dipteran
pests (Delia radicum and Musca domestica) [
]. Lei et al. studied the eﬀects of black 10–15 days-aged
garlic extracts on the lifespan of Drosophila through the observation of half-life time, and the mean and
maximum lifespan of organisms. The results suggested a signiﬁcant longevity extension in Drosophila
treated with black garlic extracts in a dose-dependent manner .
3.6. Healthspan Assays
In order to know the quality of life of the Drosophila treated in the longevity assays, we studied
the 25% of individual survival at the top of the lifespan curves obtained in the previous test for each
substance and concentration tested. This part of the lifespan is considered the healthspan of a curve
and is characterized by low and more or less constant age-speciﬁc mortality rate values [
]. The results
are shown in Table 3.
Only white and 0C1 black garlic induced a signiﬁcant increase of healthspan in D. melanogaster
compared to the control, with an average of 8 and 11.5 days, respectively. In contrast, 1C2 and 2C1
black garlic induced a signiﬁcant reduction of healthspan in Drosophila at moderate concentrations,
with a value of 7.3 and 9 days, respectively, with respect to the control. No previous studies about the
eﬀects that white and black garlic exert on quality of life have been reported.
All the substances assayed showed cytotoxic activity against HL-60 tumor cells (Figure 3). White
and black garlic showed a dose-dependent response, with an increase in the cytotoxicity level as the
concentration of garlic increased. White garlic showed the highest cytotoxic eﬀect against the tumor
cells, the inhibitory concentration 50 (IC50) being under 0.03 mg/mL.
Viability of HL-60 cells treated with diﬀerent concentrations of black and white garlic for 72 h.
) White garlic, (
) 0C1: black garlic with 13 days aging, (
) 1C2: black garlic with 32 days aging, and
(D) 2C1: black garlic with 45 days aging.
The cytotoxicity curve of 0C1 black garlic showed a dose-dependent increase with an IC
equal to 1 mg/mL. In relation to 1C2 and 2C1 black garlic, no inhibition was observed at the lowest
Foods 2019,8, 220 13 of 18
concentration tested, but contrarily, a strong tendency to increase cell growth is observed with an IC
value of 0.7 and 0.9 mg/mL, respectively. Moreover, an eventual cell-growth inhibition was observed
in 1C2 and 2C1 black garlic at 2 mg/mL.
A number of studies have demonstrated the chemopreventive activity of garlic by using diﬀerent
garlic preparations, including fresh garlic extract, aged garlic, garlic oil, and a number of organosulfur
compounds derived from garlic [
]. Such a chemopreventive activity has been attributed to the
presence of organosulfur compounds in garlic. Therefore, the consumption of garlic may provide
some kind of protection against tumor cell proliferation [
]. Studies on the preventive eﬀects of
black garlic extracts also show an induction of
inhibition in gastric cancer cell
growth, chemopreventive eﬀects in rat colon tumors, and an increase in anti-tumor activity in a mouse
3.8. DNA Internucleosomal Fragmentation
The HL-60 cell line belongs to the undiﬀerentiated immortal lines, as they are tumor cells. It is
widely investigated as a model for purposes of inducible cell diﬀerentiation. This phenomenon
might aﬀect the cell’s ability to proliferate and therefore their immortality, with the appearance of
apoptosis. Compounds capable of inducing diﬀerentiation and apoptosis are candidates to act as a
chemopreventive agents or cancer chemotherapeutics.
Figure 4shows the electrophoresis of the genomic DNA of HL-60 cells when treated with diﬀerent
concentrations of white, 0C1, 1C2, and 2C1 black garlic.
DNA internucleosomal fragmentation is represented by a DNA laddering, and it is associated
with the activation of the apoptotic way in cancer cells, a hallmark of the genomic integrity [
of the assayed concentrations (4 mg/mL to 0.25 mg/mL) induced internucleosomal fragmentation by
the diﬀerent black garlic treatments, but a slight fragmentation was observed in the lowest assayed
concentration of white garlic (0.25 mg/mL). Hence, the cytotoxic activity observed is only induced in a
proapoptotic way in the white garlic.
Internucleosomal DNA fragmentation in HL-60 cells treated for 5 h with diﬀerent
concentrations of black and white garlic. DNA fragmentation was detected following electrophoresis in
agarose gels and staining with ethidium bromide. M: indicates DNA size marker; C: indicates control
(lane 1); 0.25 mg/mL (lane 2); 0.50 mg/mL (lane 3); 1 mg/mL (lane 4); 2 mg/mL (lane 5), and 4 mg/mL
(lane 6) of garlic sample. (
) White garlic, (
) 0C1: black garlic with 13 days aging, (
) 1C2: black
garlic with 32 days aging, and (D) 2C1: black garlic with 45 days aging.
Foods 2019,8, 220 14 of 18
Our results demonstrate that only white garlic has a strong cytotoxic eﬀect and induces slight
DNA proapoptotic internucleosomal fragmentation against HL-60 cells. These results agree with
several reports demonstrating that garlic exerts a chemopreventive eﬀect by increasing apoptosis
in lung cancer cells (NCI-H1299) [
]. On the other hand, our results do not agree with the results
obtained by Wang et al., who detected a dose-dependent apoptosis in aged black garlic extract in
in vitro studies .
It is the ﬁrst time that an investigation of the relationship between the physicochemical
characterization and the biological activities of white and black garlic has been carried out. Multifocal
studies integrating the toxicity, antitoxicity, genotoxicity, antigenotoxicity, longevity, cytotoxicity, and
proapoptotic properties of diﬀerent types of garlic were followed in order to propose black garlic as a
nutraceutical or functional food.
Black garlic aged for thirteen days showed qualitative improved physicochemical characteristics
with respect to white garlic and to the other processed black garlic as well. The 0C1 black garlic (13
days aged) showed similar weight and soluble solids content (
Brix) to the raw garlic. All of the black
garlic samples had an improved the polyphenol content and inhibition percentage with respect to the
All types of garlic were safe, not showing toxicity in the D. melanogaster model, except for the
white one, although only black garlic aged for 13 days showed slight protection against the oxidative
toxicant at the three highest concentrations. Genotoxicity assays revealed that all raw and processed
garlic were not genotoxic, with the exception of the higher concentration of white garlic, and exhibit
moderate antigenotoxic eﬀects when the imaginal discs are treated with the genotoxin hydrogen
peroxide. The longevity assays in D. melanogaster yielded a signiﬁcant extension of lifespan results
in several concentrations of white and 0C1 and 1C2 black garlic. Finally, the results achieved in the
experiments for garlic cytotoxicity were hopeful. All studied garlic induced a decrease in
leukemia cells growth. However, no type of garlic was able to induce proapoptotic internucleosomal
Important information is added to the agrifood industry as our data suggest that short-aged
fermented black garlic (13 days) has higher biological activities than the longer-fermented ones,
and even more than white garlic. This could have important industrial and economics consequences.
Taking both the physicochemical and biological data, the black garlic aged for 1 days has shown itself
to have the best nutraceutical properties. Our ﬁndings are relevant for black-garlic-processing agrifood
companies as the economical and timing incomes are signiﬁcantly reduced to 13 days aging.
M.A.T.M., J.P.-A., and A.M.-O. performed all physicochemical analyses. Z.F.-B., T.M.-A.,
R.F., and M.d.R.-C. carried out all genotoxicological and antigenotoxicological analyses. R.M.-R. and A.A.-M.
designed this study and revised the manuscript. Z.F.-B., T.M.-A., R.F., and M.d.R.-C. wrote this manuscript.
T.M.-A. performed the longevity assays. Z.F.-B., T.M.-A., and A.A.-M. performed all
assays. All the listed
authors have read and approved the submitted manuscript.
Funding: This research received no external funding.
Conﬂicts of Interest: The authors declare no conﬂict of interest.
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