Received: 10 October 2019 | Revised: 18 December 2019
DOI: 10.1111/jfbc.13165
FULL ARTICLE
Characterization of phenolic profile by LC-ESI-MS/MS and
enzyme inhibitory activities of two wild edible garlic: Allium
nigrum L. and Allium subhirsutum L.
Ahmet Emir1 | Ceren Emir1
1
Department of Pharmacognosy, Faculty of
Pharmacy, Ege University, İzmir, Turkey
2
Department of Biology, Botany Section,
Faculty of Science, Ege University, İzmir,
Turkey
Correspondence
Ahmet Emir, Department of Pharmacognosy,
Faculty of Pharmacy, Ege University,
Bornova, İzmir, Turkey.
Email: ahmet.emir@ege.edu.tr,
ahmetemir84@gmail.com
Funding information
Türkiye Bilimsel ve Teknolojik Araştirma
Kurumu, Grant/Award Number: 217S341;
TUBITAK, Grant/Award Number: 217S341
J Food Biochem. 2020;44:e13165.
https://doi.org/10.1111/jfbc.13165
| Accepted: 13 January 2020
| Hasan Yıldırım2
Abstract
In our study, Allium nigrum L. and Allium subhirsutum L. were investigated in terms
of phenolic profile, acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and
tyrosinase inhibitory potentials. The colorimetric analysis revealed that the highest
levels of total phenol (45.6, 15.8 mg GAE/g extract, respectively) and total flavo-
noid contents (8.2, 5.7 mg QE/g extract, respectively) were found in the bulbs of
both plants. About 30 compounds were determined by LC-ESI-MS/MS with vali-
dated method and 3-hydroxybenzoic acid (2,188.4 μg/g extract) and p-coumaric
acid (1,700.8 μg/g extract) were major phenolic acids. (−)-Epigallocatechin gallate
(998.3 µg/g extract) and genistein (159.3 μg/g extract) which are neuroprotective
compounds were the predominant flavonoids for A. nigrum and A. subhirsutum,
respectively. Enzyme inhibitory activities of samples were performed by spectro-
photometrically with 96-well microplate reader. All samples showed anti-AChE, anti-
BuChE, and anti-tyrosinase activities and the aerial part of A. nigrum was the most
potent (IC50 6.1, 3.27, 22.31 µg/ml, respectively).
Practical applications
Many Allium species, especially those cultivated, are consumed in different countries
as food in different ways. In the literature, studies on these species have generally
focused on organosulfur compounds of the species. In our present study, phenolic
compounds having a wide range of biological activities were determined in differ-
ent parts of the two Allium species consumed as food. We also investigated in vitro
cholinesterases and tyrosinase inhibition activities of these species. A correlation
was observed between phenolic compounds and enzyme inhibition activities. These
results were further explored and confirmed by principal component analysis (PCA).
PCA revealed that samples were discriminated from each other according to phenolic
compounds and enzyme inhibitory potencies. Conclusively, this study determines
that the chemical profiles and biological activities of A. nigrum and A. subhirsutum.
KEYWORDS
Allium, cholinesterase inhibition, LC-MS/MS, phenolics, tyrosinase inhibition
wileyonlinelibrary.com/journal/jfbc © 2020 Wiley Periodicals, Inc. | 1 of 14
2 of 14 | EMIR et al.
1 | I NTRO D U C TI O N
The functional food term was first presented in Japan in the 1980s,
as the foods containing ingredients that support bodily functions
as well as being nutritious. The description of functional food has
been recommended by different authorities, academics, and indus-
tries, until today (Kaur & Das, 2011). Also, Functional Food Science
in Europe (FUFOSE) offered a description: “food that beneficially af-
fects one or more target functions in the body beyond adequate nu-
tritional effects” (Europe, 1999). Among the functional foods, Allium
species, particularly cultivated forms, namely A. sativum (garlic) and
A. cepa (onion) have been used since ancient times as vegetables,
spices, and folk remedies due to their health beneficial properties
(Iciek, Kwiecien, & Włodek, 2009). Also, Allium subhirsutum, the local
name is known as “körmen,” is the ingredient of a traditional Turkish
flatbread (Ertug, 2004; Kosar, Koyuncu, & Başer, 2006) and Allium
nigrum which is an ornamental plant used as a food spice (Lentini &
Venza, 2007).
Alzheimer’s disease (AD), the most common cause of dementia
around the world, is a neurodegenerative disease that causes irre-
versible destruction of mental functions. About 74.7 million people
in 2030 and 131.5 million people in 2050 are expected to be affected
by AD (Cummings et al., 2016). The cholinesterase inhibitors (ChEIs)
used in the treatment of AD do not affect mortality, but increase the
quality of life and stabilize the disease stage by enhancing cholin-
ergic efficacy. In this way, FDA (US Food and Drug Administration)
approved five ChEIs including tacrine, donepezil, rivastigmine,
galantamine, and memantine (NMDA receptor antagonist). Among
them donepezil (IC50 nmol/L AChE: 3,900, BuChE: 18,600) and
galantamine (an Amaryllidaceae alkaloid, IC50 nmol/L AChE: 33,
BuChE: 988) more selective for acetylcholinesterase (AChE), others
are effective both on AChE and BuChE (butyrylcholinesterase) en-
zymes (Weinstock, 1999). Clinical trials showed that in 6 months or
less duration, cholinesterase inhibitors improved cognitive function
in a major percentage of patients with Alzheimer’s disease or slowed
the rate of its decline (Antuono, 1995; Canal & Imbimbo, 1996;
Corey-Bloom, Anand, Veach, Anand, & Corey, 1998; Rogers, Farlow,
Doody, Mohs, & Friedhoff, 1998; Thal, Fergusen, Mintzer, Raskin, &
Targum, 1999). But these drugs have many adverse effects such as
nausea, vomiting, diarrhea, dizziness, abdominal pain, and headache
(Weinstock, 1999). Phytotherapeutics are hopeful agents for neu-
rodegenerative diseases like Alzheimer’s and Parkinson’s diseases,
owing to their wide range of biological activities and the least side
effects. Therefore, many types of research are increasing day by day
on the anticholinesterase activities of the plants (Rasool et al., 2014).
Tyrosinase mainly provides the synthesis of melanin by the hy-
droxylation of tyrosine and the oxidation of 3,4-dihydroxyphenyl-
alanine, in the human body. Although melanin is a photoprotective
pigment in human skin, the abnormal increases of melanin in differ-
ent parts of the skin might occur melanoma and other many skin dis-
orders (Van Kempen, Redpath, Robert, & Spatz, 2014; Zolghadri et
al., 2019). Also, tyrosinase causes the enzymatic browning of fruits
and vegetables, which affects negatively their color, taste, flavor, and
nutritional importance (Martinez & Whitaker, 1995). In addition, ty-
rosinase may oxidize dopamine and levodopa to form neuromelanin,
in the brain. Neuromelanin interacts with α-synuclein protein which
is thought to be responsible for familial Parkinson’s disease (PD) and
also, making neurons in the substantia nigra pars compact more sus-
ceptible to toxic effects (Asanuma, Miyazaki, & Ogawa, 2003). Thus,
not only anticholinesterase studies but also antityrosinase studies
are increasing in neurodegenerative diseases.
Phenolic compounds are one of the most significant groups of
natural products and most researched secondary metabolite groups
due to their pharmacological and biological effects. Many studies
on neurodegenerative diseases have shown that these compounds
cross the blood-brain barrier and create protective effects on neu-
ron cells by different mechanisms (Ishige, Schubert, & Sagara, 2001;
Nie, Cao, & Zhao, 2002; Zeng, Chen, & Zhao, 2004). And therewithal
acknowledgment of literature about Alliums extracts and its flavo-
noids, focus on especially garlic and onion, improved perspectives in
the clinic (Farooqui & Farooqui, 2018) For instance, a study by Yang,
Kim, Kim, & Song, 2013 has suggested that quercetin isolated from
onion has neuroprotective effects against HT22 cells and another
research has been reported potential of neuroprotection about the
hydromethanolic extract of A.cepa in rats (Ogadinma, Chuemere,
Ikechukwu, & Samuel, 2018). Moreover, A. sativum L., powdered in
a capsule, elevated visual memory and cognitive activities in healthy
human volunteers in a condition that both genders volunteers
and over 5 weeks (Tasnim et al., 2015). In this case of developing
skilled agents for protecting diseases likel Alzheimer, Parkinson,
and Huntington, etc. these properties are exclusively important.
Therefore, in this study, we aimed to investigate the potential of
utilization with preliminary experiments other wild Allium species.
Of these, A. nigrum has been carried out for saponin glycosides and
cysteine sulphoxides whereas A. subhirsutum has been reported to
only in vitro antioxidant activities (Mostafa et al., 2013; Nencini,
Menchiari, Franchi, & Micheli, 2011). However, qualitative and quan-
titative determination, using LC-ESI-MS/MS technique, of polyphe-
nolic composition with screening bioactivity assays of A. nigrum and
A. subhirsutum, consumed as food and grow naturally in Turkey, is a
first report to the best of our knowledge.
2 | M ATE R İ A L A N D M E TH O DS
2.1 | Chemicals
Reference standards as (+)-catechin (purity = 99%), (−)-epicatechin
(98%), (−)-epigallocatechin gallate (98%), 3-hydroxybenzoic acid (99%),
4-hydroxybenzoic acid (99%), benzoic acid (99.5%), catechol (95%),
chrysin (98%), ferulic acid, 3-hydroxyflavone (98%), galangin (95%),
genistein (98%), isorhamnetin (95%), kaempferol (97%), luteolin (97%),
p-coumaric acid (98%), morin (98%), myricetin (98%), naringenin (95%),
phenyl acetate (99%), daidzein (97%), vitexin (95%), quercetin (95%),
3-O-methylquercetin (97%), syringic acid (98%), vanillic acid (97%), fi-
setin (98%), hesperidin (97%), rutin (95%), gallic acid (98%) used in the
EMIR et al.
LC-MS/MS analysis and AChE (from Electrophorus electricus), BuChE
(from equine serum), acetylthiocholine/butyrylthiocholine iodide,
DTNB (Ellman’s reagent) [5,5′-dithio-bis-(2-nitrobenzoic acid)], tyrosi-
nase (from mushroom), galanthamine (94%), L-Dopa and kojic asid
(99%) used for enzyme inhitory activities were purchased from Sigma-
Aldrich. All other chemicals were LC grade.
2.2 | Plant material
Allium nigrum L. was collected in July 2018 from Nif mountain
(İzmir/Turkey) and Allium subhirsutum was collected in May 2018
from Menderes (İzmir/Turkey). The plants were identified by Hasan
Yıldırım. Voucher specimens (No: H.Yıldırım 7390, H.Yıldırım 7427)
have been deposited in the Herbarium of the EGE University.
2.3 | Sample preparation
The extracts were prepared from the bulbs and aerial parts of A. ni-
grum and A. subhirsutum with efficient and quick extraction method.
One gram of air-dried, powdered plant material weighted in the fal-
con tube and extracted 3 times with methanol. For the extraction,
the tube was placed in the rotator (ISOLAB Laborgeräte Gm bH) and
shook throughout 1 hr, and then centrifugation was performed to
remove the particulate matter from the solution. The supernatant
was evaporated by rotary (Buchi) under reduced pressure. Extracts
were kept at 4°C and then filtered through a 0.22 μm PTFE syringe
filter into a vial for LC-MS/MS analysis.
2.4 | Total phenolic content (TPC)
For the total phenolic content of the samples, a modified method
(Singleton, Orthofer, & Lamuela-Raventos, 1999) was performed.
1 ml of extracts at a concentration of 1 mg/ml, Folin–Ciocalteu
reagent (diluted ten-fold) and 4 ml of a sodium carbonate solution
(7.5%) were added in glass vials. After incubation at 25°C for 20 min,
the absorbance was measured at 765 nm. A similar procedure was
adopted for the calibration curve as above described in the prepara-
tion of extracts. Gallic acid was a positive control and so the results
were expressed as gallic acid equivalents (mg GAE/g extract).
2.5 | Total flavonoid content (TFC)
The total flavonoid content of extracts was evaluated by AlCl3
method (Dziri et al., 2012) with slight modifications. About 2 ml of
extract, 0.1 ml of a 10% AlCl3 solution, 0.1 ml of potassium acetate
(1 M), and 2.8 ml of distilled water were added in a glass vial. After
incubation at room temperature for 30 min, the absorbance was
measured at 415 nm. Quercetin was a positive control and so the
results were expressed as quercetin equivalents (mg QE/g extract).
| 3 of 14
2.6 | Identification of phenolic compounds
Determination and quantitation of the 30 selected phenolic com-
pounds were performed by TSQ Quantum™ Access MAX Triple
Quadrupole Mass Spectrometer (Thermo Scientific™). Separation
of compounds was applied using a GL Sciences ODS C18 column
(150 mm × 4.6 mm × 5 µm) with a gradient mobile phase. LC-MS
grade water (solvent A) with 0.1% formic acid and methanol (solvent
B) with 0.1% formic acid as follows: 5% to 35% in 4 min, 35% to 65%
from 4 to 7 min, 65 to 80% from 7 to 15 min, and finally to reach
95% in 20 min at a flow rate of 1.0 ml/min. And the injection volume
was set to 5 μL. ESI parameters were as follows: capillary tempera-
ture 400°C, vaporizer temperature 500°C the flow rate of sheath
gas, aux gas, and sweep gas were kept at 75 arb, 20 arb, and 0 arb,
respectively. Also, phenolic compounds were detected by both neg-
ative and positive ion modes of LC-MS/MS (Table 1). Quantitative
determination of phenolics was carried out by an external standard
method and results expressed as μg per gram of extracts.
2.7 | Method validation
For the validation of the method (ICH, 2005), linearity, precision,
accuracy, limits of detection (LOD), and quantitation (LOQ) studies
were performed.
2.7.1 | Linearity
The linearity of the method was shown using an external standard
calibration curve with nine known concentrations for each analyte by
injecting of the standards in the range of 0.5–3000 (µg/ml). Standard
compounds were studied in triplicate and then the calibration curves
were achieved from the peak areas versus the concentrations.
2.7.2 | Precision
For the determination of precision of the present method, intra-day
and inter-day variations were assessed. Intra-day precision was car-
ried out by applying in triplicate of three concentrations for each
phenolic on the same day. Also, inter-day variation was determined
by two different days. Results were expressed as the relative stand-
ard deviation (RSD) for precision.
2.7.3 | Recovery
The recovery of the method was performed by the standard addition
analysis. Three known amounts (low, medium, and high), selected
from the concentrations used in the calibration curve, for each com-
pound and the mixtures were analyzed by the method used in the
analysis of phenolics.
4 of 14 | EMIR et al.

TA B L E 1 Names of the analytes, molecular formula, polarity, the m/z values of their precursor and product ions, and collision energies
used for fragmentation

Collision Precursor ion

No Compound name Molecular formula energy (V) Ion. mode [m/z] Product ion [m/z]

1 Benzoic acid C 7 H 6 O2 15 Neg. 121.18 77.23

2 3-Hydroxybenzoic acid C 7H6 O3 15 Neg. 137.18 93.18
3 4-Hydroxybenzoic acid C 7H6 O3 15 Neg. 137.15 93.15
4 p-Coumaric acid C 9H 8 O 3 13 Neg. 163.01 119.08
5 Vanillic acid C 8H8O 4 7 Neg. 167.10 152.6
6 Gallic acid C7H6O5 15 Neg. 169.07 125.02
7 Ferulic acid C10H10O 4 15 Neg. 193.04 134.10
8 Syringic acid C9H10O5 15 Neg. 19.11 182.18
9 Daidzein C15H10O 4 34 Neg. 252.84 223.10
10 Chrysin C15H10O 4 35 Neg. 253.18 143.06
11 Kaempferol C15H10O6 35 Neg. 285.10 238.95
12 Luteolin C15H10O6 30 Neg. 284.96 238.95
13 Fisetin C15H10O6 30 Neg. 285.10 135.01
14 Morin C15H10O7 23 Neg. 301.22 150.94
15 Quercetin C15H10O7 26 Neg. 301.20 150.94
16 3-O-methylquercetin C16H12O7 20 Neg. 315.10 290.99
17 Isorhamnetin C16H12O7 23 Neg. 315.09 300.02
18 Galangin C15H10O5 35 Pos. 271.20 153.11
19 Myricetin C15H10O8 25 Neg. 317.05 191.00
20 Vitexin C21H20O10 20 Neg. 431.29 310.96
21 Hesperidin C28H34O15 20 Neg. 609.40 300.88
22 3-Hydroxyflavone C15H10O3 34 Pos. 239.10 165.08
23 Naringenin C15H12O5 22 Neg. 271.05 151.01
24 Genistein C15H10O5 30 Pos. 271.09 153.11
25 Rutin C27H30O16 35 Neg. 609.24 299.91
26 Phenyl acetate C 8 H 8 O2 10 Pos. 137.12 95.15
27 Catechol C 6 H 6 O2 27 Neg. 109.22 108.10
28 (+)-Catechin C15H14O6 22 Neg. 289.10 203.07
29 (−)-Epicatechin C15H14O6 15 Pos. 291.22 139.10
30 (−)-Epigallocatechin gallate C22H18O11 15 Pos. 459.18 139.10

2.7.4 | Limits of detection (LOD) and quantitation
(LOQ)
LOD and LOQ were determined at a signal-to-noise ratio (S/N) of 3
and 10, respectively. Also, analyses were experimentally determined
by 10 injections of each analyte.
(Ellman, Courtney, Andres, & Featherstone, 1961). Concentrations
were between 0.001 and 1,000 mg/ml and also, galanthamine was
a positive standard.
2.9 | Tyrosinase inhibitory activity

Tyrosinase inhibitory activity of the extracts was carried out ac-

2.8 | Anticholinesterase activity
Cholinesterase inhibitory activity of the samples was performed
by the described method (López, Bastida, Viladomat, & Codina,
2002), using 96-well microplate, modified from the Ellman’s method
cording to the modified dopachrome method (Likhitwitayawuid &
Sritularak, 2001; Masuda, Yamashita, Takeda, & Yonemori, 2005).
Seven different concentrations of methanol extracts (1,000, 750,
500, 250, 100, 10, and 1 μg/mL) were studied and kojic acid was a
positive control.
EMIR et al. | 5 of 14

Enzyme inhibitory assays were achieved in a 96-well microplate 3 | R E S U LT S A N D D I S CU S S I O N

using ELISA microplate reader (Varioskan Flash Multimode Reader,
Thermo Scientific, USA). The IC50 values of samples were calculated
by the software GraphPad Prism V5.0 (GraphPad Software, San
Diego, CA, USA).
2.10 | Statistical analysis
Pearson’s correlation coefficients were calculated to establish the
potency of the linear relationships between phenolic compounds
and enzyme inhibitory activity and principal component analysis
(PCA) was applied to decrease the number of variables in the data
matrix in order to select the most selective parameters by SPSS 25
software.
3.1 | Method validation
Results of method validation are given in Tables 2 and 3. Excellent
linearity was obtained for compounds showing a great correlation
between the phenolic concentration and the peak area. Also, the
determination coefficients (r2) of the calibration equations were
higher than 0.9919 for all analytes. The intraday assay precision (ex-
pressed as % RSD) ranged from 0.14 for 3-O-methylquercetin to 2.40
for myricetin. The interassay precision was from 0.29 for catechol
to 2.63 for genistein. The LOD and LOQ values obtained ranged
from 2 to 39 ng/ml and from 7 to 112 ng/ml, respectively. Recovery
values of 30 phenolic compounds were between 88.34% for 3-O-
methylquercetin and 111.85% for galangin. These method validation

TA B L E 2 Results of analysis on calibration curves, LOD, and LOQ

Compound Ranges (µg/ml) Linear equation R2 LOD (ng/ml) LOQ (ng/ml)

1 10–500 y = 2.4286 × −12.857 0.9982 29 96

2 10–3000 y = 8.209 × −372.34 0.9971 22 76
3 10–2000 y = 16.742 × +323.71 0.9995 28 91
4 10–2000 y = 34.595 × −229.27 0.9971 10 33
5 10–2000 y = 3.5095 × +2.2973 0.9959 24 76
6 10–2000 y = 1.9542 × +23.556 0.9986 12 35
7 10–2000 y = 8.1419 × −15.689 0.9948 7 24
8 10–500 y = 1.9951 × +4.5122 0.9979 6 19
9 0.5–100 y = 195.59 × −20.339 0.9943 4 15
10 0.5–10 y = 146.03 × +1.5753 0.9958 15 49
11 10–500 y = 1.0625 × +0.4675 0.9951 12 38
12 0.5–100 y = 407.93 × −12.425 0.9937 23 74
13 0.5–20 y = 40.393 × +5.1872 0.9985 28 87
14 0.5–20 y = 38.537 × +1.0732 0.9942 2 7
15 0.5–10 y = 34.89 × +15.233 0.9923 11 37
16 0.5–200 y = 13.177 × +92 0.9921 25 78
17 0.5–20 y = 204.1 × −2.0038 0.9986 29 92
18 0.5–200 y = 178 × −400 0.9974 21 52
19 0.5–20 y = 82.316 ×  +0.4737 0.9965 6 22
20 0.5–20 y = 259.58 × +25.035 0.9988 19 64
21 0.5–20 y = 148.86 × −0.5114 0.9996 27 81
22 0.5–20 y = 6500 × −800 0.9986 5 19
23 0.5–20 y = 640 × +540 0.9919 39 103
24 0.5–200 y = 177.5 × +650 0.9939 9 33
25 0.5–20 y = 1878 × +29.831 0.9950 36 98
26 0.5–200 y = 406.59 × −91.463 0.9993 17 49
27 10–500 y = 0.3291 × +1.7917 0.9976 8 22
28 0.5–200 y = 68.488 × +15.488 0.9987 27 85
29 0.5–20 y = 79.569 × +21.277 0.9984 33 95
30 0.5–2000 y = 3.4817 × +220.83 0.9948 36 112
6 of 14 | EMIR et al.

TA B L E 3 Precision and recoveries for the phenolic compounds

Intra-day precision (RSD, %) Inter-day precision (RSD, %) Mean recovery (%) ± SD

Amount (μg/ml) Amount (μg/ml) Amount (μg/ml)

Compound 5 10 15 5 10 15 Low Medium High

1 0.19 0.56 0.33 1.20 0.85 0.94 105.27 89.35 99.24

2 0.98 1.45 1.09 2.15 0.99 1.14 89.16 94.46 95.48
3 1.06 0.69 0.88 0.87 1.20 0.65 95.78 97.63 108.51
4 1.64 0.95 1.41 1.55 0.47 0.84 110.36 109.42 105.33
5 0.52 0.67 0.29 0.86 1.61 1.58 88.53 102.77 111.25
6 0.87 0.58 0.46 1.43 0.96 0.79 103.41 106.89 89.16
7 0.65 0.58 0.63 1.26 0.57 0.83 99.12 105.11 96.27
8 0.74 0.71 0.48 1.38 0.86 1.39 105.47 89.29 97.35
9 0.69 0.92 0.85 0.32 1.07 0.55 91.62 93.58 99.68
10 1.54 1.03 0.96 2.43 0.87 0.32 94.25 98.35 95.46
11 0.44 0.49 0.87 0.75 1.42 0.57 102.43 99.17 88.49
12 2.39 1.66 0.99 0.64 0.38 0.78 97.95 97.56 102.73
13 0.49 0.81 0.68 1.87 0.45 0.91 93.58 96.12 108.23
14 0.87 0.55 0.22 1.21 1.87 0.27 106.69 108.99 96.41
15 0.21 0.46 0.38 0.89 1.49 0.88 103.28 110.52 97.87
16 0.14 0.35 0.16 0.76 2.24 1.59 100.96 88.34 97.59
17 0.33 0.74 0.56 1.19 1.68 0.66 89.59 96.25 101.22
18 0.98 1.25 0.37 0.47 0.16 0.78 97.15 95.74 111.85
19 2.40 2.14 1.68 0.84 0.57 1.47 107.60 101.87 99.64
20 0.77 1.01 0.86 0.77 0.65 0.98 88.36 105.23 97.21
21 0.68 0.79 0.45 1.65 0.17 0.74 93.55 91.14 89.91
22 0.29 0.36 0.69 1.31 0.35 0.79 98.72 96.58 91.77
23 0.73 0.54 0.33 2.51 0.45 1.26 102.58 103.24 95.55
24 0.15 0.39 0.65 2.06 2.63 1.95 108.65 107.61 99.34
25 0.49 0.77 0.88 0.56 1.24 0.33 95.33 89.62 108.23
26 0.31 0.62 0.74 0.49 0.67 0.85 89.07 107.55 104.68
27 0.55 0.87 0.58 1.47 0.98 0.29 104.84 102.19 99.35
28 0.83 1.26 1.65 1.49 1.85 0.48 98.67 95.63 98.81
29 0.33 0.47 0.78 0.36 0.69 1.60 99.23 91.52 92.15
30 2.17 1.49 1.34 0.89 2.33 1.71 110.57 99.57 91.86

results indicated that the developed LC-MS/MS method was accept-
able for the quantitative analysis of phenolic compounds of A. nigrum
and A. subhirsutum.
3.2 | Sample analysis
An efficient, quick, sensitive, and with no changes in the structure
of the extracted compounds extraction method was performed to
different parts of A. nigrum. Extraction yield, the total phenolic, and
total flavonoid contents of extracts are shown in Table 4. All extracts
were found rich in phenolic composition and aerial part extracts had
higher values of TPC and TFC for both plants. A previous comparative
study of the total phenolic content of the ethanol extracts of the bulb,
leaf, and root of A. nigrum showed that the leaf was rich in phenolics
(Mostafa et al., 2013). To the best of our knowledge, this is the first
report of determination of phenolic compounds by LC-MS/MS in and
evaluation of its cholinesterase and tyrosinase inhibitory activities of
A. nigrum and A. subhirsutum. Identification of the phenolic compounds
in the extracts was carried out by comparing retention times and MS/
MS fragments with reference standards. Totally, 30 compounds were
quantified and the LC-MS/MS results revealed that generally, the aerial
part extracts had higher concentrations of phenolic compounds than
bulb extracts and also, phenolic acids were major compounds for all
samples (Table 5). Similarly, there are quantitative studies on different
Allium species (Kutlu, Takım, Karaaslan, & Yılmaz, 2018; Mollica, Zengin,
Locatelli, Picot-Allain, & Mahomoodally, 2018; Park, Je, & Ahn, 2016;
Parvu, Toiu, Vlase, & Alina, 2010; Simin et al., 2013) where phenolic
EMIR et al. | 7 of 14

TA B L E 4 Extraction yield, total

TPC (mg GAEa /g TFC (mg QEb /g
phenolic, and total flavonoid contents
Sample Plant Part Yield (%) extract) extract)
of different extracts of A. nigrum and A.
subhirsutum A. nigrum Aerial parts 45.3 29.1 ± 2.3 5.4 ± 1.8
Bulbs 30.1 45.6 ± 1.9 8.2 ± 0.7
A. subhirsutum Aerial parts 31.2 13.3 ± 1.7 3.6 ± 0.3
Bulbs 37.5 15.8 ± 0.9 5.7 ± 0.8

Note: Values expressed are means ± SD of three parallel measurements.

a
Gallic acid equivalent.
b
Quercetin equivalent.

TA B L E 5 Concentrations of phenolic
A. nigrum A. subhirsutum
compounds (µg/g of extract) of different
parts of A. nigrum and A. subhirsutum Aerial parts
Compound Bulbs (µg/ml) Aerial parts (µg/ml) Bulbs (µg/ml) (µg/ml)

1 313.2 ± 1.62 356.3 ± 1.95 39.2 ± 2.83 146.7 ± 0.95

2 1963.9 ± 2.38 2188.4 ± 2.63 518.6 ± 1.98 430.1 ± 2.63
3 402.6 ± 1.194 4475 ± 0.47 976.7 ± 3.44 314.2 ± 1.78
4 196.5 ± 1.20 228.9 ± 0.93 1700.8 ± 1.52 1042.4 ± 2.97
5 58.9 ± 0.34 28.8 ± 1.51 903.8 ± 2.31 621.6 ± 1.87
6 613.2 ± 0.55 756.3 ± 2.65 44.5 ± 0.93 52.3 ± 2.21
7 22.9 ± 0.97 19.5 ± 1.18 787.2 ± 1.18 1352.0 ± 3.16
8 78.0 ± 0.62 115.5 ± 1.73 30.2 ± 0.87 40.2 ± 1.85
9 0.9 ± 0.13 T 81.0 ± 0.68 94.5 ± 1.68
10 T 1.2 ± 0.15 3.1 ± 1.067 T
11 27.7 ± 0.47 381.6 ± 0.24 T T
12 1.2 ± 0.10 T 39.6 ± 2.75 64.0 ± 1.80
13 5.8 ± 0.25 7.8 ± 0.48 T T
14 3.6 ± 0.19 1.8 ± 0.17 6.1 ± 0.32 10.7 ± 1.15
15 7.6 ± 0.34 5.0 ± 0.39 T T
16 97.8 ± 0.87 76.5 ± 1.97 1.2 ± 0.22 T
17 8.3 ± 0.11 6.9 ± 0.44 6.5 ± 1.40 5.5 ± 0.43
18 53.0 ± 0.72 67.4 ± 1.13 68.8 ± 2.19 85.2 ± 1.59
19 1.3 ± 0.21 T 8.6 ± 0.87 7.8 ± 0.71
20 T 1.4 ± 0.38 T T
21 2.4 ± 0.18 1.6 ± 0.18 2.4 ± 0.33 T
22 6.7 ± 0.39 5.4 ± 0.26 T T
23 14.4 ± 0.57 13.9 ± 0.84 2.2 ± 0.59 T
24 49.6 ± 0.86 31.0 ± 1.65 130.6 ± 1.42 159.3 ± 2.76
25 T T 4.8 ± 0.63 5.5 ± 1.22
26 91.1 ± 2.13 128.3 ± 2.62 13.4 ± 2.77 11.5 ± 1.39
27 224.5 ± 2.27 121.3 ± 1.87 30.9 ± 1.39 27.7 ± 0.73
28 ND 1.2 ± 0.11 72.8 ± 2.49 43.9 ± 0.35
29 6.0 ± 0.48 9.6 ± 0.35 7.6 ± 0.92 6.9 ± 0.10
30 49.9 ± 1.86 998.3 ± 2.17 2.8 ± 0.41 5.2 ± 0.89

Note: Values expressed are means ± SD of three parallel measurements. nd: not detected, T: trace
amounts.

acids were detected as dominant phenolics. 3-hydroxybenzoic acid (−)-Epigallocatechin gallate (EGCG), 998.3 µg/g extract found in A.
(2188.4 μg/g extract) for A. nigrum and p-coumaric acid (1700.8 μg/g nigrum from our analysis, has been investigated for whether EGCG
extract) for A. subhirsutum were the most dominant phenolic acids. could reduce 6-hydroxydopamine-induced neurotoxicity on cultured
8 of 14 | EMIR et al.
neuronal cell lines and this agent displayed significant in vitro protec-
tive effect in a model of PD (Nie et al., 2002). Also, another in vivo
research implied that EGCG alleviated biochemical changes, including
enhanced AChE activity, oxidative stress, cytokines, of ethanol-treated
rat pumps (Tiwari, Kuhad, & Chopra, 2010). Moreover, genistein, a
known isoflavonoid, was observed for major flavonoid and quantified
as 159.3 µg/g extract in A. subhirsutum. Genistein has neuroprotective
effects against Aβ1-42-induced rats and Aβ25-35-induced cultured hip-
pocampal neuronal cells via different mechanisms in searching for AD
treatment (Devi, Shanmuganathan, Manayi, Nabavi, & Nabavi, 2017).
In addition, rutin was determined in trace amounts in the extracts of
A. nigrum and while kaempferol was second major flavonoid for A. ni-
grum, detected in trace amounts in A. subhirsutum extracts. MS/MS
fragments and chromatograms of major phenolics are shown in Figures
1‒4. Also, total ion chromatogram (TIC) of the aerial part extract of A.
nigrum which had the highest biological activity is shown in Figure 5.
3.3 | Cholinesterase inhibitory activity
Modified Ellman’s method was performed for AChE and BuChE
inhibitory activities of samples (Table 6). Galanthamine was a posi-
tive control and IC50 values of galanthamine were 0.106 μg/ml
and 1.04 μg/ml in anti-AChE and anti-BuChE assays, respectively.
Figure 6 shows the concentration ranges used to generate the IC50
FIGURE 1 Chromatogram and ms fragmentation of 3-hydroxybenzoic acid as a major phenolic acid of A. nigrum
data and the inhibitor response curves of extracts. The aerial parts
extract of A. nigrum exhibited lower IC50 values 6.1 μg/ml for AChE
and 3.27 μg/ml for BuChE inhibition assay hence it was found to have
greater anticholinesterase activity compared to other samples. In a
detailed acetylcholinesterase inhibition activity study with 23 differ-
ent Allium species, performed by the Ellman’s method and reported
by Hadacova, Vackova, Klozova, Kutacek, and Pitterova (1983), anti-
AChE activity was observed in all samples and A. obliquum showed
the highest activity. Also, according to Mollica, Zengin, Locatelli, &
Picot-Allain, 2018, the bulb, stem, and flower parts of A. scorodo-
prasum subsp. rotundum had potent anti-AChE (1.80, 2.17, 1.98 mg
GALAE/g extract, respectively) and anti-BuChE (2.01, 1.95, 3.16 mg
GALAE/g extract, respectively) activities. In the present study,
all extracts were observed cholinesterase inhibition capacity and
anti-BuChE activity was detected higher than anti-AChE activ-
ity. Also, data indicated that the aerial parts of A. nigrum were the
most potent sample. Higher levels of phenolic compounds, espe-
cially (−)-epigallocatechin-3-gallate (EGCG), galangin, kaempferol
and 3-O-methylquercetin, may well contribute to the cholinesterase
inhibitory activity of aerial part. According to Katalinić et al., (2010)
galangin and kaempferol were the potent anticholinesterase com-
pounds with greater anti-BuChE activities. Also, Salazar, de Athayde
Moncorvo Collado, Canal-Martínez, & Minahk, 2017) and Jung and
Park, (2007) reported that EGCG and 3-O-methylquercetin had a po-
tent anti-AChE activity, respectively.
EMIR et al.
FIGURE 2 Chromatogram and ms fragmentation of (−)-epigallocatechin gallate as a major flavonoid of A. nigrum
3.4 | Antityrosinase activity
The tyrosinase inhibitory potentials of the samples were determined
spectrophotometrically by a microplate assay with a 96-well micro-
plate reader and kojic acid was used as a positive control (IC50: 7.9 μg/
ml) (Table 6). In the literature, the extracts of A. ursinum (Nikkhahi,
Souri, Sarkhail, Baeeri, & Mohammadhosseini, 2018) and A. scoro-
doprasum subsp. rotundum (Mollica et al., 2018) were determined as
potent tyrosinase inhibitors. In our study, all extracts of Allium sp.
showed anti-tyrosinase activity harmoniously and the aerial part ex-
tract of A. nigrum (IC50: 22.31 μg/ml) was more potent owing to its
phenolics with higher levels. Besides, the same extract was found
rich in content of kaempferol and 4-hydroxybenzoic acid, reported
being intense inhibitors of tyrosinase (Kim et al., 2006; Shaikh, Khan,
& Choudhary, 2011).
3.5 | Statistical analysis
To interrelate and to easily visualize the Allium samples with
the outcomes of this investigation PCA was performed and a
| 9 of 14
two-dimensional PCA scatter plot (based on two first PCs) was con-
structed (Figure 7). The total variance explained by the two prin-
cipal components (PC1 and PC2) and data resulted in 86.54. The
first (PC1) and second principal component (PC1) were explained as
62.49% and 24.04% of the variability, respectively. Owing to pos-
sessed similar phenolics, while the samples of A. nigrum presented
on the negative side, the samples of A. subhirsutum was on the posi-
tive side of the first component. Loading plot of the variables dem-
onstrated that quercetin, 3-hydroxyflavone for bulbs of A. nigrum,
3-hydroxybenzoic acid for aerial parts of A. nigrum, chrysin, (+)-cat-
echin for bulbs of A. subhirsutum and daidzein, luteolin for aerial
parts of A. subhirsutum are the most distinguishing phenolics. Also,
in Figure 7, biological activities had a closer relationship with aerial
parts of A. subhirsutum, which had the highest IC50 values, indicat-
ing the lowest enzyme inhibitory activity. In addition, correlations
between phenolic compounds and AChE, BuChE and tyrosinase
inhibitory potencies are shown in Figure 8. 4-Hydroxybenzoic acid
(r = 1.000, 0.800 and 0.800, respectively), (−)-epicatechin (r = 0.600,
0.800 and 0.800, respectively), and chrysin (r = 0.944, 0.674 and
0.674, respectively) had significant positive correlations with en-
zyme inhibitory activities.
10 of 14 | EMIR et al.

FIGURE 3 Chromatogram and ms fragmentation of p-coumaric acid as a major phenolic acid of A. subhirsutum

FIGURE 4 Chromatogram and ms fragmentation of genistein as a major flavonoid of A. subhirsutum

EMIR et al. | 11 of 14

FIGURE 5 TIC of the aerial part extract of A. nigrum

TA B L E 6 Enzyme inhibitory properties

AChE ınhibition BuChE ınhibition Tyrosinase
of the studied samples
Plant Sample (IC50 µg/ml) (IC50 µg/ml) (IC50 µg/ml)

A. nigrum Aerial parts 6.1 ± 0.03 3.27 ± 0.02 22.31 ± 0.09

Bulbs 26.12 ± 0.06 14.08 ± 0.04 51.66 ± 0.05
A. subhirsutum Aerial parts 42.35 ± 0.09 19.25 ± 0.05 63.77 ± 0.08
Bulbs 8.35 ± 0.04 7.46 ± 0.02 49.21 ± 0.05
Galanthamine (std) 0.106 ± 0.01 1.04 ± 0.01 –
Kojic acid (std) – – 7.9 ± 0.02

Note: Values expressed are means ± SD of three parallel measurements. std: Standard

FIGURE 6 The concentration ranges (µg/ml) and the inhibitor response (inhibition %) curves
12 of 14 | EMIR et al.

F I G U R E 7 Principal component analysis of phenolic compounds and enzyme inhibitory potencies in samples. (1) Bulb extract of A.
nigrum, (2) Aerial part extract of A. nigrum, (3) Bulb extract of A. subhirsutum, and (4) Aerial part extract of A. subhirsutum

FIGURE 8 Pearson’s correlation coefficients of the phenolic compounds and enzyme inhibitory potencies

4 | CO N C LU S I O N S the major flavonoids. All samples showed significant cholinesterase

Bulb and aerial part extracts of two edible Allium species were in-
vestigated in terms of the TPC, TFC, and phenolic composition
by LC-MS/MS that is quick, fast, sensitive and with the validated
method, anti-cholinesterase, and anti-tyrosinase activities for the
first time. Outcomes of the present study were analyzed by PCA.
Our findings demonstrated that all extracts rich in phenolic acids
and also, EGCG and genistein, neuroprotective compounds, were
and tyrosinase inhibitory activities, and aerial part extract of A. ni-
grum showed higher activity due to its higher levels of phenolics.
Owing to not only possessed diverse bioactive substances but also
to be consumed these Allium species so that they have significant
nutraceutical potential. In this regard, A. nigrum and A. subhirsutum
might be functional foods sources of biologically effective phenolics,
in addition, to further in vitro and in vivo experiments are needed to
be used effectively in the treatment.
EMIR et al. | 13 of 14

AC K N OW L E D G M E N T S Ishige, K., Schubert, D., & Sagara, Y. (2001). Flavonoids protect neuro-
nal cells from oxidative stress by three distinct mechanisms. Free
This study was financially supported by TUBITAK (217S341). We
Radical Biology & Medicine, 30(4), 433–446. https​://doi.org/10.1016/
also thank the Pharmaceutical Sciences Research Centre (FABAL) S0891-5849(00)00498-6
of the Ege University Faculty of Pharmacy for equipmental support. Jung, M., & Park, M. (2007). Acetylcholinesterase inhibition by flavo-
noids from Agrimonia pilosa. Molecules, 12(9), 2130–2139. https​://doi.
C O N FL I C T O F I N T E R E S T org/10.3390/12092130
Katalinić, M., Rusak, G., Domaćinović, B. J., Šinko, G., Jelić, D.,
The authors declare no conflict of interest.
Antolović, R., & Kovarik, Z. (2010). Structural aspects of flavonoids
as inhibitors of human butyrylcholinesterase. European Journal of
ORCID Medicinal Chemistry, 45(1), 186–192. https​://doi.org/10.1016/j.
Ahmet Emir https://orcid.org/0000-0002-0971-7716 ejmech.2009.09.041
Kaur, S., & Das, M. (2011). Functional foods: An overview. Food Science
Ceren Emir https://orcid.org/0000-0001-8516-9830
and Biotechnology, 20, 861–875.
Hasan Yıldırım https://orcid.org/0000-0003-3951-4343 Kim, D., Park, J., Kim, J., Han, C., Yoon, J., Kim, N., … Lee, C. (2006).
Flavonoids as mushroom tyrosinase inhibitors: A fluorescence
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How to cite this article: Emir A, Emir C, Yıldırım H.
Characterization of phenolic profile by LC-ESI-MS/MS and
enzyme inhibitory activities of two wild edible garlic: Allium
nigrum L. and Allium subhirsutum L.. J Food Biochem.
2020;44:e13165. https​://doi.org/10.1111/jfbc.13165​