INTERNATIONAL JOURNAL OF FOOD PROPERTIES

2020, VOL. 23, NO. 1, 1097–1109

https://doi.org/10.1080/10942912.2020.1778027

Evaluation of bioactive components and antioxidant capacity of

four celery (Apium graveolens L.) leaves and petioles
De-Kun Liu, Cong-Cong Xu, Lu Zhang, Hui Ma, Xu-Jie Chen, Yu-Cui Sui, and Hong-Zhi Zhang
School of Life Science, Qufu Normal University, Qufu, Shandong Province, PR China

ABSTRACT ARTICLE HISTORY

The natural bioactive components and antioxidant capacity of leaves and Received 29 April 2019
petioles from four celery cultivars (“Benqin”, “Western”, “Baoqin”, and Revised 25 May 2020
“Majiagou”) were compared and correlated through a nontargeted metabo­ Accepted 28 May 2020
lomics analysis and partial least squares regression (PLS) analysis. A total of 18, KEYWORDS
12, and 5 antioxidant components in the celeries investigated showed sig­ Celery; leaf; petiole;
nificant (P < 0.05) positive correlations with the DPPH and O2∙ – free radical antioxidants; antioxidant
scavenging activities and ferric ion reducing power (FRP), respectively. Apiin, capacity; partial least squares
apigenin, rutin and α-ionone (standard partial least square regression coeffi­ regression analysis
cients of 0.064–0.071) made the largest contributions to the DPPH scavenging
activity, while oleic acid, α-linolenic acid, pantothenic acid, and vitamin
E succinate (coefficients of 0.084–0.110) made the largest contributions to the
O2∙ – scavenging activity. Comparing the levels of major antioxidants and
antioxidant capacity in four celery leaves and petioles showed that celery
leaves were a better source of natural antioxidants and scavenging free
radicals than petioles. Among the leaves, “Majiagou” celery leaves contained
the most types, and highest (P < 0.05) levels, of natural antioxidants (especially
vitamins, flavonoids, and unsaturated fatty acids), resulting in the largest O2∙ –
scavenging activity. Among the petioles, “Baoqin” and “Benqin” celery petioles
contained the most types, and highest (P < 0.05) levels, of dominant antiox­
idants that were responsible for their larger O2∙ – and DPPH scavenging
activities and FRP. These results provided a scientific basis for assessing the
quality and application value of these four celery cultivars.

Introduction
Celery (Apium graveolens L.) is an annual or biennial herb species that has been cultivated and
consumed worldwide. In recent years, celery consumption has increased steadily, largely owing to
the appreciable amounts of natural bioactive components contained within celeries, such as phenolic
acids, flavonoids, vitamins, coumarin, and essential oils.[1,2] Frequent consumption of these natural
components might reduce the risk of oxidative damage, inflammatory response, cardiovascular
disease, and tumors, among other conditions.[1,3]
However, these components can vary considerably among different celery cultivars, and even among
different tissues of the same cultivar. For instance, celery leaves have higher contents of ascorbic acid[4]
and essential oils[5] than celery petioles. Furthermore, the essential oil contents of celery seeds are up to
7%, while those of celery leaves and roots are only 1%.[6] Consequently, the bioactivities of these tissues
differ. Yao et al. (2010) found that Apium graveolens L. species present higher free radical scavenging
capacities compared with Cryptotaenia japonica Hassk species, which is strongly correlated with the
former having higher levels of caffeic acid, ferulic acid, apigenin, luteolin, and kaempferol.[7]
Furthermore, an increasing number of studies have reported that various components have different

CONTACT Cong-Cong Xu 18801900840@163.com School of Life Science, Qufu Normal University, Qufu, Shandong Province
273165, PR China
© 2020 De-Kun Liu, Cong-Cong Xu, Lu Zhang, Hui Ma, Xu-Jie Chen, Yu-Cui Sui and Hong-Zhi Zhang. Published by Informa UK Limited, trading as Taylor
& Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
1098 D.-K. LIU ET AL.

effects on free radical species and scavenging capacities. For example, among phenols, catechol has the
largest effect on the antioxidant activity, while resorcinol has the smallest effect.[8] Vitamin E effectively
intercepts lipid peroxyl radicals (LOO∙), but inefficiently scavenges hydroxyl radicals (∙OH) and alkoxyl
radicals (∙OR) in vivo.[9] Phenolic compounds are more efficient than aniline compounds at inhibiting
2,2-diphenyl-1-picrylhydrazyl radicals (DPPH), owing to the lower bond dissociation energy (BDE) of
hydroxyls (O–H) compared with that of amidogens (N–H).[10] Therefore, a comprehensive exploration
of the bioactive components within various celery cultivars and tissues, and their contribution to the
antioxidant capacity is necessary. To our knowledge, many studies have concentrated on some kind of
compositions, such as celery essential oils,[5] phenolic compounds,[7] polysaccharides,[11,12] carotenoids,
and tannins,[2] and their effects. However, a comprehensive study for celery has yet to be reported.
Celery varieties “Benqin” (a Chinese celery) and “Western” (a celery introduced from Western
countries) have been widely cultivated throughout China. Meanwhile, “Majiagou” and “Baoqin” celeries
are high-end cultivars that have been exclusively produced in Shandong, China. By comparison of their
appearance, “Majiagou” celery possesses thick, hollow, and crisp petioles, and its petioles and leaves
appear light yellow, while other cultivars have green petioles and leaves. The petioles of “Western” celery
appear thicker and stronger than those of both “Baoqin” and “Benqin” celeries, while “Baoqin” petioles
are solid and stronger than “Benqin” petioles. Therefore, investigating the profiles of bioactive compo­
nents and their antioxidant activities within these cultivars might be of interest. Recently, high-
performance liquid chromatography–mass spectrometry (HPLC-MS) is a potential method for such
an investigation. HPLC coupled with any type of MS, such as single quadrupole, triple quadrupole, time-
of-flight, ion trap, or matrix-assisted laser desorption/ionization MS, provides a high sensitivity, speci­
ficity and separation efficiency, versatility, and user-friendly handling. HPLC-MS can be used to achieve
almost any separation and applied to the qualitative/quantitative analysis of known/unknown com­
pounds in various matrices.[13] Accordingly, HPLC-MS has been widely applied to quality assessment
and variety discrimination in natural products.[14]
From this background, the natural bioactive component profiles in the petioles and leaves of four
celery cultivars were analyzed by HPLC-MS, and their antioxidant activities determined. Furthermore,
differences in the major antioxidants and antioxidant activities among these celery cultivars and tissues
were compared. This study could offer a scientific basis for assessing the quality and application value of
these four celery cultivars.

Materials and Methods

Materials and sample preparation
In the study, four celery cultivars, namely, “Benqin” (No. II9B0209), “Western” (temporary No.
II9B0518), “Baoqin” (temporary No. II9B0517), and “Majiagou” (temporary No. II9B0516) celeries
(Figure 1), were selected and harvested from the place of origin on March 15, 2018, in Shandong,
China. After harvest, celery petioles were separated from leaves and cut into cylinders (40 ± 2 mm in
length). The petiole and leaf samples from each cultivar were then randomly divided into three groups
for replicate experiments. For preventing the loss of bioactive components and antioxidant capacity of
as-prepared samples from the freezing/thawing process, they were immediately frozen in liquid
nitrogen and then stored at – 80°C until further analysis.

Antioxidant capacity assay

After freezing in liquid nitrogen and crushing, celery (5 g) was extracted with 95% ethanol (100 mL)
for 5 h and then centrifuged at 10,000 rpm and 4°C for 20 min, after which the supernatant was
retained. An aliquot (100 μL) of the supernatant extract was used for antioxidant capacity analysis,
including DPPH and superoxide anion radical (O2∙ –) inhibition activities and the ferric ion reducing
power (FRP). The assays were performed using assay kits (Congyi Bio., Shanghai, China) in
a microplate reader (SynergyH1, Biotek, Winooski, USA). Absorbance was measured at 517 nm
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES 1099

a b c d

Figure 1. Images of four celery varieties: (a) “Benqin”; (b) “Western”; (c) “Baoqin”; and (d) “Majiagou”.

(DPPH), 560 nm (O2∙ –), and 700 nm (FRP). The inhibition activity of DPPH and O2∙ – were calculated
as the free radical inhibition ratios. The FRP analysis is based on the principle of increase in the
absorbance of the reaction mixtures, the absorbance increases the antioxidant activity increases.[15]
Hence, it was expressed as Abs detected.

Bioactive component profile analysis

A nontargeted metabolomics analysis was performed according to the method proposed by Dai
et al. (2017) with modification.[16] For extraction, absolute methanol (800 μL) and internal
standard (10 μL, 3 mg mL–1, DL-o-chlorophenylalanine) were added to the sample (50 mg).
The mixture was extracted using a Scientz-48 grinder (Ningbo Xinzhi Biotechnology Co. Ltd.,
Zhejiang, China) at 65 kHz for 90 s and then centrifuged at 12,000 rpm and 4°C for 15 min. The
as-obtained supernatant (200 μL) was transferred into a vial for further analysis. Initial data were
acquired using an UltiMate 3000 HPLC system (Thermo Fisher Scientific, USA) coupled to an
Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, USA). Chromatographic separation
of the metabolites was conducted on a Hypersil GOLD C18 column (100 × 4.6 mm, 3 μm,
Thermo Fisher Scientific, USA). The column temperature was maintained at 40°C and the
injection volume was 4 μL. Binary mobile phases (solvent A: water + 0.1% (v/v) formic acid;
solvent B: acetonitrile + 0.1% (v/v) formic acid) were applied to the elution at a flow rate of
0.3 mL min–1. The gradient elution profile was shown in Table 1. The mass spectrometer with
a dual jet stream electrospray ionization (ESI) source was operated in both negative (ESI–) and
positive (ESI+) ion modes. The MS conditions were as follows: (ESI+) heater temperature, 300°
C; spray voltage, 3.0 kV; capillary temperature, 350°C; S-lens RF level, 30%; (ESI–) heater
temperature, 300°C; spray voltage, 3.2 kV; capillary temperature, 350°C; S-lens RF level, 60%.
A mass scan range of m/z 50–1000 was applied for full scan analysis. A reference ion at m/z
199.04000 (dichlorobenzene) was continuously infused during data acquisition for online cali­
bration to ensure accurate MS. All of ppm values (expressing mass accuracy) were less than 10
ppm. Compounds were identified by comparison of their retention times with those of authentic
standards, accurate masses, and fragmentation patterns based on metabolomics database searches
(Metlin and Human Metabolome Database).
1100 D.-K. LIU ET AL.

Table 1. The gradient of mobile phase of high-

performance liquid chromatography–mass
spectrometry (HPLC-MS).
Time (min) A (%) B (%)
0–1 95 5
1–2 95 5
2–7 60 40
7–11 20 80
11–15 5 95
15–15.5 5 95
15.5–19.5 95 5
A: solvent A, water + 0.1% (v/v) formic acid; B:
solvent B, acetonitrile + 0.1% (v/v) formic acid

Statistical analysis
Extraction and preprocessing of the detection data were performed using SIEVE software (Thermo
Fisher Scientific, USA). After conversion, the data were further normalized and edited using Microsoft
Excel 2010, providing a two-dimensional data matrix with parameters including retention time,
metabolite molecular weight, and peak intensity. The relative contents of metabolites were then
calculated from the ratios of metabolite areas to the internal standard. The resulting data were
imported into SIMCA-p software 14.1 (MKS Umetrics AB, Umeå, Sweden) for multivariate statistical
analysis. After weight normalization and unit variance (Uv) scaling, partial least squares regression
(PLS) analysis was performed to assess the contribution of x–variables (36 components) to y-variables
(antioxidant capacity) by combining all data from the four celery petioles and leaves. One-way analysis
of variance (ANOVA) was performed to determine the significant difference (P < 0.05) in the levels of
major antioxidants and antioxidant capacities among all groups. All data are presented as means ±
standard deviation (SD). Statistical analysis was performed with SPSS 24.0 software (SPSS Inc.,
Chicago, IL).

Results and Discussion

Bioactive component profile
The typical total ion current chromatograms provided abundant fingerprint information on the meta­
bolomes of petioles and leaves of “Benqin”, “Western”, “Baoqin”, and “Majiagou” celeries. After peak
alignment, 1420 and 1640 metabolite ion features were detected in ESI+ and ESI – modes, respectively.
A total of 36 bioactive components, including nine vitamins, seven phenolic compounds (six flavonoids
and chlorogenic acid), five pigments, four carotenoids, four terpenes, three coumarins, three unsaturated
fat acids, and one methoxybenzene, were structurally identified in both the petioles and leaves of the four
selected celeries (Table S1). Among them, the presence of certain vitamins (niacin, riboflavin, pantothe­
nic acid, choline, phylloquinone, dehydroascorbic acid, and vitamin E succinate), phenolic compounds
(cyanidin, chlorogenic acid, apigenin, apiin, and rutin), pigments (chlorophyll b), and unsaturated fatty
acids (oleic acid, linoleic acid, and α-linolenic acid) within the petioles and leaves of these four varieties
was consistent with those reported previously in celery leaves, petioles, or roots .[2,17–20] Furthermore, to
our knowledge, the profiles showed the presence of 20 considerable bioactive constituents, including
coniferin, flavonoids (3,7-dihydroxyflavonoid and citrusin A), coumarins (peucedanin, citropten, and
osthenol), carotenoids (xanthophyll, diadinoxanthin, and violaxanthin), terpenoids (artemisinin, gano­
deric acid V, ganoderic acid R, and α-ionone), pigments (diosmetin, tomatine, corticrocin, and safflomin
A), vitamins (vitamin D3 glucosiduronate and acitretin), and methoxybenzene (elemicin), for the first
time in the petioles and leaves of the four selected varieties.
Contrary to expectations, compounds reported previously, including a flavonol (kaempferol), phe­
nolic acids (such as caffeic, p-coumaric, and ferulic acids), volatile constituents (such as limonene,
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES 1101

myrcene, and α-selinene), and tannins,[2,21] were not detected in the petioles or leaves of the four varieties
in our study. The absence of these compounds might be due to discrimination by the extraction methods,
measurement techniques, cultivars, and harvest times used, in addition to other environmental factors.
For example, celery cultivars harvested in Taiyuan (Shanxi, China) in 2008 were extracted with a mixture
of 80% ethanol, 6 M HCl, and ascorbic acid, with kaempferol and caffeic, p-coumaric, and ferulic acids
detected by HPLC at 254 and 280 nm.[7] Furthermore, 28 components (such as 4-chloro-4,4-dimethyl-
3-(1-imidazolyl)-valerophenone and 1-dodecanol) were identified in the essential oil of Apium grave­
olens leaves using gas chromatography–mass spectrometry.[22] Finally, white celery, purchased on
January 12, 1995, was found to contain luteolin (36 μg g–1) and apigenin (104 μg g–1), while neither of
these flavonoids was detected in the same samples purchased 11 days later.[23]

Contribution of bioactive components to antioxidant activity

Based on PLS analysis, the loading plot (Figure 2) showed that the first (Comp1) and second (Comp2)
principal components accounted for 60.3% of total variance in the entire dataset. The contribution
rates of Comp1 and Comp2 are 44.9% and 15.4%, respectively. Figure 2 showed the corresponding
distributions and correlations of antioxidant components and antioxidant capacity. The influence of
bioactive components (x–variable) on the antioxidant capacities (y-variable) was transferred through
standard partial lease square regression coefficients (referred to as simply ‘coefficients’ hereafter) from
PLS analysis (Figure 3a–c). These coefficients express how strongly the y-variable is correlated to the
x–variable. If the coefficients are high values, the influence of the x–variable on the y-variable is large.
Coefficients with positive and negative signs indicate positive and negative relationships, respectively,
between the x–variable and y-variable. Furthermore, the error bars indicate the confidence intervals of
the coefficients. The coefficient is significant when the confidence interval does not contain zero. In the
study, the coefficients showed that 18, 12, and 5 antioxidant components had significantly (P < 0.05)
positive correlations with the DPPH and O2∙ – scavenging activities and FRP, respectively.
Among them, flavonoids (apiin, apigenin, and rutin) and terpenes (α-ionone) made the largest
contributions to the DPPH scavenging activities (coefficients of 0.064–0.071), followed by vitamins
(riboflavin, phylloquinone, and choline), pigments (safflomin A), and carotenoids (3-hydroxy-3ʹ-oxo-β,
ε-carotene) (coefficients of 0.052–0.057). Finally, coumarins (peucedanin and elemicin), flavonoids

Figure 2. Loading plot of 36 bioactive components and antioxidant activities of four celery petioles and leaves from partial least
squares regression (PLS) analysis. R2X = 0.929, Q2 = 0.736. Range of red, purple, and blue imaginary lines show a strong correlation
between antioxidant components and DPPH and O2∙ – scavenging activities, and ferric ion reducing power, respectively.
1102 D.-K. LIU ET AL.

Figure 3. Standard partial least square regression coefficients (PLS coefficients) between 36 natural bioactive components and
antioxidant activities of four celery petioles and leaves: (a) DPPH and (b) O2∙ – scavenging activities, and (c) ferric ion reducing power,
respectively. *PLS coefficients are significant when the error bars (confidence intervals) do not contain zero (P < 0.05).

(3,7-dihydroxyflavone and cyanidin), pigments (diosmetin), vitamins (pantothenic acid), carotenoids

(diadinoxanthin and violaxanthin), and unsaturated fatty acids (oleic acid) made the smallest contribu­
tions (coefficients of 0.035–0.049) to the DPPH scavenging activities (Figure 3a). As previously reported,
DPPH is a stable N-atom-centered free radical (NOS) at room temperature and has been widely applied
for the determination of primary antioxidant activity. DPPH scavenging is based on two main pathways,
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES 1103

involving acceptance of either an electron or a hydrogen atom from an antioxidant.[24] The second
pathway has been shown to be more favorable for DPPH scavenging.[25] The natural antioxidants
detected showed different DPPH scavenging capacities. Apiin, apigenin, and rutin had the highest
capacities, mainly due to the lower BDE of O–H bonds in their B-rings, which enhanced their H-atom-
donating ability. Bendary et al. (2013) compared the DPPH and H2O2 scavenging capacity of anilines
and phenols (including flavonoids), finding that phenols were more effective at scavenging DPPH than
anilines, owing to their ability to lose hydrogen atoms more easily.[10] Furthermore, the site and number
of O–H bonds are crucial factors in enhancing the DPPH scavenging capacity. Rutin possesses two
active O–H groups in ortho-positions, which are weaker O–H bonds compared with para- and meta-
dihydroxy functionalities, resulting in the strongest DPPH scavenging capacity .[26] Furthermore,
according to the flavonoid chemical structures, the combination of the C2 = C3 double bond and
4-carbonyl group in the C-ring can efficiently shift B-ring electrons through resonance effects, which
assists in enhancing the radical scavenging capacity.[27] Apigenin and apiin also showed a strong FRP.
The contributions to FRP were in the order safflomin A (0.035) < riboflavin (0.039) < choline (0.047) <
apigenin (0.055) < apiin (0.060) (Figure 3c). Similarly, DPPH scavenging capacity and FRP were linearly
correlated with the phenolics content.[28] Apigenin significantly enhance the FRP in rats fed a high-fat
fructose diet.[29] The inhibition effect of apiin in celery leaf on DPPH (IC50 value, 68.0 μg mL–1) has
been shown to be higher than that on O2∙ – (IC50 value, 0.39 mg mL–1).[30]
Figure 3b showed that unsaturated fatty acids (oleic acid and α-linolenic acid) and vitamins (pan­
tothenic acid and vitamin E succinate) made the largest contributions (coefficients of 0.084–0.110) to the
radical scavenging activities, followed by flavonoids (3,7-dihydroxyflavone and apigenin), coumarins
(peucedanin), vitamins (choline and riboflavin), and pigments (safflomin A) (0.062–0.081), while
flavonoids (cyanidin and apiin) made the smallest contribution (coefficients of 0.032–0.040). O2∙ – is
a reactive oxygen species (ROS) that can directly attack biological macromolecules (such as DNA,
proteins, and membrane lipids) and, therefore, induce tissue damage. Furthermore, O2∙ – can directly
produce hydroxy radicals (∙OH), which are the most dangerous ROS, being capable of modifying almost
every molecule in living cells.[31] O2∙ – can also react with NO to form a peroxynitrite anion, which causes
more damage to cells than O2∙ – and NO alone.[32] Therefore, the O2∙ – scavenging capacity can serve as
a significant indicator of the potential antioxidant activities of celery samples. ROS radicals (such as O2∙ –)
mainly attack unsaturated fatty acids (UFAs) within cell membranes. More seriously, the lipid peroxida­
tion of UFAs can undergo a self-perpetuating chain reaction.[24] The oxidation of UFAs produces fatty
acid radicals (R∙), which rapidly capture oxygen to form fatty acid peroxyl radicals (ROO∙). ROO∙ further
accept hydrogen atoms from other UFAs, generating lipid hydroperoxides (ROOH). Subsequently,
ROOH breaks down into more radical species. Exogenous UFAs can also exert strong O2∙ – scavenging
capacities.[33] Furthermore, vitamin E succinate can be hydrolyzed to form free vitamin E. Vitamin E,
which serves as a strong liposoluble “chain breaker”, can intercept ROO∙ and terminate the lipid
peroxidation chain reactions.[26] Pantothenic acid is also an important factor in the prevention of
oxidative stress in tissues. Pantothenic acid, as a precursor and building block of coenzyme A (CoA),
can stimulate the biosynthesis of CoA. CoA can reduce the tert-butyl hydroperoxide (a ROS generator)
content to almost zero and increase the glutathione (converting ROOH to stable ROH) content and the
GSH/GSSG ratio by more than 50% in Ehrlich ascites cells.[34] Therefore, these mechanisms might be
responsible for the strong relationship of UFAs (α-linolenic acid and oleic acid) and vitamins (pantothe­
nic acid and vitamin E succinate) in celery samples with the O2∙ – scavenging capacity.

Comparison of major antioxidants and antioxidant capacities among four celery leaves and
petioles
Based on the PLS analysis, the relative contents of 21 major antioxidants, including five flavonoids
(Table 2), five vitamins (Table 2), and other components (two coumarins, four carotenoids, two
pigments, one terpene, and two unsaturated fatty acids, Table 3), were compared among the four
celery leaves and petioles. The relative contents of all investigated antioxidants in the leaves of four
1104 D.-K. LIU ET AL.

celery varieties were significantly higher than those in the respective petioles (P < 0.05).
Photosynthetically active leaves of celery act as the main photoassimilate source to support other
tissues (sinks), such as developing seeds, tubers, and stalks. Therefore, photoassimilate accumulation
and reallocation in the leaves of celeries might account for the higher levels of antioxidant components
in the leaves than in the petioles. The DPPH and O2∙ – inhibition ratios, and FRP, of the celery leaves
were significantly (P < 0.05) larger than those of the corresponding petioles (Table 4), collectively
demonstrating that the antioxidant activity of celery leaves were stronger than those of petioles. This
was probably due to the larger contents of major antioxidant components existing in the leaves.
Furthermore, the differences in the species and contents of antioxidant components among both
leaves and petioles from the four cultivars were relatively complicated and dominated by various
antioxidants. For leaves, “Majiagou” celery contained the most types of antioxidants (12 species) with
the highest contents, including vitamins (choline, pantothenic acid, riboflavin, and vitamin E succinate),
flavonoids (apiin, apigenin, and 3,7-dihydroxyflavone), coumarins (peucedanin and elemicin), safflomin
A, and unsaturated fatty acids (α-linolenic acid and oleic acid). The antioxidants with the highest (P <
0.05) contents in “Baoqin” celery included vitamins (pantothenic acid), flavonoids (apiin and rutin), and
carotenoids (diadinoxanthin, violaxanthin, and 3-hydroxy-3ʹ-oxo-β,ε-carotene). The antioxidants with
the highest (P < 0.05) contents in “Western” celery included vitamins (phylloquinone), flavonoids (rutin,
cyanidin, and 3,7-dihydroxyflavone), coumarins (peucedanin), carotenoids (diadinoxanthin, violax­
anthin, and xanthophyll), and pigments (diosmetin). Finally, the antioxidants with the highest (P <
0.05) contents in “Benqin” celery included vitamins (phylloquinone), flavonoids (apiin and rutin),
carotenoids (xanthophyll), and terpenes (α-ionone). For petioles, “Baoqin” celery contained 15 antiox­
idants at the highest (P < 0.05) levels (choline, pantothenic acid, riboflavin, phylloquinone, rutin,
elemicin, diadinoxanthin, violaxanthin, 3-hydroxy-3ʹ-oxo-β,ε-carotene, xanthophyll, diosmetin, safflo­
min A, α-ionone, α-linolenic acid, and oleic acid)). “Benqin” had the second largest number of abundant
antioxidants, with 12 present at the highest (P < 0.05) levels (choline, phylloquinone, vitamin E succinate,
apiin, apigenin, 3,7-dihydroxyflavone, peucedanin, diadinoxanthin, violaxanthin, 3-hydroxy-3ʹ-oxo-β,ε-
carotene, diosmetin, and α-ionone). Meanwhile, “Majiagou” and “Western” celeries contained compara­
tively few dominant antioxidants (P < 0.05), with only α-ionone and diosmetin found at the highest levels
in “Majiagou” and “Western” celeries, respectively.
These features could be responsible for the changes in antioxidant capacities among the leaves and
petioles of the four cultivars. Among the leaves, the O2∙ – scavenging activity was in the order “Majiagou” >
“Baoqin” > “Western” > “Benqin” (P < 0.05), while no significant differences among the DPPH scavenging
activity and FRP were observed (P > 0.05). For petioles, owing to many more antioxidants being present at
high levels in “Baoqin” and “Benqin” celeries compared with “Majiagou” and “Western” celeries, the O2∙ –
and DPPH scavenging activities and FRP of “Baoqin” and “Benqin” celeries were significantly (P < 0.05)
higher than those of “Majiagou” and “Western” celeries. No significant (P > 0.05) difference was observed
between “Baoqin” and “Benqin” celeries, nor between “Majiagou” and “Western” celeries.

Conclusion
A total of 36 bioactive components were identified in each celery leaf and petiole. PLS analysis combining all
data from four celery petioles and leaves showed that 18, 12, and 5 bioactive components in celery were
significantly correlated to the DPPH and O2∙ – radical scavenging capacity and FRP enhancement,
respectively. Among them, apiin, apigenin, rutin, and α-ionone made the highest contributions to the
DPPH scavenging activity, while oleic acid, α-linolenic acid, pantothenic acid, and vitamin E succinate made
the highest contributions to the O2∙ – scavenging activity. Furthermore, comparing the levels of major
antioxidants and antioxidant activity in the four celery leaves and petioles showed that leaves of “Majiagou”
celery and petioles of “Baoqin” and “Benqin” celeries were better sources of natural antioxidants and free
radical scavengers compared with the other species. These results provided a scientific basis for assessing the
quality and application value of four celery varieties.
Table 2. Relative concentration of major vitamins and flavonoids within the leaves and petioles of four celery varieties (μg/g)*.
Antioxidants Petiole 1 Petiole 2 Petiole 3 Petiole 4 Leaf 1 Leaf 2 Leaf 3 Leaf 4
Vitamins
Choline 626 ± 70e 405 ± 44f 626 ± 57e 463 ± 44f 1251 ± 112d 1547 ± 61c 1833 ± 97b 2224 ± 202a
Pantothenic acid 19 ± 3d 16 ± 3d 245 ± 4c 17 ± 3d 26 ± 3c 37 ± 5b 95 ± 7a 92 ± 8a
Riboflavin 4.9 ± 0.5d 4.4 ± 0.5d 12.2 ± 2.1c 5.9 ± 0.9d 37 ± 3b 42 ± 6b 39 ± 4b 79 ± 8a
Phylloquinone 0.33 ± 0.03d 0.17 ± 0.01e 0.34 ± 0.03d 0.18 ± 0.02e 33 ± 4a 28 ± 3a 2.0 ± 0.4c 14.9 ± 1.3b
Vitamin E succinate 0.46 ± 0.04c 0.25 ± 0.03d 0.31 ± 0.05d 0.26 ± 0.01d 5.47 ± 0.43b 0.54 ± 0.06c 0.49 ± 0.04c 16.8 ± 2.5a
Flavonoids
Apiin 67 ± 5c 40 ± 5d 36 ± 5d 19 ± 2e 3273 ± 79a 2343 ± 68b 3163 ± 45a 2950 ± 231a
Apigenin 41 ± 5d 24 ± 4e 22 ± 4e 10 ± f 2368 ± 213c 2189 ± 111c 3053 ± 124b 3650 ± 134a
Rutin 7.76 ± 0.87d 6.75 ± 0.52d 35 ± 5c 7.4 ± 0.6d 267 ± 15a 244 ± 19a 252 ± 16a 143 ± 17b
Cyanidin 0.23 ± 0.03d 0.16 ± 0.03d 0.17 ± 0.01d 0.19 ± 0.02d 0.94 ± 0.04 c 5.65 ± 0.78a 2.86 ± 0.43b 2.83 ± 0.35b
3,7-Dihydroxyflavone 0.83 ± 0.03c 0.36 ± 0.02e 0.45 ± 0.06d 0.48 ± 0.045d 1.72 ± 0.24b 5.53 ± 0.67a 2.03 ± 0.45b 5.60 ± 0.77a
*Data are expressed as means ± standard deviation (SD) of triplicate samples. a–fMeans in the same row without a common superscript are different (P < 0.05).
Petioles 1–4: Petioles of “Benqin”, “Western”, “Baoqin”, and “Majiagou” celeries, respectively. Leaves 1–4: Leaves of “Benqin”, “Western”, “Baoqin”, and “Majiagou” celeries, respectively.
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Table 3. Relative concentration of major coumarins, carotenoids, pigments, terpenes, and unsaturated fatty acids within the leaves and petioles of four celery cultivars (μg/g)*.
Antioxidants Petiole1 Petiole 2 Petiole 3 Petiole 4 Leaf 1 Leaf 2 Leaf 3 Leaf 4
Coumarins
Peucedanin 0.59 ± 0.06c 0.38 ± 0.01d 0.40 ± 0.02d 0.45 ± 0.01d 13 ± 2b 73 ± 6a 14 ± 2b 81 ± 9a
Elemicin 2.5 ± 0.4e 0.55 ± 0.04f 5.8 ± 0.4d 2.0 ± 0.4e 74 ± 4b 71 ± 6b 9.9 ± 0.6c 177 ± 20a
Carotenoids
Diadinoxanthin 4.7 ± 0.4d 2.0 ± 0.4e 5.4 ± 0.4d 1.6 ± 0.4e 75 ± 9b 152 ± 20a 178 ± 19a 9.6 ± 0.8c
Violaxanthin 7.2 ± 0.8d 4.0 ± 0.6e 6.1 ± 0.7d 1.3 ± 0.2f 91 ± 9b 159 ± 12a 172 ± 14a 39 ± 5c
3-Hydroxy 5.8 ± 0.7d 2.4 ± 0.5e 4.8 ± 0.7d 1.7 ± 0.1e 44 ± 8b 40 ± 7b 55 ± 6a 8.0 ± 0.7c
Xanthophyll 2.0 ± 0.3e 4.3 ± 0.4d 12 ± 2c 1.3 ± 0.3e 55 ± 6a 47 ± 8a 20 ± 2b 3.0 ± 0.3d
Pigments
Diosmetin 1.9 ± 0.3e 2.2 ± 0.5e 1.8 ± 0.1e 0.23 ± 0.01f 117 ± 12c 382 ± 34a 75 ± 6d 221 ± 18b
Safflomin A 6.2 ± 0.8e 3.8 ± 0.4f 10 ± 1d 4.0 ± 0.6f 362 ± 21c 703 ± 70b 416 ± 56c 886 ± 79a
Terpene
α-Ionone 0.35 ± 0.03c 0.18 ± 0.02d 0.32 ± 0.02c 0.43 ± 0.02c 16 ± 3a 7.2 ± 1.0b 6.1 ± 0.7b 7.5 ± 0.3b
Unsaturated fatty acids
α-Linolenic acid 19 ± 3g 35 ± 4f 51 ± 4e 22 ± 3g 108 ± 10b 87 ± 7c 69 ± 5d 421 ± 32a
Oleic acid 0.20 ± 0.02f 0.17 ± 0.02f 0.36 ± 0.01e 0.20 ± 0.02f 3.6 ± 0.4d 14 ± 2b 7.1 ± 0.6c 21 ± 3a

*Data are expressed as means ± standard deviation (SD) of triplicate samples. a–fMeans in the same row without a common superscript are different (P < 0.05).
3-Hydroxy: 3-Hydroxy-3ʹ-oxobeta, epsilon-carotene; Petioles 1–4: Petioles of “Benqin”, “Western”, “Baoqin”, and “Majiagou” celeries, respectively. Leaves 1–4: Leaves of “Benqin”, “Western”, “Baoqin”,
and “Majiagou” celeries, respectively.
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Table 4. Antioxidant capacities of leaves and petioles of four celery cultivarsa.

Antioxidant capacity DPPH inhibition ratio (%) O2∙ – inhibition ratio (%) Ferric ion reducing power
Petiole 1 38 ± 2b 23 ± 0d 0.13 ± 0.03b
Petiole 2 23 ± 3c 18 ± 0e 0.07 ± 0.00 c
Petiole 3 39 ± 2b 25 ± 1d 0.15 ± 0.02b
Petiole 4 23 ± 5c 14 ± 2e 0.05 ± 0.00 c
Leaf 1 86 ± 1a 24 ± 1d 0.40 ± 0.02a
Leaf 2 87 ± 1a 46 ± 9c 0.37 ± 0.04a
Leaf 3 84 ± 1a 68 ± 2b 0.46 ± 0.06a
Leaf 4 90 ± 0a 97± 0a 0.38 ± 0.05a
a
Data are expressed as means ± standard deviation (SD) of triplicate samples. a–eMeans in the same column without a common
superscript are different (P < 0.05).
3-Hydroxy: 3-Hydroxy-3ʹ-oxobeta, epsilon-carotene; Petioles 1–4: Petioles of “Benqin”, “Western”, “Baoqin”, and “Majiagou” celeries,
respectively. Leaves 1–4: Leaves of “Benqin”, “Western”, “Baoqin”, and “Majiagou” celeries, respective

Acknowledgments
The authors wish to thank the National Natural Science Foundation of China (No. 31701661), the Research Project of
Experimental Technology of Qufu Normal University (No. SJ201717 and No. SJ201503), and the Project of
Constructing High-level Applied Specialty (group) of Shandong-Biological Engineering Specialty Group (No.
[2016] 11-10).

Funding
This work was supported by the the National Natural Science Foundation of China [31701661]; the Research Project of
Experimental Technology of Qufu Normal University [SJ201717]; the Project of Constructing High-level Applied
Specialty (group) of Shandong-Biological Engineering Specialty Group [[2016] 11-10]; the Research Project of
Experimental Technology of Qufu Normal University [SJ201503].

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