Seasonal Dynamic Changes in the Nutrient Elements and Antioxidant Activity of Ilex vomitoria Leaf

Academic Editor: Marcello Salvatore
Lenucci
Received: 6 May 2025
Revised: 21 May 2025
Accepted: 18 June 2025
Published: 23 June 2025
Citation: Sha, L.; Yin, Y.; Xue, Y.; Zou,
X.; Zheng, B.; Zhang, J.; Yan, D.
Seasonal Dynamic Changes in the
Nutrient Elements and Antioxidant
Activity of Ilex vomitoria Leaf. Plants
2025, 14, 1919. https://doi.org/
10.3390/plants14131919
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
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licenses/by/4.0/).
Article
Seasonal Dynamic Changes in the Nutrient Elements and
Antioxidant Activity of Ilex vomitoria Leaf
Luqiong Sha 1
, Yanyan Yin 1
, Yilin Xue 1
, Xue Zou 1
, Bingsong Zheng 1
, Jianhong Zhang 2 and Daoliang Yan 1,*
1 National Key Laboratory for Development and Utilization of Forest Food Resources, Zhejiang A&F
University, Hangzhou 311300, China; lqsha@stu.zafu.edu.cn (L.S.); yinyan@stu.zafu.edu.cn (Y.Y.);
xyl@stu.zafu.edu.cn (Y.X.); 19157738591@163.com (X.Z.); bszheng@zafu.edu.cn (B.Z.)
2 Ningbo Academy of Agricultural Sciences, Ningbo 315000, China; nbjianhong@163.com
* Correspondence: liangsie@zafu.edu.cn
Abstract
Ilex vomitoria Ait. is a star substitute for “tea” in recent years. At present, research on
I. vomitoria mainly focuses on its breeding and cultivation, and there are few reports on
the seasonal changes of important components such as leaf nutrients. This study focuses
on the leaves of the topmost annual branches of I. vomitoria. Leaves were harvested at
different stages, and the nutrient elements, antioxidant substances, antioxidant capacity,
and aroma components in the leaves were measured and analyzed. The results showed
that the content of mineral elements, soluble sugars, vitamin C, amino acids, flavonoids,
polyphenols, saponins, caffeine, and catechins, as well as the DPPH free radical scavenging
ability, ABTS cation free radical scavenging ability, and FRAP iron ion reduction ability in
the leaves of I. vomitoria showed significant differences with seasonal changes. The mineral
element content in spring leaves is relatively high. Flavonoids and polyphenols are the
main antioxidant substances in the leaves of I. vomitoria, indicating that the antioxidant
capacity of spring leaves is the strongest. The content of aroma components in the leaves of
I. vomitoria in spring is the highest, with alcohols ranging from 54.93% to 66.08%, followed
by ketones from 17.63% to 48.07%, and aldehydes from 21.27% to 38.51%. Overall, spring
leaves are more suitable for harvesting, development, and utilization.
Keywords: Ilex vomitoria; nutritional components; aroma components; antioxidant;
development and utilization
1. Introduction
China’s abundant leaf resources provide an important material foundation for the
cycle of material of ecosystems and human survival. However, the overall utilization rate
of leaves by humans is relatively low, with most leaves left to fall or abandoned in forest
areas after harvesting, and a small portion used as fuel, fertilizer, or feed. Therefore, the
rational development and utilization of abundant leaf resources is to meet the needs of
human continuous development, especially the development of a green economy.
The leaves of many trees are rich in nutritional value and have a wide range of
uses [1]. For example, the leaves of Diospyros kaki L. are rich in flavonoids, vitamin C,
amino acids, etc. They are often used to make tea and have the effects of clearing heat,
lowering blood pressure, and lowering blood lipids. They can also be used as medicine
or topically to stop bleeding [2,3]. Crataegus pinnatifida Bge. leaves contain a high amount
of polyphenols and flavonoids, which play an important role in lowering blood sugar
Plants 2025, 14, 1919 https://doi.org/10.3390/plants14131919
Plants 2025, 14, 1919 2 of 18
and preventing cardiovascular diseases [4]. Sapium sebiferum L. leaves have the effects of
reducing swelling, dispersing blood stasis, clearing heat, and detoxifying [5]. Leaves are
also an important source of feed. Research has shown that the leaves of Pteroceltis tatarinowii
Maxim. contain high levels of protein, fat, crude fiber, and other components, which can be
used as livestock feed [6]. Meanwhile, Souilem et al. [7] have extracted and separated the
material components of olive leaves, and the obtained extracts were used in nutritional and
beauty products. Liao et al. have converted pineapple leaves into renewable fuel through
processes such as ethanol fermentation for utilization [8].
Ilex vomitoria Ait. is an evergreen shrub belonging to the Aquifoliaceae family, native
to the southeastern United States. Its leaves and tender branches are traditionally used to
make healthy drinks [9,10]. Camellia sinensis L. leaves contain abundant caffeine and are a
classic beverage in China. However, in recent years, people have been actively seeking new
caffeine-rich plant resources to replace conventional beverage plants such as tea. Due to
its unique active substance caffeine, I. vomitoria has become a star substitute for “tea” in
recent years, with huge economic and social benefits, and broad prospects for development
and utilization.
At present, research on I. vomitoria mainly focuses on breeding and cultivation [11,12],
and there are few reports on the seasonal changes of important components such as leaf
nutrients during its annual growth cycle. This study thus represents the first comprehensive exploration of the nutritional components, antioxidant components, and aroma
components of leaves of different seasons of I. vomitoria, and further evaluates the influence
of seasons on them. The research results indicate that the nutritional components, antioxidant compounds, and aromatic compounds in I. vomitoria leaves vary significantly with
the seasons.
2. Results
2.1. Analysis of Mineral Nutrients in the Leaves of I. vomitoria
The analysis of elemental content in I. vomitoria leaves is summarized in Table 1. The
difference between the highest and lowest contents of macroelements among the three
seasons was 2.87 mg/g DW for potassium (K). Among the macroelements, the variation
ranges were 5.14 g/kg for N, 1.17 g/kg for P, 1.58 g/kg for K, 1.19 g/kg for Mg, and
1.73 g/kg for Ca, respectively. The difference between the highest and lowest contents of
microelements among the three seasons were 14.27 mg/kg for Fe and 94.17 mg/kg for
Zn, respectively.
Table 1. Content of mineral nutrient elements and nutritional components in leaves of Ilex vomitoria
in different seasons (x ± s, n = 3).
Season
Macroelement (g/kg) Microelement
(mg/kg) Soluble Sugar
(mg/g)
Vitamin C
(mg/g)
N P K Mg Ca Fe Zn
Spring 17.00
±0.15 a
1.89
±0.05 a
14.09
±0.14 a
2.94
±0.02 a
3.74
±0.04 b
81.09
±2.76 a
232.03
±2.05 a
447.79
±4.45 a
17.11
±0.43 c
Summer 11.90
±0.45 b
0.72
±0.03 c
13.16
±0.26 a
1.75
±0.19 b
4.07
±0.55 b
66.82
±2.45 b
182.00
±24.35 b
269.81
±0.17 c
22.30
±0.33 b
Autumn 11.86
±0.17 b
1.15
±0.02 b
12.51
±0.17 a
2.69
±0.02 a
5.47
±0.02 a
71.73
±0.07 b
276.17
±0.39 a
324.66
±10.30 b
29.10
±0.54 a
Note: Different lowercase letters indicate the differences in element content among seasons (p < 0.05).
The contents of N, P, and Fe in spring leaves of I. vomitoria leaves were significantly
higher than those in summer and autumn (p < 0.05), with N content reaching as high as
17.00 g/kg; the Ca content in autumn was significantly higher than that in spring and
Plants 2025, 14, 1919 3 of 18
summer; there was no significant difference in the content of K, Mg, and Zn between spring
and autumn (p > 0.05); there was no significant difference in the content of K in leaves
between spring, summer, and autumn (p > 0.05). The above results indicate that tender
leaves harvested in spring have abundant mineral elements.
2.2. Analysis of Soluble Sugar and Vitamin C Content in Leaves of I. vomitoria
As shown in Table 1, the leaf soluble sugar content was highest in spring and significantly greater than in summer and autumn (p < 0.05), with the lowest levels observed in
summer. The soluble sugar content in spring and autumn was 65% and 20% higher than in
summer, respectively.
The leaf vitamin C content was highest in autumn and significantly greater than in
spring and summer (p < 0.05), with the lowest levels observed in spring. The vitamin C
content in autumn was 70% and 30% higher than in spring and summer, respectively.
2.3. Analysis of Amino Acid Content and Its Flavor in Leaves of I. vomitoria
As shown in Table 2, in spring, the highest amino acid was Arg, which was 402.81 µg/g,
and the lowest was Cys, which was 20.36 µg/g. In summer, Asp had the highest content
at 324.17 µg/g, while Cys had the lowest content at 11.96 µg/g. In autumn, Ile had the
highest content at 116.34 µg/g, while His had the lowest content at 9.28 µg/g. The highest
total amino acid content in leaves was in spring, at 1645.14 µg/g, followed by summer
at 1425.10 µg/g, and the lowest was in autumn, at 554.81 µg/g. The highest content of
essential amino acids (EAAs) in I. vomitoria leaves was observed in spring (571.62 µg/g
dry weight), followed by summer (394.81 µg/g) and autumn (225.19 µg/g) In summary,
the content of 17 amino acids in the leaves of I. vomitoria was highest during spring
harvesting and lowest during autumn harvesting, with significant differences between the
two (p < 0.05).
Table 2. Amino acid of Ilex vomitoria leaves in different seasons.
Amino Acid
Content(µg/g)
Spring Summer Autumn
Asp 72.87 ± 7.92 b 324.17 ± 6.66 a 21.88 ± 3.63 c
Glu 157.45 ± 5.72 b 323.71 ± 4.59 a 89.85 ± 1.95 b
Ser 36.62 ± 9.51 ab 66.56 ± 1.41 a 11.22 ± 0.91 b
Gly 39.68 ± 2.06 a 12.15 ± 0.83 b 9.378 ± 0.82 b
His 28.70 ± 3.10 b 121.97 ± 1.67 a 9.28 ± 0.66 b
Arg 402.81 ± 6.85 a 16.31 ± 0.28 c 106.76 ± 0.33 b
Thr 37.33 ± 0.82 a 33.02 ± 1.86 b 9.39 ± 0.79 c
Ala 130.62 ± 1.37 a 88.44 ± 3.47 b 36.50 ± 0.79 c
Pro 76.40 ± 3.93 a 23.03 ± 1.01 b 11.71 ± 0.50 c
Tyr 73.06 ± 2.49 a 42.00 ± 2.03 b 17.56 ± 0.76 c
Val 78.62 ± 2.81 a 33.23 ± 1.98 b 14.71 ± 0.37 c
Met 21.62 ± 1.98 a 20.15 ± 1.06 a 9.48 ± 0.05 b
Cys 20.36 ± 1.17 a 11.96 ± 1.12 b 15.09 ± 0.31 a
Ile 169.92 ± 8.81 a 110.05 ± 3.13 b 116.34 ± 4.38 b
Leu 174.84 ± 8.24 a 99.53 ± 3.05 b 41.45 ± 4.31 c
Phe 89.91 ± 3.64 a 61.17 ± 2.59 b 21.81 ± 0.93 c
Lys 36.35 ± 0.50 a 37.66 ± 2.80 a 12.01 ± 0.40 b
Essential amino acid 571.62 394.81 225.19
Total amino acids 1645.14 1425.10 554.81
Note: Different lowercase letters in the same line indicate significant differences. (p < 0.05). Asp: Aspartic acid;
Glu: Glutamic acid; Ser: Serine; Gly: Glycine; His: Histidine; Arg: Argnine; Thr: Threonine; Ala: Alanine;
Pro: Proline; Tyr: Tyrosine; Val: Valine; Met: Methionine; Cys: Cysteine; Ile: Isoleucine; Leu: Leucine; Phe:
Phenylalanine; Lys: Lysine.
Plants 2025, 14, 1919 4 of 18
According to Table 3, in spring, the total TAVs of various flavor amino acids were
in the order of umami amino acids > bitter amino acids > sweet amino acids > aromatic
amino acids. The total content of amino acids in the spring flavor of I. vomitoria leaves
was 1601.13 µg/g, with Arg having the highest content at 402.81 µg/g and the highest
TAV at 4.03. During spring, the primary contributor to the sweet taste among amino acids
was His; to the bitter taste, Arg; to the fresh taste, Glu and Asp; and to the aromatic taste,
Cys. Collectively, these five free amino acids (His, Arg, Glu, Asp, and Cys) were the key
determinants of the unique flavor profile in spring I. vomitoria leaves.
Table 3. Flavor amino acid of Ilex vomitoria leaves in spring, summer, and autumn.
Class Type of
Amino Acids
Taste Threshold
(µg/g)
Spring Summer Autumn
Content
(µg/g) TAV Content
(µg/g) TAV Content
(µg/g) TAV
Sweet
amino acids
Ala 600 130.62 0.3 88.44 0.15 36.50 0.06
Pro 3000 76.40 0.03 23.03 0.01 11.71 0.01
His 200 28.70 1.43 121.97 0.61 9.28 0.05
Thr 2600 37.33 0.01 33.02 0.01 9.39 0.01
Ser 1500 36.62 0.02 66.56 0.04 11.22 0.01
Gly 1100 39.68 0.04 12.15 0.01 9.38 0.01
Bitter
amino acids
Val 1500 78.62 0.05 33.23 0.02 14.71 0.01
Leu 3800 174.84 0.04 99.53 0.03 41.45 0.01
Ile 900 169.92 0.18 110.05 0.12 116.34 0.13
Met 300 21.62 0.07 20.15 0.07 9.48 0.31
Thr 2600 37.33 0.01 33.02 0.01 9.39 0.01
Arg 100 402.81 4.03 16.31 0.16 106.76 1.06
Umami
amino acids
Lys 500 36.35 0.07 37.66 0.07 12.01 0.02
Asp 30 72.87 2.43 324.17 10.81 21.88 0.73
Glu 50 157.45 3.15 323.71 6.47 89.85 1.8
Aromatic
amino acids
Phe 1500 89.91 0.06 61.17 0.01 21.81 0.01
Tyr 2600 73.06 0.03 42.00 0.02 17.56 0.01
Cys 20 20.36 1.01 11.96 0.05 15.09 0.01
Total - - 1601.13 - 1458.48 - 526.8 -
Note: Asp: Aspartic acid; Glu: Glutamic acid; Ser: Serine; Gly: Glycine; His: Histidine; Arg: Argnine; Thr:
Threonine; Ala: Alanine; Pro: Proline; Tyr: Tyrosine; Val: Valine; Met: Methionine; Cys: Cysteine; Ile: Isoleucine;
Leu: Leucine; Phe: Phenylalanine; Lys: Lysine.
In summer, the order of total TAVs for various flavor amino acids were umami amino
acids > sweet amino acids > bitter amino acids > aromatic amino acids. The total content
of amino acids in the summer leaves of I. vomitoria was 1458.48 µg/g, with the highest
content of Asp at 324.17 µg/g. Its TAV was also the highest at 10.81, indicating that it
contributes the most to the presentation of amino acid flavor in the summer leaves of
I. vomitoria. The amino acids that present a fresh flavor in the summer leaves of I. vomitoria
mainly include Glu and Asp, so these two free amino acids were the main amino acids that
present a unique flavor in the summer leaves of I. vomitoria.
In autumn, the order of total TAVs for various flavor amino acids was umami amino
acids > bitter amino acids > sweet amino acids > aromatic amino acids. The total content of
amino acids in the autumn leaves of I. vomitoria was 526.8 µg/g, with Ile having the highest
content at 116.34 µg/g. However, its TAV was not the highest, with Glu having the highest
TAV at 1.8. Therefore, Glu contributes the most to the presentation of amino acid flavor in
the autumn leaves of I. vomitoria. The main amino acids that present a bitter taste in the
autumn leaves of I. vomitoria were Arg, and the main amino acids that present a fresh taste
Plants 2025, 14, 1919 5 of 18
were Glu. Therefore, these two free amino acids were the main amino acids that present a
unique flavor in the autumn leaves of I. vomitoria.
2.4. Analysis of Antioxidant Content and Antioxidant Activity in Leaves of I. vomitoria
According to Table 4, the leaf flavonoid content was highest in spring and significantly
greater than in summer and autumn (p < 0.05), with the lowest levels observed in autumn.
The flavonoid content in spring was 38% and 208% higher than in summer and autumn,
respectively. The leaf polyphenol content was highest in spring and significantly greater
than in summer and autumn (p < 0.05), with the lowest levels observed in autumn. The
polyphenol content in spring was 33% and 77.7% higher than in summer and autumn,
respectively. The leaf total saponin content was highest in summer and significantly greater
than in spring and autumn (p < 0.05), with the lowest levels observed in autumn. The
polyphenol content in summer was 11% and 31% higher than in spring and autumn,
respectively. The leaf caffeine content was highest in spring and significantly greater
than in summer and autumn (p < 0.05), with the lowest levels observed in autumn. The
caffeine content in spring and summer was 219.97% and 206.21% higher than in autumn,
respectively. The leaf catechins content was highest in spring and significantly greater than
in summer and autumn (p < 0.05), with the lowest levels observed in autumn. The catechins
content in spring was 5 times and 9 times higher than in summer and autumn, respectively.
Table 4. Physiologically active substance contents of Ilex vomitoria leaves in different seasons.
Season Flavonoid
(mg/g)
Polyphenol
(mg/g)
Saponins
(mg/g)
Caffeine
(µg/g)
Catechins
(µg/g)
Spring 64.86
±1.10 a
35.45
±0.34 a
130.45
±6.95 ab
2982.30
±41.07 a
1083.84
±37.53 a
Summer 46.48
±0.28 b
26.64
±1.68 b
144.56
±13.06 a
2853.98
±33.07 a
215.31
±18.32 b
Autumn 20.40
±0.47 c
19.94
±0.49 c
110.10
±1.06 b
932.03
±93.05 b
115.17
±1.38 c
Note: Different lowercase letters in the same column indicate significant differences. (p < 0.05).
DPPH is a synthetic organic free radical commonly used to determine the antioxidant
activity of plants. According to Table 5, the DPPH free radical scavenging ability of leaves
gradually decreased with seasonal changes. The DPPH free radical scavenging ability
of spring leaves was the strongest, at 305.58 µmol TE/g, significantly higher than that
of summer and autumn, and the lowest in autumn, at 96.84 µmol TE/g. The DPPH
free radical scavenging ability in spring was 1.08 and 3.15 times that of summer and
autumn, respectively.
Table 5. Antioxidant evaluation method of Ilex vomitoria leaves in different seasons.
Season
DPPH Free Radical
Scavenging Ability
(µmol TE/g)
ABTS+ Free Radical
Scavenging Ability
(µmol TE/g)
FRAP Iron Ion
Reducing Ability
(µmol TE/g)
Spring 305.58 ± 9.73 a 43.04 ± 1.93 a 375.23 ± 2.82 a
Summer 146.85 ± 3.24 b 42.23 ± 0.51 a 342.95 ± 4.90 c
Autumn 96.84 ± 10.50 c 36.72 ± 0.39 b 329.54 ± 3.50 b
Note: Different lowercase letters indicate the differences in element content among seasons (p < 0.05).
ABTS cations are oxidized by oxidants such as K2S2O8 to produce blue-green ABTS
cationic free radicals. The absorbance of the reaction mixture at a specific wavelength
(typically 734 nm), which is directly proportional to the ABTS+
concentration, is measured
Plants 2025, 14, 1919 6 of 18
to assess antioxidant capacity of the sample. The ABTS+
free radical scavenging ability
of spring leaves was determined to be 43.04 µmol TE/g, and the lowest in autumn was
36.72 µmol TE/g.
The reducing ability of I. vomitoria leaves was determined using the FRAP iron ion
reducing ability method. This method determines the reduction ability of leaves based
on the absorbance when Fe3+ is reduced to Fe2+ by antioxidant substances. The higher
the absorbance value, the stronger the reducing ability. From Table 5, it can be seen that
the FRAP iron ion reducing ability of leaves gradually decreases with seasonal changes in
the three seasons. The metal ion reducing capacity in spring leaves was the strongest, at
375.23 µmol TE/g, significantly higher than that in summer and autumn (p < 0.05), and
the lowest in autumn, at 329.54 µmol TE/g. The metal ion reducing capacity in spring was
9.41% and 13.86% stronger than that in summer and autumn, respectively.
As shown in Table 6, by comparing various correlation factors, it can be seen that in
spring, the content of polyphenols, flavonoids, caffeine, catechins, and saponins in the
leaves of I. vomitoria was significantly more correlated with DPPH free radical scavenging
ability, ABTS cation free radical scavenging ability, and FRAP iron ion reducing ability than
in summer and autumn. In spring leaves, flavonoids had the highest correlation with ABTS
cation free radical scavenging ability, polyphenols had the highest correlation with FRAP
iron ion reducing ability, and flavonoids had the highest correlation with DPPH free radical
scavenging ability, with correlation coefficients of 0.996, 0.985, and 0.999, respectively. It
can be inferred that the strongest ABTS cation free radical scavenging ability and DPPH
free radical scavenging ability in spring may be related to the highest flavonoid content
in spring.
Table 6. Correlation analysis between components of Ilex vomitoria leaves in different seasons.
Correlation Factors
ABTS FRAP DPPH
Spring Summer Autumn Spring Summer Autumn Spring Summer Autumn
Caffeine 0.956 ** 0.946 ** 0.947 ** 0.955 ** 0.954 ** 0.832 ** 0.953 ** 0.976 ** 0.859 **
Catechins 0.976 ** 0.937 ** 0.954 ** 0.975 ** 0.956 ** 0.954 ** 0.975 ** 0.955 ** 0.943 **
Polyphenol 0.989 ** 0.969 ** 0.979 ** 0.985 ** 0.988 ** 0.973 ** 0.988 ** 0.976 ** 0.974 **
Flavonoid 0.996 ** 0.975 ** 0.969 ** 0.984 ** 0.983 ** 0.974 ** 0.999 ** 0.998 ** 0.995 **
Saponins 0.865 ** 0.863 ** 0.820 ** 0.893 ** 0.853 ** 0.840 ** 0.854 ** 0.831 ** 0.820 **
DPPH 0.885 ** 0.856 ** 0.856 ** 0.775 * 0.735 * 0.765 *
FRAP 0.893 ** 0.913 ** 0.934 **
ABTS
Note: ABTS represent ABTS cationic free radical scavenging ability, FRAP represent FRAP iron reducing ability,
DPPH represent free radical scavenging ability. Asterisks (*, **) denote statistical significance of mean differences
among seasons, corresponding to p < 0.05 and p < 0.01 levels, respectively.
2.5. Analysis of Aroma Components in Leaves of Different Seasons of I. vomitoria
Seven types of aromatic compounds were isolated from the leaves of I. vomitoria,
mainly including alcohols, ketones, and aldehydes, with average contents of 58.70%,
29.75%, and 27.50%, respectively. The remaining components were esters (6.40%), other
types (ether, benzene, alkane) (5.83%), olefins (4.76%), and acids (1.38%). The relative
content of various aroma components in leaves varied greatly in different seasons, with
the proportion of alcohol, ketone, aldehyde, lipid, other types, alkene, and acid aromas
reaching the highest level in spring (Table 7).
Plants 2025, 14, 1919 7 of 18
Table 7. Composition and relative content of aroma components (%) of Ilex vomitoria leaves in
different seasons.
Season Alcohols Ketones Aldehydes Esters Other Types Olefins Acids
Spring 66.08 48.07 38.51 9.91 9.00 6.58 1.63
Summer 55.04 17.63 22.74 4.89 4.91 2.99 1.22
Autumn 54.98 23.57 21.27 4.40 4.90 4.72 1.03
Average contents 58.70 29.75 27.50 6.40 5.83 4.76 1.38
A total of 82 aroma components were reported in the leaves of I. vomitoria in Table 8.
In spring, the highest content of aroma compounds in the leaves of I. vomitoria were alcohol
compounds, accounting for 66.08%. In addition, ketones and aldehydes account for 48.07%
and 38.51%, respectively. Esters, other compounds (ether, benzene, alkane), alkenes, and
acids account for 9.91%, 9.00%, 6.58%, and 1.63%, respectively. Among the alcohols, (2E)-
3,7-dimethylocta-2,6-dien-1-ol was the predominant compound, which was 14.06%. The
contents of 2,6-dimethylcyclohexan-1-ol, 2-ethylhexan-1-ol, pentan-1-ol, hept-6-en-1-ol, and
butan-1-ol reached 13.62%, 13.06%, 12.77%, 11.26%, and 11.21%, respectively; the highest
content of ketone compounds was 1-hydroxypropan-2-one, with a content of 12.10%. octan2-one, heptan-2-one, hept-1-en-3-one, and hexane-2,3-dione reach 11.46%, 7.63%, 5.56%,
and 4.95%, respectively; the 2-phenylacetaldehyde with the highest content in aldehyde
compounds reached 14.29%, while hexanal, (E)-hex-2-enal, (E)-pent-2-enal, (E)-hept-4-enal,
and (E)-hex-3-enal reached 7.76%, 4.95%, 2.65%, 1.52%, and 1.45%, respectively.
Table 8. Analysis of the differences in aroma components of Ilex vomitoria leaves in different seasons.
Serial Number CAS The Name of the Compound Molecular
Formula
Relative Content(%) R.T.
(Minutes) Spring Summer Autumn
1 87-44-5 4,11,11-trimethyl-8-methylidenebicyclo [7.2.0]undec-4-ene C15H24 0.19 0.16 0.16 20.894
2 78-85-3 2-methylprop-2-enal C4H6O 2.43 0.68 1.60 2.643
3 55683-21-1 3,4,5-trimethylcyclopent-2-en-1-one C8H12O 0.39 0.27 0.34 3.387
4 80-56-8 2,6,6-trimethylbicyclo [3.1.1]hept-2-ene C10H16 2.14 0.95 1.03 5.101
5 106-24-1 (2E)-3,7-dimethylocta-2,6-dien-1-ol C10H18O 14.06 13.68 18.53 26.622
6 600-14-6 pentane-2,3-dione C5H8O2 1.24 1.25 0.90 6.155
7 122-78-1 2-phenylacetaldehyde C8H8O 14.29 11.08 10.66 21.807
8 104-93-8 1-methoxy-4-methylbenzene C8H10O 0.86 0.09 0.49 11.58
9 71-41-0 pentan-1-ol C5H12O 12.77 12.41 8.26 11.951
10 41519-23-7 [(Z)-hex-3-enyl] 2-methylpropanoate C10H18O2 0.36 0.24 0.28 28.649
11 142-92-7 hexyl acetate C8H16O2 0.51 0.49 0.50 12.407
12 586-62-9 1-methyl-4-propan-2-ylidenecyclohexene C10H16 0.26 0.11 0.08 12.514
13 111-13-7 octan-2-one C8H16O 11.46 5.32 9.21 12.725
14 106-72-9 2,6-dimethylhept-5-enal C9H16O 1.78 0.38 1.36 14.612
15 4132-48-3 1-methoxy-4-propan-2-ylbenzene C10H14O 0.59 0.11 0.44 14.919
16 111-11-5 methyl octanoate C9H18O2 0.32 0.08 0.19 15.695
17 31081-18-2 3-methyl-5-propylnonane C13H28 0.54 0.20 0.19 16.016
18 104-76-7 2-ethylhexan-1-ol C8H18O 13.06 25.12 13.35 18.43
19 4117-10-6 hept-6-en-1-ol C7H14O 11.26 1.15 4.59 18.922
20 932-66-1 1-(cyclohexen-1-yl)ethanone C8H12O 0.25 0.09 0.10 7.913
21 629-50-5 tridecane C13H28 2.74 0.96 1.17 13.205
22 5337-72-4 2,6-dimethylcyclohexan-1-ol C8H16O 13.62 6.41 7.43 20.98
23 4412-91-3 furan-3-ylmethanol C5H6O2 1.69 0.39 1.41 22.397
24 106-68-3 octan-3-one C8H16O 1.02 0.77 0.50 11.841
25 116-09-6 1-hydroxypropan-2-one C3H6O2 12.10 3.06 10.86 13.054
26 502-47-6 3,7-dimethyloct-6-enoic acid C10H18O2 0.89 0.49 0.40 25.29
27 1576-95-0 (Z)-pent-2-en-1-ol C5H10O 0.59 0.00 0.49 13.89
28 543-49-7 heptan-2-ol C7H16O 0.50 0.30 0.43 13.955
29 4363-93-3 quinoline-4-carbaldehyde C10H7NO 0.23 0.12 0.21 37.265
30 13894-63-8 methyl (E)-hex-2-enoate C7H12O2 2.12 1.72 2.31 12.843
31 591-93-5 penta-1,4-diene C5H8 1.25 0.75 1.3 1.631
32 74-93-1 methanethiol CH4S 0.39 0.62 0.40 1.64
33 75-07-0 acetaldehyde C2H4O - 0.12 0.14 1.683
34 110-00-9 furan C4H4O 0.02 0.3 0.08 2.03
35 142-82-5 heptane C7H16 0.03 0.21 0.63 1.69
36 78-93-3 butan-2-one C4H8O - - 0.16 2.86
37 111-84-2 nonane C9H20 - - 0.08 2.877
38 75-18-3 methylsulfanylmethane C2H6S 0.85 0.31 1.85 1.834
39 123-38-6 propanal C3H6O - - 1.16 1.999
40 111-65-9 octane C8H18 - 0.33 0.59 2.056
41 78-84-2 2-methylpropanal C4H8O - - - 2.12
Plants 2025, 14, 1919 8 of 18
Table 8. Cont.
Serial Number CAS The Name of the Compound Molecular
Formula
Relative Content(%) R.T.
(Minutes) Spring Summer Autumn
42 107-02-8 prop-2-enal C3H4O 0.49 0.31 0.92 2.338
43 123-72-8 butanal C4H8O - - 0.14 2.603
44 106-61-6 2,3-dihydroxypropyl acetate C5H10O4 - 1.92 0.02 9.08
45 67-56-1 methanol CH4O - - 0.01 2.847
46 96-17-3 2-methylbutanal C5H10O - 0.47 0.15 3.023
47 590-86-3 3-methylbutanal C5H10O - 0.02 0.17 3.081
48 109-86-4 2-methoxyethanol C3H8O2 0.95 0.01 0.56 3.319
49 64-17-5 ethanol C2H6O 0.77 - - 3.402
50 78-94-4 but-3-en-2-one C4H6O 0.14 0.26 - 3.504
51 110-62-3 pentanal C5H10O 0.84 0.60 - 4.139
52 124-18-5 decane C10H22 0.06 - - 4.69
53 1629-58-9 pent-1-en-3-one C5H8O 0.34 - 0.08 5.077
54 78-92-2 butan-2-ol C4H10O 0.28 0.09 - 5.393
55 71-23-8 propan-1-ol C3H8O 0.19 - 0.04 5.717
56 872-05-9 dec-1-ene C10H20 0.02 0.11 0.17 5.766
57 7452-79-1 ethyl 2-methylbutanoate C7H14O2 0.14 - - 6.013
58 66-25-1 hexanal C6H12O 7.76 4.43 0.52 6.711
59 497-03-0 (E)-2-methylbut-2-enal C5H8O 0.12 0.04 - 6.982
60 1120-21-4 undecane C11H24 - - 0.03 7.104
61 5878-19-3 1-methoxypropan-2-one C4H8O2 - 0.76 0.43 7.317
62 78-83-1 2-methylpropan-1-ol C4H10O 1.37 0.45 - 7.493
63 1576-87-0 (E)-pent-2-enal C5H8O 2.65 1.34 0.76 8.041
64 3848-24-6 hexane-2,3-dione C6H10O2 4.95 0.35 - 8.129
65 4440-65-7 (E)-hex-3-enal C6H10O 1.45 0.89 - 8.324
66 6032-29-7 pentan-2-ol C5H12O 0.48 0.43 0.06 8.317
67 71-36-3 butan-1-ol C4H10O 11.21 16.26 - 8.947
68 616-25-1 pent-1-en-3-ol C5H10O 0.78 0.67 - 9.336
69 108-11-2 4-methylpentan-2-ol C6H14O - 1.28 - 9.607
70 110-43-0 heptan-2-one C7H14O 7.63 2.49 - 9.67
71 112-40-3 dodecane C12H26 - - 0.42 10.124
72 6728-26-3 (E)-hex-2-enal C6H10O 4.95 0.15 0.02 10.75
73 111-90-0 2-(2-ethoxyethoxy)ethanol C6H14O3 0.78 0.5 0.01 21.516
74 929-22-6 (E)-hept-4-enal C7H12O 1.52 2.11 3.46 11.246
75 928-68-7 6-methylheptan-2-one C8H16O 2.31 3.08 - 11.339
76 6728-31-0 (Z)-hept-4-enal C7H12O 0.25 0.05 0.13 11.415
77 1120-06-5 decan-2-ol C10H22O 4.72 2.21 1.39 11.658
78 763-32-6 3-methylbut-3-en-1-ol C5H10O 9.65 3.27 0.03 11.833
79 513-86-0 3-hydroxybutan-2-one C4H8O2 2.28 0.36 1.34 12.683
80 13894-63-8 methyl (E)-hex-2-enoate C7H12O2 0.03 1.25 - 12.843
81 2918-13-0 hept-1-en-3-one C7H12O 5.56 0.35 0.02 13.181
82 4798-45-2 4-methylpent-1-en-3-ol C6H12O 0.03 0.21 0.45 13.655
Note: Other categories include ether, benzene, and alkane compounds; - represents undetected.
In summer, the highest content of aromatic compounds in the leaves of I. vomitoria was
alcohol compounds, accounting for 55.04%, followed by aldehydes and ketones, accounting
for 22.74% and 17.63%, respectively. The content of other compounds, esters, alkenes, and
acids was 4.91%, 4.89%, 2.99%, and 1.22%, respectively. The highest content of alcohol
compounds was 2-ethylhexan-1-ol, accounting for 25.12%. The contents of butan-1-ol,
(2E)-3,7-dimethylocta-2,6-dien-1-ol, and pentan-1-ol reached 16.26%, 13.68%, and 12.41%,
respectively. The highest content of ketones in summer was octan-2-one, which accounts
for 5.32%, while the content of other ketones was relatively low. The aldehyde compounds
with higher content were 2-phenylacetaldehyde, which accounts for 11.08%, and hexanal,
which accounts for 4.43%.
In autumn, the highest content of aromatic compounds in the leaves of I. vomitoria
was also alcohol compounds, accounting for 54.98%, followed by ketone compounds and
aldehyde compounds, with contents of 23.57% and 21.27%, respectively. The contents
of other compounds, alkenes, esters, and acids were 4.90%, 4.72%, 4.40%, and 1.03%,
respectively. The highest content in alcohol compounds was (2E)-3,7-dimethylocta-2,6-dien1-ol, which was 18.53%, followed by 2-ethylhexan-1-ol with a content of 13.35%; the highest
content of ketone compounds in autumn leaves was 1-hydroxypropan-2-one, accounting
for 10.86%, followed by octan-2-one, accounting for 9.21%; the highest content of aldehydes
in autumn leaves was hexanal, with a content of 10.66%.
In summary, the content of alcohols, aldehydes, esters, other compounds, and acids in
the leaves of I. vomitoria was in the order of spring>summer>autumn, while the content
of ketones and alkenes was in the order of spring>autumn>summer. Spring was the
Plants 2025, 14, 1919 9 of 18
season with the highest proportion of most aromatic substances, followed by summer, and
finally autumn.
3. Discussion
The content of N and Fe in leaves of Ilex vomitoria showed an overall decreasing
trend with seasonal changes, while the content of Ca showed an increasing trend, and
the content of P, Mg, and Zn showed a first decreasing and then increasing trend. The
contents of N and P in spring leaves of I. vomitoria leaves were significantly higher than
those in summer and autumn, and our findings are consistent with a recent study [13].
Research has shown that N absorption is closely related to the active growth of young tea
buds [14]. High content of N significantly increases tea yield and the levels of polyphenols
and caffeine [15,16]. P has been proven to alter the metabolism of minerals and metabolites
in tea plants, thus affecting tea yield and quality [17]. P contributes to improving the
aroma and flavor of tea [18]. Both N and P increase can enhance tea yield and growth. N
and P are also essential elements in the formation of enzymes like nitrate reductase (NR),
adenosine triphosphate (ATP), and nicotinamide adenine dinucleotide phosphate (NADP),
thereby boosting the antioxidant activity of tea [19]. The content of Mg and Fe in the
leaves of I. vomitoria in spring was the highest, and higher than that in the I. paraguariensis
A.St.-Hil. [20]. Mg is an essential component of chlorophyll, which plays a crucial role in
photosynthesis and many other metabolic processes. [21]. The content of Mg was highest
in spring, coinciding with the highest synthesis of carbohydrates through photosynthesis,
resulting in the highest levels of soluble sugars [22]. Fe is involved in key processes such as
DNA synthesis, respiration, photosynthesis, and nitrogen degradation [23]. Studies have
shown that increased Ca supply induces stomatal closure and mediates stress responses,
aiding in cold injury recovery and adaptation to cold stress [24]. In this study, the highest
content of Ca was observed in the autumn, likely reflecting physiological adjustments to
cooler temperatures. Research has also shown that Zn is related to the amino acid content
in tea leaves [25]. Zn plays a critical role in nucleic acid and protein synthesis and aids in
the utilization of N and P [26]. Compared to summer and autumn tea, spring tea leaves
contain higher levels of essential mineral nutrients, thus leading to higher concentrations
of amino acids, polyphenols, and caffeine—all of which contribute to better tea quality [27].
Spring provides favorable weather conditions with steadily rising temperatures and high
humidity, ideal for the growth of new tea buds [28]. Therefore, considering these factors,
spring tea leaves of I. vomitoria have the richest nutritional content and are recommended
for further development and utilization.
Soluble sugars are important carbohydrates in photosynthesis and are also one of the
consumables in respiration. Tea leaves predominantly accumulate sugar conjugates, whose
biosynthesis is closely linked to N metabolism. N is essential for chlorophyll’s porphyrin
structure, and enhances photosynthetic efficiency to drive sugar production via the Calvin
cycle, serving as precursors for sugar conjugates [29]. The content of N in the leaves of
I. vomitoria is highest in spring, which corresponds to the highest levels of soluble sugars
synthesized. Soluble sugars are key determinants of the sweet taste quality in tea [30].
In this experiment, the soluble sugar content in the leaves of I. vomitoria in spring was
significantly higher than the other two seasons.
The vitamin C content in the leaves of I. vomitoria varied with the seasons. This study
showed that the highest vitamin C content occurs in autumn. Based on these experimental
results, we would recommend harvesting leaves in autumn for the preparation of I. vomitoria
tea and its development into health beverages, due to its higher water-soluble vitamin
C content. In addition, vitamin C is a natural oxidant with a natural oxidizing effect,
Plants 2025, 14, 1919 10 of 18
which can be developed and utilized into compound medicines and beauty and skincare
products [31].
The unique flavor, quality, and health benefits of tea are closely linked to a variety
of secondary metabolites. Key secondary metabolites influencing tea quality include
polyphenols, total amino acids, and aroma compounds. Among them, polyphenols and
amino acids directly affect the taste, while polyphenol oxidation products contribute to the
color of tea, and aroma compounds determine its fragrance quality. The synthesis of these
metabolites is regulated during different growth stages of tea plants [32]. Seasonal climatic
conditions have a significant impact on the quality of tea before harvest. For instance,
spring tea is obviously higher in quality compared to summer or autumn tea [33]. The
differences in quality between spring and summer teas are primarily due to variations in
environmental conditions, which affect the synthesis of secondary metabolites [34,35].
The content of free amino acids is considered an important indicator for ensuring
tea quality, as they contribute to the overall taste and color [36]. High amino acid levels
increase the freshness of tea infusions [32]. In spring, the total amount of free amino acids
in the leaves of I. vomitoria is the highest, higher than the summer congou black tea [37], and
much higher than the amino acid content of C. sinensis tea [38], and is consistent with the
findings in green tea [39]. N is essential for the biosynthesis of amino acids, and increased
N content may help elevate the total amino acid levels in tea leaves [40]. Accordingly,
our study found the highest N and total amino acid contents in the leaves of I. vomitoria
in spring.
From a flavor perspective, amino acids play a crucial role as they are essential substances that contribute to aroma and flavor. According to the different taste characteristics
of amino acids, they are divided into four types: fresh, sweet, bitter, and aromatic. Due to
the relatively low abundance and higher detection threshold of bitter amino acids, they
generally do not contribute to tea flavor [39]. Studies suggest that Glu and Asp contribute
to the umami taste [41]. Sweet amino acids and umami amino acids have a synergistic effect [42], masking bitterness while also increasing aroma and umami taste. In this study, the
highest contents in summer leaves of I. vomitoria were Glu and Asp, and these two umami
amino acids contribute to enhancing their nutritional and flavor quality. Consequently, the
summer leaves exhibited the highest amino acid nutritional quality among tested seasons.
From the perspective of amino acid nutritional quality, it is recommended to utilize summer leaves for developing and processing high-umami flavored tea, theanine-functional
beverages, natural umami enhancers, and amino acid-fortified foods.
The overall trend of the content of flavonoids, polyphenols, caffeine, and catechins
in the leaves of I. vomitoria shows that the content is highest in spring. In spring, the
leaves of I. vomitoria have stronger DPPH free radical scavenging ability, ABTS cation free
radical scavenging ability, and FRAP iron ion reducing ability than in summer and autumn,
which may be related to the fact that spring leaves contain the highest antioxidant active
substances such as flavonoids, polyphenols, catechins and caffeine [43,44].
Flavonoids are known for their therapeutic effects on various conditions such as
cancer, atherosclerosis, and Alzheimer’s disease [45,46], which plays an important role in
plant defense against oxidative stress. They serve as UV filters, protecting plants from
various biotic and abiotic stresses, acting as signaling molecules, detoxifying agents, and
antimicrobial compounds, and are responsible for the color and fragrance of fruits and
flowers [47]. The highest flavonoid content in spring leaves of I. vomitoria indicates strong
antioxidant properties and positions these leaves as a promising plant for medical use.
Polyphenols, another crucial component in tea, are regarded as important compounds
contributing to the health benefits of tea [48]. They are also considered the most significant
antioxidants [49]. The antioxidant activity is well correlated with the total polyphenol
Plants 2025, 14, 1919 11 of 18
content in tea, and teas with higher polyphenol levels exhibit stronger antioxidant activities. As an essential mineral nutrient for plants, P indirectly participates in the synthesis
and activity regulation of phenylalanine ammonia-lyase (PAL) by modulating the transcription or translation processes of the PAL gene, thereby influencing the biosynthesis of
polyphenolic compounds in plants such as tea trees. [17]. Therefore, the highest content
of P in the leaves of I. vomitoria in spring may lead to the highest polyphenol levels and
the strongest antioxidant activity. Saponins have been increasingly recognized for their
anticancer, antiviral, antibacterial, anti-inflammatory, anti-Alzheimer’s, antioxidant, and
immunomodulatory activities, as well as their ability to inhibit α-glucosidase [50]. This
suggests that teas with higher saponin content may provide more health benefits [51].
In this study, the summer leaves of I. vomitoria were found to have the highest saponin
content. Therefore, developing and processing these leaves into functional foods or dietary
supplements rich in saponins represents a promising approach to utilize their bioactive
composition for promoting human health. Caffeine exhibits blood pressure-lowering and
anxiolytic effects [32]. Studies have also shown that increased nitrogen metabolism in
spring is beneficial for caffeine synthesis, resulting in significantly higher caffeine content
in spring tea compared to summer tea [32], consistent with our findings. Catechins, which
contribute to the bitterness and astringency of tea, are essential for tea’s biological activity and health benefits [52]. They are involved in a variety of pharmacological actions,
particularly their powerful antioxidant properties [53,54]. In this study, the spring leaves
of I. vomitoria had the highest levels of antioxidant compounds, suggesting that spring
tea leaves are the most suitable for development and utilization. Based on these findings,
potential applications include the formulation of antioxidant functional beverages, tea
polyphenol-enriched functional foods, antioxidant probiotic formulations, and antioxidant
skincare products, leveraging the antioxidant activity of these compounds for health and
cosmetic purposes.
GC-MS technology was used in this study to analyze and identify the aroma components in the leaves of I. vomitoria in different seasons. It was found that the aroma
components in the leaves I. vomitoria in spring, summer, and autumn were mainly composed of alcohols, ketones, and aldehydes. This is consistent with the results of the aroma
components of Viscum articulatum Burm.f. [55]. The aroma components such as alcohols,
ketones, and aldehydes in the leaves are highest in spring, which is inconsistent with
the results of previous studies on black tea [56,57]. This may be attributed to the significant influence of cultivars [58] and production seasons [59] on the volatile compounds
in tea leaves. Climatic conditions during different production seasons, particularly significant differences in temperature and sunlight [60,61], lead to substantial variations
in the aroma components of tea leaves [60]. Alcohols dominate in spring leaves, especially (2E)-3,7-dimethylocta-2,6-dien-1-ol (14.06%), 2,6-dimethylcyclohexan-1-ol (13.62%),
2-ethylhexan-1-ol (13.06%), and hept-6-en-1-ol (11.26%). The formation of alcohols is primarily related to the hydrolysis of glycosidic precursors and the biosynthesis of volatile
terpenoids, which significantly influence the fragrance quality of tea [62,63]. Among
these, (2E)-3,7-dimethylocta-2,6-dien-1-ol is the most common alcohol in tea and is considered a key aromatic compound responsible for the floral, fruity, and sweet notes in
tea’s aroma [64,65]. (2E)-3,7-dimethylocta-2,6-dien-1-ol also has antiviral, antibacterial, and
anti-inflammatory effects and is very helpful in improving blood circulation and regulating
glucose and lipid metabolism in the human body [66,67]. At the same time, it was found
that the aroma components of 2,6-dimethylcyclohexan-1-ol with rose and citrus aromas
were higher in spring leaves. Therefore, overall, the spring leaves of I. vomitoria have the
most abundant aroma components.
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4. Materials and Methods
4.1. Materials
The plant material used in this study was Ilex vomitoria, which was cultivated by the
experimenters and sourced from the Ilex plants nursery of Zhejiang A&F University. It
was identified by plant classification experts Yan Daoliang and Lou Luhuan. Six-year-old
I. vomitoria plants were selected, which were planted in organic yellow soil with a pH 6~6.5.
Tender leaves were picked which contained one bud one leaf in spring (April), mature
leaves in summer (July), and old leaves in autumn (October), placed in a low-temperature
box, and quickly brought back to the laboratory. After freeze-drying and crushing with
liquid nitrogen, they were used to detect the content of mineral nutrients, soluble sugar,
amino acids, and antioxidant substances in the leaves, and we analyzed indicators such as
antioxidant activity. At the same time, the vitamin C content and aroma components of
fresh leaves during the same period were measured.
4.2. Methods
4.2.1. Determination of Nutrient Elements in Leaves
The nitrogen (N) content in the leaves was determined using the Kjeldahl method.
First, the leaf powder (0.2 ± 0.001 g) was placed into a 100 mL digestion tube. CuSO4·5H2O
was used as a Kjeldahl catalyst, followed by 8 mL of 98% concentrated sulfuric acid. The
leaf powder was digested at 280 ◦C for 60 min using a graphite digestion instrument. After
cooling, 2 mL of hydrogen peroxide was added, and the tube was returned to the digestion
instrument, where it was heated at 300 ◦C for 60 min until the solution turned a brownblack color. The tube was removed, cooled, and the nitrogen content was measured using
an automatic Kjeldahl nitrogen analyzer (Hanon K9860, Hanon Advanced Technology
Group Co., Ltd, Jinan, China).
For phosphorus(P) content determination, the molybdenum–antimony colorimetric
method was adopted. The leaf powder (0.5 g) was placed into a 50 mL digestion tube. To
this, 2 mL of 68% concentrated nitric acid, 0.5 mL of hydrogen peroxide, and 0.5 mL of
deionized water were added. The leaf powder underwent pre-reaction for 1 h, and the leaf
powder and solution in the digestion tube were mixed and then left overnight. The leaf
powder was heated in a digestion furnace, rising to 250 ◦C within 25 min, followed by a
15 min incubation at 250 ◦C. Afterward, the sample was cooled rapidly for 20 min. The
excess acid solution was removed using a graphite heater at 160 ◦C, and the sample was
cooled to room temperature. After rinsing with deionized water and diluting to 25 mL,
the solution was clarified and the phosphorus content was determined by measuring the
absorbance at 700 nm using a UV spectrophotometer (UV754N, Shanghai INESA Analytical
Instrument Co., Ltd., Shanghai, China).
For potassium(K), magnesium(Mg), calcium(Ca), iron(Fe), and zinc(Zn) content determination, 0.5 g of the leaf powder was placed in a PTFE digestion tube. To this, 90 mL
0.80 mol/L nitric acid and 40 mL 1.05 mol/L perchloric acid were added. The mixture was
heated until red fumes dissipated. After cooling, 20 mL of deionized water was added, and
the solution was filtered for atomic absorption spectroscopy, which following the method
described by Albakaa et al. [68].
4.2.2. Determination of Soluble Sugar and Vitamin C in Leaves
For soluble sugar determination, 0.5 g of dry leaf powder was mixed with 1 mL of
80% ethanol and ethanol to form a paste. The paste was transferred to a 10 mL centrifuge
tube and subjected to a water bath at 100 ◦C for 20 min. After cooling, the leaf powder was
centrifuged at 8000 rpm for 30 min at room temperature, and the supernatant absorbance
Plants 2025, 14, 1919 13 of 18
was measured at 620 nm using a microplate reader (Thermo Fisher Scientific, Waltham,
MA, USA).
For vitamin C determination, 0.1 g of the leaf powder was placed into a centrifuge
tube and 5 mL of 2% oxalic acid-EDTA buffer solution (pH 4.0) was added. The leaf powder
was boiled in a water bath for 15 min, then cooled to room temperature. After centrifuging
at 8000 rpm for 25 min, 1 mL of the supernatant was transferred to a clean test tube. To this,
2 mL of oxalic acid–EDTA buffer, 1 mL of 2% phosphoric acid–acetic acid solution, 2 mL
of 5% sulfuric acid solution, and 1 mL of 5% ammonium molybdate solution were added.
The mixture was shaken, incubated in a water bath at 37 ◦C for 25 min, and then 1.5 mL of
deionized water was added. The absorbance was recorded at 760 nm, and the vitamin C
content was calculated using the corresponding formula [69].
4.2.3. Determination of Amino Acids in Leaves
One gram of the leaf powder was weighed, and 25 mL of 0.1% phenol solution and
5 mol/L hydrochloric acid were added for grinding. After evaporating the solvent, 2 mL
of 0.1 M hydrochloric acid solution was added, and the mixture was filtered. A 100 µL
aliquot of the filtrate and a 100 µL aliquot of amino acid standard solution were placed into
a 2 mL EP tube. To the tube, 20 µL of 0.05 mol/L N-Leucine Internal Standard Solution,
100 µL of 1.0 mol/L triethylamine acetonitrile solution (ensuring pH 7), and 100 µL of
0.2 mol/L phenyl isothiocyanate acetonitrile solution were added. The mixture was shaken
and allowed to stand for 1 h at 25 ◦C. Afterward, 1 mL of 95% n-hexane was added, the
solution mixed, and allowed to stand for 10 min. The aqueous lower phase (containing
phenylthiocarbamyl amino acid derivatives) was then diluted five times, filtered through a
0.22-µm membrane, and analyzed.
The amino acids were detected using HPLC analyzer (Agilent 1100, Agilent Technologies, Inc., Santa Clara, USA) equipped with Compass C18 column (250 mm × 4.6 mm, 5 µm,
Suzhou Saifen Technology Co., Ltd, Suzhou, China) [70]. The injection volume was 10 µL
and column temperature was 40 ◦C. Mobile phase A was a mixture of 6.6 g anhydrous
sodium acetate, 950 mL distilled water, 70 mL glacial acetic acid, and acetonitrile, and
mobile phase B was 80% acetonitrile aqueous solution. The linear gradient of the solvent
was 0–2 min, 100% A; 2–15 min, 100–90% A; 15–25 min, 90–70% A; 25–33 min, 70–55% A;
33–33.1 min, 50–0% A; 33.1–38 min, 0% A; 38–38.1 min, 0–100% A; and 38.1–45 min, 100%
A. The flow rate was 1 mL/min. The ultraviolet absorption wavelength was 254 nm. The
standard curve was constructed according to the peak area and concentration, and the
concentration was calculated.
The flavor intensity of amino acids was evaluated based on the taste active value
(TAV), calculated as the ratio of a specific amino acid content to its taste threshold. If
TAV ≥ 1, the amino acid contributed to the overall flavor; if TAV < 1, it was considered
non-contributory [71].
4.2.4. Determination of Flavonoids and Polyphenols in Leaves
Flavonoids and polyphenols were extracted from 0.4 g of the leaf powder using
20 mL of ethanol solution by ultrasonic extraction. The measurement method followed the
procedure described by TranThi et al. [72].
4.2.5. Determination of Saponins in Leaves
First, 0.5 g of dry leaf powder was mixed with water and allowed to stand for 30 min.
The mixture was filtered through a multilayer gauze, centrifuged at 1000 rpm for 15 min,
and 0.2 mL of 5% vanillin–acetic acid solution and 0.8 mL of 70% perchloric acid were added
to the supernatant. The solution was extracted at 50 ◦C for 2 h, and the resulting supernatant
Plants 2025, 14, 1919 14 of 18
was reserved for analysis. The content of saponins was determined by measuring the
absorbance at 550 nm, using oleanolic acid standard solutions ranging from 8 to 40 µg/mL.
4.2.6. Determination of Caffeine and Catechins in Leaves
Approximately 0.1 g of the leaf powder was weighed and placed into a 2 mL centrifuge
tube. Then, 1.0 mL of 70% methanol which was preheated to 70 ◦C was added, and the
mixture was subjected to a 70 ◦C water bath for 30 min. Afterward, the leaf powder was
centrifuged, and the supernatant was collected. An additional 1.0 mL of 70% methanol
was used for extraction, and the supernatants were combined and made up to 2 mL with
70% methanol. The solution was then filtered using a syringe filter (0.22 µm) and was
ready for analysis. The catechins and caffeine were detected using a HPLC analyzer
(Waters 2695, Suzhou Leiden Scientific Instrument Co., Ltd., Suzhou, China) equipped
with a Compass C18 column (250 mm × 4.6 mm, 5 µm). The injection volume was 10 µL
and column temperature was 35 ◦C. Mobile phase A consisted of DMF–methanol–glacial
acetic acid in a ratio of 40:2:1.5, and mobile phase B was water. The linear gradient of the
solvent was 0–10 min, 9–14% A; 10–15 min, 14–23% A; 15–27 min, 23–36% A; 27–35 min,
36–50% A; 35–36 min, 50–9% A; and 36–45 min, 9% A. The flow rate was 1 mL/min. The
absorbance of caffeine and catechins was measured at 278 nm, using caffeine standard
solutions in the range of 1–400 µg/mL and gallic acid standard solutions in the range of
1–200 µg/mL, respectively.
4.2.7. Determination of Antioxidant Capacity in Leaves
The antioxidant capacity of the leaves was measured by DPPH radical scavenging,
ABTS cation radical scavenging, and the ferric reducing antioxidant power (FRAP) methods,
as described by TranThi et al. [72]. 0.1 g of the leaf powder was mixed with 1 mL extraction
solution, ground in an ice bath, and then centrifuged at 12,000 rpm for 10 min at 4 ◦C. The
supernatant was used for further analysis.
4.2.8. Determination of Aroma Components in Leaves
Fresh leaves were ground into a fine powder and 3 g of the leaf powder was accurately
weighed and transferred into a 20 mL headspace vial. The leaf powder was then incubated
in a 60 ◦C water bath for 5 min, followed by the addition of 65 µL of 2-octanol. After sealing,
static headspace extraction was performed in the water bath for 50 min, then the extract
was collected for analysis.
The aroma components in the leaves were determined by gas chromatography–mass
spectrometry (Varian 450-GC/240-MS, Varian, Inc., Palo Alto, USA) with manual injection.
The gas chromatograph (GC) conditions were as follows: The leaf powder inlet and detector
temperatures were set to 250 ◦C. High-purity helium was used as the carrier gas with a
flow rate of 1.5 mL/min. The initial column temperature was set to 50.5 ◦C, which was
then gradually increased to 210 ◦C. This temperature was maintained for 3 min before
being raised to 230 ◦C. The mass spectrometry (MS) conditions were as follows: The ion
source temperature was maintained at 230 ◦C, with an electron energy of 70 eV and an
emission current of 34.6 µA. The quadrupole temperature was set to 150 ◦C, while the
switch interface temperature was 280 ◦C. The electric multiplier voltage was adjusted to
350 V, and the mass scan range was between 35 and 400 amu.
4.2.9. Statistical Analysis
All experiments were expressed as means ± standard deviation of triplicate measurements and significance analysis was performed by SPSS 26.0 software. Analysis of
variance (ANOVA) was carried out by Dunnett’s test, where p < 0.05 was assumed to be
statistically significant.
Plants 2025, 14, 1919 15 of 18
5. Conclusions
This study determined and analyzed the nutrient elements, antioxidant substances,
antioxidant capacity, and aroma components of the leaves of Ilex vomitoria. The results
showed that except for the highest content of vitamin C in autumn leaves and the highest
content of saponins in summer, all other substances had the highest content in spring. The
contents of mineral elements, soluble sugars, amino acids, flavonoids, polyphenols, caffeine,
and catechins in the leaves of I. vomitoria, as well as the DPPH free radical scavenging
ability, ABTS cation free radical scavenging ability, and FRAP iron ion reducing ability,
were all the highest in spring. A comprehensive evaluation indicated that the leaves in
spring are more suitable for picking, development, and utilization. The findings of this
study provide critical insights for guiding the development and utilization of I. vomitoria
leaves, which can be further processed into health drinks, antioxidant skincare products,
and medicinal health foods for human consumption.
Author Contributions: L.S., Y.Y. and Y.X. co-authored as the first authors. L.S. and Y.Y. were the main
writers of the paper, completing the collection and analysis of the relevant literature and the writing
of the first draft of the paper, Y.X. participated in the analysis and collation of the literature, X.Z., B.Z.
and J.Z. are the conceivers of the project, D.Y., as the correspondence author, was the project leader
and supervised the writing of the dissertation. All authors have read and agreed to the published
version of the manuscript.
Funding: 1. The project was supported by the Open Project of Ningbo Key Laboratory of Characteristic Horticultural Crops in Quality Adjustment and Resistance Breeding. (NBYYL20230003). 2. The
Key Research and Development Program of Ningbo (2024Z268). 3. Central Funded Forestry Science
and Technology Promotion Demonstration Project ([2023]TS 03-1).
Data Availability Statement: This study did not use publicly available data; all data were generated
by the experimenter through experimental design and data processing analysis. The voucher specimen of the plant material Ilex vomitoria used in this study is stored at the Herbarium of Xishuangbanna
Tropical Botanical Garden, Chinese Academy of Sciences (HITBC), with voucher number 103072. The
specimen was identified by Zhu Qiugui and Liu Qingwen.
Acknowledgments: The authors would like to thank Zhejiang A&F University. We are grateful to
the Ningbo Academy of Agricultural Sciences for providing us with the plant materials they have
studied. Thanks to the editors and reviewers for their constructive comments and suggestions, which
have improved the quality of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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