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Citation: Dai, W.; Li, B.; Xiong, Y.; Dai,
L.; Tian, Y.; Zhang, L.; Wang, Q.; Qian,
G. Non-Volatile Component and
Antioxidant Activity: A Comparative
Analysis between Litsea cubeba
Branches and Leaves. Molecules 2024,
29, 788. https://doi.org/10.3390/
molecules29040788
Academic Editors: Petko Denev,
Stela Dimitrova and Ana M. Dobreva
Received: 4 December 2023
Revised: 2 February 2024
Accepted: 6 February 2024
Published: 8 February 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
molecules
Article
Non-Volatile Component and Antioxidant Activity:
A Comparative Analysis between Litsea cubeba Branches
and Leaves
Wei Dai 1, Boyi Li 2, Yanli Xiong 2, Liping Dai 1, Yuan Tian 3, Liangqian Zhang 3, Qi Wang 3,*
and Guoqiang Qian 2, *
1Teaching and Experimental Center, Guangdong Pharmaceutical University, Guangzhou 510006, China;
dai_gdpu_2018@gdpu.edu.cn (W.D.)
2School of Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China;
xiongyanli106@163.com (Y.X.)
3Key Laboratory of Xinjiang Phytomedicine Resource and Utilization, Ministry of Education,
School of Pharmacy, Shihezi University, Shihezi 832003, China
*Correspondence: wq_pha@shzu.edu.cn (Q.W.); tgqqian@gdpu.edu.cn (G.Q.)
Abstract: Litsea cubeba, which is found widely distributed across the Asian region, functions as both
an economic tree and a medicinal plant with a rich historical background. Previous investigations into
its chemical composition and biological activity have predominantly centered on volatile components,
leaving the study of non-volatile components relatively unexplored. In this study, we employed
UPLC-HRMS technology to analyze the non-volatile components of L. cubeba branches and leaves,
which successfully resulted in identifying 72 constituents. Comparative analysis between branches
and leaves unveiled alkaloids, organic acids, and flavonoids as the major components. However,
noteworthy differences in the distribution of these components between branches and leaves were
observed, with only eight shared constituents, indicating substantial chemical variations in different
parts of L. cubeba. Particularly, 24 compounds were identified for the first time from this plant.
The assessment of antioxidant activity using four methods (ABTS, DPPH, FRAP, and CUPRAC)
demonstrated remarkable antioxidant capabilities in both branches and leaves, with slightly higher
efficacy observed in branches. This suggests that L. cubeba may act as a potential natural antioxidant
with applications in health and therapeutic interventions. In conclusion, the chemical composition and
antioxidant activity of L. cubeba provides a scientific foundation for its development and utilization in
medicine and health products, offering promising avenues for the rational exploitation of L. cubeba
resources in the future.
Keywords: Litsea cubeba; non-volatile component; antioxidant activity; UPLC-HRMS
1. Introduction
Litsea cubeba, a member of the Lauraceae family in the Litsea genus, thrives primarily
in China, India, Thailand, and various Asian countries [
1
]. Despite occupying a mere 0.04%
of China’s wild forest area, the L. cubeba tree stands out for its rich plant resources [
2
].
Recognized for its economic significance, L. cubeba has been an integral part of traditional
Chinese folk medicine for generations. Its roots, stems, leaves, flowers, and fruits have
been employed for medicinal purposes, offering remedies for dispelling wind and cold,
reducing swelling, and alleviating pain [1,3].
A considerable amount of research has delved into unraveling the chemical con-
stituents and biological activities of L. cubeba, with a predominant focus on its volatile
components, especially the essential oil. The fruit and leaves of L. cubeba, rich in essen-
tial oils, exhibit a diverse composition of monoterpenes and sesquiterpenes [
4
]. Notably,
citral predominates, widely used in toothpaste, soap, perfumes, and various pharmaceu-
tical and chemical products [
5
–
7
]. While volatile components have received significant
Molecules 2024,29, 788. https://doi.org/10.3390/molecules29040788 https://www.mdpi.com/journal/molecules
Molecules 2024,29, 788 2 of 15
attention, there remains a noteworthy gap in exploring non-volatile components. Over
150 non-volatile components, including alkaloids, lignans, flavonoids, and terpenoids, have
been identified in L. cubeba. Alkaloids and lignans, constituting over half of these compo-
nents, emerge as prominent contributors with significant pharmacological activities [
8
–
11
].
Aporphine alkaloids, in particular, have demonstrated hypoglycemic, anti-inflammatory,
and anti-cancer effects [
12
]. Despite these promising findings, research on non-volatile
components remains comparatively limited, overshadowed by the predominant focus on
essential oils. The imperative for an in-depth exploration of L. cubeba’s active ingredients
and their potential pharmacological effects is evident.
Oxidative stress, implicated in the onset and progression of numerous diseases, poses
a significant threat to human health, particularly in the context of neurodegenerative and
cardiovascular diseases [
13
]. Addressing this challenge involves exploring exogenous
antioxidants or enhancing endogenous antioxidant defenses. Plants, recognized as valuable
repositories of secondary metabolites, emerge as promising sources of natural antioxidants.
Studies underscore the potential of L. cubeba as a natural antioxidant, with its essential
oil showcasing robust antioxidant activity [
14
,
15
]. Extracts from L. cubeba bark, including
methanol, chloroform, n-butanol, and water extracts, also exhibit antioxidant activity [
16
].
Given the abundant resources of L. cubeba, and the relatively limited research on its
non-volatile components, there is a need for a more comprehensive exploration to facil-
itate the rational development and utilization of this plant resource. This is particularly
crucial for uncovering its broader medicinal value, with a specific focus on discovering
its potential as a robust antioxidant. This study endeavors to bridge this knowledge gap,
focusing on the branches and leaves of L. cubeba. Employing UPLC-HRMS, we aim to
comprehensively identify non-volatile components in the methanol extract. The antioxidant
activity will be assessed through standardized assays, including ABTS (2,2
′
-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid)), DPPH (1,1-diphenyl-2-trinitrophenylhydrazine),
FRAP (ferric reducing antioxidant power), and CUPRAC (cupric reducing antioxidant ca-
pacity). By meticulously comparing and analyzing differences in non-volatile components
and antioxidant activity, this study aspires to provide a fresh perspective for the rational
development and utilization of L. cubeba resources, potentially unlocking novel avenues in
medicinal research.
2. Results
2.1. UPLC-HRMS Analysis of Non-Volatile Components from L. cubeba Branches and Leaves
The chemical composition of the methanol extracts from L. cubeba branches and leaves
was analyzed using UPLC-Q-Orbitrap HRMS in both positive and negative ion modes.
Figures S1–S4 in the Supplementary Materials present the total ion chromatograms of
mass spectrometry analysis for the branches and leaves of L. cubeba. Detailed identification
information for the compounds is provided in Table 1. A total of 72 compounds were
identified in L. cubeba, comprising 23 alkaloids, 15 flavonoids, 14 organic acids, five amino
acids, four alcohols, three carbohydrates, two ketones, two phenols, two esters, one ether,
and one phenylpropanoid. Alkaloids, flavonoids, and organic acids emerged as the major
chemical constituents of L. cubeba. Specifically, 36 and 44 compounds were identified in the
branches and leaves of L. cubeba, respectively, with 24 compounds being identified for the
first time from this plant. The discovery of these compounds significantly contributes to
the chemical information available for L. cubeba.
Molecules 2024,29, 788 3 of 15
Table 1. Compounds identified in the methanol extract of L. cubeba branches and leaves by UPLC-HRMS.
No. RT/min Compounds Molecular
Formula Error/ppm m/zIon Mode Compound Types References
Leaves Branches
1 0.851 – 2-Aminosuccinamic acid C4H8N2O3−1.54 131.04599 [M −H]−alkaloids [17]
2 0.88 – Gluconic acid C6H12O7−1.22 195.05079 [M −H]−carbohydrates
3 0.89 – L-Threonic acid C4H8O5−1.26 135.02972 [M −H]−organic acids [18]
4 0.893 – D-(−)-Fructose C6H12O6−0.76 179.05597 [M −H]−carbohydrates [19]
5 0.904 – D-(-)-Quinic acid C7H12O6−0.89 191.05595 [M −H]−organic acids [20]
6 0.908 – L-Threonine C4H9NO3−0.3 120.06559 [M + H]+amino acids [21]
7 0.915 – D-Glucosamine C6H13NO50 180.0867 [M + H]+carbohydrates [22]
8 0.918 – (2R)-2,3-Dihydroxypropanoic acid ⋆C3H6O4−1.37 105.01919 [M −H]−organic acids [23]
9 0.940 – DL-Malic acid C4H6O5−1.53 133.01403 [M −H]−organic acids [24]
10 0.950 – D-Serine ⋆C3H7NO3−0.06 106.04986 [M + H]+amino acids [25]
11 0.982 – Proline C5H9NO20.5 116.07066 [M + H]+amino acids [26]
12 0.998 – Adenine C5H5N50.91 136.06189 [M + H]+alkaloids [27]
13 1.019 – Acetophenone C8H8O 0.54 121.06486 [M + H]+ketones [28]
14 – 1.024 4-Methoxycinnamaldehyde C10H10O2−0.18 180.10186 [M + H]+ethers
15 – 1.140 Higenamine ⋆C16 H17NO30.31 272.1282 [M + H]+alkaloids [29]
16 1.219 – Citric acid C6H8O7−0.32 191.01967 [M −H]−organic acids [30]
17 1.232 – Nicotinic acid C6H5NO20.14 124.03932 [M + NH4]+alkaloids [31]
18 1.238 – Pipecolic acid C6H11NO20.45 130.08631 [M + H]+alkaloids [32]
19 1.331 – Methylmalonic acid C4H6O4−1.03 117.01921 [M −H]−organic acids [33]
20 1.335 – Fumaric acid C4H4O4−1.05 115.00356 [M −H]−organic acids [34]
21 1.373 – Isoleucine C6H13 NO20.35 132.10195 [M + H]+amino acids [35]
22 1.456 – L-Norleucine C6H13NO20.41 132.10196 [M + H]+amino acids [36]
23 – 1.615 L-N-Acetylphenylalaninol ⋆C11H15 NO2−0.04 194.11755 [M + H]+alkaloids
24 – 1.989 (-)-Salsoline C11 H15NO2−0.17 194.11752 [M + H]+alkaloids
25 2.089 – Gentisic acid 5-O-β-glucoside ⋆C13H16O9−0.85 315.07189 [M −H]−organic acids
26 – 2.098 Indole-3-acetic acid C10H9NO2−0.03 176.07061 [M + H]+alkaloids [37]
27 – 3.396 trans-3-Indoleacrylic acid ⋆C11H9NO2−0.39 188.07054 [M + H]+alkaloids [38]
28 – 5.066 Norisoboldine C18H19NO40.42 314.13882 [M + H]+alkaloids [39]
29 5.091 – 3-(4-Hydroxyphenyl)-3-oxopropyl
β-D-glucopyranoside C15H20 O8−0.82 327.10827 [M −H]−phenols [40]
30 5.888 5.261 Boldine C19H21 NO4−0.07 328.15432 [M + H]+alkaloids [41]
31 5.486 – Dihydrophaseic acid ⋆C15 H22O5−0.41 281.13933 [M −H]−organic acids [42]
32 6.213 6.072 (S)-Boldine C19 H21NO40.26 328.15442 [M + H]+alkaloids [41]
33 6.636 6.487 Glaufinine C19 H21NO40.07 328.15436 [M + H]+alkaloids [43]
Molecules 2024,29, 788 4 of 15
Table 1. Cont.
No. RT/min Compounds Molecular
Formula Error/ppm m/zIon Mode Compound Types References
Leaves Branches
34 6.604 – Kaempferol 3-sophoroside ⋆C27H30O16 −0.28 609.14594 [M −H]−flavonoids [44]
35 – 6.765 Isocorydine C20H23NO40.26 342.17007 [M + H]+alkaloids [45]
36 7.526 7.817 Rutin C27H30 O16 −0.69 609.14558 [M −H]−flavonoids [41]
37 – 7.117 Isoquercetin C21H20 O12 −0.28 463.08796 [M −H]−flavonoids [46]
38 7.316 – Kaempferitrin C27H30 O14 −0.31 577.15588 [M −H]−flavonoids [47]
39 – 7.551 laurotetanine C19H21 NO40.07 328.15436 [M + H]+alkaloids [41]
40 7.557 – 5-(4-Hydroxypentyl)-1,3-benzenediol ⋆C11 H16O3−0.11 197.1172 [M + H]+phenols
41 – 7.589 Corydine ⋆C20H23 NO4−0.21 342.16991 [M + H]+alkaloids [43]
42 7.631 – Trifolin ⋆C21H20O11 −0.34 447.09298 [M −H]−flavonoids [48]
43 7.951 – Cynaroside C21H20 O11 −0.26 447.09299 [M −H]−flavonoids [49]
44 – 8.048 Quercitrin C21 H20O11 −0.36 447.09312 [M −H]−flavonoids [41]
45 8.246 – Cassythicin C19H19NO40.33 326.13879 [M + H]+alkaloids [50]
46 – 8.349 Diosmin ⋆C28 H32O15 −0.77 607.16638 [M −H]−flavonoids [51]
47 – 8.503 Neohesperidin ⋆C28H34 O15 −0.78 609.18202 [M −H]−flavonoids [52]
48 – 8.644 Hesperetin C16H14 O60.57 303.08649 [M + H]+flavonoids [53]
49 – 8.648 Hesperidin C28H34O15 0.65 611.19739 [M + H]+flavonoids [54]
50 9.196 – Afzelin C21H20 O10 −0.63 431.0981 [M −H]−flavonoids [55]
51 9.443 – Kaempferol ⋆C15H10O60.05 287.05503 [M + H]+flavonoids [56]
52 – 9.930 N-p-Coumaroyltyramine C17 H17NO30.32 284.12821 [M + H]+phenylpropanoids [57]
53 – 10.391 Crebanine C20 H21NO40.3 340.15444 [M + H]+alkaloids [58]
54 10.576 10.454 Moupinamide C18H19NO40.24 314.13876 [M + H]+alkaloids [59]
55 10.869 – Tiliroside ⋆C30 H26O13 −0.92 593.12952 [M −H]−flavonoids [60]
56 14.447 14.029 15-Hexadecynoic acid ⋆C16H28 O2−0.2 253.21616 [M + H]+organic acids
57 14.508 –
3-[[6-Deoxy-3,4-bis-O-[(2E)-3-(4-
hydroxyphenyl)-1-oxo-2-propen-1-yl]-
α-L-mannopyranosyl]oxy]-5,7-
dihydroxy-2-(4-hydroxyphenyl)-4H-1-
benzopyran-4-one
C39H32 O14 −0.9 723.17128 [M −H]−flavonoids [61]
58 15.609 15.177 Lauryldimethylamine oxide ⋆C14H31NO −0.09 230.24782 [M + H]+alkaloids [62]
59 – 16.099 Aurantiamide acetate C27 H28N2O40.05 445.21222 [M + H]+alkaloids [63]
60 – 16.286 2-Amino-1,3,4-octadecanetriol ⋆C18H39NO30.55 318.30045 [M + H]+alcohols [64]
61 – 16.290 4-Ethylbenzaldehyde C9H10O 0.59 135.08052 [M + H]+esters [65]
62 – 16.297 4-Ethoxy ethylbenzoate C11H14 O3−0.12 195.10155 [M + H]+esters [66]
63 16.529 – Schinifoline ⋆C17 H23NO −0.02 258.18524 [M + H]+alkaloids [67]
Molecules 2024,29, 788 5 of 15
Table 1. Cont.
No. RT/min Compounds Molecular
Formula Error/ppm m/zIon Mode Compound Types References
Leaves Branches
64 16.882 16.919
1,3:2,4-Di(p-ethylbenzylidene)sorbitol
⋆C24H30 O60.51 415.2117 [M + H]+alcohols
65 – 17.097 9-Oxo-ODE C18H30 O30.09 295.2268 [M + H]+organic acids [68]
66 – 17.368 13(S)-HOTrE C18H30O30.08 293.21224 [M −H]−organic acids [69]
67 – 18.395 9-Oxo-10(E),12(E)-octadecadienoic acid C18H30 O30.4 295.22687 [M + H]+organic acids [70]
68 – 19.147 12-Oxo-10,15(Z)-phytodienoic acid ⋆C18H28O30.45 293.21124 [M + H]+organic acids
69 – 19.440 Linoleoyl ethanolamide ⋆C20 H37NO20.28 324.28979 [M + H]+alcohols [71]
70 – 20.267 Sphingosine (d18:1) ⋆C18H37 NO20.6 300.28989 [M + H]+alcohols [72]
71 21.806 – Muscone C16H30O−0.21 239.23689 [M + H]+ketones [73]
72 21.829 – Pheophorbide A ⋆C35H36 N4O50.14 593.27594 [M + H]+alkaloids
–: not detected, ⋆: indicates compounds identified for the first time from L. cubeba.
Molecules 2024,29, 788 6 of 15
2.1.1. Analysis of Non-Volatile Components in Branches
Within the L. cubeba branches, a total of 36 non-volatile compounds were identified,
encompassing 16 alkaloids (No. 15, 23, 24, 26, 27, 28, 30, 32, 33, 35, 39, 41, 53, 54, 58, and
59). These alkaloids comprised 11 isoquinoline alkaloids (No. 15, 23, 24, 28, 30, 32, 33, 35,
39, 41, and 53), two indole alkaloids (No. 26 and 27), two amide alkaloids (No. 54 and 59),
and one other alkaloid (No. 59). Additionally, seven flavonoids (No. 36, 37, 44, 46, 47, 48,
and 49), five organic acids (No. 56, 65, 66, 67, and 68), four alcohols (No. 60, 64, 69, and 70),
two esters (No. 61 and 62), one ether (No. 14), and one phenylpropanoid (No. 52) were
identified. Alkaloids constituted the major component, accounting for nearly 45% of the
identified compounds. The sum of flavonoids, organic acids, and alcohol compounds also
represented approximately 45% of the total identified compounds in the branches.
2.1.2. Analysis of Non-Volatile Components in Leaves
In L. cubeba leaves, a total of 44 non-volatile compounds were identified, including
12 alkaloids (No. 1, 12, 17, 18, 30, 32, 33, 45, 54, 58, 63, and 72). Among the alkaloids,
four were isoquinoline alkaloids (No. 30, 32, 33, and 45), two were amide alkaloids (No. 1
and 54), two were pyridine alkaloids (No. 17 and 18), one was a purine alkaloid (No. 12),
one was a pyrrolidine alkaloid (No. 72), one was a quinoline alkaloid (63), and one was
classified as other (No. 58). Additionally, 10 organic acids (No. 3, 5, 8, 9, 16, 19, 20, 25,
31, and 56), nine flavonoids (No. 34, 36, 38, 42, 43, 50, 51, 55, and 57), five amino acid
compounds (No. 6, 10, 11, 21, and 22), three carbohydrates (No. 2, 4, and 7), two phenols
(No. 29 and 40), two ketones (No. 29 and 40), and one alcohol were identified. Alkaloids,
organic acids, and flavonoids were the major constituents of L. cubeba leaves, constituting
over 70% of the total identified compounds.
2.2. Comparative Analysis of Non-Volatile Components in Branches and Leaves
The comparative analysis of non-volatile components in the methanol extracts of L.
cubeba branches and leaves revealed substantial differences in their chemical compositions
(Figure 1). Out of the 72 identified compounds in L. cubeba, only eight (No. 30, 32, 33, 36,
54, 56, 58, and 64) were common to both branches and leaves. Leaves displayed 36 unique
compounds (No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 19, 20, 21, 22, 25, 29, 31, 34,
38, 40, 42, 43, 45, 50, 51, 55, 57, 63, 71, and 72), while branches had 28 unique compounds
(No. 14, 15, 23, 24, 26, 27, 28, 35, 37, 39, 41, 44, 46, 47, 48, 49, 52, 53, 59, 60, 61, 62, 65, 66, 67,
68, 69, and 70). These distinctive compounds underscore pronounced differences in their
chemical profiles.
Molecules 2024, 29, x FOR PEER REVIEW 5 of 14
(No. 14, 15, 23, 24, 26, 27, 28, 35, 37, 39, 41, 44, 46, 47, 48, 49, 52, 53, 59, 60, 61, 62, 65, 66, 67,
68, 69, and 70). These distinctive compounds underscore pronounced differences in their
chemical profiles.
Figure 1. Venn diagram of the non-volatile components of L. cubeba branches and leaves.
Alkaloids constituted a major component in both branches and leaves, sharing sev-
eral compounds such as No. 30, 32, 33, 54, and 58. However, variations exist in the types
of alkaloids, with branches primarily featuring isoquinoline alkaloids and leaves encom-
passing isoquinoline, amide, and other alkaloid categories. Organic acids were prevalent
in both branches and leaves, with compound 56 being the sole shared compound (15-Hex-
adecynoic acid). Nevertheless, other organic acid components exhibited significant differ-
ences. Flavonoids, another major class, were identified in both parts, with only Rutin
(compound 36) being a shared compound. Other flavonoids were unique to either
branches or leaves. Notable distinctions were observed in alcohol and ester content, with
branches featuring a higher representation of compounds (No. 60, 64, 69, 70, 61, and 62).
Amino acids were exclusively found in leaves (No. 6, 10, 11, 21, and 22). Phenols and ke-
tones were unique to leaves (No. 29 and 40).
In conclusion, while alkaloids, organic acids, and flavonoids were major components
in both branches and leaves, the number of shared components was limited. The presence
of unique compounds in each part contributed significantly to their distinct chemical pro-
files, enhancing our understanding of the chemical diversity of L. cubeba and the potential
biological activities associated with its various components.
2.3. Antioxidant Activity
Results of Four In Vitro Antioxidant Activity Tests
The evaluation of in vitro antioxidant activity involves various methods based on
different principles, with the most commonly utilized ones being ABTS, DPPH, FRAP,
and CUPRAC. ABTS and DPPH assess the antioxidant capacity of samples through meas-
uring absorbance change resulting from the reduction of their respective free radicals un-
der the influence of oxidants. In contrast, FRAP and CUPRAC characterize the activity of
antioxidants through reducing metal ions to lower oxidation states and measuring their
generation. The comprehensive application of these methods provides a nuanced under-
standing of antioxidant potential within samples. In this study, we assessed the in vitro
antioxidant activity of methanol extracts from both branches and leaves of L. cubeba using
ABTS, DPPH, FRAP, and CUPRAC methods. For the ABTS, DPPH, and FRAP experi-
ments, antioxidant capacity was expressed as TEAC µmol/g, where higher values signify
greater antioxidant capability. For the FRAP method, results were indicated as Fe (Ⅱ)
Figure 1. Venn diagram of the non-volatile components of L. cubeba branches and leaves.
Molecules 2024,29, 788 7 of 15
Alkaloids constituted a major component in both branches and leaves, sharing several
compounds such as No. 30, 32, 33, 54, and 58. However, variations exist in the types
of alkaloids, with branches primarily featuring isoquinoline alkaloids and leaves encom-
passing isoquinoline, amide, and other alkaloid categories. Organic acids were prevalent
in both branches and leaves, with compound 56 being the sole shared compound (15-
Hexadecynoic acid). Nevertheless, other organic acid components exhibited significant
differences. Flavonoids, another major class, were identified in both parts, with only Rutin
(compound 36) being a shared compound. Other flavonoids were unique to either branches
or leaves. Notable distinctions were observed in alcohol and ester content, with branches
featuring a higher representation of compounds (No. 60, 64, 69, 70, 61, and 62). Amino
acids were exclusively found in leaves (No. 6, 10, 11, 21, and 22). Phenols and ketones were
unique to leaves (No. 29 and 40).
In conclusion, while alkaloids, organic acids, and flavonoids were major components
in both branches and leaves, the number of shared components was limited. The presence of
unique compounds in each part contributed significantly to their distinct chemical profiles,
enhancing our understanding of the chemical diversity of L. cubeba and the potential
biological activities associated with its various components.
2.3. Antioxidant Activity
Results of Four In Vitro Antioxidant Activity Tests
The evaluation of
in vitro
antioxidant activity involves various methods based on
different principles, with the most commonly utilized ones being ABTS, DPPH, FRAP, and
CUPRAC. ABTS and DPPH assess the antioxidant capacity of samples through measuring
absorbance change resulting from the reduction of their respective free radicals under
the influence of oxidants. In contrast, FRAP and CUPRAC characterize the activity of
antioxidants through reducing metal ions to lower oxidation states and measuring their
generation. The comprehensive application of these methods provides a nuanced under-
standing of antioxidant potential within samples. In this study, we assessed the
in vitro
antioxidant activity of methanol extracts from both branches and leaves of L. cubeba using
ABTS, DPPH, FRAP, and CUPRAC methods. For the ABTS, DPPH, and FRAP experiments,
antioxidant capacity was expressed as TEAC
µ
mol/g, where higher values signify greater
antioxidant capability. For the FRAP method, results were indicated as Fe (II)
µ
mol/g, with
higher values reflecting stronger reducing power and thus greater antioxidant potential.
The methanol extracts from L. cubeba branches exhibited the following results for the four
antioxidant assays, as shown in Table 2. These antioxidant activity data indicate that both
branches and leaves of L. cubeba possess considerable antioxidant capabilities, with a slight
predominance in the antioxidant capacity of the branches over the leaves.
Table 2. Antioxidant activity of methanol extracts from branches and leaves of L. cubeba.
Parts ABTS
(TEAC µmol/g)
DPPH
(TEAC µmol/g)
FRAP
(Fe(II) µmol/g)
CUPRAC
(TEAC µmol/g)
Branches 42.75 ±3.94 7.55 ±0.43 22.21 ±0.53 42.65 ±0.35
Leaves 57.78 ±1.87 9.29 ±0.39 28.68 ±0.78 45.89 ±0.27
3. Discussion
In this investigation, UPLC-HRMS technology facilitated the rapid and precise analysis
of non-volatile components within L. cubeba branches and leaves. The established analytical
method for non-volatile components offered insights into the diverse compounds present
in these plant parts, contributing detailed information to enhance our understanding of the
plant’s chemical characteristics. The identification of 72 compounds, including alkaloids,
flavonoids, and organic acids, underscored the chemical diversity of L. cubeba. Moreover,
methanol extracts from both branches and leaves exhibited noteworthy antioxidant activity
in vitro.
Molecules 2024,29, 788 8 of 15
While the results align with some previously reported chemical compositions, this
study uncovered 24 compounds not documented before, enriching our comprehensive un-
derstanding of L. cubeba compounds and supporting its chemical taxonomy, development,
and utilization [
74
]. Alkaloids, especially isoquinoline alkaloids, emerged as significant
components in the non-volatile fraction of L. cubeba, aligning with prior research [
74
]. In-
triguingly, a higher abundance of isoquinoline alkaloids in branches compared to leaves
was observed. Further analysis of alkaloid differences identified eight shared compounds,
five of which belonged to the alkaloid category (compounds 30, 32, 33, 54, and 58). The
predominance of isoquinoline alkaloids in branches, and the greater diversity of alkaloid
types in leaves, suggest potential variations in pharmacological activities. Known for
their diverse biological activities, especially in pharmaceutical research, the abundance of
isoquinoline alkaloids in L. cubeba implies their essential role in its medicinal effects.
Flavonoids, renowned for their antioxidant properties, were identified as major compo-
nents in both branches and leaves of L. cubeba. The presence of the shared compound rutin
(compound 36) suggests common antioxidant capabilities. However, unique flavonoid
compositions in each part may contribute to subtle differences in antioxidant activity. The
study also confirmed the presence of organic acids, a less explored aspect of L. cubeba chem-
istry. The only shared organic acid between the branches and leaves was 15-hexadecynoic
acid (compound 56), indicating potential variations in their health-promoting properties.
Beyond the primary compound categories, differences in alcohols, esters, amino acids,
and phenols between the branches and leaves add layers to the plant’s chemical diversity,
potentially influencing related biological activities and the potential medicinal value of
L. cubeba.
The assessment of antioxidant potential using ABTS, DPPH, FRAP, and CUPRAC
assays demonstrated significant antioxidant capabilities in both the branch and leaf extracts,
with a slightly higher antioxidant capacity observed in the branches. In comparison with
antioxidant capacities of traditional medicinal plants, such as Angelica dahurica,Fritillaria cir-
rhosa, and Perilla frutescens,L. cubeba branches and leaves exhibited superior or comparable
antioxidant abilities [
75
]. The plant’s rich content of alkaloids, flavonoids, and organic acids,
known for their antioxidant properties, likely contributes to its robust antioxidant activity,
emphasizing the potential health benefits of L. cubeba in traditional herbal applications.
In summary, this comparative analysis highlights the diverse chemical characteristics
and antioxidant capabilities of L. cubeba branches and leaves. As a botanical resource, L.
cubeba exhibits unique features in terms of chemical composition and antioxidant activity,
providing a theoretical foundation for its applications in medicine and the food industry.
Future research could delve deeper into the biological activities and pharmacological effects
of L. cubeba compounds to explore its potential health benefits. This study offers valuable
information for the development and utilization of L. cubeba, laying a scientific foundation
for its diverse applications.
4. Materials and Methods
4.1. Plant Material and Extraction
4.1.1. Plant Material
L. cubeba was sourced from Yunfu, China, in April 2023, and Dr. Xinger Ye, affiliated
with the College of Traditional Chinese Medicine Resources at Guangdong Pharmaceutical
University, authenticated the plant materials.
4.1.2. Preparation of Liquid-Sample Solution for Mass Spectrometry
The dried leaves and stems of L. cubeba were individually pulverized using the DFY–
300C Swing Crusher from Wenling Linda Machinery Co., Ltd, Wenling, China. Approx-
imately 1.0 g of each sample powder was precisely weighed into a 100 mL conical flask.
Analytical methanol (50 mL) was added accurately, and the mixture was re-weighed. The
Q-500DE ultrasound equipment (Dongguan Keqiao Ultrasonic Equipment Co., Ltd., Dong-
guan, China) was used for ultrasonic extraction at 50
◦
C with 500 W power and 40 kHz
Molecules 2024,29, 788 9 of 15
frequency for 30 min. After cooling, the sample was re-weighed, and any weight loss was
compensated by adding methanol. Ten milliliters of the upper clear liquid were transferred
to a centrifuge tube, and centrifuged at 2200
×
gfor 15 min using a high-speed centrifuge.
Two hundred microliters of the supernatant were extracted, mixed with 800
µ
L of chro-
matographic methanol, diluted to 1 mL, thoroughly mixed, and then filtered through a
0.22
µ
m microporous membrane. The resultant filtered solution served as the liquid–sample
solution for mass spectrometry.
4.1.3. Preparation of Samples for Antioxidant Activity Testing
For antioxidant activity testing, 1.0 g of L. cubeba leaves and branches powder was
precisely weighed and combined with 40 mL of methanol. Ultrasonic extraction was
conducted at 50
◦
C using an ultrasonic power of 500 W (frequency, 40 kHz) for 50 min,
followed by filtration and rotary evaporation to obtain the respective extracts. The yield
was 117.7 mg for leaf extract and 61.4 mg for branch extract. The extracts were dissolved
in 15 mL of methanol. Based on preliminary experiments, the leaf methanol extract was
diluted 10 times, and the branch methanol extract was diluted 5 times to produce the
test solutions.
4.2. The Main Chemicals and Reagents
Analytical grade methanol from Da Mao Chemical Reagent Co., Ltd., Tianjin, China;
chromatography-grade acetonitrile from Honeywell Trading (Shanghai) Co., Ltd.,
Shanghai
,
China; and distilled water from Watsons, Hong Kong, China. The total antioxidant ca-
pacity assay kits (ABTS, DPPH, FRAP methods) were obtained from Shanghai Macklin
Biochemical Technology Co., Ltd., Shanghai, China, and the liquid sample total antioxidant
capacity assay kit (Cu
2+
method) was sourced from Beijing Applygen Technology Co., Ltd.,
Beijing, China.
4.3. UPLC-HRMS Analysis
4.3.1. Instrumentation and Conditions
The analysis employed the Vanquish Flex UPLC system, coupled with the Orbitrap
Exploris 120 quadrupole electrostatic field orbital well high-resolution mass spectrometer
for UPLC-Q-Orbitrap HRMS, sourced from Thermo Fisher Scientific (Waltham, MA, USA).
A Hypersil GOLD C
18
column (100 mm
×
2.1 mm, 1.9
µ
m) was used for chromatographic
analysis with a flow rate of 0.3 mL
·
min
−1
, maintaining the column temperature at 35
◦
C.
A 2.00
µ
L sample was injected into the system. The mobile phase comprised acetonitrile
(A) and a 0.1% formic acid solution (B). The gradient elution profile was programmed as
follows: starting at 95% B for 5 min, gradually decreasing to 80% B from 5 to 8 min, further
decreasing to 75% B from 8 to 20 min, reducing to 5% B from 20 to 22 min, holding at 5% B
from 22 to 22.001 min, and finally increasing to 95% B from 22.001 to 25 min.
The mass spectrometry analysis utilized positive and negative ion switching scan
modes (Full scan/dd-MS
2
). H-ESI ionization was employed with an electrospray voltage
of 3.5 kV (+)/2.8 kV (
−
). The ion transfer tube temperature was set at 325
◦
C, and the
auxiliary gas temperature was maintained at 350
◦
C, RF Lens at 70%. Nitrogen served
as nebulizing gas, auxiliary gas, and sheath gas. The flow rates for sheath gas, auxiliary
gas, and sheath gas were 50 Arb, 8 Arb, and 1 Arb, respectively. Primary and secondary
resolutions were set at 60,000 (MS) and 15,000 (MS
2
), respectively. The ion scan range (m/z)
was 100–1500, and the collision energy was normalized. The gradient for collision energy
was set at 20%, 40%, and 60%.
4.3.2. Data Analysis and Identification of Compounds
The original data files were processed using Compound Discoverer 3.3 software
provided by Thermo Fisher Scientific (Waltham, MA, USA), employing a compound identi-
fication method template. This involved peak area extraction, alignment, and matching
secondary fragment spectra against the mzCloud network database and the local database
Molecules 2024,29, 788 10 of 15
mzVault. Filtering criteria included mass deviation control, optimal scores, and comparison
with compound information in the compound library. Subsequent analysis of compounds
included cross-referencing with literature and online databases (PubChem, CNKI, PubMed)
for enhanced understanding.
4.4. Antioxidant Activity
4.4.1. ABTS Radical Scavenging Assay
The ABTS assay followed the Total Antioxidant Capacity Assay Kit. ABTS solution
was combined with an oxidizing agent, and the resulting mixture served as the working
solution. After 12–16 h of dark storage at room temperature, the ABTS working solution
underwent dilution with 80% ethanol, and the absorbance at 734 nm was measured using
an Agilent Synergy H1 microplate reader (Agilent Technologies, Inc., VT, USA) until
reaching 0.7
±
0.05 absorbance units. Test and standard solutions were mixed with the
ABTS working solution in a 96-well plate, and absorbance at 734 nm was recorded after
incubation. Trolox served as the standard, and a series of Trolox standard solutions (0.15,
0.3, 0.6, 0.9, 1.2, 1.5 mM) were prepared. The total antioxidant capacity of each sample was
expressed as µmol Trolox/g of herbal medicine dry weight [76].
4.4.2. DPPH Radical Scavenging Assay
The DPPH assay followed the Total Antioxidant Capacity Assay Kit. Test and standard
solutions were mixed with the reagent, and after a 20-min dark reaction at room tempera-
ture, absorbance was measured at 515 nm using a microplate reader. Trolox served as the
standard, and a series of Trolox standard solutions (0.18, 0.15, 0.12, 0.09, 0.06, 0.03 mM) were
prepared. The total antioxidant capacity of each sample was expressed as
µ
mol Trolox/g of
herbal medicine dry weight [77].
4.4.3. Ferric Reducing Ability of Plasma (FRAP) Assay
The FRAP assay followed the Total Antioxidant Capacity Assay Kit. The FRAP
working solution was prepared by mixing TPTZ (2,4,6-tris(2-pyridyl)-s-triazine) diluent,
TPTZ solution, and detection buffer solution at a ratio of 10:1:1 (v/v/v). After incubation
at 37
◦
C and using within 1–2 h, test and standard solutions were mixed with the FRAP
working solution in a 96-well plate and incubated at 37
◦
C for 3–5 min. After cooling to
room temperature, the absorbance of the mixture was recorded at 593 nm using a microplate
reader. FeSO
4
served as the standard, and a series of FeSO
4
standard solutions (0.15, 0.3,
0.6, 0.9, 1.2, 1.5 mM) were prepared. The total antioxidant capacity of each sample was
expressed as µmol Fe(II)/g of herbal medicine dry weight [78].
4.4.4. Cupric Reducing Antioxidant Capacity (CUPRAC) Assay
The CUPRAC assay followed the Total Antioxidant Capacity Assay Kit (Cu
2+
method).
The Cu
2+
working solution was prepared by mixing Cu
2+
chelating agent solution and
Cu
2+
solution at a ratio of 50:1 (v/v). Test and standard solutions were mixed with the
Cu
2+
working solution in a 96-well plate, incubated at room temperature for 30 min, and
absorbance was measured at 570 nm using a microplate reader. Trolox served as the
standard, and a series of Trolox standard solutions (1, 0.5, 0.25, 0.125, 0.062, 0.031 mM) were
prepared. The total antioxidant capacity of each sample was expressed as
µ
mol Trolox/g of
herbal medicine dry weight [79].
4.4.5. Construction of Standard Curves
In this study, standard curves were constructed for the ABTS, DPPH, FRAP, and
CUPRAC assays. The ABTS assay’s standard curve correlated absorbance values with
known Trolox (6-hydroxy-2,5,7,8-etramethylchroman-2-carboxylic acid) concentrations,
facilitating the calculation of Trolox Equivalent Antioxidant Capacity (TEAC) using a linear
regression equation. Similarly, the DPPH assay utilized absorbance values correlated with
various Trolox concentrations to generate a standard curve, enabling the conversion of
Molecules 2024,29, 788 11 of 15
experimental sample absorbance readings to TEAC values through a linear equation. For
the FRAP assay, we created a standard curve by plotting absorbance against different con-
centrations of Fe(II). The resulting linear regression equation was then applied to compute
the FRAP of the tested samples. Likewise, the CUPRAC assay employed a standard curve
established with known Trolox concentrations, utilizing the linear equation to quantify
the CUPRAC of the samples. These standard curves not only formed the foundation for
quantification and data analysis but also enhanced the accuracy and reliability of antiox-
idant activity assessments. Refer to Figure 2for the standard curves, and Table 3for the
corresponding equations. The R
2
values, ranging from 0.99133 to 0.99987, indicate excellent
linearity, meeting the required standards.
Molecules 2024, 29, x FOR PEER REVIEW 10 of 15
Figure 2. Standard curves for four antioxidant activity methods. ((A) Standard curve for Trolox in
the ABTS method; (B) Standard curve for Trolox in the DPPH method; (C) Standard curve for Fe (Ⅱ)
in the FRAP method; (D) Standard curve for Trolox in the CUPRAC method).
Table 3. Standard curve equations for four antioxidant activity methods.
Method
Equation
R2
ABTS
y = −0.32338x + 0.76745
0.99755
DPPH
y = −1.23810x + 0.57000
0.99133
FRAP
y = 0.20124x + 0.10870
0.99987
CUPRAC
y = 0.43161x + 0.18027
0.99903
4.4.6. Statistical Analysis
The experiments were conducted in triplicate, and the results were reported as the
mean ± standard deviation.
5. Conclusions
Through a comparative analysis of the non-volatile components and antioxidant ac-
tivity in the branches and leaves of the traditional medicinal plant L. cubeba, we unveiled
its diverse chemical composition, encompassing alkaloids, avonoids, and organic acids
across various categories. This underscores its potential as a valuable resource in tradi-
tional herbal medicine and the food industry. The distinct variations in alkaloids, organic
acids, and avonoids between the branches and leaves suggest potential dierences in
their suitability for various applications. Supporting our observations, UPLC-HRMS tech-
nology identied 72 constituents, unveiling signicant chemical heterogeneity between
the branches and leaves, with only eight components shared. This comprehensive charac-
terization contributes to our understanding of L. cubeba’s chemical diversity. Regarding
antioxidant activity, both the branches and leaves exhibited signicant capabilities, with
the branches demonstrating slightly higher ecacy. Consistent support from ABTS,
0.0 0.5 1.0 1.5
0.2
0.4
0.6
0.8
Absorbance
Concentration （mM）
A
0.00 0.05 0.10 0.15 0.20
0.2
0.3
0.4
0.5
0.6
0.7
Absorbance
Concentration (mM)
B
0.0 0.5 1.0 1.5
0.1
0.2
0.3
0.4
Absorbance
Concentration (mM)
C
0.0 0.5 1.0
0.2
0.4
0.6
Absorbance
Concentration (mM)
D
Molecules 2024, 29, x FOR PEER REVIEW 10 of 15
Figure 2. Standard curves for four antioxidant activity methods. ((A) Standard curve for Trolox in
the ABTS method; (B) Standard curve for Trolox in the DPPH method; (C) Standard curve for Fe (Ⅱ)
in the FRAP method; (D) Standard curve for Trolox in the CUPRAC method).
Table 3. Standard curve equations for four antioxidant activity methods.
Method
Equation
R2
ABTS
y = −0.32338x + 0.76745
0.99755
DPPH
y = −1.23810x + 0.57000
0.99133
FRAP
y = 0.20124x + 0.10870
0.99987
CUPRAC
y = 0.43161x + 0.18027
0.99903
4.4.6. Statistical Analysis
The experiments were conducted in triplicate, and the results were reported as the
mean ± standard deviation.
5. Conclusions
Through a comparative analysis of the non-volatile components and antioxidant ac-
tivity in the branches and leaves of the traditional medicinal plant L. cubeba, we unveiled
its diverse chemical composition, encompassing alkaloids, avonoids, and organic acids
across various categories. This underscores its potential as a valuable resource in tradi-
tional herbal medicine and the food industry. The distinct variations in alkaloids, organic
acids, and avonoids between the branches and leaves suggest potential dierences in
their suitability for various applications. Supporting our observations, UPLC-HRMS tech-
nology identied 72 constituents, unveiling signicant chemical heterogeneity between
the branches and leaves, with only eight components shared. This comprehensive charac-
terization contributes to our understanding of L. cubeba’s chemical diversity. Regarding
antioxidant activity, both the branches and leaves exhibited signicant capabilities, with
the branches demonstrating slightly higher ecacy. Consistent support from ABTS,
0.0 0.5 1.0 1.5
0.2
0.4
0.6
0.8
Absorbance
Concentration （mM）
A
0.00 0.05 0.10 0.15 0.20
0.2
0.3
0.4
0.5
0.6
0.7
Absorbance
Concentration (mM)
B
0.0 0.5 1.0 1.5
0.1
0.2
0.3
0.4
Absorbance
Concentration (mM)
C
0.0 0.5 1.0
0.2
0.4
0.6
Absorbance
Concentration (mM)
D
Figure 2. Standard curves for four antioxidant activity methods. ((A) Standard curve for Trolox in the
ABTS method; (B) Standard curve for Trolox in the DPPH method; (C) Standard curve for Fe (II) in
the FRAP method; (D) Standard curve for Trolox in the CUPRAC method).
Table 3. Standard curve equations for four antioxidant activity methods.
Method Equation R2
ABTS y = −0.32338x + 0.76745 0.99755
DPPH y = −1.23810x + 0.57000 0.99133
FRAP y = 0.20124x + 0.10870 0.99987
CUPRAC y = 0.43161x + 0.18027 0.99903
Molecules 2024,29, 788 12 of 15
4.4.6. Statistical Analysis
The experiments were conducted in triplicate, and the results were reported as the
mean ±standard deviation.
5. Conclusions
Through a comparative analysis of the non-volatile components and antioxidant ac-
tivity in the branches and leaves of the traditional medicinal plant L. cubeba, we unveiled
its diverse chemical composition, encompassing alkaloids, flavonoids, and organic acids
across various categories. This underscores its potential as a valuable resource in traditional
herbal medicine and the food industry. The distinct variations in alkaloids, organic acids,
and flavonoids between the branches and leaves suggest potential differences in their
suitability for various applications. Supporting our observations, UPLC-HRMS technol-
ogy identified 72 constituents, unveiling significant chemical heterogeneity between the
branches and leaves, with only eight components shared. This comprehensive charac-
terization contributes to our understanding of L. cubeba’s chemical diversity. Regarding
antioxidant activity, both the branches and leaves exhibited significant capabilities, with
the branches demonstrating slightly higher efficacy. Consistent support from ABTS, DPPH,
FRAP, and CUPRAC methods underscores the robust antioxidant potential within L. cubeba.
This suggests L. cubeba’s promising role as a natural antioxidant with implications for health
and therapeutic interventions. In summary, L. cubeba stands out as a botanical resource
with unique chemical composition and potent antioxidant properties, providing theoretical
support for its applications in medicine and the food industry. The identified chemical
diversity and robust antioxidant activity lay a solid scientific foundation for further ex-
ploration. Future research endeavors could delve deeper into the biological activities and
pharmacological effects of L. cubeba compounds, enhancing our understanding of its poten-
tial health benefits. This study contributes valuable insights for the ongoing exploration
and utilization of L. cubeba in various fields.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules29040788/s1, Figures S1–S4: UPLC-Q-Orbitrap HRMS
chromatograms of Litsea cubeba.
Author Contributions: Conceptualization, W.D., Q.W. and G.Q.; methodology, L.D. and Q.W.;
software, Y.T. and B.L.; validation, W.D. and Y.T.; formal analysis, L.Z. and Y.X.; investigation, Y.T.
and L.Z.; resources, W.D.; data curation, Y.T. and L.Z.; writing—original draft preparation, W.D. and
B.L.; writing—review and editing, G.Q. and Q.W.; funding acquisition, W.D. and G.Q. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the following: Guangzhou Basic Research Program Fund
Project (No. 202102020630); Scientific research project of Guangdong Provincial Administration of
Traditional Chinese Medicine (No. 20231208); Guangdong Medical Research Fund (No. B2021324).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article and supplementary materials.
Conflicts of Interest: The authors declare no conflicts of interest.
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