PLOS ONE

RESEARCH ARTICLE

Flavonoids and antioxidant activity of rare and

endangered fern: Isoetes sinensis
Xin Wang ID1, Guohua Ding1*, Baodong Liu1*, Quanxi Wang2,3
1 College of Life Science and Technology, Harbin Normal University, Harbin, China, 2 College of Life and
Environmental Sciences, Shanghai Normal University, Shanghai, China, 3 Shanghai Key Laboratory of Plant
Functional Genomics and Resources, Chinese Academy of Sciences, Shanghai Chenshan Botanical
Garden, Shanghai, China

* [email protected] (GD); [email protected] (BL)

a1111111111
a1111111111 Abstract
a1111111111
a1111111111 Isoetes sinensis Palmer is a critically endangered, first-class protected plant in China. Until
a1111111111 now, researchers have primarily focused on the ultrastructure, phylogeny, and transcrip-
tomes of the plant. However, flavonoid profiles and bioactivity of I. sinensis have not been
extensively investigated. To develop the endangered I. sinensis for edible and medicinal
purposes, flavonoid content, chemical constitution, and antioxidant activities were investi-
OPEN ACCESS gated in this study. Results revealed the following. 1) The total flavonoid content was deter-
Citation: Wang X, Ding G, Liu B, Wang Q (2020) mined as 10.74 ± 0.25 mg/g., 2) Antioxidant activities were stronger than most ferns,
Flavonoids and antioxidant activity of rare and especially ABTS free radical scavenging activities. 3) Four flavones, containing apigenin,
endangered fern: Isoetes sinensis. PLoS ONE 15
apigenin-7-glucuronide, acacetin-7-O-glcopyranoside, and homoplantageninisoetin; four
(5): e0232185. https://doi.org/10.1371/journal.
pone.0232185 flavonols, namely, isoetin, kaempferol-3-O-glucoside, quercetin-3-O-[6”-O-(3-hydroxy-3-
methylglutaryl)-β-D-glucopyranoside], and limocitrin-Neo; one prodelphinidin (procyani-
Editor: Mohammad Ansari, University of Delhi,
INDIA dins;) and one nothofagin (dihydrochalcone) were tentatively identified in the mass spec-
trometry-DAD (254nm) chromatograms. This study was the first to report on flavonoid
Received: October 15, 2019
content and antioxidant activities of I. sinensis. Stronger antioxidant activity and flavonoid
Accepted: April 8, 2020
content suggests that the endangered I. sinensis is an important and potentially edible and
Published: May 12, 2020 medicinal plant.
Copyright: © 2020 Wang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Introduction
Data Availability Statement: All relevant data are
within the manuscript. Evidence based on epidemiological and pharmacological data has shown that flavonoids play
an important role in preventing and managing modern diseases [1–5]. Total flavonoid content
Funding: The work was sponsored by Shanghai
and antioxidant capacities have been the main focus of research on medicinal and food appli-
Engineering Research Center of Plant Germplasm
Resources (No. 17DZ2252700). The funder had no
cations of natural phytochemicals [6–8].
role in study design, data collection and analysis, Isoetes are considered rare living fossils. With the rapid development of the economy result-
decision to publish, or preparation of the ing in habitat degradation, wild populations have declined dramatically. Thus far, resource uti-
manuscript. lization of Isoetes has yet to be reported, especially for the specific species, Isoetes sinensis
Competing interests: The authors have declared Palmer. I. sinensis was a common species in the wetlands of An’hui, Jiangxi, Jiangsu, and Zhe-
that no competing interests exist. jiang Provinces before 1980. In the last 20 years, this species has all but disappeared [9]. Due to

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PLOS ONE Flavonoids and antioxidant activity of rare and endangered fern: Isoetes sinensis

this dramatic decline, I. sinensis has been considered a critically endangered, first-class pro-
tected plant in China since 1999 [10–12]. Fortunately, our lab has propagated tens of thou-
sands of I. sinensis by way of creative spore propagation, which has allowed for the
development of Isoetes and its continued use in present and future research.
As of today, the ultrastructure, transcriptome, numerous sequences, and functional genes
of I. sinensis have been reported [12–16]. However, the flavonoids and antioxidant activity
from Isoetes have been scantily studied. The goal of this study was to report on flavonoid con-
tent and antioxidant activities of I. sinensis for edible and medicinal development

Materials and methods

Plant materials
Plants were collected from “Fern Garden” in the Botanical Garden of Harbin Normal Univer-
sity. “Fern Garden” was a greenhouse which served for scientific research and teaching. After
overcoming various reproductive difficulties, Isoetes sinensis was cultivated successfully in
“Fern Garden” with proper temperature (18˚C -35˚C), luminous intensity (1500Lx-3000Lx)
and relative humidity (35%-80%). Isoetes sinensis were cultivated,. So, no specific permissions
were required for this location. Plants were identified by Prof. Liu who worked in Harbin Nor-
mal University and engaged in the reproductive development of ferns and the propagation of
endangered ferns for 30 years in China. Voucher specimens were deposited in the College of
Life Science & Technology at Harbin Normal University.

Chemicals and reagents

Refer to our previous report [17] for an overview of the chemicals and reagents used in this
study. Rutin (purity > 99.0%), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azinobis-(3-ethyl-
benzothiazoline-6-sulfonic acid) (ABTS), Nitrotetrazolium blue chloride (NBT), phenazine
methosulfate (PMS), nicotinamide adenine dinucleotide (NADH), 5, 5’-dithiobis-(2-nitroben-
zoic acid) (DTNB) and 2,4,6-tri-2-pyridyl-s-triazine (TPTZ) were purchased from Sigma Co.
(Shanghai, China). Acetonitrile was purchased from Thermo Fisher Scientific (Shanghai,
China).

Preparation of plant extracts

Fresh plant materials were cleaned by distilled water and then dried under the outdoor shady
conditions, finally, at roughly 75˚C for 48 h in an electro- thermostatic blast oven (Bluepard
Instruments CO., LTD, Shanghai, China). Materials were then powdered by the pulverizer and
filtered through a 40-mesh screen. 1g dried sample was separately extracted with 25 mL of 60%
ethanol for 2 h at 50˚C in the polyscience. Ultrasound-assisted extraction was performed for
20 min. The extraction process was repeated twice. The mixture was then filtered via a vacuum
suction filter pump and the extract solutions were collected. The extract solution was measured
at 8 mL and was extracted twice with 8 mL petroleum ether, and then the residue solution was
extracted with dichloromethane, ethyl acetate, and n-butanol, respectively. Each fraction was
concentrated to dryness by evaporation on a rotary evaporator and dissolved with 1 mL etha-
nol, respectively. Before testing, the solutions were filtered through a 0.45-μm membrane
(Millipore, Billerica, MA, USA). Samples were then prepared for HPLC analysis.

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PLOS ONE Flavonoids and antioxidant activity of rare and endangered fern: Isoetes sinensis

Determination of total flavonoids content

Total flavonoid content was measured by a colorimetric assay. Rutin was used to draw a cali-
bration curve [17]. The following formula was used:
Total flavonoid content ð%Þ ¼ ½ðOD1 þ OD2 þ OD3 Þ=3 A�=B�10=2�Volume=1000�100%:

Antioxidant activity DPPH assay. 1 mL 0.1 mM DPPH and extracts were mixed for 30
min. The optical density was measured and recorded at 517 nm. For the control group, 60%
methanol was used. Experiments were performed in triplicate with similar results
(RSD < 5.0%). DPPH free radical scavenging activity is determined with:
ð%Þ ¼ ð1 Asample 517 =Acontrol 517 Þ�100:

Please refer to [17] for an overview of the DPPH assay used in this study.
ABTS assay. 150 μL extracts and 3 mL ABTS solutions with an optical density of ± 0.9
mixed for 6 min. The absorbance value was determined to be 734 nm. Experiments were per-
formed in triplicate with similar results (RSD < 5.0%). ABTS free radical scavenging activity is
determined with:
ð%Þ ¼ ð1 Asample 734 =Acontrol 734 Þ�100:

Please refer to [17] for an overview of the ABTS assay used in this study.
Superoxide anion (O2-) scavenging activity. 1 mL NBT (150 μM), 1 mL NADH
(468 μM), and 1 mL PMS (60 μM) were consecutively added to 1 mL the mixture of extracts
and sodium phosphate buffer. After incubation for 5 min at 25˚C, the optical density was
determined to be 560 nm. For the control group, 60% methanol was used. Superoxide anion
scavenging activity was determined by:

ðO2 Þ scavenging activity ð%Þ ¼ ð1 Asample 560 =Acontrol 560 Þ�100:

Experiments were performed in triplicate with similar results (RSD < 5.0%). Please refer to
[17] for an overview of the scavenging activity assay used in this study.
Reducing power assay. 1 mL extracts, 2.5 mL phosphate buffer, and 2.5 mL potassium
ferricyanide were mixed and then placed in a water bath at 50˚C for 20 min. 2.5 mL TCA was
added to terminate the reaction. After centrifuging for 10 min, 2.5 mL supernatant was added
to 2 mL distilled water and 0.5 mL 0.1% ferric chloride. 2.5 mL supernatant was added to 3 mL
distilled water as part of the control group. The optical density was recorded at 700 nm and
reflected the reducing power. The experiments were performed in triplicate with similar
results (RSD < 5.0%). Please refer to [17] for an overview of the reducing power assay used in
this study.
FRAP assay. The FRAP reagent, which was made up with 10 mM TPTZ in 40 mM HCl
solution and 20 mM FeCl3 in 250 mL acetate buffer, pH 3.6. 50 μL Extracts were mixed with
the 1.5 mL FRAP reagent for 4 mins. The optical density of the mixture was recorded at 593
nm. The FRAP reagent was mixed with 50 μL distilled water as part of the control group. The
experiments were performed in triplicate with similar results (RSD < 5.0%). The FRAP assay
was used in this study as detailed in [17].
Flavonoids analysis of I. sinensis by HPLC-ESI-TOF-MS. Lastly, an Agilent 1100 HPLC
system (Agilent Technologies, USA) was used to perform the chromatographic separation.
The system was equipped with an abinary pump, amicrodegasser, Hi-performance well-pla-
teauto sampler, thermostated column compartment, and diode-array detector (DAD). The

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PLOS ONE Flavonoids and antioxidant activity of rare and endangered fern: Isoetes sinensis

UV Spectrum was recorded between 190–400 nm; the UV detector was set at 254 nm. SHI-
SEIDO MG-C18 (1003.3 mm; i.d. 3.0 mm) column using a gradient elution [methanol (A)/
water (0.1% HCOOH)(B)] was chosen. All of the MS experiments were conducted on an Agi-
lent 6220 Time-of-Flight mass spectrometry (TOF) equipped with an electrospray ionization
(ESI) interface (Agilent Technologies, USA).
The gradient condition was 0–15 min at 15–45% A, 15–25 min at 45–55% A, and 25–35
min at 55–90% A. The column temperature was set at 25˚C, the flow rate was kept at 0.4 mL/
min, and the injection volume was 10 μL. Both the auxiliary and nebulizer gases consisted of
nitrogen with a flow rate of 10 L/min. The MS analysis was performed in both positive and
negative scan modes under the following operation parameters: the nebulizer pressure was set
at 45 psi, the dry gas temperature was set at 350˚C, and the voltage was set at 160 V. Full scan
data acquisition and dependent scan event data acquisition were performed from m/z 100–
1200.

Results and discussion

Total flavonoid content of I. sinensis
The concentration and total flavonoid content of I. sinensis were determined as 0.043 mg/ml,
10.74 ± 0.25 mg/g, respectively. Although far lower than most species of filicinae, a little higher
than that of selaginella and equisetums [18], and higher than the total flavonoid content of
bryophytes [19]. Primitive plant groups might have lower flavonoid contents than their mod-
ern counterparts.

Antioxidant activity
With the increasing of the concentration, the DPPH free radical scavenging potential observ-
ably increased (Fig 1). Roughly 111.9 μL of the extracts could scavenge about 50% of the free
radicals. The IC50 of DPPH scavenging activity was recorded as 4.8 mg/μL, 10 mg/μL extracts
could scavenge 100% DPPH free radicals.
ABTS free radical scavenging potential noticeably increased with the increasing of the con-
centration (Fig 2). Roughly 11 μL of the extracts could scavenge about 50% of the free radicals.
The IC50 of ABTS scavenging activity was recorded as 3.2 mg/μL.
Superoxide radicals scavenging potential noticeably increased was relative to the increasing
concentration (Fig 3). Roughly 991.5 μL of the extracts could scavenge about 50% of the free
radicals. The IC50 of superoxide radicals scavenging activity was recorded as 42.6 mg/μL.
The results from the FRAP and reducing power assays illustrated that the extracts from I.
sinensis possessed antioxidant and reductive activity of Fe3+ (Figs 4 & 5). With increasing vol-
ume, the activity clearly improved.
Based on the results noted above, the antioxidant activities of I. sinensis were obviously
stronger than most reported ferns and bryophytes, especially in terms of ABTS-free radical
scavenging activities [19, 20]. This showed that I. sinensis had clear medicinal implications. I.
debii Sinha was commonly used in Indian cuisines [21] and roasted rhizomes are used in
cough and cold medication [22]. With stronger antioxidant activity than other species of its
kind, I. sinensis has potentially important applications in both food and medicine.

Flavonoids analysis of I. sinensis by HPLC-ESI-QTOF-MS

HPLC-ESI-QTOF-MS was utilized in the qualitative analysis of flavonoid of I. sinensis
(Table 1). In comparison with the known chromatograms and mass spectral data in literature,

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PLOS ONE Flavonoids and antioxidant activity of rare and endangered fern: Isoetes sinensis

Fig 1. DPPH free radical scavenging activity observed in extracts from I. sinensis.
https://doi.org/10.1371/journal.pone.0232185.g001

a total of 22 peaks were tentatively identified from 4 extracts: petroleum ether fraction (A),
dichloromethane fraction (B), ethyl acetate fraction (C), and n-butanol fraction (D) (Fig 6).
Four flavones contain apigenin, apigenin-7-glucuronide, acacetin-7-O-glcopyranoside, and
homoplantageninisoetin; four flavonols, namely, isoetin, kaempferol-3-O-glucoside, querce-
tin-3-O-[6”-O-(3-hydroxy-3-methylglutaryl)-β-D-glucopyranoside], and limocitrin-Neo; one

Fig 2. ABTS free radical scavenging activity observed in extracts from I. sinensis.
https://doi.org/10.1371/journal.pone.0232185.g002

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PLOS ONE Flavonoids and antioxidant activity of rare and endangered fern: Isoetes sinensis

Fig 3. Superoxide radical scavenging activity observed in extracts from I. sinensis.

https://doi.org/10.1371/journal.pone.0232185.g003

prodelphinidin (procyanidins); and one nothofagin (dihydrochalcone) were tentatively identi-

fied in the mass spectrometry-DAD (254nm) chromatograms of extracts from I. sinensis. Api-
genin was an edible natural flavonoid found in several dietary plant foods such as vegetables
and fruits and showed potential antioxidant, anti-inflammatory and anticancer properties
[23]. Hepatoprotective effects of kaempferol-3-O-glucoside had been proved [24]. Prolonged

Fig 4. The reducing power observed in extracts from I. sinensis.

https://doi.org/10.1371/journal.pone.0232185.g004

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PLOS ONE Flavonoids and antioxidant activity of rare and endangered fern: Isoetes sinensis

Fig 5. Antioxidant power by FRAP assay observed in extracts from I. sinensis.

https://doi.org/10.1371/journal.pone.0232185.g005

diuretic and saluretic effect of nothofagin had been proved [25]. It was inferred that I. sinensis
was potential edible and medical plant.
Flavonoid content of Isoetaceae was poorly known but appears to contain mainly flavone,
apigenin, luteolin, chrysoeriol, selgin, tricin, and isoetin (as the 5’-glucoside), which had been
reported in other species of Isoetes [26]. Expectisoetin, apigenin procyanidins and dihydro-
chalcone might exist in I. sinensis as well. Flavones and flavonols were the main flavonoids in
most species from filicinae [18]. Flavones, flavonols, flavanones, aurones, and dihydrochal-
cones were the main flavonoids in bryophytes [27]. The dihydrochalcones observed in this
study suggested that Isoetaceae and bryophytes may be phylogenetically similar.
Effects of various ecological factors on the secondary metabolite profile have also been
observed in angiospermae [28]. In a previous study [18, 20], a similar impact of ecological fac-
tors on the flavonoids found in the same species. The lower flavonoid content of aquatic ferns
was attributed to specific environmental factors, which typically require no need to produce
flavonoids in self-defense. Pilularia globulifera, a marsileaceous fern, was previously reported
to accumulate quercetin and kaempferol glycosides [29], which was similar to the content
found within I. sinensis. An aquatic macrophyte, Stratiotes aloides, accumulated luteolin and
chrysoeriol glycosides [30], which were different from I. sinensis. Thus, it was speculated that
the phytogroup was also influencing factors on the secondary metabolite profile, in addition to
the ecological factors.

Conclusion
This study is the first to report on the phytochemistry and biological activities of I. sinensis.
The results showed that I. sinensis was with stronger antioxidant activity than some fern and
bryophytes and higher flavonoid content (10.74 ± 0.25mg/g), So the endangered I. sinensis
should be an important and potentially edible and medicinal plant.

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PLOS ONE Flavonoids and antioxidant activity of rare and endangered fern: Isoetes sinensis

Table 1. Analysis of I. sinensis by HPLC-ESI-QTOF-MS. a) petroleum ether fraction; b) dichloromethane fraction; c) ethyl acetate fraction; and d) n-butanol fraction.
No. RT Compound type Formula MW Observed Calculated Mass UV λ Identification Part Ref
mass[M-H]- mass[M-H]- error max/
(ppm) nm
1. 37.02 benzoic acid C15H22O2 234.1615 233.1542 233.1547 2.02 245 4-Octylbenzoic acid A [17]
2. 37.91 fattyacid C16H28O3 268.2039 267.1966 267.1966 -0.04 250 3(z)-Hydroxy-hexadeca-4(E),6(Z)- A [31]
dienoic acid
3. 38.72 fattyacid C18H30O3 294.2197 293.2124 293.2122 -0.63 250, 9-Hydroxy-10E-octadecen-12-ynoic acid A [32]
295
4. 39.43 fattyacid C18H32O3 296.2349 295.2276 295.2279 0.76 255 9-,13-Hydroxyoctadecadienoic acid A [33]
5. 40.75 fattyacid C20H34O2 306.2562 305.2489 305.2486 -1.05 245 Dihomolinolenic A [34]
6. 41.31 ketone C30H48O5 488.3505 487.3432 487.3429 -0.57 255, Ganodermanontetrol A [35]
275,
320
7. 47.03 fattyacid C20H34O2 306.2562 305.2489 305.2486 -1.05 245 8,11,14-Eicosatrienoic acid A [36]
8. 9.72 benzoic acid C7H6O3 138.0317 137.0244 137.0244 0.23 258 P-Hydroxybenzoic acid BB [37]
9. 20.67 benzoic acid C14H12O3 228.0781 227.0708 227.0714 2.35 238, 4-(Phenoxymethyl)benzoic acid B [38]
305,
320
10. 22.20 flavone C21H18O11 446.0853 445.078 445.0776 -0.85 240, Apigenin-7-glucuronide B [31]
268,
340
11. 24.35 flavone C22H22O10 446.1208 445.1135 445.114 1.08 240, Acacetin-7-O-glcopyranoside B [39]
260,
335
12. 28.48 flavonol C15H10O6 286.0473 285.04 285.0405 1.62 252, Isoetin B [40]
270,
352
13. 31.69 flavone C15H10O5 270.0523 269.0451 269.0455 1.79 238, Apigenin B [41]
269,
340
14. 35.57 coumarin C16H12O5 284.0681 283.0608 283.0612 1.45 240, Tomenin B [42]
268,
340
15. 15.50 dihydrochalcone C21H24O10 436.1376 435.1303 435.1297 -1.46 238, Nothofagin C [43]
310
16. 17.52 flavonol C21H20O11 448.1007 447.0934 447.0933 -0.28 260, Kaempferol-3-O-glucoside C [44]
340
17. 12.40 lactic acid C9H10O3 166.0634 165.0561 165.0557 -2.35 240, Phenyllactic acid D [45]
300,
320
18. 12.64 coumarin C16H18O9 354.0955 353.0882 353.0878 -1.05 245, Scopolin D [46]
295,
326
19. 16.12 procyanidins C27H30O15 594.1591 593.1518 593.1512 -1.01 245, Prodelphinidin D [47]
272,
330
20. 17.27 flavonol C27H28O16 608.1388 607.1315 607.1305 -1.75 240, Quercetin-3-O-[6@-O-(3-hydroxy- D [48]
268, 3-methylglutaryl)-β-d-glucopyranoside]
320
21. 17.89 flavonol C29H34O17 654.1795 653.1722 653.1723 0.16 245, Limocitrin-neo D [49]
265,
325
22. 21.91 flavone C21H18O12 462.0808 461.0735 461.0725 -2.1 255, Homoplantagenin D [50]
268,
350
https://doi.org/10.1371/journal.pone.0232185.t001

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PLOS ONE Flavonoids and antioxidant activity of rare and endangered fern: Isoetes sinensis

Fig 6. DAD (254nm) chromatograms of flavonoid extracts from I. sinensis: a) Petroleum ether fraction; b)
dichloromethane fraction; c) ethyl acetate fraction; and d) n-butanol fraction.
https://doi.org/10.1371/journal.pone.0232185.g006

Acknowledgments
We are grateful to Prof. Chengjian Zheng from Second Military Medical University for excel-
lent technical assistance. We thank LetPub (www.letpub.com) for its linguistic assistance dur-
ing the preparation of this manuscript.

Author Contributions
Conceptualization: Xin Wang, Quanxi Wang.
Data curation: Xin Wang.
Formal analysis: Xin Wang.
Funding acquisition: Baodong Liu, Quanxi Wang.
Methodology: Xin Wang.
Project administration: Quanxi Wang.
Resources: Baodong Liu.
Writing – original draft: Xin Wang.
Writing – review & editing: Guohua Ding, Baodong Liu.

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