Int. J. Mol. Sci. 2015, 16, 2497-2516; doi:10.3390/ijms16022497
International Journal of
Chemical Constituents Analysis and Antidiabetic Activity
Validation of Four Fern Species from Taiwan
Chen-Yu Chen 1,2, Fu-Yu Chiu 3, Yenshou Lin 3, Wei-Jan Huang 1,2, Po-Shiuan Hsieh 4,†
and Feng-Lin Hsu 1,2,†,*
1 College of Pharmacy, School of Pharmacy, Taipei Medical University, 250 Wuxing St., Taipei 110,
Taiwan; E-Mails: email@example.com (C.-Y.C.); firstname.lastname@example.org (W.-J.H.)
2 Graduate Institute of Pharmacognosy, School of Pharmacy, Taipei Medical University,
250 Wuxing St., Taipei 110, Taiwan
3 Department of Life Science, National Taiwan Normal University, No. 162, Sec. 1, Heping E. Rd.,
Taipei 106, Taiwan; E-Mails: email@example.com (F.-Y.C.);
4 Department of Physiology and Biophysics, National Defense Medical Center, No. 161, Sec. 6,
Minquan E. Rd., Taipei 114, Taiwan; E-Mail: firstname.lastname@example.org
† These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: email@example.com;
Tel.: +886-2-2736-1661 (ext. 6132); Fax: +886-2-2737-0903.
Academic Editor: Chang Won Choi
Received: 25 November 2014 / Accepted: 13 January 2015 / Published: 22 January 2015
Abstract: Pterosins are abundant in ferns, and pterosin A was considered a novel activator
of adenosine monophosphate-activated protein kinase, which is crucial for regulating
blood glucose homeostasis. However, the distribution of pterosins in different species
of ferns from various places in Taiwan is currently unclear. To address this question,
the distribution of pterosins, glucose-uptake efficiency, and protective effects of pterosin A
on β-cells were examined. Our results showed that three novel compounds, 13-chloro-spelosin
3-O-β-D-glucopyranoside (1), (3R)-Pterosin D 3-O-β-D-(3'-p-coumaroyl)-glucopyranoside
(2), and (2R,3R)-Pterosin L 3-O-β-D-(3'-p-coumaroyl)-glucopyranoside (3), were isolated
for the first time from four fern species (Ceratopteris thalictroides, Hypolepis punctata,
Nephrolepis multiflora, and Pteridium revolutum) along with 27 known compounds.
We also examined the distribution of these pterosin compounds in the mentioned fern
species (except N. multiflora). Although all pterosin analogs exhibited the same effects
Int. J. Mol. Sci. 2015, 16 2498
in glucose uptake assays, pterosin A prevented cell death and reduced reactive oxygen
species (ROS) production. This paper is the first report to provide new insights into
the distribution of pterosins in ferns from Taiwan. The potential anti-diabetic activity
of these novel phytocompounds warrants further functional studies.
Keywords: pterosin; reactive oxygen species (ROS); rat pancreatic insulin-secreting
Ferns are a group of approximately 12,000 species belonging to the botanical group known
as Pteridophyta. Certain fern species are consumed as food or as folk medicine in several countries
to treat various ailments. Ferns primarily contain flavonoids, alkaloids, phenols, steroids, and triterpenoids;
exhibit various bioactivities such as antibacterial, antiosteoporosis, and anti-Alzheimer’s disease
activity; and possess hypolipidemic and hypoglycemic activities . Therefore, ferns are a major
medicinal resource in ethnopharmacy.
Pterosin, sesquiterpenes with 1-indanone skeletons, were first isolated from the bracken fern
Pteridium aquilinum var. latiusculum (Pteridaceae) . Approximately 31 pterosins have been isolated
from several fern species (Table S1) and exhibit anticancer, smooth-muscle relaxation, and leishmanicidal
activities . Pterosin A was expressed against type 1 and type 2 diabetes in an animal model. In addition,
further research has indicated that pterosin A can promote glucose uptake, improve insulin sensitivity,
and enhance adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, which
regulates carbohydrate and fatty acid metabolisms . Therefore, pterosin compounds may be useful
for treating metabolic disease in future studies.
Oxidative stress damages several cellular functions in the pathophysiology of various diseases.
Reportedly, reactive oxygen species (ROS) were produced by macrophages and were responsible
for apoptosis or necrosis of insulin-secreting cells . β-Cell compensation for insulin resistance occurs
by increased insulin secretion or cell mass, and lack of compensation causes glucose intolerance .
ROS production has been associated with β-cell dysfunction and cell death in both type 1 and type 2
diabetes . Chronic exposure to long-chain saturated fatty acids is another major inducer of type 2
diabetes. Accelerated free fatty acid (FFA) production will promote oxidative process in mitochondria,
which may also enhance ROS production. Moreover, with an irregular protein synthesis rate,
the endoplasmic reticulum accumulates with increasing unfolded protein levels in the lumen, which
is associated with abnormal oxidation. Aggregated misfolding proteins may cause excess ROS
production, inducing gradual apoptosis of pancreatic β-cells .
AMPK is a cellular sensor that regulates energy and metabolic homeostasis; it activates in response
to increased ratio of AMP to adenosine triphosphate and calcium ion content. AMPK is a master
regulator in the physiology of several organs, regulating carbohydrate, lipid, and protein metabolism.
AMPK activity primarily maintains the glucose content within the physiological range in various cells,
particularly β-cells . However, increased AMPK activity can suppress insulin secretion to prevent
exhausted β-cells . Impaired functional β-cell production after chronic compensation reduces
Int. J. Mol. Sci. 2015, 16 2499
insulin secretion and AMPK activation, which may potentiate glycolipotoxicity-induced cell death .
Therefore, the AMPK pathway is crucial for regulating glucose homeostasis and is a major target
of therapy for type 2 diabetes.
However, the actual distribution and content of pterosin analogues in certain ferns from Taiwan
remains unclear. In the present study, we isolated 30 phytochemicals from four fern species:
Hypolepis punctata (Thumb.) Mett, Ceratopteris thalictroides (L.) Brongn, Nephrolepis multiflora
(Roxb.) Jarret ex Morton and Pteridium revolutum (BI.) Nakai. Among these, 13-chloro-spelosin
3-O-β-D-glucopyranoside (1), (3R)-pterosin D 3-O-β-D-(3'-p-coumaroyl)-glucopyranoside (2),
and (2R,3R)-pterosin L 3-O-β-D-(3'-p-coumaroyl)-glucopyranoside (3) are novel compounds. Here we
describe the structural elucidations of 1, 2, and 3. These pterosin compounds were evaluated for their
antidiabetic activity. In addition, we developed and validated a sensitive and specific method involving
liquid chromatography-tandem mass spectrometry (LC–MS–MS) for analysis of these pterosins.
2.1. Structural Elucidation
Fresh fern material from H. punctata, C. thalictroides, N. multiflora, and P. revolutum was
extracted using organic solvent. Repeated chromatography on silica gel and highly porous polymer
gel produced three new compounds (Figure 1) in addition to 27 known compounds, which were
determined by comparing their physicochemical and spectroscopic data with published reports.
Figure 1. Structures of Compounds 1–3.
Compound 1 was obtained as a colorless oil. The IR spectra at 1598 and 1697 cm−1 indicated
the presence of a benzene ring and carbonyl group. Characteristic 1H-NMR spectra revealed signals
assignable to gem-dimethyl (δ 1.07, 1.61 (each 3H, s, H-10, 11)), two aromatic methyl groups at δ 2.50
(3H, s, H-15) and 2.73 (3H, s, H-14), one chloroethyl group (δ 3.93 (2H, m, H-13), 5.40 (1H, dd,
J = 5.4, 5.2 Hz, H-12)), one allylic oxygenated methylene at δ 4.84 (1H, s, H-3), and one aromatic
proton (δ 7.53 (1H, s, H-5)). In addition, the 1H-NMR shifts at δ 3.27–4.56 suggested one sugar
moiety. These signals indicated the presence of a pteroside skeleton. On the basis of the correlation
spectroscopy (COSY) and heteronuclear multiple quantum coherence (HMQC) spectra, the glycosidic
moieties were assigned as a glucopyranose. The configuration of the anomeric position (δ 4.56) was
confirmed as a β-configuration by the coupling constant (J = 7.7 Hz). The heteronuclear multiple bond
coherence (HMBC) correlations between glucopyranose H-1' and aglycone C-3 suggested that glucose
Int. J. Mol. Sci. 2015, 16 2500
was substituted at C-3. Moreover, ESI-MS revealed isotopic [M + H]+ ion peaks at m/z 443/445, and
the molecular formula of Compound 1 was suggested as C21H29ClO8. A comparison of this aglycone
with spelosin  revealed an upfield shift of the C-13 spectra; thus, the chlorine group was attached
at C-13. Acid hydrolysis of 1 gave the aglycone and glucopyranose, rescpectively, and their structures
were confirmed by comparison of the 13C-NMR spectra with those of references. The absolute
configuration of aglycone was determined by the specific rotation with a value of [α]D24 + 82.6 (c = 0.7,
MeOH) similar to that of spelosin ([α]D22 + 83.3 (c = 0.7, MeOH)) . Consequently, Compound 1 was
determined as 13-chloro-spelosin 3-O-β-D-glucopyranoside.
The molecular formula of Compound 2 was C30H36O10Na, as determined from HR-ESI-MS
m/z 556.2312 [M + Na]+. The 1H-NMR showed gem-dimethyl at δ 1.08 (3H, s, H-10), 1.29 (3H, s,
H-11), two aromatic methyl groups at δ 2.46 (3H, s, H-15) and 2.63 (3H, s, H-14), two coupled
methylenes of a hydroxyethyl group (δ 3.30 (2H, t, J = 7.7 Hz, H-12) and 3.60 (2H, t, J = 7.7 Hz,
H-13)), one allylic oxygenated methylene at δ 4.85 (1H, s, H-3), and one aromatic protons (δ 7.57 (1H,
s, H-5)) for a pterosin D skeleton, along with a p-coumaroyl group (δ 6.40 (1H, d, J = 15.8 Hz),
δ 7.66 (1H, d, J = 15.8 Hz), δ 6.80 (2H, d, J = 8.4 Hz), and δ 7.46 (2H, d, J = 8.4 Hz)), except
for the presence of sugar signals. According to the COSY and HMQC spectra, the glycosidic moiety
was assigned as a glucopyranose. The HMBC correlation between glucopyranose H-1' and aglycone
C-3 suggested that glucose was substituted at C-3 of pterosin D. A comparison of the 1H-NMR spectra
for Compound 2 with (3R)-pterosin D 3-O-β-D-glucopyranoside revealed a downfield shift of H-3'
(δ 5.12) of the glucose moiety, which supported together with the HMBC signal H-3'/C-9" the linkage
of the p-coumaroyl group to C-3' (Figure 2). Comparison of the specific rotation of pterosin D
([α]D24 + 4.8 (c = 0.5, MeOH)), which was obtained by acid hydrolysis of 2, with that of previously
isolated (3R)-pterosin D ([α]D22 + 5 (c = 0.35, MeOH))  led to the (3R)-configuration of 2.
Additionally, based on the result of NOESY correlation of H-10/H-3 and H-11/H-1', the absolute
configuration of 2 was suggested to be the same as that of (3R)-pterosin D. Accordingly, Compound 2
was identified as (3R)-pterosin D 3-O-β-D-(3'-p-coumaroyl)-glucopyranoside.
Figure 2. HMBC (heteronuclear multiple bond coherence) correlations for Compound 2.
The molecular formula of Compound 3 was determined as C30H36O11Na m/z 572.2263 [M + Na]+
by HR-ESI-MS. The 1H- and 13C-NMR data (Table 1) were similar to those of Compound 2, except for
the one hydroxymethyl group at H-2 of the 1-indanone skeleton. Compound 3 revealed a p-coumaroyl
moiety, a glucopyranose unit, and pterosin L as determined by the NMR data . From the HMBC
data, the correlation of H-3 with anomeric carbon (C-1') suggested that glucose was substituted at C-3
Int. J. Mol. Sci. 2015, 16 2501
of pterosin L. In addition, the substantial downfield shift of H-3' indicated the connection site
of the coumaroyl group. The HMBC correlation demonstrated that the H-3' linkage was located
at the conjugated carbonyl of p-coumaroyl. Acid hydrolysis of 3 yielded pterosin L, p-coumaric acid,
and glucopyranose. The optical rotation of pterosin L with a value of [α]D24 + 19.5 (c = 1.1, MeOH)
were consistent with literature values ([α]D22 + 20 (c = 0.25, MeOH)) . Thus, Compound 3
was structurally elucidated as (2R,3R)-pterosin L 3-O-β-D-(3'-p-coumaroyl)-glucopyranoside.
Table 1. 1H- and 13C-NMR spectra for compounds 1, 2 and 3.
Position 1 2 3
δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz) δC
1 211.1 211.3 207.9
2 52.8 52.6 55.9
3 4.84, s 86.0 4.85, s 86.5 4.74, s 84.1
4 153.1 151.8 145.2
5 7.53, s 128.0 7.57, s 126.8 7.54, s 125.0
6 146.0 146.2 136.5
7 140.2 138.3 132.3
8 139.0 138.6 137.2
9 131.5 130.9 131.3
10 1.07, s 22.7 1.08, s 22.0 1.22, s 17.2
11 1.61, s 22.2 1.29, s 22.8 3.56, m 65.7
12 5.40, dd (5,4, 5.2) 71.9 3.30, t (7.7) 33.1 3.00, t (7.7) 31.7
13 3.93, m 47.8 3.60, t (7.7) 61.6 3.58, t (7.7) 60.2
14 2.73, s 15.1 2.63, s 14.1 2.65, s 12.7
15 2.50, s 22.0 2.46, s 21.4 2.47, s 20.0
1' 4.56, d (7.7) 105.9 4.70, d (7.7) 105.8 4.64, d (7.9) 104.3
2' 3.27–3.43, m 75.3 3.00–4.00, m 73.8 3.00–4.00, m 72.2
3' 3.27–3.43, m 78.2 3.00–4.00, m 79.1 3.00–4.00, m 77.3
4' 3.27–3.43, m 71.7 3.00–4.00, m 69.9 3.00–4.00, m 68.4
5' 3.27–3.43, m 78.0 3.00–4.00, m 77.9 3.00–4.00, m 76.7
6' 3.72–3.75, m 62.9 3.72–3.93, m 62.5 3.70–3.80, m 60.9
1" 127.3 125.9
2",6" 7.46, d (8.4) 131.1 7.50, d (8.6) 129.7
3",5" 7.64, d (8.4) 116.8 6.80, d (8.6) 115.4
4" 161.2 159.9
7" 7.66, d (15.8) 146.6 7.67, d (16.0) 144.9
8" 6.40, d (15.8) 115.6 6.41, d (16.0) 114.1
9" 169.1 167.6
2.2. LC-MS-MS of Pterosins A, I, and Z
We analyzed the isolated pterosins by LC–MS–MS. Figure 3 presents the MRM and daughter ion
chromatograms obtained for analyzing the pterosin mixture of the analytes.
Int. J. Mol. Sci. 2015, 16 2502
Figure 3. LC–MS–MS chromatography of pterosins. (A) High-performance liquid
chromatography of pterosins A, I, and Z and piromidic acid (internal standard);
(B) Multiple reaction monitoring chromatography corresponding to the LC–MS–MS
analyses of pterosins and piromidic acid and (C) daughter ion chromatograms.
Int. J. Mol. Sci. 2015, 16 2503
Fragmentation patterns of the precursor ions were observed for pterosins (A, Z, and I) when these
were analyzed using ESI with a triple quadrupole MS. After CID, the [M + H]
of the aforementioned
pterosins produced a major fragment ion at m/z 249.43, 233.36, and 247.41, respectively. Each [M + H]
pterosin of the parent ion was screened based on the first paragraph. The cleavage fragments (daughter
ions) were detected by a second mass analysis. Pterosins of daughter ion mass spectra revealed
collision energies of 18 eV (pterosin A and I) and 28 eV (pterosin Z) (Figure 4). Each of the three
components exhibited fractured fragments, and the relative strength of the various peaks of fragments
can be used to identify the features of the constituents.
Figure 4. First mass scan analysis and daughter ion mass spectra of three pterosins:
(A) pterosin A; (B) pterosin Z; and (C) pterosin I.
Int. J. Mol. Sci. 2015, 16 2504
2.3. Biology Activity
2.3.1. Pterosins Increased Cellular Uptake of Glucose
We investigated the glucose uptake activities of pterosins in C2C12 myocytes based on the
2-deoxyglucose uptake levels after a 20-min treatment with 1 µM of the aforementioned pterosin
compounds. 2-Hydroxypterosin C and (2S,3S)-pterosin C significantly increased glucose uptake
(p < 0.01), as indicated by the mild elevation with pterosins A, I, and Z (p < 0.05) (Figure 5).
Figure 5. Effects of the isolated pterosins on glucose uptake in C2C12 myocytes. The cells
were treated with the test compounds (1 μM) and 2-deoxy-D-[3H] glucose was added
to determine the glucose uptake activity. * p < 0.05, ** p < 0.005, *** p < 0.001.
2.3.2. Pterosin A Protected H2O2-induced Reactive Oxygen Species (ROS) through Adenosine
Monophosphate-Activated Protein Kinase (AMPK) Activation
The generation of ROS, including hydroxyl radicals (·OH), H2O2, and superoxide anion (O2−),
and the concomitant formation of NO was associated with β-cell dysfunction and cell death .
The RINm5f β-cells were incubated with various concentrations of pteroisn A with and without 40 μM
H2O2; subsequently, the cell viability and ROS levels were determined using MTT and NBT assays,
respectively. Pterosin A exhibited a mild protective effect through H2O2-induced cell death,
and the scavenging capacity effect of ROS was dose-dependent (Figure 6A,B); therefore, pterosin A
may, as an antioxidant, reduce oxidative stress-induced cell death in β-cells.
Pterosin A was found to be a novel AMPK activator. In addition, AMPK phosphorylation inhibits
NO-induced apoptosis . Therefore, we examined the protective effects of pterosin A on cells
through AMPK activation. The AMPK activation was more substantial with H2O2 pretreatment than
that with pterosin A or H2O2 alone (Figure 6C); however, Compound C attenuated the protective
effects of pterosin A on H2O2-induced oxidative stress (Figure 6D). Thus, the cytoprotective effects
of pterosin A might be partially mediated through AMPK activation.
(Fold of control)
Int. J. Mol. Sci. 2015, 16 2505
Figure 6. Pterosin A protected against H2O2-induced reactive oxygen species (ROS) cell
damage through adenosine monophosphate-activated protein kinase (AMPK) activation.
(A) RINm5f cells were treated with 40 μM H2O2 for 2 h and then incubated with various
doses of pterosin A for 18 h; (B) The cells were coincubated with H2O2 (40 μM)
and various doses of pterosin A for 18 h. The ROS levels were determined using an NBT
assay; (C) Cell viability on incubation with H2O2 with and without pterosin A (100 μM)
and Compound C (AMPK inhibitor) for 2 h; (D) The cells were incubated with H2O2
for 2 h and exposed to pterosin A, followed by western blot analysis of phospho-T172
AMPK and total AMPK levels. Data are presented as mean ± SEM. * p < 0.05.
2.3.3. AMPK Activation Avoided Palmitate-Induced Lipotoxicity by Pterosin A
H2O2 is produced by oxidative stress, which may result from excess glucose or lipid intake.
In the present study, the RINm5f β-cells were pretreated with Compound C before incubation
with palmitate and pterosin A cotreatment. Cell viability decreased with antioxidant palmitate,
and palmitate with Compound C also reduced cell viability, but this diminished cell viability was
dose-dependently reversed by pterosin A (Figure 7A,B). Pterosin A dose-dependently enhanced
the AMPK phosphorylation in the palmitate-stimulated β-cells by at least 24 h (Figure 7C). Therefore,
pterosin A might play a protective role in reducing lipotoxicity-induced cell death in β-cells through
Int. J. Mol. Sci. 2015, 16 2506
Figure 7. Effects of pterosin A on palmitate-induced lipotoxicity and AMPK expression
in RINm5F cells. (A) Cell viability on cotreatment with various doses of pterosin A and
palmitate (250 μM) for 24 h; (B) The cells were pretreated with Compound C (20 μM)
for 2 h, followed by cotreatment with plamitate (250 μM) with and without pterosin A
for 24 h; (C) Western blot analysis of total and phospho-AMPK (A: 5-aminoimidazole-4-
carboxamide ribonucleotide (AICAR) was used as a positive control). Data are presented
as mean ± SEM. * p < 0.05; *** p < 0.001.
2.3.4. Pterosin A Inhibition in Palmitate-Induced ROS Production
A recent study indicated that inhibition of ROS plays a protective role in palmitate-induced
β-cell apoptosis . We assessed ROS generation by 2',7'-dichlorofluorescein diacetate (DCFH-DA)
staining in β-cells. The palmitate-treated RINm5f cells revealed increased ROS levels at 24 h
(Figure 8). Moreover, pterosin A revealed a dose-dependent reduction in ROS production.
Int. J. Mol. Sci. 2015, 16 2507
Figure 8. Effects of pterosin A on palmitate-induced ROS in RINm5F cells. The cells
were exposed to various doses of pterosin A with palmitate (250 μM) (A: AICAR was
used as a positive control). Fluorescent microscopy to determine the ROS levels
by 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining (original magnification 200×),
Hoechst staining of nuclei.
Pterosins comprise a large group of sesquiterpenes, and these compounds occur widely
in the Dennstaediaceae and Pteridaceae families. We isolated the seasonal variations of pterosins
compounds and other components from four fern species, including nine pterosins, five pterosides,
six lignans, three flavonoids, six phenolics, and one carbohydrate, along with photochemicals from
C. thalictroides and N. multiflora. In addition, Compounds 21 to 23 were identified for the first time
in H. punctata. Moreover, seven compounds (Compounds 10 to 12, 14, and 25 to 27) were identified
in P. revolutum for the first time. Furthermore, the results revealed that the distributions of the pterosin
compounds and pterosin A in the three aforementioned species (H. punctata, C. thalictroide,
and P. revolutum), except N. multiflora (Nephrolepdiaceae), were higher than the corresponding
distributions of the other pterosin analogs (Table S2). Several previous studies have isolated several
Int. J. Mol. Sci. 2015, 16 2508
triterpenes and steroids from Nephrolepdiaceae . These findings clearly indicated the presence
of nonpterosin-type components in N. multiflora.
However, whether pterosin A has protective effects on pancreatic β-cells against oxidative stress
remains unknown. Therefore, the present study assessed the possible beneficial effects of pterosin A
on cell survival and ROS production in insulin-secreting cells subjected to oxidative stress
or lipotoxicity. In this study, pterosin A effectively reduced the ROS-induced cell damage in the
insulin-secreting cells through the AMPK signaling pathway. Reportedly, pancreatic abnormal glucose
metabolism and long-term treatment with FFA can cause defects in mitochondrial function and gradual
increase of ROS production, which leads to β-cell dysfunction [16–18]. We observed that pterosin A
could not reverse the ROS-reduced cell viability but could reduce ROS production. Additional studies
focused on detecting the activity of antioxidant enzymes under pterosin A treatment may be required
to confirm this indication.
In the present study, pterosin A protected cells against oxidative stress or lipotoxicity-induced
damage through AMPK activation. Cotreatment with Compound C inhibited the AMPK activation
and eliminated the protective effects of pterosin A on cell viability, with consequent cell injury
induced by palmitate or H2O2. AMPK activation exhibited positive effects on the functional
impairment and cell mass of β-cells because of glucotoxicity . Tuberous sclerosis complex 2
(TSC2), downstream of AMPK, can protect against cell death through various signal pathways that
regulate cell size, translation, and apoptosis in adverse growth environments . In addition, AMPK
activity may be useful to promote the physiological functions of β-cells. Therefore, the protective
effects of pterosin A against oxidative damage through AMPK activation presented in our preliminary
data may be explained by the aforementioned mechanisms; however, further research is required
to confirm these findings.
As described previously, pterosin A is a major compound of pterosins that has antidiabetic
and protective effects against β-cell damage. Therefore, pterosin A may be used as a lead compound
in the development of drugs for type 2 diabetes. However, the impaired glucose transport in skeletal
muscles observed in patients with type 2 diabetes was considered as a major factor responsible
for reduced overall glucose uptake in the body . Both insulin stimulation  and AMPK
activation  enhance glucose uptake. In addition, AMPK activation is insulin independent.
Moreover, a previous study demonstrated that pterosin A increased the glucose uptake in skeletal
muscle cells . In the present study, we screened other pterosin-type compounds to determine
whether these pterosins analogs promoted glucose uptake as well, and these pterosins exhibited
the same effects in the glucose uptake assays. These findings indicate that pterosins influence various
Only few studies have investigated the ptaquiloside content in the products of milk, soil,
and groundwater  but never the pterosin detection methods. LC-MS-MS is a powerful technique
with extremely high sensitivity and selectivity and is thus useful in various applications.
In our previous study, we investigated the concentrations of pterosins A, I, and Z present in various
fern samples collected from H. punctata, C. thalictroide, and P. revolutum which revealed the same
effects in glucose uptake assays. Therefore, the present study is the first to establish an LC–MS–MS
method to determine three compounds: pteroisns A, I, and Z. In addition, the present study proposed
Int. J. Mol. Sci. 2015, 16 2509
a method for pterosin detection that presented a clear separation on chromatograms, indicating that this
method may be useful to determine the pterosin content in ferns in Taiwan.
4. Experimental Section
4.1. Chemicals and Reagents
RPMI-1640 and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from
Gibco-BRL-Life Technologies (Grand Island, NY, USA); fetal bovine serum from Thermo Scientific
(South Logan, UT, USA); (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) MTT, H2O2,
nitrotetrazolium blue chloride (NBT), and 2',7'-dichlorofluorescein diacetate (DCFH-DA) from Sigma
Chemical Company (St. Louis, MO, USA); 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-
pyrazolo[1,5-a]pyrimidine dihydrochloride (Compound C) from Tocris Bioscience (Bristol, UK);
antibodies for phospho-AMPK-α (Thr172) and total AMPK from Cell Signaling Technology (Beverly,
MA, USA); and horseradish peroxidase-conjugated antirabbit secondary antibody from Jackson
(West Grove, PA, USA). The solvents used for column chromatography, including methanol, i-PrOH,
n-BuOH, dichloromethane, chloroform, n-hexane, ethyl acetate, acetonitrile, acetone, and formic acid,
were purchased from Merck (Darmstadt, Germany).
4.2. General Experimental Procedures
The optical rotations were measured using a JASCO P-2000 polarimeter. Infrared (IR) spectra were
measured using an Avatar-320-FT-IR spectrometer. In addition, 1D and 2D NMR spectra were
obtained by using a Bruker AM-500 (500 MHz) FT-NMR spectrometer with tetramethylsilane
as an internal standard. A VG Platform Electrospray mass spectrometer was used for high-resolution
electrospray ionization mass spectrometry (HR-ESI-MS). Column chromatography involved the use
of Diaion HP 20 (100–200 mesh, Mitsubishi Chemical Industries, Tokyo, Japan), MCI-gel CHP 20P
(75–150 μm, Mitsubishi Chemical Industries, Japan), and Cosmosil C18-OPN (75 μm, Nacalai
Tesque, Kyoto, Japan). Thin-layer chromatography involved silica gel plates (70–230 mesh, Merck),
in which a 10% sulfuric acid solution was used as a visualizing agent during heating.
4.3. Plant Material
H. punctata, C. thalictroides, N. multiflora, and P. revolutum were collected from Hehuan
Mountain, Sun Lake, Jinquashi, and Siyuan Wind Gap, Taiwan, respectively, and were identified
by Chen-Meng Kuo (Institute of Ecology and Evolutionary Biology, National Taiwan University,
Taiwan). Voucher specimens were deposited at the Department of Medicinal Chemistry, College
of Pharmacy, Taipei Medical University.
4.4. Extraction and Isolation
(1) C. thalictroides (L.) Brongn: Fresh whole plants (50 kg) were extracted three times with MeOH
at room temperature. The MeOH extract (490 g) was partitioned between with n-hexane/H2O
and EtOAc/H2O. The EtOAc fraction (265.7 g) was chromatographed on a Sephadex LH-20 with 95%
Int. J. Mol. Sci. 2015, 16 2510
EtOH to yield four fractions (CT1–4). The fraction CT1 (30 g) was further applied to MCI gels with
an H2O-MeOH gradient to yield caffeic acid methyl ester  (15, 11.7 mg), quercetin
3-O-β-D-glucopyranoside  (22, 174.2 mg), and kaempherol 3-O-β-D-glucopyranoside 
(23, 356.9 mg). The CT2 (20 g) fraction was chromatographed on an MCI-gel CHP 20P with
an H2O-MeOH gradient to produce CT2.1–2.5; subsequently, each subfraction was further purified
on silica gel with a CH2Cl2-MeOH gradient and a reverse-C18 silica gel column with an H2O–MeOH
gradient to yield p-coumaric acid  (16, 23.8 mg) and p-coumaric acid methyl ester 
(17, 7.5 mg). The CT3 (20 g) fraction was chromatographed on silica gel with a CH2Cl2–MeOH
gradient to produce pterosin A  (6, 6 mg) and pterosin Z  (7, 3.5 mg). The CT4 (30 g) fraction
was repeatedly chromatographed on a Sephadex LH-20 with an H2O–MeOH gradient to produce
CT4.1–4.4; subsequently, each subfraction was further purified on silica gel with a CH2Cl2–MeOH
gradient to yield Compound 1 (3.6 mg), Compound 2 (2 mg), Compound 3 (3 mg), pterosin D
3-O-β-D-glucopyranoside  (4, 38 mg), pteroside Z  (5, 14 mg), and 6-O-p-coumaroyl-D-
glucopyranoside  (18, 3.5 mg).
(2) H. punctata (Thumb.) Mett: Fresh whole plants (20 kg) were extracted three times with
MeOH at room temperature. The methanolic extract (1.2 kg) was evaporated and partitioned using
n-hexane/H2O to yield n-hexane (350 g) and water fractions. The water fraction was further partitioned
using EtOAc/H2O to obtain EtOAc (230 g) and water fractions (640 g). The EtOAc fraction
was chromatographed on a Sephadex LH-20 with an H2O–MeOH gradient to yield HP factions 1 to 3.
The HP-2 (35.6 g) fraction was subfractionated to HP2.1–2.3 on MCI gels with an H2O–MeOH
gradient. The HP2.2 fractions were purified using a silica gel column with n-hexane–EtOAc (3:1
to 2:1) to yield pterosin A  (6, 4 g), pterosin Z  (7, 2.3 g), pterosin D  (8, 50 mg),
and pterosin I  (9, 187 mg). The fraction HP1 (42 g) was chromatographed on a MCI-gel CHP 20P
with an H2O–MeOH gradient to produce subfractions HP1.1–1.5; each subfraction was then further
purified on silica gel with a CH2Cl2–MeOH gradient, Sephadex LH-20 with acetone, and a reverse-C18
silica gel column with an H2O–MeOH gradient to yield quercetin  (21, 1.2 g), quercetin
3-O-β-D-glucopyranoside  (22, 394 mg), and kaempherol 3-O-β-D-glucopyranoside  (23, 355 mg).
(3) N. multiflora (Roxb.) Jarret ex Morton: Fresh whole plants (20 kg) were extracted three times
with MeOH and then concentrated to a residue (460.8 g) under vacuum at 40 °C, dissolved in H2O,
and partitioned between n-hexane/H2O, EtOAc/H2O, and CH2Cl2/H2O to produce four layers.
The concentrated EtOAc extract (35.5 g) was subjected to column chromatography on a Sephadex
LH20 column and silica gel eluted with a CH2Cl2–MeOH gradient to produce kaempherol
3-O-β-D-glucopyranoside  (23, 23.6 mg), matairesinoside  (29, 15.8 mg), shikimic acid 
(19, 95.5 mg), ethyl shikimate  (20, 25 mg), and ethyl β-D-fructopyranoside  (30, 16.8 mg).
Subsequently, the CH2Cl2 fraction (22.1 g) was chromatographed on Sephadex LH-20 with 95% EtOH
to yield three fractions, which were further purified on silica gel and n-hexane-EtOAc along
with an MCI-gel column with an H2O–MeOH gradient to yield arctigenin  (24, 412.1 mg)
and arctiin  (28, 88 mg).
(4) P. revolutum (BI.) Nakai: Fresh whole plants (50 kg) were extracted with MeOH at room
temperature; after evaporation of the organic solvent, the extract was subjected to Celite CC sequential
elute with n-hexane, CH2Cl2, and MeOH to produce three fractions. The CH2Cl2 extract (151.1 g)
underwent column chromatography on the MCI gel eluted with an H2O–MeOH gradient to produce
Int. J. Mol. Sci. 2015, 16 2511
three fractions. The PR1 (12.4 g) fraction was purified on silica gel (CH2Cl2/MeOH 14:1) to produce
three subfractions PR1–3. The PR1.1 (8.7 g) fraction was applied on silica gel (n-hexane/EtOAc 1:2)
and reverse-C18 silica gel (CH3CN/H2O 20:80–30:70) to produce pterosin A  (6, 238 mg),
(2R,3R)-pterosin L  (10, 179 mg), and pterosin G  (11, 73 mg). The PR1.2 (1.5 g) fraction
was then chromatographed on silica gel (n-hexane/EtOAc 1:2) and a reverse-C18 silica gel
(CH3CN/H2O 10:90) to produce 2-hydroxypterosin C  (12, 4.3 mg). The PR2 (4.8 g) fraction
was purified on silica gel (n-hexane/EtOAc 1:2) to produce eight fractions. The PR2.5 fraction was
further purified on silica gel with hexane–EtOAc to yield (2S,3S)-pterosin C  (13, 162 mg),
(2R,3S)-pterosin C  (14, 13 mg), balanophonin  (25, 45.3 mg), pinoresinol  (26, 251 mg),
and lariciresinol  (27, 7 mg).
13-Chloro-spelosin 3-O-β-D-glucopyranoside (1): colorless oil; [α]D25 + 9.08 (c = 1.0, MeOH);
IR (KBr) vmax : 3387, 1697, and 1598 cm−1; UV (MeOH) λmax: 211, 227, and 311 nm; 1H- and
13C-NMR spectra (Table 1); HR-ESI-MS m/z: 445.1631 [M + H]+ (calcd. for C21H29ClO8, 445.1629).
(3R)-pterosin D 3-O-β-D-(3'-p-coumaroyl)-glucopyranoside (2): colorless oil; [α]D25 − 11.04
(c = 1.0, MeOH); IR (KBr) vmax: 3408, 2962, 1697, and 1603 cm−1; UV (MeOH) λmax: 218, 260,
and 310 nm; 1H- and 13C-NMR spectra (Table 1); HR-ESI-MS m/z: 556.2312 [M + Na]+ (calcd.
for C30H36O11Na, 556.2308).
(2R,3R)-pterosin L 3-O-β-D-(3'-p-coumaroyl)-glucopyranoside (3): colorless oil; [α]D25 − 26.0
(c = 1.0, MeOH); UV (MeOH) λmax: 218, 260, and 310 nm; IR (KBr) vmax: 3419, 2931, 1695,
and 1605 cm−1; 1H- and 13C-NMR spectra (Table 1); HR-ESI-MS m/z: 572.2263 [M + Na]+
(calcd. for C30H36O11Na, 572.2258).
Acid hydrolysis of compounds 1–3. Compounds 1–3 (2 mg) were treated with 2 N HCl in aqueous
MeOH (2 mL) for 4 h, and the reaction mixture was further extracted with EtOAc. The EtOAc
layer was removed in vacuo and the residue was passed through the silica gel with eluent of
n-hexane/EtOAc to yield 13-chloro-spelosin, (3R)-pterosin D and (2R,3R)-pterosin L, respectively.
The sugar was analyzed by silica gel TLC [i-PrOH–Me2CO–H2O (5:3:1)] comparison with
an authentic sample.
4.5. Pterosin Analysis by LC–MS–MS
Three pterosin compounds (pterosins A, I, and Z, 120 μg/mL and internal standard stock solution
(piromidic acid, 11.1 μg/mL) were prepared. Separation involved a reverse-phase C18 column
(Cosmosil MS-II, 3C18, 4.6 × 100 mm) under gradient elution. The mobile phase comprised a mixed
solvent system of acetonitrile/H2O/0.25% formic acid (A/B/C) at a 220-nm wavelength. The elution
conditions were maintained at 20/60/20 to 80/0/20 (A/B/C) for 0 to 25 min (linear gradient) and 80/0/20
(A/B/C) for 5 min, set at a flow rate of 0.5 mL/min with a split ratio of 1:1 in a photodiode array
and a tandem mass spectrophotometer. ESI was used for operating the ion source in the positive mode,
which was monitored using multiple reaction monitoring (MRM). The source and desolvation
temperatures were set at 120 and 350 °C, respectively. The desolvation gas flow (N2) was 600 L/h,
and the cone gas flow (N2) was 60 L/h. The capillary and cone voltages were 3.0 kV and 80 V,
respectively. The collision energies were optimized for each compound. Qualitative analysis
was achieved by daughter ion analysis.
Int. J. Mol. Sci. 2015, 16 2512
4.6. Cell Culture
C2C12 myoblast and rat pancreatic insulin-secreting (RINm5F) cells were obtained from
the American Type Culture Collection (Rockville, MD, USA). The cells were maintained in DMEM
and RPMI-1640 medium at 37 °C in an atmosphere of 5% CO2.
4.7. Biological Validation
4.7.1. Determination of Glucose Uptake in C2C12 Myocytes
Glucose uptake was determined based on the uptake of the radioactive glucose analogue
2-deoxy-D-[3H] glucose (Sigma-Aldrich, St. Louis, MO, USA) as described previously .
The C2C12 myocytes were washed with phosphate-buffered saline (PBS) and incubated in serum-free
DMEM and then treated with pterosin compounds (1 μM) at 37 °C for 1 h. The glucose uptake
was determined by adding 0.5 μCi 2-deoxy-D-[3H] glucose for 20 min. The reaction was terminated using
ice-cold PBS. After centrifugation, the cells were washed twice with ice-cold PBS to remove extrinsic
glucose and lysed with 0.1% SDS; the glucose uptake was then estimated using a scintillation counter.
4.7.2. Measurement of ROS and Cell Viability
ROS levels were determined by NBT analysis as described previously . The cells were seeded
in 24-well plates at 2 × 105 cell/well and then treated with pterosin A at various doses and incubated
for 18 h. The absorbance was recorded at 630 nm. Cell viability was measured by MTT assay.
The RINm5F cells were seeded in 24-well plates at 2 × 105 cell/0.5 mL and grown for 3 days
for adherence. Subsequently, 50 μL of MTT solution (1 mg/mL in PBS) were added to each well
for 2 h at 37 °C. The medium was aspirated, and 200 μL of DMSO were added. After the formazan
product was dissolved, the absorbance at 570 nm was measured using a spectrophotometer.
4.7.3. Immunofluorescence Study
Intracellular oxidation was analyzed using a fluorometric assay with DCFH-DA. DCFH-DA
transports across the cell membrane and deacetylates by cellular esterases to nonfluorescent DCFH,
which quickly oxidizes to highly fluorescent DCF by ROS . The RINm5F cells (3 × 105 cell/well
in 12 wells) were exposed to different treatments for varying durations after adhering for 3 days. In
total, 10 μM of DCFH-DA was added with no serum medium for 20 min. The cells were washed two
times with PBS and then subjected to DCF fluorescence by using fluorescence microscopy at 488-nm
excitation (argon laser) and 515-nm long-pass emission.
4.7.4. Western Blot Analysis
Total cellular proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride
membranes for immunoblotting. Nonspecific binding was blocked using a blocking buffer containing
5% fat-free milk powder in Tris-buffered saline with 1% Tween-20 for 1 h at room temperature.
The lysates were incubated with monoclonal antibodies against phospho-AMPK and total AMPK.
Int. J. Mol. Sci. 2015, 16 2513
The protein expression was determined using an enhanced chemiluminescence kit (Amersham
International, Amersham, UK).
4.8. Statistical Analysis
The significance of various treatments was determined by one-way analysis of variance. Data were
expressed as mean ± SEM. Statistically significant differences were considered at p < 0.05.
This paper reports the isolation of pterosin-type compounds (discovered in three fern species:
H. punctata, C. thalictroides, and P. revolutum), that have the same effects on glucose uptake assays
as known isolated pterosins. In addition, three new compounds were isolated from the C. thalictroides
fern. Moreover, the present study is the first to demonstrate that pterosin A has protective effects
on insulin secretion in cells against ROS- and palmitate-induced cell damage. We provide information
regarding these signals with pterosin-like UV spectra in the chromatographic system, which is vital to
determine the pterosin-type constituents in ferns.
Supplementary materials can be found at http://www.mdpi.com/1422-0067/16/02/2947/s1.
We thank Hsien-Chang Chang for help in identifying the desired plant material. The authors
are grateful to Shu-Yun Sun (Taipei Regional Analytical Instrumentation Center, NSC) for measuring
the HR-ESI-MS spectra and Shwu-Hui Wang (Core Facility Center, Office of Research and
Development, Taipei Medical University) for measuring the NMR spectra.
Feng-Lin Hsu designed the experiment and contributed to manuscript preparation; Chen-Yu Chen
carried out the experiment and wrote the manuscript; Wei-Jan Huang, Po-Shiuan Hsieh, Yenshou Lin
and Fu-Yu Chiu performed and analyzed the bioassay and LC-MS-MS.
Conflicts of Interest
The authors declare no conflict of interest.
1. Ho, R.; Teai, T.; Bianchini, J.-P.; Lafont, R.; Raharivelomanana, P. Working with ferns: Issues
and applications. In Ferns: From Traditional Uses to Pharmaceutical Development, Chemical
Identification of Active Principles; Fernández, H., Revilla, M.A., Kumar, A., Eds.; Springer:
New York, NY, USA, 2010; pp. 321–346.
Int. J. Mol. Sci. 2015, 16 2514
2. Hikino, H.; Takahashi, T.; Arihara, S.; Takemoto, T. Structure of pteroside B, glycoside
of Pteridium aquilinum var latiusculum. Chem. Pharm. Bull. 1970, 18, 1488–1489.
3. Yoshihira, K.; Fukuoka, M.; Kuroyannagi, M.; Natori, S.; Umeda, M.; Morohoshi, T.;
Enomoto, M.; Saito, M. Chemical and toxicological studies on bracken fern, Pteridium aquilinum
var. latiusculum. I. Introduction, extraction and fractionation of constituents, and toxicological
studies including carcinogenicity tests. Chem. Pharm. Bull. (Tokyo) 1978, 26, 2346–2364.
4. Hsu, F.L.; Huang, C.F.; Chen, Y.W.; Yen, Y.P.; Wu, C.T.; Uang, B.J.; Yang, R.S.; Liu, S.H.
Antidiabetic effects of pterosin A, a small-molecular-weight natural product, on diabetic mouse
models. Diabetes 2013, 62, 628–638.
5. Xiong, F.L.; Sun, X.H.; Gan, L.; Yang, X.L.; Xu, H.B. Puerarin protects rat pancreatic islets from
damage by hydrogen peroxide. Eur. J. Pharmacol. 2006, 529, 1–7.
6. Kaneto, H.; Kawamori, D.; Matsuoka, T.-A.; Kajimoto, Y.; Yamasaki, Y. Oxidative stress
and pancreatic β-cell dysfunction. Am. J. Ther. 2005, 12, 529–533.
7. Shimabukuro, M.; Ohneda, M.; Lee, Y.; Unger, R.H. Role of nitric oxide in obesity-induced β cell
disease. J. Clin. Investig. 1997, 100, 290–295.
8. Fonseca, S.G.; Gromada, J.; Urano, F. Endoplasmic reticulum stress and pancreatic β-cell death.
Trends Endocrinol. Metab. 2011, 22, 266–274.
9. Rutter, G.A.; da Silva Xavier, G.; Leclerc, I. Roles of 5'-AMP-activated protein kinase (AMPK)
in mammalian glucose homoeostasis. Biochem. J. 2003, 375, 1–16.
10. Eto, K.; Yamashita, T.; Matsui, J.; Terauchi, Y.; Noda, M.; Kadowaki, T. Genetic manipulations
of fatty acid metabolism in β-cells are associated with dysregulated insulin secretion. Diabetes
2002, 51, S414–S420.
11. Richards, S.K.; Parton, L.E.; Leclerc, I.; Rutter, G.A.; Smith, R.M. Over-expression
of AMP-activated protein kinase impairs pancreatic β-cell function in vivo. J. Endocrinol. 2005,
12. Kuraishi, T.; Murakami, T.; Taniguchi, T.; Kobuki, Y.; Maehashi, H.; Tanaka, N.; Saiki, Y.;
Chen, C.M. Chemical and chemotaxonomical studies of ferns. LIV. Pterosin derivatives
of the genus Microlepia (Pteridaceae). Chem. Pharm. Bull. 1985, 33, 2305–2312.
13. Nyblom, H.K.; Sargsyan, E.; Bergsten, P. AMP-activated protein kinase agonist dose dependently
improves function and reduces apoptosis in glucotoxic β-cells without changing triglyceride
levels. J. Mol. Endocrinol. 2008, 41, 187–194.
14. Lin, N.; Chen, H.; Zhang, H.; Wan, X.; Su, Q. Mitochondrial reactive oxygen species (ROS)
inhibition ameliorates palmitate-induced INS-1 β cell death. Endocrine 2012, 42, 107–117.
15. Banerjee, J.; Datta, G.; Duita, C.P.; Som, U.K. Chemical constituents of Nephrolepis tuberosa.
J. Indian Chem. Soc. 1988, 65, 881–882.
16. Carlsson, C.; Håkan Borg, L.A.; Welsh, N. Sodium palmitate induces partial mitochondrial
uncoupling and reactive oxygen species in rat pancreatic islets in vitro. Endocrinology 1999, 140,
17. Evans, J.L.; Goldfine, I.D.; Maddux, B.A.; Grodsky, G.M. Are oxidative stress-activated signaling
pathways mediators of insulin resistance and β-cell dysfunction? Diabetes 2003, 52, 1–8.
Int. J. Mol. Sci. 2015, 16 2515
18. Wang, X.; Li, H.; de Leo, D.; Guo, W.; Koshkin, V.; Fantus, I.G.; Giacca, A.; Chan, C.B.; Der, S.;
Wheeler, M.B. Gene and protein kinase expression profiling of reactive oxygen species-associated
lipotoxicity in the pancreatic β-cell line MIN6. Diabetes 2004, 53, 129–140.
19. Inoki, K.; Zhu, T.; Guan, K.-L. TSC2 mediates cellular energy response to control cell growth
and survival. Cell 2003, 115, 577–590.
20. Zierath, J.R.; He, L.; Guma, A.; Odegoard Wahlstrom, E.; Klip, A.; Wallberg-Henriksson, H.
Insulin action on glucose transport and plasma membrane GLUT4 content in skeletal muscle from
patients with NIDDM. Diabetologia 1996, 39, 1180–1189.
21. Wang, Q.; Somwar, R.; Bilan, P.J.; Liu, Z.; Jin, J.; Woodgett, J.R.; Klip, A. Protein kinase B/Akt
participates in GLUT4 translocation by insulin in L6 myoblasts. Mol. Cell Biol. 1999, 19,
22. Czech, M.P.; Corvera, S. Signaling mechanisms that regulate glucose transport. J. Biol. Chem.
1999, 274, 1865–1868.
23. Jensen, P.H.; Jacobsen, O.S.; Hansen, H.C.; Juhler, R.K. Quantification of ptaquiloside
and pterosin B in soil and groundwater using liquid chromatography-tandem mass spectrometry
(LC–MS/MS). J. Agric. Food Chem. 2008, 56, 9848–9854.
24. Kelley, C.J.; Harruff, R.C.; Carmack, M. Polyphenolic acids of Lithospermum ruderale. II.
Carbon-13 nuclear magnetic resonance of lithospermic and rosmarinic acids. J. Org. Chem. 1976,
25. Zhang, H.L.; Nagatsu, A.; Okuyama, H.; Mizukami, H.; Sakakibara, J. Sesquiterpene glycosides
from cotton oil cake. Phytochemistry 1998, 48, 665–668.
26. Hikino, H.; Takahashi, T.; Takemoto, T. Structure of pteroside A and C, glycosides of
Pteridium aquilinum var latiusculum. Chem. Pharm. Bull. 1972, 20, 210–212.
27. Tanaka, N.; Satake, T.; Takahashi, A.; Mochizuki, M.; Murakami, T.; Saiki, Y.; Yang, J.Z.;
Chen, C.M. Chemical and chemotaxonomical studies of ferns. XXXIX. Chemical studies
on the constituents of Pteris bella Tagawa and Pteridium aquilinum subsp. wightianum (Wall)
Shich. Chem. Pharm. Bull. 1982, 30, 3640–3646.
28. Hikino, H.; Takahashi, T.; Takemoto, T. Structure of pterosides Z and D, glycosides of
Pteridium aquilinum var latiusculum. Chem. Pharm. Bull. 1971, 19, 2424–2425.
29. Shimomura, H.; Sashida, Y.; Adachi, T. Phenylpropanoid glucose esters from Prunus buergeriana.
Phytochemistry 1988, 27, 641–644.
30. Markham, K.R.; Ternai, B.; Stanley, R.; Geiger, H.; Mabry, T.J. Carbon-13 NMR studies
of flavonoids. III. Naturally occurring flavonoid glycosides and their acylated derivatives.
Tetrahedron 1978, 34, 1389–1397.
31. Abe, F.; Yamauchi, T. Lignans from Trachelospermum asiaticum (Tracheolospermum. II).
Chem. Pharm. Bull. 1986, 34, 4340–4345.
32. Talapatra, B.; Das, A.K.; Talapatra, S.K. On the chemistry of Indian Orchidaceae plants. Part V.
Defuscin, a new phenolic ester from Dendrobium fuscescens: Conformation of shikimic acid.
Phytochemistry 1988, 28, 290–292.
33. Xie, C.; Li, Z.; Qu, J.; Sun, B.; Lou, H. Chemical constituents of two liverworts Dumortiera hirsute
and Pallavicinia ambigua. Chin. Pharm. J. 2007, 42, 1706–1708.
Int. J. Mol. Sci. 2015, 16 2516
34. Kuang, H.X.; Kasai, R.; Ohtani, K.; Liu, Z.S.; Yuan, C.S.; Tanaka, O. Chemical constituents
of pericarps of Rosa davurica Pall., a traditional Chinese medicine. Chem. Pharm. Bull. 1989, 37,
35. Rahman, M.M.A.; Dewick, P.M.; Jackson, D.E.; Lucas, J.A. Lignans of Forsythia intermedia.
Phytochemistry 1990, 29, 1971–1980.
36. Yoshihira, K.; Fukuoka, M.; Kuroyanagi, M.; Natori, S. Further characterization of 1-indanone
derivatives from bracken, Pteridium aquilinum var latiusculum. Chem. Pharm. Bull. 1972, 20,
37. Tanaka, N.; Murakami, T.; Saiki, Y.; Chen, C.M.; Gomez, P.L.D. Chemical
and chemotaxonomical studies of ferns. XXXVII. Chemical studies on the constituents of Costa
Rican ferns. 2. Chem. Pharm. Bull. 1981, 29, 3455–3463.
38. Warashina, T.; Nagatani, Y.; Noro, T. Further constituents from the bark of Tabebuia impetiginosa.
Phytochemistry 2005, 66, 589–597.
39. Nishibe, S.; Tsukamoto, H.; Hisada, S. Effects of O-methylation and O-glucosylation on carbon-13
nuclear magnetic resonance chemical shifts of matairesinol, (+)-pinoresinol and (+)-epipinoresinol.
Chem. Pharm. Bull. 1984, 32, 4653–4657.
40. El Gamal, A.A.; Takeya, K.; Itokawa, H.; Halim, A.F.; Amer, M.M.; Saad, H.E.A. Lignan
bis-glucosides from Galium sinaicum. Phytochemistry 1997, 45, 597–600.
41. Park, C.E.; Kim, M.J.; Lee, J.H.; Min, B.I.; Bae, H.; Choe, W.; Kim, S.S.; Ha, J. Resveratrol
stimulates glucose transport in C2C12 myotubes by activating AMP-activated protein kinase.
Exp. Mol. Med. 2007, 39, 222–229.
42. Choi, H.S.; Kim, J.W.; Cha, Y.N.; Kim, C. A quantitative nitroblue tetrazolium assay for determining
intracellular superoxide anion production in phagocytic cells. J. Immunoass. Immunochem. 2006, 27,
43. Rosenkranz, A.R.; Schmaldienst, S.; Stuhlmeier, K.M.; Chen, W.; Knapp, W.; Zlabinger, G.J.
A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescein-diacetate.
J. Immunol. Methods 1992, 156, 39–45.
© 2015 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 license