Chemical Constituents Analysis and Antidiabetic Activity Validation of Four Fern Species from Taiwan

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 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.

insulin secretion and AMPK activation, which may potentiate glycolipotoxicity-induced cell death [11]. Therefore, the AMPK pathway is crucial for regulating glucose homeostasis and is a major target of therapy for type 2 diabetes.

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. 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 1 H-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 1 H-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 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 [12] 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 13 C-NMR spectra with those of references. The absolute configuration of aglycone was determined by the specific rotation with a value of [α]D 24 + 82.6 (c = 0.7, MeOH) similar to that of spelosin ([α]D 22 + 83.3 (c = 0.7, MeOH)) [12]. Consequently, Compound 1 was determined as 13-chloro-spelosin 3-O-β-D-glucopyranoside.

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. 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.

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).

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 [7]. 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 [13]. 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. 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.

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 AMPK activation. 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-4carboxamide ribonucleotide (AICAR) was used as a positive control). Data are presented as mean ± SEM. * p < 0.05; *** p < 0.001.

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 [14]. 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.

Discussion
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 triterpenes and steroids from Nephrolepdiaceae [15]. 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][17][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 [13]. 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 [19]. 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 [20]. Both insulin stimulation [21] and AMPK activation [22] 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 [4]. 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 biological processes.
Only few studies have investigated the ptaquiloside content in the products of milk, soil, and groundwater [23] 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 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.

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.

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.

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.

Determination of Glucose Uptake in C2C12 Myocytes
Glucose uptake was determined based on the uptake of the radioactive glucose analogue 2-deoxy-D-[ 3 H] glucose (Sigma-Aldrich, St. Louis, MO, USA) as described previously [41]. 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-[ 3 H] 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.

Measurement of ROS and Cell Viability
ROS levels were determined by NBT analysis as described previously [42]. The cells were seeded in 24-well plates at 2 × 10 5 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 × 10 5 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.

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 [43]. The RINm5F cells (3 × 10 5 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.

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. The protein expression was determined using an enhanced chemiluminescence kit (Amersham International, Amersham, UK).

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.

Conclusions
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.