Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
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Selaginellin and biflavonoids as protein tyrosine phosphatase 1B inhibitors from Selaginella tamariscina and their glucose uptake stimulatory effects Phi-Hung Nguyen a,d, , Da-Jung Ji a, , Yu-Ran Han b, Jae-Sue Choi b, Dong-Young Rhyu c, Byung-Sun Min a,⇑, Mi-Hee Woo a,⇑ a
College of Pharmacy, Drug Research and Development Center, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea Department of Food Science & Nutrition, Pukyong National University, 599-1 Daeyeon-3 Dong, Namgu, Busan 608-737, Republic of Korea c Department of Oriental Medicine Resources, Mokpo National University, Muan-gun 534-729, Republic of Korea d Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Hanoi, Viet Nam b
a r t i c l e
i n f o
Article history: Received 14 January 2015 Revised 30 March 2015 Accepted 2 April 2015 Available online xxxx Keywords: Selaginella tamariscina Biflavonoids PTP1B inhibitors 2-NBDG
a b s t r a c t As part of an ongoing search for new antidiabetic agents from medicinal plants, the methanol extract of the aerial parts of Selaginella tamariscina was found to possess stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. Thus, bioassay-guided isolation of this active extract yielded two new compounds (1 and 2) along with five known biflavonoids (3–7). Their structures were elucidated by extensive analysis of spectroscopic and physicochemical data. The absolute configuration of compound 2 was determined by specific rotation and CD data analysis. All isolates exhibited potent inhibitory effects on PTP1B enzyme with IC50 values ranging from 4.5 ± 0.1 to 13.2 ± 0.8 lM. Furthermore, the isolates (1–7) showed significant stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. Of these, compounds (1, 6, and 7) which exhibited mixed-competitive inhibition modes against PTP1B, showed potent stimulatory effects on 2-NBDG uptake. This result indicated the potential of these biflavonoids as lead molecules for development of antidiabetic agents and the beneficial use of S. tamariscina against hyperglycemia. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Diabetes mellitus, long regarded as a disease of minor consequence to world health, occupies now one of the main causes of serious maladies in the 21st century. The number of people with diabetes anticipate rising from current estimate of 150–220 million in 2010, and 300 million in 2025.1 Among the diseases, type 2 diabetes is characterized by chronic hyperglycemia associated with abnormalities in carbohydrate, fat, and protein metabolism. The American Diabetes Association reported that about 22.3 million people in the United States have diabetes, accounting for 7% of the population.2a Thus, great efforts are being made in the search for new therapeutic agents to stem its progress.2b Protein tyrosine phosphatase (PTP) superfamily coordinates with protein tyrosine kinases to regulate a vast array of cellular
⇑ Corresponding authors. Tel.: +82 53 850 3613 (B.-S.M); tel.: +82 53 850 3620; fax: +82 53 850 3602 (M.-H.W). E-mail addresses: [email protected] (B.-S. Min), [email protected] (M.-H. Woo). Authors contributed equally to this work.
functions, including proliferation, differentiation, apoptosis and motility. Of the various PTPs, PTP1B plays a critical role in regulating glucose homeostasis and body weight by acting as a key negative regulator of insulin and leptin signaling pathway, respectively.3 Its overexpression has been shown to inhibit the insulin receptor (IR) signaling cascade, and increased expression of PTP1B occurs in insulin-resistant states,4 while PTP1B knockout mice have been shown to increase insulin sensitivity and obesity resistance.5 Leptin, which is secreted from adipose tissue, also controls food intake and weight loss in the hypothalamus. Furthermore, recent evidence has shown that the leptin signaling pathway can be attenuated by PTPs, and there is evidence that PTP1B is also involved in this process.6 PTP1B was shown subsequently to bind and dephosphorylate Janus kinase 2 (JAK2), which is downstream of leptin receptor. Thus, the resistance to diet-induced obesity observed in PTP1B/ mice is likely to be associated with increased energy expenditure owing to enhanced leptin sensitivity.7 Moreover, pretreatment of leptin-resistant rats with a potent and selective PTP1B inhibitor results in a marked improvement in leptin-dependent suppression of food intake.8
http://dx.doi.org/10.1016/j.bmc.2015.04.007 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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Thus, PTP1B inhibitors could be useful in treating type 2 diabetes as well as obesity.9 Selaginella, also known as spikemoss, is the only surviving genus within the plant family Selaginellaceae. Selaginella includes more than 700 species widely distributed around the globe.10 Among the genus, Selaginella tamariscina (Beauv.) Spring was first recorded by ‘Shen Nong Ben Cao Jing’ (The Divine Farmer’s Materia Medica) in 2737 BC. It has been used in oriental medicine to treat inflammation, amenorrhea, dysmenorrhea, metrorrhagia, hematuria, prolapse of the anus, abdominal lumps in women, chronic hepatitis and hyperglycemia.11 Moreover, S. tamariscina has been reported to lower blood glucose levels and to facilitate the repair of pancreatic islet b-cells injured by alloxan.12 A number of flavonoids, lignans, selaginellins, and phenols were reported as chemical constituents of S. tamariscina.13 As part of an ongoing investigation on the discovery of new antidiabetic agents from medicinal plants, the methanol extract of the aerial parts of S. tamariscina was found to increase in vitro glucose uptake in 3T3-L1 adipocytes (1.13-fold induction at a concentration of 10 lg/mL). Thus, phytochemical investigation of this medicinal plant using chromatographic methods led to the isolation of two new natural products (1 and 2) and five known biflavones (3–7). Furthermore, compounds 1–7 were evaluated for their stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells as well as their inhibitory effects on PTP1B enzyme. In this Letter, the purification, structural determination, and potential antidiabetic properties of these isolates are discussed.
2. Results and discussion Bioassay-guided isolation of the methanol extract of the aerial part of S. tamariscina has led to the isolation of two new compounds (1 and 2) and five known (3–7) biflavonoid derivatives as active principles (Fig. 1). The known compounds were elucidated as amentoflavone (3),14 robustaflavone (4),14 cupressuflavone (5),15 taiwaniaflavone (6),16 and 3,800 -biapigenin (7)17 based on NMR spectroscopic and MS data analysis and comparison with literature values. Compound 1 was obtained as a red amorphous powder. Its IR spectrum indicated absorption bands for hydroxy group (3394 cm1), alkynyl (2203 cm1), unsaturated carboxyl (1674 cm1), and aromatic ring (1077–1045 cm1). A molecular formula of C22H16O5 was determined from the molecular ion peak at m/z 360.1026 [M]+ obtained by HRFABMS. The UV spectrum exhibited absorption maxima at 264, 300, 322, and 420 nm, characteristic values for a selaginellin analogue.18 The assignment of the 1H and 13C NMR data (Table 1) was supported by 2D-NMR techniques (Supplementary data). In the 1H and 13C NMR spectra of 1, the typical signals of an alkynyl substituent at dC 84.7 and 100.7, an ABspin system at dH 7.69 (1H, d, J = 8.0 Hz, H-3) and 7.31 (1H, d, J = 8.0 Hz, H-4) for the ortho-tetrasubstituted A-ring, and two AA0 BB0 systems with [dH 6.97 and 6.62 (each 2H, dd, J = 2.0, 8.8 Hz)] and [dH 6.75 and 6.53 (each 2H, dd, J = 2.0, 8.4 Hz)] for the para-substituted B- and C-rings, were apparent. These assignment resembled those of selaginellin K and selaginellin L,19 however, signals for the aldehyde or methyl group was not presented, instead of that, a carboxyl carbon signal (dC 165.5) was found in the 13C NMR spectrum of compound 1 (Supplementary data). The alkynyl group was the linkage between the B-ring and the A-ring at C-1 based on HMBC correlations between H-20 /H-60 and C-10, and H-3 and C-1/C-9. The C-ring was connected to the A-ring at C-5 as supported by the HMBC correlations between H-200 / H-600 and C-5 and H-4 and C-100 (Fig. 2). A hydroxymethyl group [dH 4.91 (2H, s, H-8) and dC 63.7 (C-8)] was assigned to C-2 by the aid of HSQC and HMBC
experiments showing correlations from H-3 to C-8 and from H-8 to C-1, C-2, and C-9 (Fig. 2). Hence, C-6 (142.4) in the A-ring was the only position left for the carboxylic group (dC 165.5, C-7). On the basis of the above evidence, compound 1 (selariscinin D) was thus characterized as 40 -hydroxy-4-(hydroxymethyl)-3-[(4-hydroxyphenyl)ethynyl]biphenyl-2-carboxylic acid. Compound 2 was obtained as a yellow amorphous powder. Its HRFABMS showed a molecular ion peak at m/z 540.1060 [M]+ (calcd for m/z 540.1056), consistent with a molecular formula of C30H20O10. The IR spectrum exhibited absorption bands at 3331 cm1 (OH), 1670 cm1 (C@O), 1593, and 1242–1045 cm1 (aromatic ring). Its UV spectrum showed absorption maxima at 221, 286, and 329 nm, characteristic values for a biflavonoid analogue.20 A negative optical rotation value 6.3 (c 0.25, MeOH) was observed for compound 2. The 1H and 13C NMR spectra of 2 displayed characteristic signals assignable for an AMX spin system of a flavanone skeleton21 at dH 5.42 (1H, dd, J = 12.8, 2.8 Hz, H-2), 3.18 (1H, dd, J = 12.8, 16.8 Hz, H-3ax), and 2.76 (1H, dd, J = 2.8, 16.8, H-3eq), with corresponding carbon signals for C-2 (dC 80.6) and C-3 (dC 44.1), and the ketone carbon for C-4 (dC 198.0). The olefinic proton at dH 6.60 (1H, s), with its corresponding carbon (dC 103.4, C-300 ), showed HMBC correlations with an oxygenated quaternary carbon at dC 166.3 (C-200 ) and a carbonyl carbon at dC 184.5 (C-400 ) (Table 1 and Fig. 2), indicating the presence of a flavone skeleton.20 The 13C NMR spectrum together with MS data indicated a flavone–flavanone based biflavonoid structure22 for 2 with 30 carbon resonances representing two carbonyl (dC 184.5 and 198.0), two aliphatic (dC 80.6 and 44.1), and 26 olefinic and aromatic carbons (Table 1). Furthermore, an AB spin system at dH 7.41 and 7.05 (each 1H, d, J = 8.4 Hz, H-500 /H-600 ) for the ortho-substituted A0 -ring and a AA0 BB0 spin system at dH 7.55 and 6.79 (each 2H, dd, J = 2.4, 8.8 Hz) for the para-substituted B0 -ring of the flavone skeleton were apparent. In addition, two meta-coupled proton signals at H-6 and H-8 in ring A at dH 5.87 and 5.90 (each 1H, d, J = 2.0 Hz) with two corresponding carbons at dC 97.2 and 96.4, respectively, and two singlet proton signals at dH 6.38 (1H, s, H-50 ) and 7.44 (1H, s, H-60 ), assignable to two para-coupled aromatic protons in ring B of 2 were presented (Table 1 and Supplementary data). All of these assignments and the arrangement of the rings were confirmed by 1H–1H gCOSY, 1 H–13C gHMQC and 1H–13C gHMBC experiments (Fig. 2 and Supplementary data). In the HMBC spectrum, the correlations between H-2 (dH 5.42) and C-10 (dC 131.2) and C-20 (dC 162.4), between H-30 (dH 6.38) and C-20 (dC 162.4), C-40 (dC 163.8), and C-50 (dC 103.4), and between H-60 (dH 7.44) and C-2 (dC 80.6), C-10 (dC 131.2), and C-20 (dC 162.4), indicated a 1,2,4,5-tetrasubstitued B-ring with two hydroxy groups attached at C-20 (162.4) and C-40 (163.8), respectively. Ring B was shown to be connected to ring A at C-50 3C-800 axis via three bond-coupled correlation between H60 (dH 7.44) and C-800 (dC 120.8) as well as four bond-coupled correlations between H-60 and C-700 (dC 157.4) and C-900 (dC 156.6) (Fig. 2 and Supplementary data). The absolute configuration at C2 was deduced to be (S) by the presence of a negative Cotton effect (8.7) at 286 nm and a positive Cotton effect (+4.5) at 330 nm in its CD spectrum.22,23 Thus, compound 2 (selariscinin E) was established as (2S)-2,3-dihydro-5,7,700 ,20 ,40 ,4000 -hexahydroxy-(50 –800 )biflavone, which is a new natural biflavonoid. As insulin resistance is one of critical causes in the pathogenesis of type 2 diabetes, reconstitution of insulin sensitivity is one of the key strategies for the treatment of this disease.24 However, some thiazolidinediones as a representative class of insulin sensitizers have been withdrawn from the market due to severe side effects, which include edema, weight gain, and the increased risk of heart failure.25 Therefore, there is an urgent demand to discover new anti-diabetic agents acting as insulin mimics and/or insulin sensitizers. 2-NBDG has been reported as an useful fluorescent-tagged
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HO
3' 3''
HO 4''
C
5''
6''
OH
2'
HO 1''
7
O
5 4
3
9
1
A
B
1'
6
4' 5'
7 6
6'
10
HO
8
9
A 5
O
2'''
B'
1'''
2''
A'
O
5''
10'' 4''
HO
O HO
O HO O
O O
6'''
OH
5'''
O
3
4
OH
OH
O HO
HO
OH
O
2 O
OH OH
OH
1
OH OH
6''
C' 3''
OH
7''
8'' 9''
3''' 4'''
HO
5'
O
O
OH OH
4'
6'
3 4
8
OH
3'
B
2
C
OH
2
O
10
2' 1'
OH
HO
OH
OH O HO
OH
OH
O
O
O OH
OH
O
HO
O
OH
OH
O O
O HO
5
OH O
6
HO
7
Figure 1. Chemical structure of compounds 1–7 isolated from Selaginella tamariscina.
Table 1 H (400 MHz) and and 2 in CD3OD 1
Position
1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 100 200 300 400 500 600 700 800 900 1000 1000 2000 3000 4000 5000 6000
13
C (100 MHz) NMR spectroscopic data for the new compounds 1
Selariscinin D (1)
Selariscinin E (2)
dH (J in Hz)
dC
7.69 (1H, d, 8.0)
123.8 143.0 129.0
7.31 (1H, d, 8.0)
4.91 (2H, s)
6.97 (1H, dd, 8.8, 2.0) 6.62 (1H, dd, 8.8, 2.0) 6.62 (1H, dd, 8.8, 2.0) 6.97 (1H, dd, 8.8, 2.0) 6.75 (1H, dd, 8.4, 2.0) 6.53 (1H, dd, 8.4, 2.0) 6.53 (1H, dd, 8.4, 2.0) 6.75 (1H, dd, 8.4, 2.0)
131.0 143.6 142.7 165.5 63.7 84.7 100.7 114.6 134.2 116.5 159.6 116.5 134.2 133.1 131.2 115.9 158.0 115.9 131.2
dH (J in Hz)
dC
5.42 (1H, dd, 12.8, 2.8) 3.18 (1H, dd, 12.8, 16.8) 2.76 (1H, dd, 2.8, 16.8)
80.6 44.1
5.87 (1H, d, 2.0) 5.90 (1H, d, 2.0)
6.38 (1H, s)
7.44 (1H, s)
6.60 (1H, s) 7.41 (1H, d, 8.4) 7.05 (1H, d, 8.4)
7.55 (1H, dd, 8.8, 2.4) 6.79 (1H, dd, 8.8, 2.4) 6.79 (1H, dd, 8.8, 2.4) 7.55 (1H, dd, 8.8, 2.4)
198.0 168.5 97.2 165.6 96.4 165.1 103.5 131.2 162.4 100.2 163.8 103.4 128.9 166.3 103.4 184.5 131.3 117.0 157.4 120.8 156.6 106.4 123.4 129.6 117.0 162.7 117.0 129.6
glucose probe for discovering insulin mimetic compounds.26 Thus, the stimulatory effects of compounds 1–7 were further evaluated on glucose uptake using 2-NBDG in 3T3-L1 adipocyte cells.27 As presented in Figure 4, all the isolates showed dose-dependent stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. At a concentration of 10 lM, compounds 1, 6, and 7 significantly stimulated 2-NBDG uptake by 1.24 ± 0.05-, 1.47 ± 0.08-, and 1.44 ± 0.06-fold inductions as compared with the control (DMSO), respectively. In this assay, the positive control (insulin) showed
an induction of 1.54 ± 0.05-fold at a concentration of 100 nM. The cytotoxic effects of the isolated compounds 1–7 were also evaluated in the absence of 2-NBDG using MTT assay. All isolates did not show cytotoxicity to the cells at both concentrations of 5 and 10 lM (Supplementary data). Thus, the stimulatory effects of these compounds on 2-NBDG uptake were not affected by any cytotoxicity. The biapigenins, taiwaniaflavone (6) and 3,800 -biapigenin (7) with a linkage at C-3 position, showed more potent effects than the other biflavonoids (2–5). GLUT4 glucose transport translocation is essential for inducible glucose uptake into the plasma membrane in muscle and adipocyte cells. This process depends mainly on the regulation of two physiological pathways: the AMP-activated protein kinase (AMPK) pathway and the insulin signaling pathway.28a In the insulin signaling pathway, binding of insulin to the insulin receptor on adipocytes and muscle cells triggers the recruitment and phosphorylation of insulin receptor substrate (IRS), which forms a docking site for PI3K at the membrane. When docked, PI3K activates phosphoinositide-dependent protein kinase 1 (PDK1), which in turn mediates the phosphorylation of both Akt (also known as protein kinase B) at Thr308 and ribosomal protein 70 kDa S6 kinase 1 (p70s6k).28 Thus, activated Akt facilitates glucose uptake in adipocytes and muscle cells by allowing the translocation of GLUT4, the insulin-responsive isoform of the glucose transporter located in intracellular storage vesicles, to the plasma membrane.28 As a negative regulator, PTP1B dephosphorylates IR and IRS in the case of insulin signaling and also tyrosine kinase JAK2 (Janus kinase 2) in the leptin signaling pathway.29 Therefore, inhibition of the activity or the expression level of PTP1B can facilitate the insulin signaling pathway, leading to an increase in glucose uptake in adipocytes. In this regard, compounds 1–7 were tested for their inhibitory effects on PTP1B enzyme using ursolic acid and RK-682 as positive controls30 (Table 2). All isolates potentially inhibited PTP1B activity in a dose-dependent manner with IC50 values ranging from 4.5 ± 0.1 to 13.2 ± 0.8 lM. Among the isolates, compounds 6 and 7 exhibited equivalent activities with IC50 values of 5.4 ± 0.2 and 4.5 ± 0.1 lM to the positive controls, ursolic acid (IC50 = 3.4 ± 0.1 lM) and RK682 (IC50 = 4.5 ± 0.2 lM). As second potent inhibitors, the biapigenin analogues (3–5) exhibited IC50 values of 7.4 ± 0.5, 6.1 ± 0.1, and 9.6 ± 0.3 lM, respectively. The new selaginellin derivative (1) and flavone–flavanone based biflavonoid (2) displayed significant activity with IC50 values of 13.2 ± 0.8 and 9.8 ± 0.6 lM, respectively.
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HO
OH HO
O
HO
OH OH
O
HO
O OH OH
O
O
HO
1
2 1H-1H COSY
correlations
1H-13C HMBC
correlations
1H-13C four-bond
HMBC correlations
Figure 2. 1H–1H COSY (bold lines) and 1H–13C HMBC correlations (arrows) for the new compounds, selariscinins D (1) and E (2).
Table 2 Inhibitory effects of isolated compounds (1–7) on PTP1B enzyme Compound
Inhibitory effect (IC50, lM)a
(Ki value, lM)
Inhibition type
1 2 3 4 5 6 7 Ursolic acidb RK-682b
13.2 ± 0.8 9.8 ± 0.6 7.4 ± 0.5 6.2 ± 0.1 9.6 ± 0.3 5.4 ± 0.2 4.5 ± 0.1 3.4 ± 0.1 4.5 ± 0.2
14.5 ± 0.7 10.4 ± 0.6 7.8 ± 0.5 6.8 ± 0.3 10.7 ± 0.4 4.5 ± 0.3 4.6 ± 0.1 6.4 ± 0.2 —c
Mixed-competitive Uncompetitive Mixed-competitive Mixed-competitive Competitive Mixed-competitive Mixed-competitive Mixed-competitive —
a Results are expressed as IC50 values (lM), determined by regression analysis and expressed as the means ± SD of three replicates. b Positive controls. c Data not determined.
In kinetic study, the Dixon plots and Lineweaver-Burk plots (Corish-Bowden 1974 and Dixon 1953) were used in order to determine the type of enzyme inhibition and the dissociation or inhibition constant (Ki) for an enzyme–inhibitor complex.. The results showed that the inhibition modes of compounds 1, 3, 4, 6, and 7, as determined using Lineweaver–Burk and Dixon plots (Fig. 3 and Supplementary data), were mixtures of inhibition types because the Vmax values decreased with concentration without changing the Km for the substrate, and the lines did not intersect on the y-axis in the Lineweaver–Burk plot and the x-axis in Dixon plot. However, both experiments showed that compound 5 exhibited competitive inhibition because the pattern of straight lines with intersecting y intercepts (Fig. 3A) is characteristic of competitive inhibitor. This indicated that 5 may directly bind to the active binding site of the enzyme to inhibit the activity of PTP1B. In contrast, increasing the substrate concentrations resulted in a series of lines that did not intersect on the y-axis in the Lineweaver–Burk plot and the x-axis in Dixon plots, but paralleled each other, confirming that compound 2 shows uncompetitive inhibition. Similar structure–activity relationships were observed in PTP1B and 2-NBDG assays for the biflavonoid 2–7. Taiwaniaflavone (6) and 3,800 -biapigenin (7), with the linkage axis rising from C-3 position, possessed stronger inhibitory effects on PTP1B activity than amentoflavone (C-30 3C-800 ), robustaflavone (C-50 3C-600 ), and cupressuflavone (C-80 3C-800 ) (Table 2 and Fig. 4). Thus, it is suggested that the linkage between two apigenin skeletons through C-3 position may result in an increase of the inhibitory effect on PTP1B enzyme and a stimulatory effect on 2-NBDG uptake of these biflavones. Regarding to the insulin signaling pathway, this result indicates that the glucose uptake stimulatory effects of the isolates
(1–7) may be related to the inhibitory effects on the PTP1B enzyme. Selaginella tamariscina has been reported to possess various biological properties such as cytotoxic, antihyperglycemic, antimicrobial, antifungal, anti-inflammatory, and antidiabetic activity.15,17,31 Compounds 1–7 from S. tamariscina not only showed stimulatory potency on 2-NBDG uptake but also showed potent inhibitory effects on PTP1B enzyme activity, which suggested the potential of the isolates as insulin mimetics for developing antidiabetic agents. The results reveal a molecular mechanism for the potential beneficial effects of biflavonoids on type-2 diabetes and obesity, and that these active biflavonoids may be the compounds responsible for the antihyperglycemic and antidiabetic properties of S. tamarisscina.31 Furthermore, the 2-NBDG uptake stimulatory and PTP1B inhibitory effects by the biflavonoids have not been previously reported. Thus, further confirmation of the antidiabetic effects of these natural products and evaluation of their in vivo efficacy in a diabetic model are necessary. 3. Experimental 3.1. General experimental procedures The specific rotations were determined on a JASCO DIP-1000 digital polarimeter (JASCO Corp., Tokyo, Japan) using a 100 mm glass microcell. UV spectra were recorded in MeOH using a Shimadzu UV spectrometer (Shimadzu Corp., Kyoto, Japan). IR spectra (KBr) were recorded on a Nicolet 6700 FT-IR spectrometer (Thermo electron Corp., Tokyo, Japan). The NMR spectra were recorded in methanol-d4 (CD3OD) on Varian Oxford-AS 400 MHz instrument (Palo Alto, CA, USA), with TMS as the internal standard, at the Department of Pharmacy, Catholic University of Daegu, Korea. All mass spectrometric experiments were performed on a Quattro II mass spectrometer at Korea Basic Science Institute Daegu Center (KBSI, Daegu, Korea). Silica gel (63–200 lm particle size) and RP-C18 (40–63 lm particle size) for column chromatography were from Merck (Darmstadt, Germany). Thin-layer chromatography (TLC) was carried out on precoated Merck silica gel 60 F254 and RP-C18 F254 plates from Merck. HPLC was carried out using a Gilson system (Middleton, WI, USA) with a UV detector and an Optima Pak C18 column (10 250 mm, 10 lm particle size, RS Tech Corporation, Seoul, Korea). HPLC solvents were obtained from Fisher Scientific Korea Ltd (Seoul, Korea). All other reagents were of the highest analytical grade. 3.2. Plant material The aerial parts of Selaginella tamariscina were purchased from Yeongcheon market, Gyeongbuk, Republic of Korea, in January
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Figure 3. Graphical determination of inhibition type for the isolated compounds 2–7. (A) Lineweaver–Burk plots for the inhibition of isolated compounds (2–7) on the PTP1Bcatalyzed hydrolysis of p-NPP. (B) Dixon plots for isolated compounds (2–7) used for determining the inhibition constant Ki. Ki values were determined from the negative xaxis value at the point of the intersection of the three lines. Data are expressed as the mean reciprocal of initial velocity for n = 3 replicates at each substrate concentration.
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Figure 4. Stimulatory effects of the isolated compounds (1–7) on glucose uptake in 3T3-L1 adipocyte cells (Insulin: positive control; Control: DMSO). Confluent 3T3-L1 preadipocytes were differentiated into adipocytes for 8 days (from days 0 to 8). Fully differentiated 3T3-L1 adipocytes were treated with 5 and 10 lM of each compound and insulin (100 nM) followed by treatment with 2-NBDG, a fluorescent derivative of glucose used as a glucose analogue, for 30 min. Glucose uptake was measured using a fluorescence reader. Bars with different signals are significantly different at p
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Selaginellin and biflavonoids as protein tyrosine phosphatase 1B inhibitors from Selaginella tamariscina and their glucose uptake stimulatory effects Phi-Hung Nguyen a,d, , Da-Jung Ji a, , Yu-Ran Han b, Jae-Sue Choi b, Dong-Young Rhyu c, Byung-Sun Min a,⇑, Mi-Hee Woo a,⇑ a
College of Pharmacy, Drug Research and Development Center, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea Department of Food Science & Nutrition, Pukyong National University, 599-1 Daeyeon-3 Dong, Namgu, Busan 608-737, Republic of Korea c Department of Oriental Medicine Resources, Mokpo National University, Muan-gun 534-729, Republic of Korea d Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Hanoi, Viet Nam b
a r t i c l e
i n f o
Article history: Received 14 January 2015 Revised 30 March 2015 Accepted 2 April 2015 Available online xxxx Keywords: Selaginella tamariscina Biflavonoids PTP1B inhibitors 2-NBDG
a b s t r a c t As part of an ongoing search for new antidiabetic agents from medicinal plants, the methanol extract of the aerial parts of Selaginella tamariscina was found to possess stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. Thus, bioassay-guided isolation of this active extract yielded two new compounds (1 and 2) along with five known biflavonoids (3–7). Their structures were elucidated by extensive analysis of spectroscopic and physicochemical data. The absolute configuration of compound 2 was determined by specific rotation and CD data analysis. All isolates exhibited potent inhibitory effects on PTP1B enzyme with IC50 values ranging from 4.5 ± 0.1 to 13.2 ± 0.8 lM. Furthermore, the isolates (1–7) showed significant stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. Of these, compounds (1, 6, and 7) which exhibited mixed-competitive inhibition modes against PTP1B, showed potent stimulatory effects on 2-NBDG uptake. This result indicated the potential of these biflavonoids as lead molecules for development of antidiabetic agents and the beneficial use of S. tamariscina against hyperglycemia. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Diabetes mellitus, long regarded as a disease of minor consequence to world health, occupies now one of the main causes of serious maladies in the 21st century. The number of people with diabetes anticipate rising from current estimate of 150–220 million in 2010, and 300 million in 2025.1 Among the diseases, type 2 diabetes is characterized by chronic hyperglycemia associated with abnormalities in carbohydrate, fat, and protein metabolism. The American Diabetes Association reported that about 22.3 million people in the United States have diabetes, accounting for 7% of the population.2a Thus, great efforts are being made in the search for new therapeutic agents to stem its progress.2b Protein tyrosine phosphatase (PTP) superfamily coordinates with protein tyrosine kinases to regulate a vast array of cellular
⇑ Corresponding authors. Tel.: +82 53 850 3613 (B.-S.M); tel.: +82 53 850 3620; fax: +82 53 850 3602 (M.-H.W). E-mail addresses: [email protected] (B.-S. Min), [email protected] (M.-H. Woo). Authors contributed equally to this work.
functions, including proliferation, differentiation, apoptosis and motility. Of the various PTPs, PTP1B plays a critical role in regulating glucose homeostasis and body weight by acting as a key negative regulator of insulin and leptin signaling pathway, respectively.3 Its overexpression has been shown to inhibit the insulin receptor (IR) signaling cascade, and increased expression of PTP1B occurs in insulin-resistant states,4 while PTP1B knockout mice have been shown to increase insulin sensitivity and obesity resistance.5 Leptin, which is secreted from adipose tissue, also controls food intake and weight loss in the hypothalamus. Furthermore, recent evidence has shown that the leptin signaling pathway can be attenuated by PTPs, and there is evidence that PTP1B is also involved in this process.6 PTP1B was shown subsequently to bind and dephosphorylate Janus kinase 2 (JAK2), which is downstream of leptin receptor. Thus, the resistance to diet-induced obesity observed in PTP1B/ mice is likely to be associated with increased energy expenditure owing to enhanced leptin sensitivity.7 Moreover, pretreatment of leptin-resistant rats with a potent and selective PTP1B inhibitor results in a marked improvement in leptin-dependent suppression of food intake.8
http://dx.doi.org/10.1016/j.bmc.2015.04.007 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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Thus, PTP1B inhibitors could be useful in treating type 2 diabetes as well as obesity.9 Selaginella, also known as spikemoss, is the only surviving genus within the plant family Selaginellaceae. Selaginella includes more than 700 species widely distributed around the globe.10 Among the genus, Selaginella tamariscina (Beauv.) Spring was first recorded by ‘Shen Nong Ben Cao Jing’ (The Divine Farmer’s Materia Medica) in 2737 BC. It has been used in oriental medicine to treat inflammation, amenorrhea, dysmenorrhea, metrorrhagia, hematuria, prolapse of the anus, abdominal lumps in women, chronic hepatitis and hyperglycemia.11 Moreover, S. tamariscina has been reported to lower blood glucose levels and to facilitate the repair of pancreatic islet b-cells injured by alloxan.12 A number of flavonoids, lignans, selaginellins, and phenols were reported as chemical constituents of S. tamariscina.13 As part of an ongoing investigation on the discovery of new antidiabetic agents from medicinal plants, the methanol extract of the aerial parts of S. tamariscina was found to increase in vitro glucose uptake in 3T3-L1 adipocytes (1.13-fold induction at a concentration of 10 lg/mL). Thus, phytochemical investigation of this medicinal plant using chromatographic methods led to the isolation of two new natural products (1 and 2) and five known biflavones (3–7). Furthermore, compounds 1–7 were evaluated for their stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells as well as their inhibitory effects on PTP1B enzyme. In this Letter, the purification, structural determination, and potential antidiabetic properties of these isolates are discussed.
2. Results and discussion Bioassay-guided isolation of the methanol extract of the aerial part of S. tamariscina has led to the isolation of two new compounds (1 and 2) and five known (3–7) biflavonoid derivatives as active principles (Fig. 1). The known compounds were elucidated as amentoflavone (3),14 robustaflavone (4),14 cupressuflavone (5),15 taiwaniaflavone (6),16 and 3,800 -biapigenin (7)17 based on NMR spectroscopic and MS data analysis and comparison with literature values. Compound 1 was obtained as a red amorphous powder. Its IR spectrum indicated absorption bands for hydroxy group (3394 cm1), alkynyl (2203 cm1), unsaturated carboxyl (1674 cm1), and aromatic ring (1077–1045 cm1). A molecular formula of C22H16O5 was determined from the molecular ion peak at m/z 360.1026 [M]+ obtained by HRFABMS. The UV spectrum exhibited absorption maxima at 264, 300, 322, and 420 nm, characteristic values for a selaginellin analogue.18 The assignment of the 1H and 13C NMR data (Table 1) was supported by 2D-NMR techniques (Supplementary data). In the 1H and 13C NMR spectra of 1, the typical signals of an alkynyl substituent at dC 84.7 and 100.7, an ABspin system at dH 7.69 (1H, d, J = 8.0 Hz, H-3) and 7.31 (1H, d, J = 8.0 Hz, H-4) for the ortho-tetrasubstituted A-ring, and two AA0 BB0 systems with [dH 6.97 and 6.62 (each 2H, dd, J = 2.0, 8.8 Hz)] and [dH 6.75 and 6.53 (each 2H, dd, J = 2.0, 8.4 Hz)] for the para-substituted B- and C-rings, were apparent. These assignment resembled those of selaginellin K and selaginellin L,19 however, signals for the aldehyde or methyl group was not presented, instead of that, a carboxyl carbon signal (dC 165.5) was found in the 13C NMR spectrum of compound 1 (Supplementary data). The alkynyl group was the linkage between the B-ring and the A-ring at C-1 based on HMBC correlations between H-20 /H-60 and C-10, and H-3 and C-1/C-9. The C-ring was connected to the A-ring at C-5 as supported by the HMBC correlations between H-200 / H-600 and C-5 and H-4 and C-100 (Fig. 2). A hydroxymethyl group [dH 4.91 (2H, s, H-8) and dC 63.7 (C-8)] was assigned to C-2 by the aid of HSQC and HMBC
experiments showing correlations from H-3 to C-8 and from H-8 to C-1, C-2, and C-9 (Fig. 2). Hence, C-6 (142.4) in the A-ring was the only position left for the carboxylic group (dC 165.5, C-7). On the basis of the above evidence, compound 1 (selariscinin D) was thus characterized as 40 -hydroxy-4-(hydroxymethyl)-3-[(4-hydroxyphenyl)ethynyl]biphenyl-2-carboxylic acid. Compound 2 was obtained as a yellow amorphous powder. Its HRFABMS showed a molecular ion peak at m/z 540.1060 [M]+ (calcd for m/z 540.1056), consistent with a molecular formula of C30H20O10. The IR spectrum exhibited absorption bands at 3331 cm1 (OH), 1670 cm1 (C@O), 1593, and 1242–1045 cm1 (aromatic ring). Its UV spectrum showed absorption maxima at 221, 286, and 329 nm, characteristic values for a biflavonoid analogue.20 A negative optical rotation value 6.3 (c 0.25, MeOH) was observed for compound 2. The 1H and 13C NMR spectra of 2 displayed characteristic signals assignable for an AMX spin system of a flavanone skeleton21 at dH 5.42 (1H, dd, J = 12.8, 2.8 Hz, H-2), 3.18 (1H, dd, J = 12.8, 16.8 Hz, H-3ax), and 2.76 (1H, dd, J = 2.8, 16.8, H-3eq), with corresponding carbon signals for C-2 (dC 80.6) and C-3 (dC 44.1), and the ketone carbon for C-4 (dC 198.0). The olefinic proton at dH 6.60 (1H, s), with its corresponding carbon (dC 103.4, C-300 ), showed HMBC correlations with an oxygenated quaternary carbon at dC 166.3 (C-200 ) and a carbonyl carbon at dC 184.5 (C-400 ) (Table 1 and Fig. 2), indicating the presence of a flavone skeleton.20 The 13C NMR spectrum together with MS data indicated a flavone–flavanone based biflavonoid structure22 for 2 with 30 carbon resonances representing two carbonyl (dC 184.5 and 198.0), two aliphatic (dC 80.6 and 44.1), and 26 olefinic and aromatic carbons (Table 1). Furthermore, an AB spin system at dH 7.41 and 7.05 (each 1H, d, J = 8.4 Hz, H-500 /H-600 ) for the ortho-substituted A0 -ring and a AA0 BB0 spin system at dH 7.55 and 6.79 (each 2H, dd, J = 2.4, 8.8 Hz) for the para-substituted B0 -ring of the flavone skeleton were apparent. In addition, two meta-coupled proton signals at H-6 and H-8 in ring A at dH 5.87 and 5.90 (each 1H, d, J = 2.0 Hz) with two corresponding carbons at dC 97.2 and 96.4, respectively, and two singlet proton signals at dH 6.38 (1H, s, H-50 ) and 7.44 (1H, s, H-60 ), assignable to two para-coupled aromatic protons in ring B of 2 were presented (Table 1 and Supplementary data). All of these assignments and the arrangement of the rings were confirmed by 1H–1H gCOSY, 1 H–13C gHMQC and 1H–13C gHMBC experiments (Fig. 2 and Supplementary data). In the HMBC spectrum, the correlations between H-2 (dH 5.42) and C-10 (dC 131.2) and C-20 (dC 162.4), between H-30 (dH 6.38) and C-20 (dC 162.4), C-40 (dC 163.8), and C-50 (dC 103.4), and between H-60 (dH 7.44) and C-2 (dC 80.6), C-10 (dC 131.2), and C-20 (dC 162.4), indicated a 1,2,4,5-tetrasubstitued B-ring with two hydroxy groups attached at C-20 (162.4) and C-40 (163.8), respectively. Ring B was shown to be connected to ring A at C-50 3C-800 axis via three bond-coupled correlation between H60 (dH 7.44) and C-800 (dC 120.8) as well as four bond-coupled correlations between H-60 and C-700 (dC 157.4) and C-900 (dC 156.6) (Fig. 2 and Supplementary data). The absolute configuration at C2 was deduced to be (S) by the presence of a negative Cotton effect (8.7) at 286 nm and a positive Cotton effect (+4.5) at 330 nm in its CD spectrum.22,23 Thus, compound 2 (selariscinin E) was established as (2S)-2,3-dihydro-5,7,700 ,20 ,40 ,4000 -hexahydroxy-(50 –800 )biflavone, which is a new natural biflavonoid. As insulin resistance is one of critical causes in the pathogenesis of type 2 diabetes, reconstitution of insulin sensitivity is one of the key strategies for the treatment of this disease.24 However, some thiazolidinediones as a representative class of insulin sensitizers have been withdrawn from the market due to severe side effects, which include edema, weight gain, and the increased risk of heart failure.25 Therefore, there is an urgent demand to discover new anti-diabetic agents acting as insulin mimics and/or insulin sensitizers. 2-NBDG has been reported as an useful fluorescent-tagged
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HO
3' 3''
HO 4''
C
5''
6''
OH
2'
HO 1''
7
O
5 4
3
9
1
A
B
1'
6
4' 5'
7 6
6'
10
HO
8
9
A 5
O
2'''
B'
1'''
2''
A'
O
5''
10'' 4''
HO
O HO
O HO O
O O
6'''
OH
5'''
O
3
4
OH
OH
O HO
HO
OH
O
2 O
OH OH
OH
1
OH OH
6''
C' 3''
OH
7''
8'' 9''
3''' 4'''
HO
5'
O
O
OH OH
4'
6'
3 4
8
OH
3'
B
2
C
OH
2
O
10
2' 1'
OH
HO
OH
OH O HO
OH
OH
O
O
O OH
OH
O
HO
O
OH
OH
O O
O HO
5
OH O
6
HO
7
Figure 1. Chemical structure of compounds 1–7 isolated from Selaginella tamariscina.
Table 1 H (400 MHz) and and 2 in CD3OD 1
Position
1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 100 200 300 400 500 600 700 800 900 1000 1000 2000 3000 4000 5000 6000
13
C (100 MHz) NMR spectroscopic data for the new compounds 1
Selariscinin D (1)
Selariscinin E (2)
dH (J in Hz)
dC
7.69 (1H, d, 8.0)
123.8 143.0 129.0
7.31 (1H, d, 8.0)
4.91 (2H, s)
6.97 (1H, dd, 8.8, 2.0) 6.62 (1H, dd, 8.8, 2.0) 6.62 (1H, dd, 8.8, 2.0) 6.97 (1H, dd, 8.8, 2.0) 6.75 (1H, dd, 8.4, 2.0) 6.53 (1H, dd, 8.4, 2.0) 6.53 (1H, dd, 8.4, 2.0) 6.75 (1H, dd, 8.4, 2.0)
131.0 143.6 142.7 165.5 63.7 84.7 100.7 114.6 134.2 116.5 159.6 116.5 134.2 133.1 131.2 115.9 158.0 115.9 131.2
dH (J in Hz)
dC
5.42 (1H, dd, 12.8, 2.8) 3.18 (1H, dd, 12.8, 16.8) 2.76 (1H, dd, 2.8, 16.8)
80.6 44.1
5.87 (1H, d, 2.0) 5.90 (1H, d, 2.0)
6.38 (1H, s)
7.44 (1H, s)
6.60 (1H, s) 7.41 (1H, d, 8.4) 7.05 (1H, d, 8.4)
7.55 (1H, dd, 8.8, 2.4) 6.79 (1H, dd, 8.8, 2.4) 6.79 (1H, dd, 8.8, 2.4) 7.55 (1H, dd, 8.8, 2.4)
198.0 168.5 97.2 165.6 96.4 165.1 103.5 131.2 162.4 100.2 163.8 103.4 128.9 166.3 103.4 184.5 131.3 117.0 157.4 120.8 156.6 106.4 123.4 129.6 117.0 162.7 117.0 129.6
glucose probe for discovering insulin mimetic compounds.26 Thus, the stimulatory effects of compounds 1–7 were further evaluated on glucose uptake using 2-NBDG in 3T3-L1 adipocyte cells.27 As presented in Figure 4, all the isolates showed dose-dependent stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. At a concentration of 10 lM, compounds 1, 6, and 7 significantly stimulated 2-NBDG uptake by 1.24 ± 0.05-, 1.47 ± 0.08-, and 1.44 ± 0.06-fold inductions as compared with the control (DMSO), respectively. In this assay, the positive control (insulin) showed
an induction of 1.54 ± 0.05-fold at a concentration of 100 nM. The cytotoxic effects of the isolated compounds 1–7 were also evaluated in the absence of 2-NBDG using MTT assay. All isolates did not show cytotoxicity to the cells at both concentrations of 5 and 10 lM (Supplementary data). Thus, the stimulatory effects of these compounds on 2-NBDG uptake were not affected by any cytotoxicity. The biapigenins, taiwaniaflavone (6) and 3,800 -biapigenin (7) with a linkage at C-3 position, showed more potent effects than the other biflavonoids (2–5). GLUT4 glucose transport translocation is essential for inducible glucose uptake into the plasma membrane in muscle and adipocyte cells. This process depends mainly on the regulation of two physiological pathways: the AMP-activated protein kinase (AMPK) pathway and the insulin signaling pathway.28a In the insulin signaling pathway, binding of insulin to the insulin receptor on adipocytes and muscle cells triggers the recruitment and phosphorylation of insulin receptor substrate (IRS), which forms a docking site for PI3K at the membrane. When docked, PI3K activates phosphoinositide-dependent protein kinase 1 (PDK1), which in turn mediates the phosphorylation of both Akt (also known as protein kinase B) at Thr308 and ribosomal protein 70 kDa S6 kinase 1 (p70s6k).28 Thus, activated Akt facilitates glucose uptake in adipocytes and muscle cells by allowing the translocation of GLUT4, the insulin-responsive isoform of the glucose transporter located in intracellular storage vesicles, to the plasma membrane.28 As a negative regulator, PTP1B dephosphorylates IR and IRS in the case of insulin signaling and also tyrosine kinase JAK2 (Janus kinase 2) in the leptin signaling pathway.29 Therefore, inhibition of the activity or the expression level of PTP1B can facilitate the insulin signaling pathway, leading to an increase in glucose uptake in adipocytes. In this regard, compounds 1–7 were tested for their inhibitory effects on PTP1B enzyme using ursolic acid and RK-682 as positive controls30 (Table 2). All isolates potentially inhibited PTP1B activity in a dose-dependent manner with IC50 values ranging from 4.5 ± 0.1 to 13.2 ± 0.8 lM. Among the isolates, compounds 6 and 7 exhibited equivalent activities with IC50 values of 5.4 ± 0.2 and 4.5 ± 0.1 lM to the positive controls, ursolic acid (IC50 = 3.4 ± 0.1 lM) and RK682 (IC50 = 4.5 ± 0.2 lM). As second potent inhibitors, the biapigenin analogues (3–5) exhibited IC50 values of 7.4 ± 0.5, 6.1 ± 0.1, and 9.6 ± 0.3 lM, respectively. The new selaginellin derivative (1) and flavone–flavanone based biflavonoid (2) displayed significant activity with IC50 values of 13.2 ± 0.8 and 9.8 ± 0.6 lM, respectively.
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HO
OH HO
O
HO
OH OH
O
HO
O OH OH
O
O
HO
1
2 1H-1H COSY
correlations
1H-13C HMBC
correlations
1H-13C four-bond
HMBC correlations
Figure 2. 1H–1H COSY (bold lines) and 1H–13C HMBC correlations (arrows) for the new compounds, selariscinins D (1) and E (2).
Table 2 Inhibitory effects of isolated compounds (1–7) on PTP1B enzyme Compound
Inhibitory effect (IC50, lM)a
(Ki value, lM)
Inhibition type
1 2 3 4 5 6 7 Ursolic acidb RK-682b
13.2 ± 0.8 9.8 ± 0.6 7.4 ± 0.5 6.2 ± 0.1 9.6 ± 0.3 5.4 ± 0.2 4.5 ± 0.1 3.4 ± 0.1 4.5 ± 0.2
14.5 ± 0.7 10.4 ± 0.6 7.8 ± 0.5 6.8 ± 0.3 10.7 ± 0.4 4.5 ± 0.3 4.6 ± 0.1 6.4 ± 0.2 —c
Mixed-competitive Uncompetitive Mixed-competitive Mixed-competitive Competitive Mixed-competitive Mixed-competitive Mixed-competitive —
a Results are expressed as IC50 values (lM), determined by regression analysis and expressed as the means ± SD of three replicates. b Positive controls. c Data not determined.
In kinetic study, the Dixon plots and Lineweaver-Burk plots (Corish-Bowden 1974 and Dixon 1953) were used in order to determine the type of enzyme inhibition and the dissociation or inhibition constant (Ki) for an enzyme–inhibitor complex.. The results showed that the inhibition modes of compounds 1, 3, 4, 6, and 7, as determined using Lineweaver–Burk and Dixon plots (Fig. 3 and Supplementary data), were mixtures of inhibition types because the Vmax values decreased with concentration without changing the Km for the substrate, and the lines did not intersect on the y-axis in the Lineweaver–Burk plot and the x-axis in Dixon plot. However, both experiments showed that compound 5 exhibited competitive inhibition because the pattern of straight lines with intersecting y intercepts (Fig. 3A) is characteristic of competitive inhibitor. This indicated that 5 may directly bind to the active binding site of the enzyme to inhibit the activity of PTP1B. In contrast, increasing the substrate concentrations resulted in a series of lines that did not intersect on the y-axis in the Lineweaver–Burk plot and the x-axis in Dixon plots, but paralleled each other, confirming that compound 2 shows uncompetitive inhibition. Similar structure–activity relationships were observed in PTP1B and 2-NBDG assays for the biflavonoid 2–7. Taiwaniaflavone (6) and 3,800 -biapigenin (7), with the linkage axis rising from C-3 position, possessed stronger inhibitory effects on PTP1B activity than amentoflavone (C-30 3C-800 ), robustaflavone (C-50 3C-600 ), and cupressuflavone (C-80 3C-800 ) (Table 2 and Fig. 4). Thus, it is suggested that the linkage between two apigenin skeletons through C-3 position may result in an increase of the inhibitory effect on PTP1B enzyme and a stimulatory effect on 2-NBDG uptake of these biflavones. Regarding to the insulin signaling pathway, this result indicates that the glucose uptake stimulatory effects of the isolates
(1–7) may be related to the inhibitory effects on the PTP1B enzyme. Selaginella tamariscina has been reported to possess various biological properties such as cytotoxic, antihyperglycemic, antimicrobial, antifungal, anti-inflammatory, and antidiabetic activity.15,17,31 Compounds 1–7 from S. tamariscina not only showed stimulatory potency on 2-NBDG uptake but also showed potent inhibitory effects on PTP1B enzyme activity, which suggested the potential of the isolates as insulin mimetics for developing antidiabetic agents. The results reveal a molecular mechanism for the potential beneficial effects of biflavonoids on type-2 diabetes and obesity, and that these active biflavonoids may be the compounds responsible for the antihyperglycemic and antidiabetic properties of S. tamarisscina.31 Furthermore, the 2-NBDG uptake stimulatory and PTP1B inhibitory effects by the biflavonoids have not been previously reported. Thus, further confirmation of the antidiabetic effects of these natural products and evaluation of their in vivo efficacy in a diabetic model are necessary. 3. Experimental 3.1. General experimental procedures The specific rotations were determined on a JASCO DIP-1000 digital polarimeter (JASCO Corp., Tokyo, Japan) using a 100 mm glass microcell. UV spectra were recorded in MeOH using a Shimadzu UV spectrometer (Shimadzu Corp., Kyoto, Japan). IR spectra (KBr) were recorded on a Nicolet 6700 FT-IR spectrometer (Thermo electron Corp., Tokyo, Japan). The NMR spectra were recorded in methanol-d4 (CD3OD) on Varian Oxford-AS 400 MHz instrument (Palo Alto, CA, USA), with TMS as the internal standard, at the Department of Pharmacy, Catholic University of Daegu, Korea. All mass spectrometric experiments were performed on a Quattro II mass spectrometer at Korea Basic Science Institute Daegu Center (KBSI, Daegu, Korea). Silica gel (63–200 lm particle size) and RP-C18 (40–63 lm particle size) for column chromatography were from Merck (Darmstadt, Germany). Thin-layer chromatography (TLC) was carried out on precoated Merck silica gel 60 F254 and RP-C18 F254 plates from Merck. HPLC was carried out using a Gilson system (Middleton, WI, USA) with a UV detector and an Optima Pak C18 column (10 250 mm, 10 lm particle size, RS Tech Corporation, Seoul, Korea). HPLC solvents were obtained from Fisher Scientific Korea Ltd (Seoul, Korea). All other reagents were of the highest analytical grade. 3.2. Plant material The aerial parts of Selaginella tamariscina were purchased from Yeongcheon market, Gyeongbuk, Republic of Korea, in January
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Figure 3. Graphical determination of inhibition type for the isolated compounds 2–7. (A) Lineweaver–Burk plots for the inhibition of isolated compounds (2–7) on the PTP1Bcatalyzed hydrolysis of p-NPP. (B) Dixon plots for isolated compounds (2–7) used for determining the inhibition constant Ki. Ki values were determined from the negative xaxis value at the point of the intersection of the three lines. Data are expressed as the mean reciprocal of initial velocity for n = 3 replicates at each substrate concentration.
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Figure 4. Stimulatory effects of the isolated compounds (1–7) on glucose uptake in 3T3-L1 adipocyte cells (Insulin: positive control; Control: DMSO). Confluent 3T3-L1 preadipocytes were differentiated into adipocytes for 8 days (from days 0 to 8). Fully differentiated 3T3-L1 adipocytes were treated with 5 and 10 lM of each compound and insulin (100 nM) followed by treatment with 2-NBDG, a fluorescent derivative of glucose used as a glucose analogue, for 30 min. Glucose uptake was measured using a fluorescence reader. Bars with different signals are significantly different at p
