Constituents of Coreopsis lanceolata Flower and Their Dipeptidyl Peptidase IV Inhibitory Effects

A new polyacetylene glycoside, (5R)-6E-tetradecene-8,10,12-triyne-1-ol-5-O-β-glucoside (1), was isolated from the flower of Coreopsis lanceolata (Compositae), together with two known compounds, bidenoside C (10) and (3S,4S)-5E-trideca-1,5-dien-7,9,11-triyne-3,4-diol-4-O-β-glucopyranoside (11), which were found in Coreopsis species for the first time. The other known compounds, lanceoletin (2), 3,2′-dihydroxy-4-3′-dimethoxychalcone-4′-glucoside (3), 4-methoxylanceoletin (4), lanceolin (5), leptosidin (6), (2R)-8-methoxybutin (7), luteolin (8) and quercetin (9), were isolated in this study and reported previously from this plant. The structure of 1 was elucidated by analyzing one-dimensional and two-dimensional nuclear magnetic resonance and high resolution-electrospray ionization-mass spectrometry data. All compounds were tested for their dipeptidyl peptidase IV (DPP-IV) inhibitory activity and compounds 2-4, 6 and 7 inhibited DPP-IV activity in a concentration-dependent manner, with IC50 values from 9.6 to 64.9 μM. These results suggest that C. lanceolata flower and its active constituents show potential as therapeutic agents for diseases associated with type 2 diabetes mellitus.


Introduction
Coreopsis is a perennial plant belonging to the Compositae family [1]. It was planted for decoration on the roadsides in North America, Asia and Oceania regions [2][3][4]. Typically, C. lanceolata, C. tinctoria and C. drummondii are generally Coreopsis plants distributed all around Korea [5]. Among them, C. lanceolate are ornamental plants commonly found in Korea in spring and summer. The shape of the yellow petals are split and jagged, and have a diameter 4-6 cm, which is larger than other species [5,6]. In the previous phytochemical studies on C. lanceolata, phenolics, flavonoids (aurones, chalcones, flavaons, flanavol) and acetylene compounds have been isolated from this plant [5][6][7][8][9][10][11][12]. The C. lanceolata flower has been reported to have diverse biological activities, such as anti-cancer [5], anti-inflammatory [6], antioxidant [6,8,10], anti-allerginc [9,10], antileukemic [11] and nematicidal [12] effects. Because of the good efficacy of C. lanceolata, it has been traditionally used as an ingredient in herbal medicine, such as folk remedies to control fever in China and East Asia [8]. This plant has been studied for a few chemical constituents and its pharmacological activity, but a literature review revealed that there is no anti-diabetes.
Asia [8]. This plant has been studied for a few chemical constituents and its pharmacological activity, but a literature review revealed that there is no anti-diabetes.
Type 2 diabetes mellitus is a chronic metabolic disorder, characterized by hyperglycemia caused by the dysregulation of blood glucose homeostasis, increased liver glucose production, insulin secretion, insulin resistance and β-cell dysfunction [13]. According to the eighth edition of the International Diabetes Federation (IDF) Diabetes Atlas, there were 425 million people with diabetes worldwide in 2017, and most of them have type 2 diabetes mellitus. This number of patients is increasing every year, and is expected to increase to about 700 million by 2045 [14]. The five factors that affect diabetes treatment are dipeptidyl peptidase-IV (DPP-IV), α-glucosidase inhibitors, aldose reductase (ALR) inhibitors, phatase 1B (PTP1B) inhibitors and peroxisome proliferator-activated receptor-γ (PPAR-γ) [15]. Recently, incretin-based therapies for the treatment of type 2 diabetes mellitus have begun to appear, one of which is DPP-IV inhibitors [16]. The incretin system includes glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), which in response to high blood sugar levels induce the release of insulin from the pancreatic β-cell [13,16]. DPP-IV is involved in the quick degradation and inactivation of GLP-1 and GIP in the incretin system [17]. As a result, DPP-IV inhibition enhances the glucose-producing effect of GLP-1, thereby improving glucose tolerance in diabetic patients [18]. As DPP-IV inhibitors, synthetic chemical compounds or natural product-derived compounds recently represent promising drug candidates [16,[19][20][21][22].
During the screening procedure to find new candidates from natural sources for the treatment of type 2 diabetes mellitus, the ethyl acetate fraction of the C. lanceolata flower showed potent inhibitory activity in a DPP-IV inhibitor screening assay, with 100% inhibition at a concentration of 100 μg/mL. Therefore, this active fraction was subjected to detailed phytochemical investigation, and 11 compounds were isolated, including a new compound 1 (Figure 1). In this study, we describe the isolation and structural elucidation of 1 and the biological results of compounds 1-11. 14

Structure Elucidation of Compound 1
Compound 1 was obtained as a brown solid, with a molecular ion peak at m/z 401.1575 [M+Na] + in the high resolution electrospray ionization mass spectrum, corresponding to an elemental formula of C 20 H 26 O 7 Na. Its specific UV spectrum showed maximum absorptions at 254, 260, 274, 290, 310 and 330 nm, predicted to be ene-triyne chromophore [23,24]. The 1 H NMR and 13 C NMR spectra of 1 (Table 1) [25,26]. In the 1 H-13 C HMBC NMR spectrum, the hemiacetal proton H-1 showed a key correlation with C-5, suggesting that the β-glucopyranoside was positioned at C-5 in the polycetylene aglycone. An absolute configuration of 1 at C-5 was assigned by comparing its experimental circular dichroism (CD) data with the literature data [27]. In the literature, polyacetylene glycosides established their absolute configurations by comparison of the calculated and experimental ECD spectra. According to the Snatzke s helicity rule, the negative Cotton effect at 300-320 nm permitted the configuration of the glucoside-attached carbon to R, while the positive Cotton effect at 300-320 nm permitted the configuration of the glucoside-attached carbon to S. Based on this observation ( Figures S8 and S9), the absolute configuration of 1 was determined as (5R). Therefore, the structure of 1 was elucidated as (5R)-6E-tetradecene-8,10,12-triyne-1-ol-5-O-β-glucoside (1), which is a new polyacetylene glycoside.

Biological Activity
The methanol extract and solvent fractions of C. lanceolata confirmed their anti-diabetic effect using an in vitro DPP-IV inhibitor screening assay. The methanol extract inhibited DPP-IV activity with 87.2% inhibition at 100 μg/mL. Thus, it was partitioned with hexanes, ethyl acetate and nbutanol, successively, and these solvent fractions were screened for their DPP-IV inhibition at 100 μg/mL concentrations. Since the ethyl acetate fraction inhibited DPP-IV activity with 100% inhibition, we conducted a detailed phytochemical investigation, leading to the isolation of 11 compounds. All isolates were evaluated for their DPP-IV inhibitory effects, and compounds 2-4, 6 and 7 inhibited DPP-IV activity in a concentration-dependent manner, with IC50 values of 9.6, 14.3, 21.6, 13.3 and 64.9 μM, respectively ( Figure 3). The positive control, sitagliptin, exhibited an IC50 of 0.071 μM.
Compounds 2-5 have the chalcone skeleton, which is an α-β-unsaturated ketone with two phenyl rings. There have been several reviews on the biological and pharmacological activities of chancones, including anti-cancer, anti-malarial, anti-microbial, anti-inflammatory, anti-protozoal and anti-HIV activities [33], as well as its role in diabetes control [15,34,35]. In a comprehensive study into modulating the therapeutic targets PPAR-γ, DPP-IV, α-glucosidase, PTP1B, ALR and insulin secretion via anti-diabetic chalcones and their structure-activity relationships [15], in particular, it was reported that the chalcone structure of which the carbonyl group was replaced with an oxime function exhibited enhanced DPP-IV inhibitory activity. In addition, there have been reports that  (7) [11], luteolin (8) [28], quercetin (9) [29], bidenoside C (10) [30] and (3S,4S)-5Etrideca-1,5-dien-7,9,11-triyne-3,4-diol-4-O-β-glucopyranoside (11) [31] by comparing their spectroscopic data with published data. Flavanone 7 showed the negative Cotton effect at 295-310 nm and the positive Cotton effect at 280-290 nm in its CD spectrum, indicating a 2R configuration [32] ( Figure S10). The absolute configuration of 11 at C-3 and C-4 was assigned by comparing its experimental CD data with the data in the literature [27], as described above for the absolute configuration determination of 1. Compound 11 was a diastereomer and the threo stereochemistry between H-3 and H-4 was deduced by their large coupling constant (J = 5.5 Hz, in methanol-d 4 ). The positive Cotton effect at 300-320 nm in the CD spectrum of 11 was attributed to a (3S,4S) configuration for 11 ( Figure S11). Although the known compounds (2-9) have been isolated from the Coreopsis species, the isolation of compounds 10 and 11 from the Coreopsis species has not been reported yet.

Biological Activity
The methanol extract and solvent fractions of C. lanceolata confirmed their anti-diabetic effect using an in vitro DPP-IV inhibitor screening assay. The methanol extract inhibited DPP-IV activity with 87.2% inhibition at 100 µg/mL. Thus, it was partitioned with hexanes, ethyl acetate and n-butanol, successively, and these solvent fractions were screened for their DPP-IV inhibition at 100 µg/mL concentrations. Since the ethyl acetate fraction inhibited DPP-IV activity with 100% inhibition, we conducted a detailed phytochemical investigation, leading to the isolation of 11 compounds. All isolates were evaluated for their DPP-IV inhibitory effects, and compounds 2-4, 6 and 7 inhibited DPP-IV activity in a concentration-dependent manner, with IC 50 values of 9.6, 14.3, 21.6, 13.3 and 64.9 µM, respectively ( Figure 3). The positive control, sitagliptin, exhibited an IC 50 of 0.071 µM.
Compounds 2-5 have the chalcone skeleton, which is an α-β-unsaturated ketone with two phenyl rings. There have been several reviews on the biological and pharmacological activities of chancones, including anti-cancer, anti-malarial, anti-microbial, anti-inflammatory, anti-protozoal and anti-HIV activities [33], as well as its role in diabetes control [15,34,35]. In a comprehensive study into modulating the therapeutic targets PPAR-γ, DPP-IV, α-glucosidase, PTP1B, ALR and insulin secretion via anti-diabetic chalcones and their structure-activity relationships [15], in particular, it was reported that the chalcone structure of which the carbonyl group was replaced with an oxime function exhibited enhanced DPP-IV inhibitory activity. In addition, there have been reports that nitrochalcones and prenylated chalcones increase insulin secretion and glucose uptake, respectively [34]. Among chalcones isolated in this study, 3-5 showed less inhibitory activity than 2, indicating that chalcones substituted with additional methoxy group and/or sugar are less effective. The most active compound, 2, has been reported as having various biological, activities such as antileukemic [11], antioxidant [4] and anti-cancer [5], however its DPP-IV potential has not been studied previously. Compounds 6 and 7, with the structures of aurone and flavanone, respectively, exhibited moderate DPP-IV inhibitory effects, while flavone (8), flavonol (9) and polyacetylenes (1, 10 and 11) did not show their activities. The evaluation of the inhibitory activities of 1-11 against DPP-IV was reported for the first time in this study. synthetic DPP-IV inhibitors diprotin A and sitagiptin, the natural products with structural diversity could be useful for discovering safer DPP-IV inhibitors. Therefore, these results suggest that compounds 2-4, 6 and 7 have the potential for the development of naturally derived DPP-IV inhibitors for the treatment of type 2 diabetes mellitus and hyperglycemia, although a further study of their mechanisms of action is necessary, using in vitro and in vivo models.

General Procedures
The optical rotations were measured by a JASCO DIP-1000 polarimeter (JASCO Co., Tokyo, Japan). The circular dichroism (CD) measurements were conducted using a JASCO J-810 CD-ORD Extensive studies on the phytochemical-based DPP-IV inhibitors were undertaken in the literature [19][20][21], and in our previous studies [16,22]. Flavonoids and phenolic compounds were found to be the DPP-IV inhibitors with potent to mild activities, showing IC 50 values in the range of 0.12 to 63.26 µM. Although naturally occurring compounds were relatively less active than the synthetic DPP-IV inhibitors diprotin A and sitagiptin, the natural products with structural diversity could be useful for discovering safer DPP-IV inhibitors. Therefore, these results suggest that compounds 2-4, 6 and 7 have the potential for the development of naturally derived DPP-IV inhibitors for the treatment of type 2 diabetes mellitus and hyperglycemia, although a further study of their mechanisms of action is necessary, using in vitro and in vivo models.

DPP-IV Inhibitory Activity Assay
The measurement of DPP-IV activity was performed using the DPP-IV inhibitor screening assay kit (Cayman Chemical, Ann Arbor, Michigan, USA), based on the manufacturer's protocols (Cayman Chemical). Briefly, a DDP assay buffer consisting of 20 mM Tris-HCL (pH 8.0), 100 nM NaCl and 1 mM EDTA was used for the assay solution. Each of the compounds were initially dissolved in DMSO at a concentration of 1 mM. Subsequently, the final concentration of compounds was diluted to 0.1, 1, 10, 25, 50 and 100 µM, respectively. The human recombinant DPP-IV enzyme and the substrate 5 mM H-Gly-Pro conjugated to aminomethylcoumarin (AMC) were put in the same buffer. Diluted assay buffer (30 µL) and diluted enzyme solution (10 µL) were added to a 96-well plate containing 10 µL of solvent (blank) or 10 µL of solvent-dissolved test compounds. The reaction by adding 50 µL of the diluted solution of the fluorogenic substrate was initiated and cleavage of the peptide bond by DPP released the free AMC group. The fluorescence was measured as an excitation wavelength of 350 nm and an emission wavelength of 450 nm using a plate reader (TECAN, Männedorf, Switzerland). The percentage inhibition was expressed as [(DPP-IV level of vehicle-treated control−DPP-IV level of test samples)/DPP-IV level of vehicle-treated control] × 100. Subsequently, the 50% inhibitory concentration (IC 50 ) was determined using GraphPad Prism software (GaraphPad Software, La Jolla, CA, USA) via a dose-response analysis.