Chemical Constituents from the Roots of Angelica reflexa That Improve Glucose-Stimulated Insulin Secretion by Regulating Pancreatic β-Cell Metabolism

The aim of this study was to discover bioactive constituents of Angelica reflexa that improve glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. Herein, three new compounds, namely, koseonolin A (1), koseonolin B (2), and isohydroxylomatin (3), along with 28 compounds (4–31) were isolated from the roots of A. reflexa by chromatographic methods. The chemical structures of new compounds (1–3) were elucidated through spectroscopic/spectrometric methods such as NMR and HRESIMS. In particular, the absolute configuration of the new compounds (1 and 3) was performed by electronic circular dichroism (ECD) studies. The effects of the root extract of A. reflexa (KH2E) and isolated compounds (1–31) on GSIS were detected by GSIS assay, ADP/ATP ratio assay, and Western blot assay. We observed that KH2E enhanced GSIS. Among the compounds 1–31, isohydroxylomatin (3), (−)-marmesin (17), and marmesinin (19) increased GSIS. In particular, marmesinin (19) was the most effective; this effect was superior to treatment with gliclazide. GSI values were: 13.21 ± 0.12 and 7.02 ± 0.32 for marmesinin (19) and gliclazide at a same concentration of 10 μM, respectively. Gliclazide is often performed in patients with type 2 diabetes (T2D). KH2E and marmesinin (19) enhanced the protein expressions associated with pancreatic β-cell metabolism such as peroxisome proliferator-activated receptor γ, pancreatic and duodenal homeobox 1, and insulin receptor substrate-2. The effect of marmesinin (19) on GSIS was improved by an L-type Ca2+ channel agonist and K+ channel blocker and was inhibited by an L-type Ca2+ channel blocker and K+ channel activator. Marmesinin (19) may improve hyperglycemia by enhancing GSIS in pancreatic β-cells. Thus, marmesinin (19) may have potential use in developing novel anti-T2D therapy. These findings promote the potential application of marmesinin (19) toward the management of hyperglycemia in T2D.

Cell viability was assessed using Ez-Cytox cell viability reagent (Daeil Lab Service Co., Seoul, Republic of Korea) with a modified protocol based on previous study [26]. After treatment, Ez-Cytox cell viability reagent was added to each well. After cultivation for 1 h, the absorbance (490 nm) was recorded using a PowerWave XS microplate reader (Bio-Tek Instruments, Winooski, VT, USA). kit (Gentaur, Shibayagi Co. Ltd., Gunma, Shibukaw, Japan) with a protocol based on supplier's instructions. ADP/ATP ratio was determined using an ADP/ATP ratio assay kit (Sigma-Aldrich, St Louis, MO, USA) with a protocol based on supplier's instructions.

Western Blot Analysis
Western blot was carried out with a modified protocol based on previous study [27]. The membranes were incubated with the relevant primary antibodies (Cell Signaling, Danvers, MA, USA) for 1 h on ice. They were further incubated with horseradish peroxidaseconjugated anti-rabbit secondary antibodies (Cell Signaling) for 1 h on ice.

Statistical Analysis
Statistical significance was performed by one-way analysis of variance (ANOVA), with the Bonferroni correction for multiple comparisons. Statistical significance was set at p < 0.05. All analyses were performed by SPSS Statistics ver. 19.0 (SPSS Inc., Chicago, IL, USA).

Structural Elucidation of Isolated Compounds
The chemical structures of the isolated compounds (1-31) were elucidated through spectroscopic/spectrometric methods such as NMR and HRMS (Figure 1), and in particular, the absolute composition of the new compounds (1 and 3) was carried out through ECD studies.
Compound 1 was isolated as an amorphous solid and had a molecular formula of C 26 Table 1). The HMBC correlations of H-1 with C-5 (δ C 148.6) /C-2 (δ C 77.4) /C-3 (δ C 71.7) and H-4 /H-5 with C-2 /C-3 indicated the presence of oxypeucedanin hydrate [28]. In addition, the downfield shifted H-2 (δ H 5.44) and the HMBC cross peak of H-2 with C-9 (δ C 166.7) indicated that the feruloyl group is linked to oxypeucedanin hydrate C-2 position ( Figure 2). The absolute configurations of C-2 position of 1 was determined by ECD study. The experimental spectrum was in good agreement with the calculated ECD spectrum of the "R"model ( Figure 3). Based on this spectroscopic evidence, the structure of compound 1 established and named koseonolin A.

Effect of Marmesinin (19) on GSIS
We determined the effect of marmesinin (19) on the GSIS and ATP/ADP ratio. As shown in Figure 6A,B, marmesinin (19) increased the GSIS and glucose-dependent ATP/ ADP ratio. Furthermore, we determined the efficiency of marmesinin (19) to modulate K + and Ca 2+ channels. As shown in Figure 6C,D, marmesinin-induced GSIS was enhanced by Bay K 8644 (L-type Ca 2+ channel agonist) and glibenclamide (K + channel blocker), while abrogated by nifedipine (L-type Ca 2+ channel blocker) and diazoxide (K + channel activator).

Discussion
Previous studies have reported that plants including Rhizophora mucronata, Cassia glauca, and Urtica dentata exhibit antidiabetic activity. These plants contain coumarin compounds and decrease blood glucose level in diabetic treated rats [51][52][53]. A previous review study of the anti-diabetic effect of coumarins reports the pharmacodynamics of simple coumarins, furanocoumarins, and pyranocoumarins, chemically classified based on the substitution of lactone and benzene rings in various experimental diabetes mellitus models [54]. In the present study, findings showed that KH2E, an extract from the roots of A. reflexa, significantly enhanced GSIS in INS-1 pancreatic β-cells. Thus, to find new active substances of KH2E, we isolated and identified 31 compounds (1-31) including 3 new compounds (1-3) from KH2E. Among the compounds 1-31, three coumarins, isohydroxylomatin (3), (−)-marmesin (17), and marmesinin (19), increased GSIS. The biological activity of a new compound, isohydroxylomatin (3), has not yet been reported. Furocoumarin, (−)-marmesin (17, nodakenetin), isolated from angelica decursiva, has been reported to have anti-diabetic and anti-Alzheimer-related activities [55]. It has also been reported that (−)-marmesin (17) exhibits anticancer effects against human leukemia cells in vitro and in vivo [56]. On the other hand, marmesin, stereoisomer of 17, isolated from Aegle marmelos, induces both pancreatic β cell regeneration and insulin secretion [57]. Marmesin has been reported to exert anti-cancer activities [58][59][60]. Further, it has been reported that marmesin directly stimulates glucose or acts like insulin to increase glucose utilization [61]. Marmesinin (19) isolated from Angelica gigas has been reported to exhibit neuroprotective and antiplasmodial effects [62,63]. Marmesinin (19) isolated from the bark of Streblus indicus has been reported to exhibit antimicrobial activity, and this compound isolated from the stem bark of Zanthoxylum leprieurii has been reported to exhibit antimycobacterial activity. [64,65]. Additionally, marmesinin (19) has been reported to significantly reduce lipidperoxide-induced myocardial damage in rats [66,67].
In the present study, marmesinin (19) was the most effective on GSIS assay. This effect was superior to treatment with gliclazide, which is often performed in patients with T2D. Marmesinin (19) is a furocoumarin glycoside. The effects of glycoside on insulin secretion have previously been reported in INS-1 cells and isolated mouse islets [68,69]. In addition, glycoside such as stevioside, rutin, and puerarin have been reported for antidiabetic activity [70].
In an additional experiment, treatment with marmesinin (19) resulted in increases in the ATP/ADP ratio. Glibenclamide and Bay K 8644 enhanced GSIS by marmesinin (19), whereas it was suppressed by nifedipine and diazoxide. Previous studies have shown that an increased ATP/ADP ratio is essential in the Ca2 + influx and closure of ATP-sensitive K +

Discussion
Previous studies have reported that plants including Rhizophora mucronata, Cassia glauca, and Urtica dentata exhibit antidiabetic activity. These plants contain coumarin compounds and decrease blood glucose level in diabetic treated rats [51][52][53]. A previous review study of the anti-diabetic effect of coumarins reports the pharmacodynamics of simple coumarins, furanocoumarins, and pyranocoumarins, chemically classified based on the substitution of lactone and benzene rings in various experimental diabetes mellitus models [54]. In the present study, findings showed that KH2E, an extract from the roots of A. reflexa, significantly enhanced GSIS in INS-1 pancreatic β-cells. Thus, to find new active substances of KH2E, we isolated and identified 31 compounds (1-31) including 3 new compounds (1-3) from KH2E. Among the compounds 1-31, three coumarins, isohydroxylomatin (3), (−)-marmesin (17), and marmesinin (19), increased GSIS. The biological activity of a new compound, isohydroxylomatin (3), has not yet been reported. Furocoumarin, (−)-marmesin (17, nodakenetin), isolated from angelica decursiva, has been reported to have anti-diabetic and anti-Alzheimer-related activities [55]. It has also been reported that (−)-marmesin (17) exhibits anticancer effects against human leukemia cells in vitro and in vivo [56]. On the other hand, marmesin, stereoisomer of 17, isolated from Aegle marmelos, induces both pancreatic β cell regeneration and insulin secretion [57]. Marmesin has been reported to exert anti-cancer activities [58][59][60]. Further, it has been reported that marmesin directly stimulates glucose or acts like insulin to increase glucose utilization [61]. Marmesinin (19) isolated from Angelica gigas has been reported to exhibit neuroprotective and antiplasmodial effects [62,63]. Marmesinin (19) isolated from the bark of Streblus indicus has been reported to exhibit antimicrobial activity, and this compound isolated from the stem bark of Zanthoxylum leprieurii has been reported to exhibit antimycobacterial activity. [64,65]. Additionally, marmesinin (19) has been reported to significantly reduce lipidperoxide-induced myocardial damage in rats [66,67].
In the present study, marmesinin (19) was the most effective on GSIS assay. This effect was superior to treatment with gliclazide, which is often performed in patients with T2D. Marmesinin (19) is a furocoumarin glycoside. The effects of glycoside on insulin secretion have previously been reported in INS-1 cells and isolated mouse islets [68,69]. In addition, glycoside such as stevioside, rutin, and puerarin have been reported for antidiabetic activity [70].
In an additional experiment, treatment with marmesinin (19) resulted in increases in the ATP/ADP ratio. Glibenclamide and Bay K 8644 enhanced GSIS by marmesinin (19), whereas it was suppressed by nifedipine and diazoxide. Previous studies have shown that an increased ATP/ADP ratio is essential in the Ca2 + influx and closure of ATP-sensitive K + (KATP) channels [71]. Diazoxide and nifedipine decrease insulin secretion, whereas glibenclamide and Bay K 8644 increase insulin secretion in pancreatic β-cells [72][73][74][75]. Taken together, our findings suggested that after treatment with marmesinin (19), the ability of the pancreatic β-cell to secrete insulin may be due to closure of ATP-sensitive K + (KATP) channels, Ca 2+ influx, and an increase in the ATP/ADP ratio. Furthermore, expression of PPARγ, PDX-1, and IRS-2 were increased and partly attributable to increased insulin secretion following treatment with marmesinin (19). Previous examples of literature have shown that PPARγ activation via full agonists increase GSIS in INS-1 cells [76]. It has been reported that PPAR-γ agonists can protect β cells from apoptosis and restore β cell functions, including GSIS [77]. It has also been described that IRS-2 knockout mice and PDX-1 knockout mice have been reported to display decreased GSIS [78,79]. Accumulating evidence suggests that GSIS from pancreatic β-cells requires an increase in protein expressions of PPARγ, IRS-2, and PDX-1. Consequently, our results show that KH2E can be considered as a potential anti-diabetic plant by increasing insulin secretion in pancreatic β-cells. Therefore, its anti-diabetic effect is attributed to the presence of marmesinin (19). Further preclinical investigations are required to fully understand the potency of marmesinin (19) as a possible antidiabetic agent.

Conclusions
Three new compounds (1-3) along with twenty-eight known compounds (4-31) were isolated from KH2E, the roots extract of A. reflexa. All the isolated compounds were evaluated for their anti-diabetic activity. The present study demonstrated that marmesinin (19) enhances GSIS in INS-1 cells. KH2E and marmesinin (19) increased phosphorylation levels of IRS-2 and activation of PPARγ and PDX-1, associated with pancreatic β-cell functions. Marmesinin (19) enhanced GSIS by shifting the intracellular ATP/ADP ratio and regulating K + and Ca 2+ channels. These findings promote potential application of marmesinin (19) toward the management of hyperglycemia in T2D, and future studies, including animal experiments, would greatly extend our understanding of the additional mechanisms of action of marmesinin (19).