A New Labdane-Type Diterpene, 6-O-Acetyl-(12R)-epiblumdane, from Stevia rebaudiana Leaves with Insulin Secretion Effect

Stevia rebaudiana (Asteraceae), commonly known as candyleaf, sweetleaf, or sugarleaf, is a branched bushy shrub whose leaves are used as a natural sweetener owing to the high content of sweet diterpenes. As part of our ongoing work to identify structurally novel and bioactive natural products, phytochemical investigation of the ethanolic extract of S. rebaudiana leaves led to the isolation of one new labdane-type diterpene, 6-O-acetyl-(12R)-epiblumdane (1), and nine known terpenoids, including six diterpenes (2–6 and 10), two monoterpenes (7 and 8), and one triterpene (9). The structure of the new compound 1 was elucidated via analysis of one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopic data and high-resolution electrospray ionization mass spectrometry data, and its absolute configuration was established using electronic circular dichroism (ECD) calculations and gauge-including atomic orbital NMR chemical shift calculations, followed by DP4 + probability analysis. The isolated compounds 1–10 were evaluated for their effects on glucose-stimulated insulin secretion in the INS-1 rat pancreatic β-cell line. The new compound 1, 6-O-acetyl-(12R)-epiblumdane, stimulated glucose-stimulated insulin secretion in INS-1 pancreatic β-cells without inducing cytotoxicity. Thus, 6-O-acetyl-(12R)-epiblumdane (1), an active compound derived from S. rebaudiana leaves, can be used as a potential therapeutic agent to prevent type 2 diabetes.


Introduction
Stevia rebaudiana Bertoni is a branched bushy shrub of the Asteraceae family, native to the Amambay region in northeast Paraguay. It also grows in neighboring regions of Brazil and Argentina. Its cultivation has recently spread to other regions of the world, including Canada, some parts of Asia, and Europe. S. rebaudiana is commonly known as candyleaf, sweetleaf, or sugarleaf, and its leaves are called "Stevia" and used as a natural sweetener due to the high content of sweet diterpene (~4-20%) in the leaf dry matter. The leaves are the source of several ent-kaurene diterpenoid glycosides (steviosides), which are responsible for the sweet taste. Among 230 species in the genus Stevia, only S. rebaudiana and S. phlebophylla are known to produce steviosides [1]. Stevia and steviosides have been used as substitutes for saccharose in the treatment of diabetes mellitus, obesity, hypertension, and caries prevention [2]. In addition, as natural sweeteners, steviosides and related compounds may offer health benefits with their anti-hyperglycemic, anti-hypertensive, antiinflammatory, anti-tumor, anti-diarrheal, diuretic, and immunomodulatory activities [1]. According to previous phytochemical studies, the leaves of S. rebaudiana possess diverse bioactive compounds, including flavonoids, alkaloids, chlorophylls, xanthophylls, hydroxycinnamic acids (caffeic acids and chlorogenic acids), oligosaccharides, and amino acids [3]. S. rebaudiana leaves are rich in diterpenes, such as sterebins A-N and 6-O-acetyl-austroinulin, Biomedicines 2022, 10, 839 2 of 11 and diterpenoid glycosides, including stevioside, steviolbioside, rebaudiosides A-F, and dulcoside [4].
As part of an ongoing research project to discover unique bioactive natural products derived from diverse medicinal plants and microbes [5][6][7][8][9][10], the leaves of S. rebaudiana were explored for bioactive phytochemicals from their ethanolic (EtOH) extracts. Chemical analysis of the EtOH extract, aided by liquid chromatography-mass spectrometry (LC/MS)based analysis equipped with an in-house UV spectra library, led to the isolation and identification of a new labdane-type diterpene (1) along with nine known compounds (2)(3)(4)(5)(6)(7)(8)(9)(10). The structure of the new labdane-type diterpene (1) was characterized by conducting one-and two-dimensional nuclear magnetic resonance (NMR) experiments and highresolution mass spectrometry (HR-MS), and its absolute configurations were elucidated using electronic circular dichroism (ECD) and gauge-including atomic orbital (GIAO) NMR chemical shift calculations, followed by DP4 + probability analysis. Herein, we describe the separation and structural characterization of the isolated compounds (1-10), including one new compound, and evaluate their effects on glucose-stimulated insulin secretion (GSIS) in an INS-1 rat pancreatic β-cell line.

General Experimental Procedures
Detailed information on the general experimental procedure is included in the Supplementary Materials.

Plant Material
S. rebaudiana leaves were collected in August 2018 from Namyangju-si, Gyeonggi-do, Republic of Korea. The plant material was verified by Prof. K. H. Kim, one of the authors. A voucher specimen, STBA-08-2018, was deposited in the herbarium of the School of Pharmacy affiliated with Sungkyunkwan University, Suwon, Republic of Korea.

Computational Analysis
All of the proposed conformers were obtained through the MacroModel (version 2019-3, Schrödinger, LLC, New York, NY, USA) module with mixed torsional/low-mode sampling implemented with the MMFF94 force field. All cases of searches were initially set in the gas phase, with a 10 kJ/mol energy window limit and a maximum of 10,000 steps, to explore all potential conformers thoroughly. The Polak-Ribiere conjugate gradient protocol was established with 10,000 maximum iterations and a 0.001 kJ (mol Å) −1 convergence threshold on the root mean square gradient to minimize conformers [11][12][13]. The conformers proposed in this study (within 5 kJ/mol found in the MMFF force field) were selected for geometry optimization by TURBOMOLE V7.2, with the density-functional theory settings of B3-LYP/6-31+G(d,p). Geometrically optimized conformers for the possible diastereomers 1a and 1b were used to calculate the GIAO magnetic shielding tensors at the B3-LYP/6-31+G(d,p) level. Chemical shift values were calculated from the magnetic shielding tensors using Equation (1) [14][15][16], where δ x calc is the calculated NMR chemical shift for nucleus x and σ o is the shielding tensor for the proton and carbon nuclei in tetramethylsilane calculated using the density-functional theory B3-LYP/6-31+G(d,p) basis set.
The calculated unscaled NMR properties of the optimized structures were averaged, and the scaled chemical shift values were obtained using Equation (2).
The DP4 + probability analysis was performed using an Excel sheet (DP4 +) provided by Grimblat et al. [15]. ECD calculations of the optimized conformers of enantiomers 1a and 1c were performed at the B3LYP/6-31+G(d,p) level. The calculated ECD spectra were simulated by overlying each transition, where σ is the width of the band at height 1/e (Equation (3)) and ∆E i and R i are the excitation and rotatory strengths of transition i, respectively. In this study, σ was 0.10 eV. The excitation energies and rotational strengths for the ECD spectra were calculated based on the Boltzmann populations of the conformers, and ECD visualization was performed using SigmaPlot 14.0 (Systat Software, Inc., San Jose, CA, USA).

Cell Viability Assay
To measure cell viability, an Ez-Cytox cell viability assay kit purchased from Daeil Lab Service Co. (Seoul, Korea) was used. INS-1 cells were incubated in 96-well plates for 24 h to determine the non-toxic concentration ranges of compounds 1-10. After treatment of the compounds, the Ez-Cytox reagent was added, and absorbance at 450 nm was measured using a microplate reader.

Glucose-Stimulated Insulin Secretion Assay
INS-1 cells were cultured in 12-well plates for 24 h to measure GSIS after treatment with compounds 1-10. After starvation for 2 h, INS-1 cells were treated with the compounds 1-10. After 2 h of incubation, glucose (2.8 and 16.7 mM as basal and stimulant, respectively) was added to each well and incubated further for 1 h. According to the manufacturer's instructions, GSIS was calculated with the secreted insulin by using a rat insulin ELISA kit (Gentaur, Shibayagi Co. Ltd., Shibukawa, Japan).

Statistical Analysis
Statistical significance was analyzed using one-way analysis of variance (ANOVA) and multiple comparisons with Bonferroni correction. All analyses were carried out using SPSS Statistics ver. 19.0 (SPSS Inc., Chicago, IL, USA), and significant differences were considered present at the 5% level (p < 0.05).

Isolation of Compounds 1-10
Leaves of S. rebaudiana were dried and extracted with 80% aqueous EtOH to obtain the crude EtOH extract via rotary evaporation. The resultant EtOH extract was subjected to solvent partitioning using four organic solvents (namely hexane, CH 2 Cl 2 , EtOAc, and n-BuOH), which yielded four main fractions. LC/MS-based analysis of the fractions with reference to our in-house UV library suggested that the hexane-soluble fraction is promising for phytochemical investigation because peaks for diterpenes without sugar moieties were detected in the hexane-soluble fraction. Repeated column chromatography and semi-preparative HPLC resulted in the isolation of 10 terpenoids, including seven diterpenes (1-6 and 10), two monoterpenes (7 and 8), and one triterpene (9) (Figure 2) from the hexane-soluble fraction. reference to our in-house UV library suggested that the hexane-soluble fraction is promising for phytochemical investigation because peaks for diterpenes without sugar moieties were detected in the hexane-soluble fraction. Repeated column chromatography and semi-preparative HPLC resulted in the isolation of 10 terpenoids, including seven diterpenes (1-6 and 10), two monoterpenes (7 and 8), and one triterpene (9) (Figure 2) Figure 2. Chemical structures of the isolated compounds 1-10.  Table 1). The 1 H NMR data (Table  1) (Table 1)  . Comprehensive analysis of the NMR spectral data suggested that the structure of 1 closely resembled that of compound 3, 6-O-acetylaustroinulin [4], which was also isolated as a labdane-type diterpene in this   Table 1). The 1 H NMR data (Table 1) (Table 1) of 1, acquired using heteronuclear single quantum coherence (HSQC) and heteronuclear multiple-bond coherence (HMBC) spectra, showed the presence of 22 carbon resonances, including two olefinic pairs (δ C 114.8/136.7 and δ C 116.5/145.1), three oxygenated methine carbons (δ C 73.1, 83.4, and 83.5), four methylene groups (δ C 17.6, 25.9, 39.5, and 43.3), two methine groups (δ C 52.9 and 56.5), four non-protonated carbons (δ C 33.4, 39.1, 77.8, and 172.1)(including a carbonyl group (δ C 172.1)), and five methyl groups (δ C 16.6, 19.8, 21.6, 21.8, and 35.7). Comprehensive analysis of the NMR spectral data suggested that the structure of 1 closely resembled that of compound 3, 6-O-acetylaustroinulin [4], which was also isolated as a labdane-type diterpene in this study. The NMR data of 1 were similar to those of 3 except for the additional presence of one hydroxyl group and an exo-methylene unit in 1. In addition, a literature survey revealed that the NMR data of 1 also have high similarity to those of (12R)-epiblumdane [17], except for the presence of an acetyl-moiety in 1. The gross planar structure of 1 was confirmed by analysis of 2D NMR experiments.

Structural Elucidation of the Isolated Compounds
The positions of the additional hydroxyl group and exo-methylene units were unambiguously assigned as C-12 and C-13/C-16, respectively, by the HMBC correlations of  (Figure 3), where the partial structure of 1 for the side chain was confirmed as a 2-hydroxy-3-methylene-4-penten-1-yl moiety. In addition, the cross-peaks of H-5/H-6/H-7 in the 1 H-1 H COSY spectrum, along with the HMBC correlations of H-6/C-1 and H-2 /C-1 , were observed, which indicated the presence of an O-acetyl-moiety at C-6. The complete interpretation of the 1 H-1 H COSY and HMBC data afforded the complete planar structure of 1 (Figure 2). one hydroxyl group and an exo-methylene unit in 1. In addition, a literature survey revealed that the NMR data of 1 also have high similarity to those of (12R)-epiblumdane [17], except for the presence of an acetyl-moiety in 1. The gross planar structure of 1 was confirmed by analysis of 2D NMR experiments.

Effect of Compounds on Glucose-Stimulated Insulin Secretion
S. rebaudiana leaves have been proven to be effective against diabetes [24,25]. However, few experimental studies have been performed to identify the antidiabetic compounds from S. rebaudiana leaf extracts. It's well-known that the progression of type 2 diabetes is characterized by the defects in glucose-stimulated insulin secretion (GSIS) [26]. Hence, we investigated the effects of compounds 1-10 on GSIS in an INS-1 rat pancreatic β cell line. To select non-toxic concentrations of compounds 1-10 for performing the GSIS assay, their effects on cell viability were assessed using the Ez-Cytox cell viability assay. Compounds 1-10 did not show any toxicity at concentrations ranging from 2.5 to 10 µM ( Figure 6). Thus, the highest concentration of all the compounds was set to 10 μM in the GSIS assay. To determine the efficacy, compounds 1-10 were screened in INS-1 rat pancreatic β cells. Among these compounds, the new compound 1 significantly increased GSIS (Figure 7). The GSI values of compound 1 were 2.51 ± 0.04 and 3.34 ± 0.05 at concentrations of 5 and 10 μM, respectively. Thus, the highest concentration of all the compounds was set to 10 µM in the GSIS assay.
To determine the efficacy, compounds 1-10 were screened in INS-1 rat pancreatic β cells. Among these compounds, the new compound 1 significantly increased GSIS (Figure 7). The GSI values of compound 1 were 2.51 ± 0.04 and 3.34 ± 0.05 at concentrations of 5 and 10 µM, respectively. Thus, the highest concentration of all the compounds was set to 10 μM in the GSIS assay. To determine the efficacy, compounds 1-10 were screened in INS-1 rat pancreatic β cells. Among these compounds, the new compound 1 significantly increased GSIS (Figure 7). The GSI values of compound 1 were 2.51 ± 0.04 and 3.34 ± 0.05 at concentrations of 5 and 10 μM, respectively.

Conclusions
In this study, chemical investigation of the extracts of S. rebaudiana leaves resulted in the isolation of one new labdane-type diterpene, 6-O-acetyl-(12R)-epiblumdane (1), and nine known terpenoids, including six diterpenes (2-6 and 10), two monoterpenes (7 and 8), and one triterpene (9). The structure of the new compound 1 was elucidated by analyzing one-and two-dimensional NMR data, HR-ESI-MS data, and NMR chemical shift calculations, followed by DP4 + probability analysis as well as quantum chemical ECD calculations. We found that 6-O-acetyl-(12R)-epiblumdane (1) increased glucose-stimulated insulin secretion in an INS-1 rat pancreatic β-cell line. Our results suggest that 6-O-acetyl-(12R)-epiblumdane (1), an active compound derived from S. rebaudiana leaves, can be applied as a potential antidiabetic agent. Further studies will be required to elucidate the exact mechanism by which the active compound 1 prevents type 2 diabetes.