Triterpenoids from the Leaves of Cyclocarya paliurus and Their Glucose Uptake Activity in 3T3-L1 Adipocytes

Four new dammarane triterpenoid saponins cypaliurusides Z1–Z4 (1–4) and eight known analogs (5–12) were isolated from the leaves of Cyclocarya paliurus. The structures of the isolated compounds were determined using a comprehensive analysis of 1D and 2D NMR and HRESIMS data. The docking study demonstrated that compound 10 strongly bonded with PTP1B (a potential drug target for the treatment of type-II diabetes and obesity), hydrogen bonds, and hydrophobic interactions, verifying the importance of sugar unit. The effects of the isolates on insulin-stimulated glucose uptake in 3T3-L1 adipocytes were evaluated and three dammarane triterpenoid saponins (6, 7 and 10) were found to enhance insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Furthermore, compounds 6, 7, and 10 exhibited potent abilities to promote insulin-stimulated glucose uptake in 3T3-L1 adipocytes in a dose-dependent manner. Thus, the abundant dammarane triterpenoid saponins from C. paliurus leaves exhibited stimulatory effects on glucose uptake with application potential as a antidiabetic treatment.


Introduction
Triterpenoids, with a wide variety of structural types and extensive biological activity, are important compounds for the prevention and treatment of various kinds of diseases, such as diabetes mellitus and metabolic syndrome, cancer, hyperlipidemia, cardiovascular and cerebrovascular disease, neurodegenerative disease, bone disease, liver disease, kidney disease, aging, gastrointestinal disease, mental illness involving depression, and skin aging [1][2][3][4][5]. Among triterpenoids, dammarane triterpenoids exhibit the most significant hypoglycemic activity [6][7][8].
Cyclocarya paliurus, an endemic plant that grows in southern China, has been widely used as an herbal tea and is commonly known as the 'sweet tea tree'. The leaves of this plant have been used in Chinese folk medicine to prevent diabetes, hypertension, inflammation, and heart disease [9,10]. There have been many reports on the significant antidiabetic effects of the extract of C. paliurus leaves [11][12][13][14][15][16][17]. An antidiabetic prescription containing the leaves of C. paliurus reduces blood glucose and improves glucose tolerance [18,19].
The leaves of C. paliurus were approved as a novel raw food by the National Health Commission of the People's Republic of China in 2013 [20]. Currently, C. paliurus is a crop for rural revitalization and an important medical and functional raw food. A variety of triterpenoids, flavonoids, and other compounds can be isolated from the leaves of this plant [9,10]. Among these constituents, dammarane triterpenoids are characteristic indicators and the key functional and active constituents of C. paliurus [9,10,21,22]. Several dammarane triterpenoids from this plant demonstrated to moderate the activity of PTP1B (a potential drug target for the treatment of type-II diabetes and obesity) and enhance insulin-stimulated glucose uptake in 3T3-L1 adipocytes [23][24][25][26]. To further investigate potential constituents for promoting glucose uptake activity in 3T3-L1 adipocytes, the active ingredients were separated from the leaves of C. paliurus to afford four novel dammarane saponins (1−4) and eight known analogs (5−12) (Figure 1). Furthermore, stimulation of glucose consumption in 3T3-L1 adipocytes by the triterpenoids isolated from C. paliurus leaves was evaluated and possible hypoglycemic mechanisms of the active compounds were studied. Herein, the isolation, purification, and determination of these isolates, as well as the assays used to determine the glucose uptake activity in 3T3-L1 adipocytes of the constituents and predictions of their mechanisms, are described.
is a crop for rural revitalization and an important medical and functional raw food. A variety of triterpenoids, flavonoids, and other compounds can be isolated from the leaves of this plant [9,10]. Among these constituents, dammarane triterpenoids are characteristic indicators and the key functional and active constituents of C. paliurus [9,10,21,22]. Several dammarane triterpenoids from this plant demonstrated to moderate the activity of PTP1B (a potential drug target for the treatment of type-II diabetes and obesity) and enhance insulin-stimulated glucose uptake in 3T3-L1 adipocytes [23][24][25][26]. To further investigate potential constituents for promoting glucose uptake activity in 3T3-L1 adipocytes, the active ingredients were separated from the leaves of C. paliurus to afford four novel dammarane saponins (1-4) and eight known analogs (5-12) (Figure 1). Furthermore, stimulation of glucose consumption in 3T3-L1 adipocytes by the triterpenoids isolated from C. paliurus leaves was evaluated and possible hypoglycemic mechanisms of the active compounds were studied. Herein, the isolation, purification, and determination of these isolates, as well as the assays used to determine the glucose uptake activity in 3T3-L1 adipocytes of the constituents and predictions of their mechanisms, are described.

Predicted Binding Modes of Compounds and PTP1B Using Molecular Docking Analysis
PTP1B is a potential drug target for the treatment of type-II diabetes and obesity. To to investigate the interactions of PTP1B with dammarane triterpenoid saponins, an independent docking run was performed and the compounds ligand with the lowest binding affinity mode was selected for analysis.
The results showed that 10 was well docked into the active site in PTP1B and the binding energy was −8.9 kcal·mol −1 , which is superior to that of the oleanolic acid (positive control, −7.2 kcal·mol −1 ). The catalytic and allosteric sites of docking compound 10 with PTP1B were further simulated. The results showed that compound 10 depicted multiple important interactions, such as Pi-sigma, alkyl and Pi-alkyl, conventional hydrogen bonds, and van der Waals interactions within the active pocket of PTP1B ( Figure 3). In visualization of the catalytic site docking results ( Figure 3A,C), various amino acid residues, such as Asp548, Ser618, Lys616, Lys620, Asp681, Gly718, Ile719, Ala717, Asp715, Gly720, Ser716, Arg721, Gln762, Phe682, Tyr546, and Val549, surrounded the active pocket of PTP1B. Hydroxyls of compound 10 mainly formed hydrogen bonds with the PTP1B residues Gly720, Asp715, Ile719, ASP681, and Gln762. Moreover, Phe628 revealed Pi-sigma and Pi-alkyl interaction with the methyl of C-6 and C-30, respectively. In visualization of the allosteric site docking results, the sugar unit of compound 10 formed hydrogen bonding with the PTP1B residue Asp789, whereas van der Waals interactions were noticed with Ser786 and Gln788. In addition, the hydrophobic part of the ligand revealed Alkyl and Pi-alkyl interaction with the Phe780 and Lys792 ( Figure 3B,D).
Thus, the extract of C. paliurus leaves (25 µg/mL) and isolated triterpenoid saponins (1-12, 10 µM) were added to differentiated 3T3-L1 adipocytes with 2-NBDG. As shown in Figure 4A, all isolated triterpenoid saponins (1-12) exhibited potency to enhance glucose uptake in 3T3-L1 adipocytes. Among the isolates, compounds 6-7 and 10 at 10 µM increased insulin-stimulated glucose uptake by approximately 37%, 35%, and 46% in 3T3-L1 adipocytes, respectively. The positive control rosiglitazone (ROS, 10 µM) increased glucose uptake in the 3T3-L1 adipocytes by approximately 55%. These triterpene saponins enhance glucose uptake in 3T3-L1 adipocytes more effectively than those of the extract of C. paliurus leaves. The active 6-7 and 10 at various concentrations (2.5, 5, and 10 µM) were further investigated for their effect on glucose uptake. As shown in Figure 4B, 6-7 and 10 significantly increase glucose uptake in the 3T3-L1 adipocytes compared with the vehicle control, which indicates that these compounds may enhance glucose uptake by improving insulin sensitivity in a concentration-dependent manner. Asp715, Gly720, Ser716, Arg721, Gln762, Phe682, Tyr546, and Val549, surrounded the active pocket of PTP1B. Hydroxyls of compound 10 mainly formed hydrogen bonds with the PTP1B residues Gly720, Asp715, Ile719, ASP681, and Gln762. Moreover, Phe628 revealed Pi-sigma and Pi-alkyl interaction with the methyl of C-6′ and C-30, respectively. In visualization of the allosteric site docking results, the sugar unit of compound 10 formed hydrogen bonding with the PTP1B residue Asp789, whereas van der Waals interactions were noticed with Ser786 and Gln788. In addition, the hydrophobic part of the ligand revealed Alkyl and Pi-alkyl interaction with the Phe780 and Lys792 ( Figure 3B,D).

EtOAc Extract and Dammarane Saponins Enhance Glucose Uptake
The 3T3-L1 preadipocyte is one of the most commonly used in vitro models for screening antidiabetic compounds [34,35]. In the present study, the enhancement of glucose uptake by the EtOAc extract (GAE) and triterpenoid saponins (1-12) of C. paliurus was investigated using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy glucose (2-NBDG) uptake model. saponins enhance glucose uptake in 3T3-L1 adipocytes more effectively than those of the extract of C. paliurus leaves. The active 6-7 and 10 at various concentrations (2.5, 5, and 10 µM) were further investigated for their effect on glucose uptake. As shown in Figure  4B, 6-7 and 10 significantly increase glucose uptake in the 3T3-L1 adipocytes compared with the vehicle control, which indicates that these compounds may enhance glucose uptake by improving insulin sensitivity in a concentration-dependent manner.

Plant Material
Dried leaves (10 kg) of C. paliurus were obtained from Xiushui County, Jiangxi, China, in July 2020 and identified by Associate Professor Qiang Xie (Guangxi Normal The differentiated 3T3-L1 adipocytes were treated with compounds 6-7 and 10 at various concentrations (2.5, 5.0, and 10 µM) for 1 h. These results are expressed as the mean ± SD (n = 3) of triplicate analysis; * p < 0.05 and ** p < 0.01.

Plant Material
Dried leaves (10 kg) of C. paliurus were obtained from Xiushui County, Jiangxi, China, in July 2020 and identified by Associate Professor Qiang Xie (Guangxi Normal University). A voucher specimen (No. ID-202070910) was deposited at the State Key Laboratory of Guangxi Normal University (GXNU), China.

Computational Analysis of Molecular Docking Simulation
Molecular docking was used to investigate the interactions between triterpenoids and PTP1B. The structure of PTP1B (PDB ID: 1EEO [36]) was obtained from the Online Protein Data Bank and the structures of the ligands were downloaded from Pubchem. AutoDock tools (ADT, version: 1.5.6) to explore the binding mode between PTP1B and the ligands. AutoDock vina (version 1.2.3) calculated scoring function and predicted binding affinity (kcal/mol), while Pymol (version 2.2.0) was used to analyze the visualization of the docking results.

Extraction and Separation of Dammarane Saponins
The leaves of C. paliurus (10 kg) were purchased from Liuhe medicine market of Guilin city in October 2020. The leaves were extracted 3 times with a 75% ethanol-H 2 O mixture (3:1, v/v, 20 L) under reflux and concentrated under vacuum to remove the ethanol, obtaining a crude extract (2.5 kg). The crude extract (2.5 kg) was suspended in distilled water and partitioned with polyethylene (PE), ethyl acetate (EtOAc), and n-butanol (n-BuOH) to obtain a PE extract (480 g), EtOAc extract (450 g), and n-BuOH extract (1090 g). The glucose uptake activity of the sub-extracts in 3T3-L1 adipocytes was assessed.

Measurement of Glucose Uptake Assay
The level of glucose uptake was assessed using a fluorescent derivative of glucose, 2-NBDG (Beijing, China, Invitrogen, OR, USA). The fully differentiated 3T3-L1 adipocytes were seeded on 96-well plates with glucose-free medium containing 10% FBS and 1 µg/mL insulin. After 24 h of incubation, the cells were treated with test extract (CPBE, 25 µg/mL) as well as compounds (10 µM) and rosiglitazone (ROS, 10 µM, as a positive control) in the presence or absence of 50 µM 2-NBDG. The cells were rinsed with phosphate-buffered saline (PBS) after 1 h of incubation, while cell lysates were treated with 70 µL of 1% Triton X-100 in PBS and 0.1 M K3PO4 for 10 min. To quantify 2-NBDG fluorescence, the fluorescence signal was measured at an excitation wavelength of 450 nm and an emission wavelength of 535 nm using a SpectraMax M5 microplate reader. The 2-NBDG signal was determined using a fluorescence microscope (Olympus ix70, Tokyo, Japan) to detect glucose uptake.

Conclusions
To identify compounds for promoting glucose uptake, the components of C. paliurus leaves were separated, resulting in the identification of twelve triterpenoid saponins (1-12), including four previously undescribed dammarane triterpenoid saponins. The docking study demonstrated that the most active compound strongly bonded with PTP1B, hydrogen bonds and hydrophobic interactions, verifying the importance of the sugar unit. Bioassays demonstrated that the dammarane triterpenoid saponins 6-7 and 10 strongly enhanced insulin-stimulated glucose uptake in 3T3-L1 adipocytes in a dose-dependent manner. Collectively, C. paliurus leaves contain abundant dammarane triterpenoid saponins that affect glucose uptake in 3T3-L1 adipocytes; this discovery could be meaningful for the development of new treatments for insulin resistance and hyperglycemia.