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

Liang, Xiaoqin; Deng, Shengping; Huang, Yan; Pan, Liwei; Chang, Yanling; Hou, Ping; Ren, Chenyang; Xu, Weifeng; Yang, Ruiyun; Li, Kanyuan; Li, Jun; He, Ruijie

doi:10.3390/molecules28083294

Open AccessArticle

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

by

Xiaoqin Liang

¹,

Shengping Deng

¹,

Yan Huang

¹,

Liwei Pan

¹,

Yanling Chang

¹,

Ping Hou

¹,

Chenyang Ren

¹,

Weifeng Xu

¹,

Ruiyun Yang

¹,

Kanyuan Li

¹,

Jun Li

^1,* and

Ruijie He

^2,*

¹

State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources/Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), Collaborative Innovation Center for Guangxi Ethnic Medicine, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004, China

²

Guangxi Key Laboratory of Plant Functional Phytochemicals and Sustainable Utilization, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006, China

^*

Authors to whom correspondence should be addressed.

Molecules 2023, 28(8), 3294; https://doi.org/10.3390/molecules28083294

Submission received: 4 March 2023 / Revised: 31 March 2023 / Accepted: 5 April 2023 / Published: 7 April 2023

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Abstract

:

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.

Keywords:

Cyclocarya paliurus; dammarane triterpenoid saponins; glucose uptake; 3T3-L1 adipocytes

1. 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.

2. Results and Discussion

2.1. Elucidation of the Chemical Structure of Cypaliurusides Z₁–Z₄ (1−4)

Cypaliuruside Z₁ (1) was deduced to have the molecular formula C₃₇H₅₈O₁₂ (Figure S1) based on its NMR (¹H, ¹³C, pyridine-d₅) data and the positive-ion peak [M+Na]⁺ at m/z 717.3811 (calculated for C₃₇H₅₈O₁₂Na, 717.3826) in its HRESIMS spectrum. Comparisons between the MS and NMR signals of compound 1 and those of cyclocarioside L (5) [27] indicated that 1 and 5 have almost identical NMR data (Table 1; Figures S2 and S3). There were only a few differences in the ¹H-and ¹³C-NMR data (pyridine-d₅) between 1 and 5 at the positions 20, 22, 23, and 24 and two sugar units. The ¹H NMR signals of H-22 and 23 shifted from δ_H 1.97 (m, H-22), 1.82 (m, H-22), 2.64 (m, H-23), and 2.49 (m, H-23) for 5 to δ_H 7.64 (d, 5.7, H-22) and 6.26 (d, 5.7, H-23) for 1, respectively (Table 1; Figure S2). The ¹³C NMR (pyridine-d₅, Table 1; Figure S3) signals of C-20, C-22, C-23, and C-24 shifted from δ_C 89.6, 32.5, 29.7, and 176.9 for 5 to δ_C 92.6, 161.2, 121.9, and 173.4 for 1, respectively (Table 1); this indicates that there is a double bond at the positions 22 and 23 in 1. Moreover, the ¹³C NMR data of the two sugar units in 1 contained ten carbon signals is compared to eleven corresponding carbon signals for 5. In the ¹H-¹H COSY spectrum of 1, the correlations of H-1/H-2/H-3, H-5/H-6/H-7 and H-9/H-11/H-12/H-13/H-15/H-16/H-17 substantiated the existence of three fragments: –CH₂CH₂CH–, –CH₂CH₂CH–, and –CHCH₂CHCHCHCH CH₂– (Figure 2A and Figure S6). In the HMBC spectrum of 1 (Figure 2A and Figure S7, pyridine-d₅), the cross peaks from H-19-CH₃ (δ_H 1.29, s) to C-1 (δ_C 36.1), C-5 (δ_C 51.3) and C-9 (δ_C 54.3), H-18-CH₃ (δ_H 0.90, s) to C-9 (δ_C 54.3) and C-14 (δ_C 41.9), and H-30-CH₃ (δ_H 0.55, s) to C-8 (δ_C 50.7) and C-15 (δ_C 31.7) indicated that CH₃-19 was linked to C-10, CH₃-18 was located at C-8, and CH₃-30 was connected to C-14, respectively. The HMBC correlations from H-21 (δ_H 1.36, s) to C-17 (δ_C 47.8) and C-22 (δ_C 121.9) suggested that CH₃-21 was situated at C-20. Furthermore, the diagnostic ROESY correlations of H-3 and H-11 to H-19 suggested that H-3, H-11, and H-19 are β-oriented, biosynthetic characteristics of dammarane trierpenoids [28], further indicating that H-19 just located at β-oriented. Similarly, the diagnostic ROESY peaks of H-17 and H-30 indicated that H-17 and H-30 are α-oriented (Figure 2B). The HMBC correlations from the anomeric protons H-1′ (δ_H 4.73, d, J = 6.8 Hz) to C-3 (δ_C 82.2) and H-1″ (δ_H 4.87, d, J = 6.5 Hz) to C-12 (δ_C 76.8) suggest that the two L-arabinoses are situated at C-3 and C-12 respectively (Figure 2A). The sugar unit was determined to be an α-L-arabinosyl moiety based on the hydrolysis of 1 with 1 M HCl and HPLC (Table 2). Therefore, 1 was deduced to have the structure (20S,24)-lactone-22-en-dammarane-(3α,12β)-12-O-α-L-arabinopyranosyl-3-O-α-L-arabinopyranoside and, therefore, is named cypaliuruside Z₁ (Figure 1).

Cypaliuruside Z₂ (2) was deduced to have the molecular formula C₃₈H₆₂O₁₃ by HRESIMS m/z 749.4075 [M + Na]⁺ (calculated for C₃₈H₆₂O₁₃Na, 749.4088) (Figure S9), suggesting eight degrees of unsaturation. Detailed analyses show that the NMR data of 2 (Table 1) were highly similar to those of 5 [27], suggesting that 2 includes a basic (20S,24)-lactone-22-en-dammarane triterpenoid skeleton and differed from those of cyclocarioside L only in the signals of the two sugar units (Table 1; Figure S10). The ¹H NMR spectrum indicated two anomeric protons at δ_H 5.11 (d, J = 7.7 Hz, H-1″) and 4.74 (d, J = 6.5 Hz, H-1′) and six methyl protons at δ_H 1.38 (s, H-19), 1.26 (s, H-29), 0.98 (s, H-18), 0.96 (s, H-28), 1.28 (s, H-21), and 0.62 (s, H-30) (Table 1). The ¹³C NMR of 38 carbon resonances confirmed the aforementioned moieties (Table 1). The ¹³C-NMR (pyridine-d₅, Table 1, Figure S11) and DEPT (distortionless enhancement by polarization transfer, pyridine-d₅) spectra indicated the presence of six methyl carbon signals at δ_C 17.5 (C-18), 17.4 (C-19), 24.9 (C-21), 23.8 (C-28), 30.0 (C-29), and 16.8 (C-30) and two glycosyl anomeric carbon signals at δ_C 101.8 (C-1′) and δ_C 102.3 (C-1″), respectively. In the ¹H-¹H COSY spectrum of 2, the correlations of H-1/H-2/H-3, H-5/H-6/H-7 and H-9/H-11/H-12/H-13/H-15/H-16/H-17 substantiated the existence of three fragments: –CH₂CH₂CH–, –CH₂CH₂CH–, and –CHCH₂CHCHCHCH₂CH₂– (Figure 2A and Figure S6). The sugar unit was determined to be α-L-arabinopyranose and β-D-glucopyranose based on the hydrolysis of 2 with 1 M HCl and HPLC (Table 2). Therefore, 2 was deduced to have the structure (20S,24)-lactone-dammarane-(3α,12β)-12- O-β-D-glucopyranosyl-3-O-α-L-arabinopyranoside and is, thus, named cypaliuruside Z₂ (Figure 1).

The molecular formula of cypaliuruside Z₃ (3) was deduced to be C₃₈H₆₂O₁₃ due to the positive sodium adduct ion HRESIMS peak at m/z 749.4096 [M + Na]⁺ (calculated for C₃₈H₆₂O₁₃Na, 749.4088) (Figure S17), which is the same as that of 2. The ¹H- and ¹³C-NMR data of 3 (pyridine-d₅, Table 3) were found to be identical to those of 2. A small difference between the ¹H- and ¹³C-NMR data of 3 and 2 (pyridine-d₅) was related to a sugar unit linked to C-3. That is, the ¹H-NMR data of the sugar unit linked to C-3 shifted from δ_H 4.74 (d, J = 6.5 Hz, H-1′), 4.45 (t, J = 8.9 Hz, H-2′), 4.27 (dd, J = 8.4, 3.5 Hz, H-3′), 4.42 (m, H-4′), 4.34 (dd, J = 12.1, 3.7 Hz, H-5′), and 3.80 (m, H-5′) for 2 (pyridine-d₅, Table 2) to δ_H 5.55 (d, J = 6.5 Hz, H-1′), 4.89 (t, J = 5.6 Hz, H-2′), 4.85 (m, H-3′), 4.76 (dd, J = 6.0, 5.0 Hz, H-4′), 4.40 (m, H-5′), and 4.29 (d, J = 9.4 Hz, H-5′) for 3 (pyridine-d₅, Table 2). Moreover, the ¹³C-NMR data of the sugar unit linked to C-3 changed from δ_C 102.3 (C-1′), 72.1 (C-2′), 75.3 (C-3′), 69.7 (C-4′), and 66.9 (C-5′) for 2 to δ_C 106.9 (C-1′), 84.6 (C-2′), 79.9 (C-3′), 86.0 (C-4′), and 63.4 (C-5′) for 3, respectively (pyridine-d₅, Table 2). Comparing the ¹H- and ¹³C-NMR (Table 2) and HRESIMS data between 3 and 2 revealed the presence of an arabinofuranose linked to C-3 in 3 instead of an arabinopyranose linked to C-3 in 2. α-L-Arabinofuranose and β-D-glucopyranose were confirmed by the acid hydrolysis solution of 3 in conjunction with comparison to authentic sugars in the HPLC assay (Table 2). Accordingly, 3 was deduced to have the structure (20S,24)-lactone-dammarane-(3α,12β)-12-O-β-D-glucopyranosyl-3-O-α-L-arabino-furanoside and is, thus, named cypaliuruside Z₃ (3) (Figure 1).

Cypaliuruside Z₄ (4) was deduced to have the molecular formula C₃₈H₆₂O₁₂ based on the positive sodium adduct ion HRESIMS peak at m/z 733.4146 [M + Na]⁺ (calculated for C₃₈H₆₂O₁₂Na, 733.4139) (Figure S25), which is 16 Da lower than that of 2. A detailed analysis of the ¹H- and ¹³C-NMR data (pyridine-d₅, Table 3) showed that 4 is very similar to 2, except that the glucopyranosyl moiety is replaced by a quinovopyranosyl moiety at C-12 in 4. Moreover, the hydrolysate of compound 4 was determined to contain L-arabinose and quinovose by comparison with an authentic sample in the HPLC test (Table 2). Accordingly, 4 was deduced to have the structure (20S,24)-lactone-dammarane-(3α,12β)-12-O-β-D-quinovopyranosyl-3-O-α-L-arabinopyranoside and is, thus, named cypaliuruside Z₄ (Figure 1).

By comparing the measured NMR (¹H and ¹³C) and MS data to those reported in the literature, the known dammarane triterpenoid saponins were identified as cyclocarioside L (5) [27], cypaliuruside M (6) [29], cyclocarioside J (7) [30], cyclocarioside Z₁₈ (8) [25], cyclocarioside Z₁₀ (9) [25], cyclocarioside H (10) [31], 2α,3α,23-trihydroxy-12,20(30)-dien-28-ursolic acid 28-O-β-D-glucopyranoside (11) [32], 1-oxo-3β,23-dihydroxy olean-12-en-28-oic acid 28-O-β-D-glucopyranoside (12) [33].

2.2. 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).

2.3. 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.

Firstly, the cell viability was measured using the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. The 3T3-L1 adipocyte cell viability test showed the extract at a glucose: 2-NBDG. Isolated compounds and rosiglitazone (ROS, the positive control) were treated to differentiate concentrations of 25 μg/mL; the triterpenoid saponins (1–12) at a concentration of 10 μM were not cytotoxic and the cells had a survival rate of up to 90%.

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.

3. Materials and Methods

3.1. General Experimental Procedures

Silica gel (200–300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China), reversed-phase C18 (50 μm, Merck, Rahway, NJ, USA), and Sephadex LH-20 (Amersham Pharmacia Biotech, Amersham, UK) columns were used for chromatographic separations. L-Arabinose, D-arabinose, L-glucose, D-glucose, L-quinovose and D-quinovose were used as sugar standards (Sigma, Munich, Germany). All solvents were of HPLC or analytical grade. Agilent 1260 semipreparative HPLC (ODS-A, 8 μm, 250 ×10.0 mm, YMC, Kyoto, Japan).

3.2. 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.

3.3. 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.

3.4. 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–H₂O 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.

The active n-BuOH fraction (1090 g) was chromatographed on a silica gel column eluted with a gradient of increasing methanol content (0–100%) in a mixture with dichloromethane to yield 8 fractions (Fr. I to Fr. VIII). The active Fr. III (220 g) was subjected to C₁₈ column chromatography (H₂O/MeOH 50:50–10:90) to provide nine fractions (Fr. III-1 to Fr. III-9). Fr. III-2 (2.3 g) was fractioned by an HW-40F column (H₂O/MeOH, 100:0−0:100) to obtain four subfractions (Fr. III-2-1 to Fr. III-2-7). Fr. III-2-1 (1000 mg) was isolated with a C₁₈ column, followed by C₁₈ semipreparative HPLC (H₂O-MeCN, 34:66, v/v, 3.0 mL/min), to yield 1 (t_R 28.43 min, 10.7 mg), 2 (t_R 18.59 min, 3.2 mg), and 3 (t_R 22.68 min, 5.1 mg). Fr. III-3 (4.0 g) was separated by silica gel (CH₂Cl₂-MeOH, 100:0–0:100) to obtain nine subfractions (Fr. III-3-1 to Fr. III-3-9). Fr. III-3-6 was purified by Sephadex LH-20 (MeOH) and C₁₈ semipreparative HPLC (H₂O-MeCN, 58:42, v/v, 3.0 mL/min) to obtain 4 (t_R 15.10 min, 3.5 mg), 11 (t_R 35.46 min, 6.8 mg) and 12 (t_R 45.12 min, 2.7 mg). Fr. III-6 (3.8 g) was further chromatographed by silica gel CC eluted with CH₂Cl₂-MeOH (12:1, 10:1, 6:1) to 100% to afford three subfractions (Fr. III-6-1 to Fr. III-6-3). Fr. III-6-2 was subjected to Sephadex LH-20 CC, eluted with MeOH, and then further purified by C₁₈ preparative HPLC (H₂O-MeOH, 15:85, v/v, 3.0 mL/min) to afford compounds 7 (t_R 25.35 min, 3.0 mg), 9 (t_R 13.05 min, 3.7 mg), and 10 (t_R 16.89 min, 2.0 mg). Fr. III-6-3 was isolated by Sephadex LH-20, eluted with MeOH, and then purified by C₁₈ preparative HPLC (H₂O-MeOH, 15:85, v/v, 3.0 mL/min) to obtain compounds 5 (t_R 16.89 min, 2.0 mg), 6 (t_R 16.78 min, 3.5 mg), and 8 (t_R 22.23 min, 10.3 mg).

3.5. Characterization of the Isolates

Cypaliuruside Z₁ (1): white amorphous powder;

{[α]}_{D}^{25}

= −18.72 (c 0.30, MeOH); HRESIMS m/z 717.3811 [M + Na]⁺ (calculated for C₃₇H₅₈O₁₂Na, 717.3826); for the ¹H (pyridine-d₅, 500 MHz) and ¹³C NMR (pyridine-d₅, 125 MHz) data, see Table 1. All significant data are given in the electronic supporting information materials (Figures S1–S8).

Cypaliuruside Z₂ (2): white amorphous powder;

{[α]}_{D}^{25}

= −20.03 (c 0.22, MeOH); HRESIMS m/z 749.4075 [M + Na]⁺ (calculated for C₃₈H₆₂O₁₃Na, 749.4088); for the ¹H (pyridine-d₅, 600 MHz) and ¹³C NMR (pyridine-d₅, 150 MHz) data, see Table 1. All significant data are given in the electronic supporting information materials (Figures S9–S16).

Cypaliuruside Z₃ (3): white amorphous powder;

{[α]}_{D}^{25}

= −20.94 (c 0.22, MeOH); HRESIMS m/z 749.4096 [M + Na]⁺ (calculated for C₃₈H₆₂O₁₃Na, 749.4088); for the ¹H (pyridine-d₅, 500 MHz) and ¹³C NMR (pyridine-d₅, 125 MHz) data, see Table 2. All significant data are given in the electronic supporting information materials (Figures S17–S24).

Cypaliuruside Z₄ (4): white amorphous powder;

{[α]}_{D}^{25}

= −19.89 (c 0.25, MeOH); HRESIMS m/z 733.4146 [M + Na]⁺ (calculated for C₃₈H₆₂O₁₂Na, 733.4139); for the ¹H (pyridine-d₅, 600 MHz) and ¹³C NMR (pyridine-d₅, 150 MHz) data, see Table 2. All significant data are given in the electronic supporting information materials (Figures S25–S32).

Cyclocarioside L (5): white amorphous powder. ¹H NMR (pyridine-d₅, 600 MHz) δ_H 2.98 (1H, m, H-1a), 2.01 (1H, m, H-1b), 2.01 (1H, m, H-2a), 1.89 (1H, m, H-2b), 3.50 (1H, m, H-3), 1.53 (1H, d, J = 11.0 Hz, H-5), 1.63 (1H, m, H-6a), 1.45 (1H, m, H-6b), 1.39 (1H, m, H-7a), 1.08 (1H, d, J = 9.2 Hz, H-7b), 4.16 (1H, m, H-12), 0.92 (3H, s, H-18), 1.35 (3H, s, H-19), 1.45 (3H, s, H-21), 0.93 (3H, s, H-28), 1.23 (3H, s, H-29), 0.65 (3H, s, H-30), 5.45 (1H, d, J = 7.0 Hz, H-1′), 4.75 (1H, dd, J = 3.1, 8.5 Hz, H-4′), 5.10 (1H, d, J = 7.5 Hz, H-1″), 1.65 (3H, d, J = 7.3 Hz, H-6″). ¹³C NMR (pyridine-d₅, 150 MHz) δ_C 37.1 (C-1), 22.4 (C-2), 80.5 (C-3), 38.9 (C-4), 50.7 (C-5), 19.3 (C-6), 36.0 (C-7), 50.6 (C-8), 57.7 (C-9), 40.3 (C-10), 34.2 (C-11), 76.1 (C-12), 41.6 (C-13), 40.5 (C-14), 31.7 (C-15), 25.7 (C-16), 50.1 (C-17), 17.0 (C-18), 17.1 (C-19), 89.6 (C-20), 21.4 (C-21), 32.1 (C-22), 30.3 (C-23), 176.2 (C-24), 23.6 (C-28), 30.4 (C-29), 16.8 (C-30), 102.4 (C-1′), 72.3 (C-2′), 75.4 (C-3′), 69.4 (C-4′), 69.8 (C-5′), 101.5 (C-1″), 75.1 (C-2″), 78.7 (C-3″), 78.1 (C-4″), 72.1 (C-5″), 18.9 (C-6″).

Cypaliuruside M (6): white amorphous powder. ¹H NMR (pyridine-d₅, 600 MHz) δ_H 3.12 (1H, m, H-1a), 2.11 (1H, m, H-1b), 1.97 (1H, m, H-2a), 1.59 (1H, m, H-2b), 3.64 (1H, t, J = 2.7 Hz, H-3), 1.68 (1H, m, H-5), 1.53 (1H, m, H-6a), 1.45 (1H, m, H-6b), 1.49 (1H, m, H-7a), 1.28 (1H, m, H-7b), 4.26 (1H, dt, J = 4.7, 10.5 Hz, H-11), 1.12 (3H, s, H-18), 1.45 (3H, s, H-19), 1.15 (3H, s, H-21), 1.45 (3H, s, H-26), 1.43 (3H, s, H-27), 1.03 (3H, s, H-28), 1.33 (3H, s, H-29), 0.65 (3H, s, H-30), 4.65 (1H, d, J = 7.5 Hz, H-1′), 4.21 (1H, dd, J = 2.1, 8.5 Hz, H-3′), 4.25 (1H, dd, J = 5.1, 9.5 Hz, H-4′), 4.35 (1H, dd, J = 5.1, 11.5 Hz, H-5′), 1.59 (3H, d, J = 6.7 Hz, H-6′), 5.05 (1H, d, J = 7.7 Hz, H-1″), 4.05 (1H, d, J = 5.3 Hz, H-2″), 4.10 (1H, dd, J = 5.1, 11.5 Hz, H-3″), 1.68 (3H, d, J = 5.3 Hz, H-6″). ¹³C NMR (pyridine-d₅, 150 MHz) δ_C 36.1 (C-1), 21.4 (C-2), 81.5 (C-3), 38.9 (C-4), 50.9 (C-5), 18.3 (C-6), 36.7 (C-7), 50.6 (C-8), 54.7 (C-9), 40.6 (C-10), 76.2 (C-11), 34.1 (C-12), 41.6 (C-13), 40.8 (C-14), 31.4 (C-15), 26.7 (C-16), 50.1 (C-17), 17.2 (C-18), 17.0 (C-19), 86.2 (C-20), 24.0 (C-21), 34.1 (C-22), 26.3 (C-23), 84.2 (C-24), 71.2 (C-25), 26.1 (C-26), 28.4 (C-27), 23.6 (C-28), 30.4 (C-29), 16.8 (C-30), 101.4 (C-1′), 75.3 (C-2′), 78.4 (C-3′), 77.4 (C-4′), 72.8 (C-5′), 18.8 (C-6′), 102.5 (C-1″), 75.3 (C-2″), 78.7 (C-3″), 77.6 (C-4″), 73.1 (C-5″), 18.9 (C-6″).

Cyclocarioside J (7): white amorphous powder. ¹H NMR (pyridine-d₅, 600 MHz) δ_H 3.11 (1H, m, H-1a), 1.98 (1H, m, H-1b), 1.87 (1H, m, H-2a), 1.69 (1H, m, H-2b), 3.65 (1H, t, J = 3.7 Hz, H-3), 1.65 (1H, m, H-5), 1.48 (1H, m, H-6a), 1.49 (1H, m, H-6b), 4.33 (1H, dt, J = 5.3, 10.8 Hz, H-11), 1.04 (3H, s, H-18), 1.42 (3H, s, H-19), 1.10 (3H, s, H-21), 1.55 (3H, s, H-26), 1.39 (3H, s, H-27), 0.98 (3H, s, H-28), 1.30 (3H, s, H-29), 0.67 (3H, s, H-30), 4.75 (1H, d, J = 6.8 Hz, H-1′), 4.42 (1H, t, J = 7.6 Hz, H-2′), 4.21 (1H, dd, J = 2.1, 8.5 Hz, H-3′), 4.25 (1H, dd, J = 5.1, 9.5 Hz, H-4′), 4.35 (1H, m, H-5a′), 3.78 (1H, dd, J = 11.0, 13.4 Hz, H-5b′), 5.01 (1H, d, J = 7.1 Hz, H-1″), 4.12 (1H, m, H-2″), 4.21 (1H, dd, J = 4.8, 10.9 Hz, H-3″), 1.65 (3H, d, J = 5.7 Hz, H-6″). ¹³C NMR (pyridine-d₅, 150 MHz) δ_C 36.2 (C-1), 21.5 (C-2), 81.5 (C-3), 38.1 (C-4), 51.2 (C-5), 18.4 (C-6), 36.5 (C-7), 50.1 (C-8), 54.4 (C-9), 40.2 (C-10), 76.5 (C-11), 34.3 (C-12), 41.2 (C-13), 41.2 (C-14), 31.2 (C-15), 26.9 (C-16), 49.9 (C-17), 17.0 (C-18), 16.9 (C-19), 85.8 (C-20), 24.4 (C-21), 34.5 (C-22), 25.8 (C-23), 84.0 (C-24), 71.1 (C-25), 26.1 (C-26), 28.6 (C-27), 23.3 (C-28), 30.4 (C-29), 16.6 (C-30), 103.4 (C-1′), 72.3 (C-2′), 75.4 (C-3′), 70.0 (C-4′), 67.8 (C-5′), 102.2 (C-1″), 76.0 (C-2″), 78.7 (C-3″), 77.1 (C-4″), 73.0 (C-5″), 19.0 (C-6″).

Cyclocarioside Z₁₈ (8): white amorphous powder. ¹H NMR (pyridine-d₅, 600 MHz) δ_H 3.05 (1H, m, H-1a), 1.84 (1H, m, H-1b), 1.77 (1H, m, H-2a), 1.67 (1H, m, H-2b), 3.55 (1H, br s, H-3), 1.56 (1H, m, H-5), 1.58 (1H, m, H-6a), 1.44 (1H, m, H-6b), 4.43 (1H, dt, J = 5.0, 10.5 Hz, H-11), 1.05 (3H, s, H-18), 1.32 (3H, s, H-19), 1.45 (3H, s, H-21), 1.35 (3H, s, H-26), 1.35 (3H, s, H-27), 0.98 (3H, s, H-28), 1.26 (3H, s, H-29), 0.80 (3H, s, H-30), 5.51 (1H, br s, H-1′), 4.45 (1H, m, H-2′), 4.63 (1H, m, H-3′), 4.75 (1H, m, H-4′), 4.61 (1H, m, H-5a′), 4.28 (1H, m, H-5b′), 5.11 (1H, d, J = 7.5 Hz, H-1″), 4.02 (1H, d, J = 8.5 Hz, H-2″), 4.26 (1H, m, H-3″), 4.03 (1H, dd, J = 4.1, 8.4 Hz, H-4″), 4.17 (1H, t, J = 8.9 Hz, H-5″), 4.65 (H, d, J = 11.2 Hz, H-6a″), 4.35 (H, m, H-6b″). ¹³C NMR (pyridine-d₅, 150 MHz) δ_C 36.2 (C-1), 21.6 (C-2), 79.5 (C-3), 38.1 (C-4), 51.2 (C-5), 18.6 (C-6), 36.5 (C-7), 50.1 (C-8), 54.4 (C-9), 40.7 (C-10), 77.5 (C-11), 34.3 (C-12), 41.4 (C-13), 41.6 (C-14), 31.7 (C-15), 26.8 (C-16), 49.6 (C-17), 17.2 (C-18), 16.9 (C-19), 86.8 (C-20), 24.5 (C-21), 34.5 (C-22), 26.8 (C-23), 84.5 (C-24), 71.1 (C-25), 26.1 (C-26), 28.3 (C-27), 23.3 (C-28), 30.4 (C-29), 16.9 (C-30), 106.4 (C-1′), 84.5 (C-2′), 80.0 (C-3′), 81.2 (C-4′), 65.8 (C-5′), 102.2 (C-1″), 76.0 (C-2″), 78.9 (C-3″), 78.1 (C-4″), 73.0 (C-5″), 19.6 (C-6″).

Cyclocarioside Z₁₀ (9): white amorphous powder. ¹H NMR (pyridine-d₅, 400 MHz) δ_H 3.08 (1H, dd, J = 2.7, 9.2 Hz, H-1a), 2.12 (1H, m, H-1b), 2.11 (1H, m, H-2a), 1.79 (1H, m, H-2b), 3.63 (1H, m, H-3), 1.83 (1H, m, H-5), 1.53 (1H, m, H-6a), 1.45 (1H, m, H-6b), 4.46 (1H, dt, J = 5.7, 9.9 Hz, H-11), 1.12 (3H, s, H-18), 1.38 (3H, s, H-19), 1.44 (3H, s, H-21), 5.30 (1H, s, H-26a), 4.95 (1H, s, H-26b), 1.90 (3H, s, H-27), 0.98 (3H, s, H-28), 1.23 (3H, s, H-29), 0.85 (3H, s, H-30), 4.95 (1H, d, J = 7.5 Hz, H-1″), 3.97 (1H, t, J = 8.5 Hz, H-2″), 2.00 (3H, s, CH₃COO), 4.96 (1H, br s, H-1′), 4.05 (1H, m, H-2′), 3.89 (1H, dd, J = 6.3, 7.5 Hz, H-3′), 4.75 (1H, m, H-4′), 4.21 (1H, dd, J = 3.3, 11.5 Hz, H-5a′), 4.28 (1H, dd, J = 6.3, 11.3 Hz, H-5b′), 4.40 (1H, d, J = 7.7 Hz, H-1″), 3.10 (1H, dd, J = 7.7, 9.0 Hz, H-2″), 3.35 (1H, dd, J = 8.7,9.0 Hz, H-3″), 2.98 (1H, dd, J = 2.7, 8.7 Hz, H-4″), 3.24 (1H, m, H-5″), 1.24 (3H, d, J = 5.8 Hz, H-6″). ¹³C NMR (pyridine-d₅, 100 MHz) δ_C 37.1 (C-1), 26.3 (C-2), 79.5 (C-3), 38.2 (C-4), 51.0 (C-5), 18.7 (C-6), 36.6 (C-7), 41.6 (C-8), 54.7 (C-9), 40.3 (C-10), 34.2 (C-11), 76.9 (C-12), 41.6 (C-13), 50.0 (C-14), 31.7 (C-15), 27.7 (C-16), 50.1 (C-17), 17.1 (C-18), 16.8 (C-19), 86.6 (C-20), 24.4 (C-21), 34.1 (C-22), 26.3 (C-23), 84.2 (C-24), 71.5 (C-25), 26.5 (C-26), 26.4 (C-27), 29.6 (C-28), 22.4 (C-29), 16.9 (C-30), 106.4 (C-1′), 83.3 (C-2′), 79.4 (C-3′), 82.0 (C-4′), 65.8 (C-5′), 170.9 (CH₃COO), 100.9 (C-1″), 75.1 (C-2″), 78.2 (C-3″), 77.1 (C-4″), 72.1 (C-5″), 18.3 (C-6″).

Cyclocarioside H (10): white amorphous powder. ¹H NMR (pyridine-d₅, 400 MHz) δ_H 2.58 (1H, dd, J = 3.7, 13.2 Hz, H-1a), 1.34 (1H, m, H-1b), 4.06 (1H, m, H-12), 1.10 (3H, s, H-18), 1.18 (3H, s, H-19), 1.14 (3H, s, H-21), 3.74 (1H, d, J = 7.5 Hz, H-24), 1.15 (3H, s, H-26), 1.19 (3H, s, H-27), 0.98 (3H, s, H-28), 0.90 (3H, s, H-29), 0.95 (3H, s, H-30), 4.95 (1H, d, J = 7.5 Hz, H-1″), 4.08 (1H, t, J = 8.5 Hz, H-2″), 4.08 (1H, d, J = 8.7 Hz, H-3″), 3.65 (1H, dd, J = 2.7, 8.7 Hz, H-4″), 3.60 (1H, d, J = 5.9 Hz, H-5″), 1.60 (3H, d, J = 5.8 Hz, H-6″). ¹³C NMR (pyridine-d₅, 100 MHz) δ_C 36.1 (C-1), 26.4 (C-2), 75.5 (C-3), 38.9 (C-4), 50.9 (C-5), 19.0 (C-6), 36.0 (C-7), 50.6 (C-8), 54.7 (C-9), 40.3 (C-10), 77.2 (C-11), 35.0 (C-12), 41.6 (C-13), 42.0 (C-14), 31.7 (C-15), 27.7 (C-16), 50.1 (C-17), 17.5 (C-18), 17.1 (C-19), 74.6 (C-20), 27.4 (C-21), 38.1 (C-22), 31.3 (C-23), 76.2 (C-24), 150.1 (C-25), 110.5 (C-26), 18.9 (C-27), 23.6 (C-28), 30.4 (C-29), 16.9 (C-30), 101.9 (C-1″), 76.1 (C-2″), 78.7 (C-3″), 77.1 (C-4″), 73.1 (C-5″), 18.9 (C-6″).

2α,3α,23-trihydroxy-12,20(30)-dien-28-ursolic acid 28-O-β-D-glucopyranoside (11): ¹H NMR (MeOD, 600 MHz) δ_H 5.28 (1H, t, J = 3.5 Hz, H-12), 4.71 (1H, br s, H-30a), 4.61 (1H, br s, H-30b), 3.78 (1H, m, H-23a), 3.70 (1H, dd, J = 11.6, 4.6 Hz, H-23b), 1.23 (s, 3H, H-26), 1.01 (3H, s, H-27), 0.99 (3H, s, H-25), 0.82 (3H, s, H-29), 0.80 (3H, s, H-24), 5.26 (1H, d, J = 8.4 Hz, H-1′), 3.85 (1H, m, H-6′a), 3.67 (1H, m, H-6′b). ¹³C NMR (MeOD, 150 MHz) δ_C 43.4 (C-1), 68.9 (C-2), 78.0 (C-3), 42.4 (C-4), 44.2 (C-5), 19.3 (C-6), 33.5 (C-7), 41.0 (C-8), 49.3 (C-9), 38.9 (C-10), 24.5 (C-11), 127.2 (C-12), 139.3 (C-13), 42.1 (C-14), 29.0 (C-15), 25.2 (C-16), 49.6 (C-17), 56.3 (C-18), 38.5 (C-19), 154.3 (C-20), 33.2 (C-21), 39.5 (C-22), 71.0 (C-23), 17.7 (C-24), 17.2 (C-25), 17.5 (C-26), 24.0 (C-27), 177.1 (C-28), 16.5 (C-29), 105.2 (C-30), 95.5 (C-1′), 74.1 (C-2′), 78.6 (C-3′), 71.3 (C-4′), 78.5 (C-5′), 62.0 (C-6′).

1-oxo-3β,23-dihydroxy-olean-12-en-28-oicacid-28-O-β-D-glucopyranosside (12): colorless amorphous power. ¹H NMR (MeOD, 400 MHz) δ_H 5.15 (1H, t, J = 4.5 Hz, H-12), 3.72 (1H, m, H-3), 1.20 (3H, s, H-27), 0.93 (3H, s, H-30), 0.92 (3H, s, H-29), 0.85 (6H, s, H-24, 26), 5.33 (1H, d, J = 8.0 Hz, H-1′), 3.48–3.69 (6H, m, H-2′, H-3′, H-4′, H-5′, H-6′). ¹³C NMR (MeOD, 100 MHz) δ_C 215.0 (C-1), 44.6 (C-2), 73.4 (C-3), 44.1 (C-4), 47.4 (C-5), 18.3 (C-6), 33.2 (C-7), 40.7 (C-8), 40.3 (C-9), 53.1 (C-10),26.2 (C-11), 124.2 (C-12), 144.1 (C-13), 43.3 (C-14), 28.9 (C-15), 24.2 (C-16), 48.3 (C-17), 43.0 (C-18), 47.1 (C-19), 31.6 (C-20), 35.1 (C-21), 33.3 (C-22), 65.7 (C-23), 13.3 (C-24), 15.8 (C-25), 18.2 (C-26), 26.2 (C-27), 178.0 (C-28), 33.0 (C-29), 23.9 (C-30), 95.6 (C-1′), 78.3 (C-5′), 78.0 (C-3′), 73.7 (C-2′), 71.1 (C-4′), 62.5 (C-6’).

3.6. Hydrolyses of Novel Compounds to Determine the Linking Sugars

Each new isolate was respectively dissolved in 1 M hydrochloric acid (HCl) and refluxed for 2 h at 80 °C. The reaction solutions were then fractionated with ethyl acetate (EtOAc, 3 times). The remaining aqueous phase was concentrated under vacuum to remove water (H₂O). Analytical HPLC was conducted on a Waters 2695 instrument (Waters Corporation, Milford, MA, USA) using Waters 2424 ELSD as a detector. A GH0525046C18AQ column (5 μm, 4.6 × 250 mm, Sil Green) was used, which used formic acid: water = 0.1: 100 (v/v) as mobile phase. The column temperature was 35 °C. The HPLC spectrum of the standards of L-arabinose, D-glucopyranose and D-quinovose are shown in the Supporting Information as Figure S33–S35. The existence of L-arabinose was determined by HPLC test with an authentic sugar in the aqueous phase of compound Z₁ (1) (Figure S36), while D-glucopyranose and the L-arabinose were determined by HPLC test with two authentic sugars in the aqueous phase of compounds Z₂ (2) and Z₃ (3) (Figure S37). The presence of L-arabinose and D-quinovose was found by HPLC test with standard sugars in the water phase of compound Z₄ (4) (Figure S38).

3.7. Assay for the Glucose Uptake Effects of the Fraction and Dammarane Saponins

3.7.1. Cell Viability Assay

The cell viability assay was determined by the 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma-Aldrich). The 3T3-L1 adipocytes were treated at a density of 3000 cells/well in 96-well plates in DMEM with 10% FBS. After 24 h of incubation, the cells were exposed to the test extract (CPBE, 25 μg/mL) and compounds (10 μM), which were dissolved in serum-free DMEM for 24 h. Each well was filled with twenty microliters of 2 mg/mL MTT solution and incubated for 4 h at 37 ℃ in the dark. The reduction product, a formazan, was dissolved with DMSO and the absorbance was measured at 550 nm with a microplate reader.

3.7.2. Differentiation of 3T3-L1 Adipocytes

3T3-L1 cells were purchased from Cell Culture Center, Institute of Basic Medical Sciences, Institute of Basic Medicine, Chinese Academy of Medical Sciences (Batch number 1101MOU-PUMC 0001551). The cells were cultured in DMEM with 10% calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (HyClone) in 5% CO₂ at 37 ℃. The growth media was changed to DMEM with 10% fetal bovine serum (HyClone) containing 1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 520 μM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), and 1 μg/mL insulin (Beijing, China, Roche, Germany) after two days. After 48 h, the cells were grown in DMEM with 10% FBS, 1 μg/mL insulin, 100 U/mL penicillin, and 100 mg/mL streptomycin for 2 days of incubation. Every two days, the medium was replenished with fresh DMEM supplemented with 10% FBS medium until adipogenesis was induced.

3.7.3. 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.

4. 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.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28083294/s1. Figure S1. HRESIMS spectrum of compound 1. Figure S2. 1H NMR spectrum of compound 1 (600 MHz, pyridine-d5). Figure S3. 13C NMR spectrum of compound 1 (150 MHz, pyridine-d5). Figure S4. DEPT spectrum of compound 1 in pyridine-d5. Figure S5. HSQC spectrum of compound 1 in pyridine-d5. Figure S6. 1H-1H COSY spectrum of compound 1 in pyridine-d5. Figure S7. HMBC spectrum of compound 1 in pyridine-d5. Figure S8. ROESY spectrum of compound 1 in pyridine-d5. Figure S9. HRESIMS spectrum of compound 2. Figure S10. 1H NMR spectrum of compound 2 (600 MHz, pyridine-d5). Figure S11. 13C NMR spectrum of compound 2 (150 MHz, pyridine-d5). Figure S12. DEPT spectrum of compound 2 in pyridine-d5. Figure S13. HSQC spectrum of compound 2 in pyridine-d5. Figure S14. 1H-1H COSY spectrum of compound 2 in pyridine-d5. Figure S15. HMBC spectrum of compound 2 in pyridine-d5. Figure S16. ROESY spectrum of compound 2 in pyridine-d5. Figure S17. HRESIMS spectrum of compound 3. Figure S18. 1H NMR spectrum of compound 3 (600 MHz, pyridine-d5). Figure S19. 13C NMR spectrum of compound 3 (150MHz, pyridine-d5). Figure S20. DEPT spectrum of compound 3 in pyridine-d5. Figure S21. HSQC spectrum of compound 3 in pyridine-d5. Figure S22. 1H-1H COSY spectrum of compound 3 in pyridine-d5. Figure S23. HMBC spectrum of compound 3 in pyridine-d5. Figure S25. HRESIMS spectrum of compound 4. Figure S26. 1H NMR spectrum of compound 4 (600 MHz, pyridine-d5). Figure S27. 13C NMR spectrum of compound 4 (150MHz, pyridine-d5). Figure S28. DEPT spectrum of compound 4 in pyridine-d5. Figure S29. HSQC spectrum of compound 4 in pyridine-d5. Figure S30. 1H-1H COSY spectrum of compound 4 in pyridine-d5. Figure S31. HMBC spectrum of compound 4 in pyridine-d5. Figure S32. ROESY spectrum of compound 4 in pyridine-d5. Figure S33. The HPLC spectrum of the standard of L-arabinose. Figure S34. The HPLC spectrum of the standard of D-glucopyranose. Figure S35. The HPLC spectrum of the standard of D-quinovose. Figure S36. The HPLC spectrum of the compound 1 of L-arabinose. Figure S37. The HPLC spectrum of the compound 2 and 3 of D-glucopyranose and L-arabinose. Figure S38. The HPLC spectrum of the compound 4 of L-arabinose and D-quinovose.

Author Contributions

J.L. conceived, designed and authored the article; W.X. and Y.H. analyzed the data; S.D. contributed the samples/reagents/materials/analysis tools; Data Curation, X.L., Y.C., P.H. and C.R.; writing—review and editing, R.Y. and R.H.; X.L., L.P. and K.L. performed the experiments. All authors have read and agreed to the published version of the manuscript.

Funding

This research work was financially supported by National Natural Science Foundation (32060097, 3206020108); The Natural Science Foundation of Guangxi Province (2018GXNSFDA050007); Guanxi Key Laboratory of Traditional Chinese Medicine Quality Standards (grant number GZZK202001, GZZK201804); The Open Research Fund program of the Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (CMEMR2020-A04).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data of the article can be obtained from the authors.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Not available.

References

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Figure 1. Structures of compounds 1–12 isolated from C. paliurus.

Figure 2. (A) Key HMBC (red arrows) and COSY (blue bold bonds) correlations of compounds 1–4. (B) ROESY correlations of compounds 1 and 4.

Figure 3. Catalytic site of three-dimensional (A) and two-dimensional (C) visualization of compound 10 against PTP1B; Allosteric sites of three-dimensional (B) and two-dimensional (D) visualization of compound 10 against PTP1B. Conventional hydrogen bond as green, Pi-sigma as purple, Pi-alkyl and alkyl interactions as light pink, and van der Waals as light green.

Figure 4. (A) The glucose uptake effects of compounds 1–12 on 3T3-L1 adipocytes using a fluorescent derivate of cells at 10 μM. (B) Compounds 6–7 and 10 increased glucose uptake in 3T3-L1 adipocytes. 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.

Table 1. ¹H and ¹³C NMR spectral data of compounds 1–2 (600 MHz, Pyridine-d₅, δ in ppm).

Position	1		2
Position	δ_H, Mult (J in Hz)	δ_C	δ_H, Mult (J in Hz)	δ_C
1	3.05, m; 2.00, m	36.1	3.13, m; 2.08, m	36.0
2	1.74, m; 1.68, m	22.2	1.87, m; 1.46, m	21.4
3	3.53, br s	81.6	3.60, br s	81.9
4		38.5		38.6
5	1.54, d, (11.2)	51.3	1.61, m	50.7
6	1.53, m; 1.40, m	18.7	1.58, m; 1.46, m	18.8
7	1.41, m; 1.06, d, (9.2)	36.8	1.48, m; 1.13, d, (11.7)	36.8
8		50.7		50.1
9	1.80, m	54.3	1.89, m	54.3
10		40.4		40.4
11	2.70, m; 1.46, m	34.1	2.65, m; 1.52, m	34.0
12	4.36, m	76.8	4.51, td, (10.6, 4.9)	76.4
13	1.80, m	42.5	1.73, m	41.6
14		41.9		41.0
15	1.32, m; 1.01, m	31.7	1.35, m; 0.97, m	31.6
16	1.64, m; 1.34, m	25.9	1.72, m; 1.43, m	26.0
17	1.53, m	47.8	1.49, m	50.6
18	0.90, s	17.5	0.98, s	17.5
19	1.29, s	17.4	1.38, s	17.4
20		92.6		89.9
21	1.36, s	23.9	1.28, s	24.9
22	7.64, d, (5.7)	161.2	2.00, m; 1.88, m	32.0
23	6.26, d, (5.7)	121.9	2.70, m; 2.59, m	29.1
24		173.4		176.8
28	0.91, s	23.5	0.96, s	23.8
29	1.20, s	30.4	1.26, s	30.0
30	0.55, s	16.9	0.62, s	16.8
1′	4.68, d, (7.2)	102.1	4.74, d, (6.5)	102.3
2′	4.38, dd, (9.2, 7.2)	72.9	4.45, t, (8.9)	72.1
3′	4.13, dd, (9.2, 4.2)	75.2	4.27, dd, (8.4, 3.5)	75.3
4′	4.19, dd, (8.2, 3.6)	70.3	4.42, m	69.7
5′	4.29, dd; (8.2, 3.6), 3.74, m	66.8	4.34, dd, (12.1, 3.7); 3.80, m	66.9
1′′	4.87, d, (6.5)	102.7	5.11, d, (7.7)	102.4
2′′	4.36, dd, (8.0, 7.2)	73.1	4.16, t, (8.2)	75.8
3′′	4.28, dd, (9.2, 4.1)	75.4	4.03, dd, (8.2, 6.5)	79.2
4′′	4.40, m	69.5	4.01, dd, (8.2, 6.5)	78.5
5′′	4.29, dd, (8.6, 3.2); 3.80, m	68.1	4.31, d, (8.9); 3.78, m	72.9
6′′			4.58, dd, (11.4, 2.8); 4.38, m	63.9

Table 2. The HPLC retention time of sugar moieties of compounds 1–4.

Compounds	Retention Times (min)	Types of the Monosaccharides
1	6.844, 6.847	L-Ara, L-Ara
2	6.842, 6.562	L-Ara, D-Glc
3	6.849, 6.561	L-Ara, D-Glc
4	6.841, 7.419	L-Ara, D-Qui
D-Glc	6.567
L-Ara	6.843
D-Qui	7.416

Table 3. ¹H and ¹³C NMR spectral data of compounds 3–4 (600 MHz, pyridine-d₅, δ in ppm).

Position	3		4
Position	δ_H, Mult. (J in Hz)	δ_C	δ_H, Mult. (J in Hz)	δ_C
1	3.01, m; 2.01, m	36.6	3.10, m; 2.06, m	36.1
2	1.91, m; 1.74, m	22.3	1.89, m; 1.78, m	21.8
3	3.53, m	81.9	3.62, d, (5.1)	79.8
4		38.6		38.3
5	1.53, (d, 11.2)	51.4	1.60, m	51.3
6	1.60, m; 1.40, m	18.8	1.58, m; 1.46, m	18.6
7	1.39, m; 1.06, d, (9.2)	35.9	1.47, m; 1.44, m	36.5
8		50.7		50.6
9	1.80, m	55.4	1.89, m	54.2
10		40.4		40.3
11	1.98, m; 1.84, m	34.0	1.98, m; 1.84, m	34.1
12	4.16, m	76.0	4.51, m	76.5
13	1.61, m	42.3	1.73, m	42.0
14		41.0		41.6
15	1.32, m; 1.03, m	31.4	1.32, m; 1.03, m	31.5
16	1.64, m; 1.34, m	25.8	1.83, m; 1.72, m	25.8
17	1.53, m	50.0	1.81, m	49.9
18	0.90, s	17.1	1.03, s	17.2
19	1.34, s	17.4	1.38, s	17.1
20		89.7		89.9
21	1.47, s	21.9	1.25, s	23.3
22	1.98, m; 1.87, m	32.7	1.98, m; 1.84, m	32.6
23	2.67, m; 2.51, m	29.7	2.66, m; 2.50, m	29.7
24		176.6		177.3
28	0.97, s	23.6	1.00, s	23.2
29	1.28, s	30,4	1.28, s	30.3
30	0.69, s	16.7	0.61, s	16.7
1′	5.55, d, (6.5)	106.9	4.75, d, (6.5)	102.5
2′	4.89, t, (5.6)	84.6	4.44, m	72.9
3′	4.85, m	79.9	4.24, dd, (8.2, 3.5)	75.3
4′	4.76, dd, (6.0, 5.0)	86.0	4.40, t, (6.0)	69.5
5′	4.40, m; 4.29, d, (9.4)	63.4	4.33, dd, (12.1, 3.7); 3.79, m	66.9
1′′	5.12, d, (7.6)	102.1	5.00, d, (7.7)	101.9
2′′	4.03, t, (8.3)	75.0	4.07, t, (9.2)	75.7
3′′	4.31, t, (9.1)	78.9	4.18, t, (8.9)	79.0
4′′	4.05, m	77.4	3.70, t, (9.0)	78.3
5′′	4.18, dd, (9.1, 3.2); 3.78, m	73.4	4.41, m, 3.79; dd, (8.9, 2.7)	72.7
6′′	4.41, m; 4.30, m	63.8	1.66, d, (7.4)	19.1

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Liang, X.; Deng, S.; Huang, Y.; Pan, L.; Chang, Y.; Hou, P.; Ren, C.; Xu, W.; Yang, R.; Li, K.; et al. Triterpenoids from the Leaves of Cyclocarya paliurus and Their Glucose Uptake Activity in 3T3-L1 Adipocytes. Molecules 2023, 28, 3294. https://doi.org/10.3390/molecules28083294

AMA Style

Liang X, Deng S, Huang Y, Pan L, Chang Y, Hou P, Ren C, Xu W, Yang R, Li K, et al. Triterpenoids from the Leaves of Cyclocarya paliurus and Their Glucose Uptake Activity in 3T3-L1 Adipocytes. Molecules. 2023; 28(8):3294. https://doi.org/10.3390/molecules28083294

Chicago/Turabian Style

Liang, Xiaoqin, Shengping Deng, Yan Huang, Liwei Pan, Yanling Chang, Ping Hou, Chenyang Ren, Weifeng Xu, Ruiyun Yang, Kanyuan Li, and et al. 2023. "Triterpenoids from the Leaves of Cyclocarya paliurus and Their Glucose Uptake Activity in 3T3-L1 Adipocytes" Molecules 28, no. 8: 3294. https://doi.org/10.3390/molecules28083294

APA Style

Liang, X., Deng, S., Huang, Y., Pan, L., Chang, Y., Hou, P., Ren, C., Xu, W., Yang, R., Li, K., Li, J., & He, R. (2023). Triterpenoids from the Leaves of Cyclocarya paliurus and Their Glucose Uptake Activity in 3T3-L1 Adipocytes. Molecules, 28(8), 3294. https://doi.org/10.3390/molecules28083294

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

Abstract

1. Introduction

2. Results and Discussion

2.1. Elucidation of the Chemical Structure of Cypaliurusides Z₁–Z₄ (1−4)

2.2. Predicted Binding Modes of Compounds and PTP1B Using Molecular Docking Analysis

2.3. EtOAc Extract and Dammarane Saponins Enhance Glucose Uptake