Two New Apotirucallane-Type Triterpenoids from the Pericarp of Toona sinensis and Their Ability to Reduce Oxidative Stress in Rat Glomerular Mesangial Cells Cultured under High-Glucose Conditions

Hyperglycemia is a strong risk factor for chronic complications of diabetes. Hyperglycemic conditions foster not only the production of reactive oxygen species (ROS), but also the consumption of antioxidants, leading to oxidative stress and promoting the occurrence and progression of complications. During our continuous search for antioxidant constituents from the pericarp of Toona sinensis (A. Juss.) Roem, we isolated two previously unreported apotirucallane-type triterpenoids, toonasinensin A (1) and toonasinensin B (2), together with five known apotirucallane-type triterpenoids (3–7) and two known cycloartane-type triterpenoids (8–9) from the pericarp. Compounds 8–9 were obtained from T. sinensis for the first time. Their structures were characterized based on interpretation of spectroscopic data (1D, 2D NMR, high-resolution electrospray ionization mass spectra, HR-ESI-MS) and comparison to previous reports. Compounds (2, 4, 6, 7, and 9) were able to inhibit proliferation against rat glomerular mesangial cells (GMCs) cultured under high-glucose conditions within a concentration of 80 μM. Compounds (2, 6, and 7) were tested for antioxidant activity attributable to superoxide dismutase (SOD), malondialdehyde (MDA), and ROS in vitro, and the results showed that compounds (2, 6, and 7) could significantly increase the levels of SOD and reduce the levels of MDA and ROS. The current studies showed that apotirucallane-type triterpenoids (2, 6, and 7) might have the antioxidant effects against diabetic nephropathy.


Introduction
Diabetic nephropathy (DN) is one of the most common complications of diabetes, and the occurrence and development of oxidative stress play an important role [1,2]. High-glucose levels can increase the accumulation of reactive oxygen species (ROS) by promoting the generation of ROS and inhibiting the activity of antioxidant enzymes in cells. Excessive oxidative stress could lead to inflammation, fibrosis, the cell apoptosis, and cell damage and death, which are also considered as an important pathological change in DN [3]. There is an urgent need to identify previously unreported antioxidant constituents.
No study has yet reported the extraction and separation of chemical components in the pericarp of T. sinensis, which is often discarded as waste. In the search for potential antioxidant constituents, we performed a chemical investigation and bioactive evaluation of compounds from the pericarp of T. sinensis. We described the isolation and structures of two previously unreported apotirucallane-type triterpenoids, toonasinensin A (1) and toonasinensin B (2), and those of five known apotirucallane-type triterpenoids (3)(4)(5)(6)(7) and two known cycloartane-type triterpenoids (8-9) (Figure 1). The reducing oxidative stress activities of compounds 1-9 were evaluated in rat glomerular mesangial cells (GMCs) cultured under high-glucose conditions.

Results and Discussion
Compound 1 was obtained as a white amorphous powder. Its molecular formula was assigned as C37H62O7 by high-resolution electrospray ionization mass spectra (HR-ESI-MS) ( Figure S7
Oxidative stress caused by high glucose is a major process in the progression of DN. In order to determine the effect of high glucose on oxidative stress of GMCs, we determined the levels of Values are expressed as mean ± SD of three independent experiments, with # p < 0.01 relative to the 5.6 mM glucose (normal group, NG), and ** p < 0.01, * p < 0.05 relative to the 25 mM high glucose (high-glucose group, HG).
Oxidative stress caused by high glucose is a major process in the progression of DN. In order to determine the effect of high glucose on oxidative stress of GMCs, we determined the levels of superoxide dismutase (SOD), malondialdehyde (MDA), and ROS. The GMCs were treated at concentrations of 10, 30, and 50 µM. The results showed that compounds 2, 6, and 7 could render the level of SOD higher than in the HG (Figure 4). Compounds 2, 6, and 7 could render the levels of MDA and ROS lower than in the HG (Figures 5 and 6). These results indicated that they could significantly reduce oxidative stress of GMCs. A preliminary structure-activity relationship indicated that apotirucallane-type triterpenoids (2, 6, and 7) showed significant antioxidant effects with respect to DN; nevertheless cycloartane-type triterpenoids (8)(9) had no antioxidant activities. Even more interesting was that compounds 6 and 7 possessed similar activities, perhaps because the stereochemistry of C-21 could not affect the strength of antioxidant activities. In this way, apotirucallane-type triterpenoids have the potential for further development and research.
Molecules 2020, 25, x 5 of 11 superoxide dismutase (SOD), malondialdehyde (MDA), and ROS. The GMCs were treated at concentrations of 10, 30, and 50 μM. The results showed that compounds 2, 6, and 7 could render the level of SOD higher than in the HG (Figure 4). Compounds 2, 6, and 7 could render the levels of MDA and ROS lower than in the HG (Figures 5 and 6). These results indicated that they could significantly reduce oxidative stress of GMCs. A preliminary structure-activity relationship indicated that apotirucallane-type triterpenoids (2, 6, and 7) showed significant antioxidant effects with respect to DN; nevertheless cycloartane-type triterpenoids (8)(9) had no antioxidant activities. Even more interesting was that compounds 6 and 7 possessed similar activities, perhaps because the stereochemistry of C-21 could not affect the strength of antioxidant activities. In this way, apotirucallane-type triterpenoids have the potential for further development and research.   superoxide dismutase (SOD), malondialdehyde (MDA), and ROS. The GMCs were treated at concentrations of 10, 30, and 50 μM. The results showed that compounds 2, 6, and 7 could render the level of SOD higher than in the HG (Figure 4). Compounds 2, 6, and 7 could render the levels of MDA and ROS lower than in the HG (Figures 5 and 6). These results indicated that they could significantly reduce oxidative stress of GMCs. A preliminary structure-activity relationship indicated that apotirucallane-type triterpenoids (2, 6, and 7) showed significant antioxidant effects with respect to DN; nevertheless cycloartane-type triterpenoids (8)(9) had no antioxidant activities. Even more interesting was that compounds 6 and 7 possessed similar activities, perhaps because the stereochemistry of C-21 could not affect the strength of antioxidant activities. In this way, apotirucallane-type triterpenoids have the potential for further development and research.

Plant Material
The pericarp of T. sinensis was collected by the Jinan Shengke Technology Company of China and identified by Prof. Chongmei Xu. A voucher specimen (voucher number: WF-YXY-1712) has been deposited at the Pharmacognosy Laboratory of the School of Pharmacy, Weifang Medical University.

Extraction and Isolation
Dried pericarp of T. sinensis (20 kg) was extracted three times with 95% EtOH (100 L × 3 times) and heated for 10 h. The combined extracts were concentrated under a vacuum to obtain a crude extract (854 g). The crude extract was suspended in H2O (3 L) and partitioned sequentially with CH2Cl2, EtOAc, and n-BuOH (3 L × 3 times in each case). The CH2Cl2 extract (147 g) was rested after evaporations. The CH2Cl2 extract was dissolved in the mixed solvent of CH2Cl2-MeOH, and silica gel (185 g) was added to conduct dry sample mixing before further fractionation. The CH2Cl2 extract

Plant Material
The pericarp of T. sinensis was collected by the Jinan Shengke Technology Company of China and identified by Prof. Chongmei Xu. A voucher specimen (voucher number: WF-YXY-1712) has been deposited at the Pharmacognosy Laboratory of the School of Pharmacy, Weifang Medical University.

Cytotoxicity Assay
Cytotoxic activity against GMCs (Keygen Biotechnology, Nanjing, China) was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium (MTT) method. GMCs (5 × 10 3 cells/well) in Dulbecco s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) were plated into 96-well plates. Then GMCs were cultured with 5.6 mM glucose (normal group, NG) or NG medium in the presence of compounds (1-9) (80 µM) for 48 h, followed by the addition of 100 µL of the MTT solution (0.5 mg/mL) to each well and further incubation for 4 h. The medium was removed and the dark blue crystals in each well were dissolved in 100 µL dimethyl sulfoxide (DMSO). The absorbance of the wells was measured with a microplate reader at test and reference wavelength of 490 nm.

Cell Proliferation Assay
GMCs were plated into 96-well plates. Then GMCs were divided into NG, NG medium in the presence of mannitol (25 mM), 25 mM high glucose (high-glucose group, HG), HG medium in the presence of epalrestat (10 µM), and HG medium in the presence of compounds (2, 4, 6, 7, and 9) (5, 10, 20, 40, and 80 µM). Cell proliferation was measured using the MTT assay. Mannitol was used as osmotic pressure group and epalrestat was used as positive control.
3.6. In Vitro Antioxidant Activity of SOD, MDA, and ROS 3.6.1. SOD Superoxide dismutase is an important antioxidant enzyme defense in all organisms [30]. SOD levels were detected by Nanjing Jiancheng Bioengineering Institute assay kit. GMCs were cultured in a 6-well plate at 3 × 10 5 cells/well and exposed to the compounds (10, 30, and 50 µM) for 48 h. Absorbance was recorded at 450 nm using microplate reader. SOD activity is expressed as (U/mL), where each unit represents the amount of enzyme. The disproportionation of 50% superoxide radicals needs to be revealed.

MDA
MDA is an indicator of lipid peroxidation. MDA was detected by Nanjing Jiancheng Bioengineering Institute assay kit [31,32]. GMCs were cultured in a 6-well plate at 3 × 10 5 cells/mL and exposed to the compounds (10, 30, and 50 µM) for 48 h. Absorbance was recorded at 532 nm using microplate reader.

ROS
Medium and high concentrations of ROS induce cell apoptosis and even necrosis through cell oxidative stress reaction [33,34]. GMCs were cultured in a 96-well plate at 5 × 10 3 cells/well and exposed to the compounds (10, 30, and 50 µM) for 48 h. The cells' supernatant was discarded and determined ROS by reactive oxygen species assay kit (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). Measured fluorescence intensity with fluorescence microplate reader at excitation and emission wavelengths of 488 and 525 nm.

Statistical Analysis
Statistical differences between two groups were analyzed by the T-test and differences between multiple groups of data were analyzed by one-way ANOVA with the prism software (GraphPad, San Diego, CA), and the data were expressed as the mean ± SD of three independent experiments. A p value of less than 0.05 was considered statistically significant.

Conclusions
Nine compounds were isolated from the pericarp of T. sinensis, including two previously unreported apotirucallane-type triterpenoids, toonasinensin A (1) and toonasinensin B (2); five known apotirucallane-type triterpenoids (3-7); and two known cycloartane-type triterpenoids (8)(9). Compounds 2, 4, 6, 7, and 9 were found to significantly inhibit high-glucose induced GMCs proliferation. Compounds 2, 6, and 7 were able to significantly increase the vitality of SOD and reduce the levels of MDA and ROS. In summary, compounds 2, 6, and 7 were able to prevent DN by reducing oxidative stress, indicating that T. sinensis is worthy of further exploration to find more novel constituents with potential bioactivity.

Author Contributions:
This study was conceived and designed by W.