Terpenoids from the Seeds of Toona sinensis and Their Ability to Attenuate High Glucose-Induced Oxidative Stress and Inflammation in Rat Glomerular Mesangial Cells

Toona sinensis (A. Juss.) Roem is an edible medicinal plant that belongs to the genus Toona within the Meliaceae family. It has been confirmed to display a wide variety of biological activities. During our continuous search for active constituents from the seeds of T. sinensis, two new acyclic diterpenoids (1–2), together with five known limonoid-type triterpenoids (3–7), five known apotirucallane-type triterpenoids (8–12), and three known cycloartane-type triterpenoids (13–15), were isolated and characterized. Their structures were identified based on extensive spectroscopic experiments, including nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectra (HR-ESI-MS), and electronic circular dichroism (ECD), as well as the comparison with those reported in the literature. We compared these findings to those reported in the literature. Compounds 5, 8, and 13–14 were isolated from the genus Toona, and compounds 11 and 15 were obtained from T. sinensis for the first time. The antidiabetic nephropathy effects of isolated compounds against high glucose-induced oxidative stress and inflammation in rat glomerular mesangial cells (GMCs) were assessed in vitro. The results showed that new compounds 1 and 2 could significantly increase the levels of Nrf-2/HO-1 and reduce the levels of NF-κB, TNF-α, and IL-6 at concentrations of 30 μM. These results suggest that compounds 1 and 2 might prevent the occurrence and development of diabetic nephropathy (DN) and facilitate the research and development of new antioxidant and anti-inflammatory drugs suitable for the prevention and treatment of DN.


Introduction
Diabetic nephropathy (DN) is a chronic microvascular complication with a high incidence and mortality. It is the principal cause of end-stage renal disease worldwide [1]. The pathogenesis of DN is complicated and obscure, and there is currently no efficient way to alleviate DN. Oxidative stress and inflammation are the major causes of DN progression [2]. The release of reactive oxygen species (ROS) and the accumulation of inflammatory mediators can be very high in rat glomerular mesangial cells (GMCs) exposed to high glucose (HG) [3]. Continuing to search for a prospective inhibitor with antioxidative and anti-inflammatory properties may reveal an effective way to halt the progression of DN.
Nuclear factor erythroid 2-related factor 2 (Nrf2) has been proven to prevent ROSinduced oxidative stress injury and is a master transcriptional regulator of genes that encode antioxidative factors, such as heme oxygenase-1 (HO-1) [4]. Nuclear factor kappa-B (NF-κB) is an important transcription factor. It plays a vital role in the inflammatory response and mediates the expression of inflammatory cytokines involved in DN [5]. TNFα and IL-6 are pro-inflammatory cytokines that play an important role in inflammatory responses, lipid metabolism, and insulin resistance. It was reported that TNF-α and IL-6 Molecules 2022, 27, 5784 2 of 12 were associated with increased oxidative stress and inflammation in DN. NF-κB, TNF-α, and IL-6 are activated in renal tissue, and they have been reported as important biomarkers of DN [6,7]. The Nrf2/NF-κB pathway could be used to modulate oxidative stress and inflammation and so affect DN [8,9]. Hence, the Nrf2/NF-κB pathway may be a suitable target for the treatment of DN.
Toona sinensis (A. Juss.) Roem, a medicinal and edible plant that belongs to the genus Toona within the Meliaceae family, is mainly distributed in Asia and Oceania [10,11]. The tender leaves and buds are not only eaten as vegetables but also possess considerable value as a folk medicine in the treatment of heliosis, vomiting, dysentery, enteritis, and itchiness [12]. The roots can be used as astringents and the stems as correctives [13]. T. sinensis is rich in a variety of active components, including terpenoids, flavonoids, lignans, and phenols, and it exerts antioxidant, anti-inflammatory, anti-diabetic, and anti-tumor effects [14,15]. At present, studies have shown that the chemical constituents of T. sinensis are mainly concentrated on the leaves and bark, and there have been a few reports on the seeds. The results showed that the seeds mainly contained terpenoids, flavonoids, and phenols [13,16,17]. Further exploration may identify the active constituents of T. sinensis seeds that improve oxidative stress and inflammation in DN.

Results and Discussion
Compound 1 was isolated as a white gum and had a molecular formula of C20H36O4 based on its high-resolution electrospray ionization mass spectra (HR-ESI-MS) ( Figure  S9), which showed a peak at m/z 341.26270 [M+H] + (calcd. 341.26864), corresponding to three degrees of unsaturation. The 1 H NMR spectrum ( Figure S2) of 1 exhibited resonances
The cytotoxicity of compounds isolated from the T. sinensis seeds was measured through a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium (MTT) assay. As shown in Figure S1, compounds 7, 8, 13, and 15 exhibited significantly more cytotoxicity than the normal group (NG) (p < 0.01), and compounds 1-6, 9-12, and 14 (80 µM) showed no cytotoxicity against GMCs. To further explore the effects of different concentrations of compounds (1-6, 9-12, and 14) on GMC proliferation, we investigated the cell proliferation induced by HG though MTT assay. As indicated in Figure 4, there was significantly more GMC proliferation in the HG group than in the NG (p < 0.01). However, the increase could be reversed by epalrestat (EPA), and the effect of proliferation was dramatically inhibited after treatment with compounds 2, 4, 9-11, and 14 in a dose-dependent manner.
Oxidative stress caused by HG plays a dominant role in the progression of DN. The Nrf2 pathway is an important defense system against oxidative stress. It plays an antioxidative stress role through the up-regulation of HO-1 and other antioxidant genes. The antioxidant effects were preliminarily evaluated based on the up-regulation of Nrf2/HO-1 in HG-stimulated GMCs. As shown in Figure 5, there was more Nrf2/HO-1 expression by GMCs increased in the HG group than by those in the NG group. In addition, Nrf2/HO-1 expression was further enhanced after incubation with compounds 1, 2, 4, 6, and 14 for 48 h. Taken together, these results proved that compounds 1, 2, 4, 6, and 14 can alleviate oxidative stress and are associated with the activation of the Nrf2/HO-1 pathway.
Increased oxidative stress is associated with the activation of inflammation. NF-κB is an important transcription factor that can regulate the expression of various inflammatory factors, such as TNF-α and IL-6. As shown in Figure 6, there was significantly more expression of inflammatory cytokines in the HG group than in the NG. Nevertheless, we found that HG-induced levels of NF-κB, IL-6, and TNF-α were suppressed by EpA and by compounds 1, 2, 4, and 14 in a dose-dependent manner. However, compound 6 exhibited the same effect on NF-κB and TNF-α factors, but the inhibition of IL-6 was not dose-dependent. In short, compounds 1, 2, 4, and 6 showed strong inhibitory activities at doses below 50 µM (p < 0.05 or p < 0.01), indicating that compounds 1, 2, 4, and 6 suppressed the activation of the NF-κB pathway to block the progression of DN.
A preliminary structure-activity relationship was established, indicating that new acyclic diterpenoids (1 and 2) exerted significant antidiabetic nephropathy through the activation of Nrf2/HO-1 and the inhibition of NF-κB pathways with respect to HG-induced GMCs. Compounds 1 and 2 exhibited similar activities at concentrations of 30 µM (p < 0.05 or p < 0.01), perhaps because the functional group of C-1 or stereochemistry of C-12, 14 could not affect the strength of the regulatory activity. These data are reported as antidiabetic nephropathy agents herein for the first time.  Oxidative stress caused by HG plays a dominant role in the progression of DN. T Nrf2 pathway is an important defense system against oxidative stress. It plays an anti idative stress role through the up-regulation of HO-1 and other antioxidant genes. T antioxidant effects were preliminarily evaluated based on the up-regulation of Nrf2/H 1 in HG-stimulated GMCs. As shown in Figure 5, there was more Nrf2/HO-1 express by GMCs increased in the HG group than by those in the NG group. In addition, Nrf2/H 1 expression was further enhanced after incubation with compounds 1, 2, 4, 6, and 14  48 h. Taken together, these results proved that compounds 1, 2, 4, 6, and 14 can allevi oxidative stress and are associated with the activation of the Nrf2/HO-1 pathway. Values are expressed as mean ± SD of three independent experiments, with ## p < 0.05 relative to the NG and * p < 0.05 or ** p < 0.01 relative to the HG group.
duced GMCs. Compounds 1 and 2 exhibited similar activities at concentrations of 30 μ (p < 0.05 or p < 0.01), perhaps because the functional group of C-1 or stereochemistry of 12, 14 could not affect the strength of the regulatory activity. These data are reported antidiabetic nephropathy agents herein for the first time.   1, 2, 4, 6, 14 at concentrations of 10, 30, and 50 µM in HG for 48 h. The expressions of Nrf2/HO-1 were analyzed using commercially available Elisa kits. Values are expressed as mean ± SD of three independent experiments, with ## p < 0.05 relative to the NG and * p < 0.05 or ** p < 0.01 relative to the HG group.
Molecules 2022, 27, x FOR PEER REVIEW 8 of 12 ± SD of three independent experiments, with ## p < 0.05 relative to the NG and * p < 0.05 or ** p < 0.01 relative to the HG group. The expressions of NF-κB, TNF-α, and IL-6 were analyzed using commercially available Elisa kits. Values are expressed as mean ± SD of three independent experiments, with ## p < 0.05 relative to the NG and * p < 0.05 or ** p < 0.01 relative to the HG group.

Plant Material
The T. sinensis seeds were collected by the Jinan Shengke Technology Company (Jinan, China) and identified by Prof. Chongmei Xu. A voucher specimen (voucher number: WF-YXY-TSS1507) was deposited at the Pharmacognosy Laboratory of the School of Pharmacy, Weifang Medical University.

Cytotoxicity Assay
GMCs (Institute of Nanjing Jiancheng) were incubated in a 5% CO 2 atmosphere at 37 • C and cultured in 5.6 mmol/L glucose DMEM, which contained 10% foetal bovine serum and a 1% penicillin streptomycin solution. Cytotoxic activity was tested with MTT. The cells were plated on 96-well plates at 4 × 10 3 cells per well, then incubated at 37 • C for 24 h. Then, the cells were cultured in NG and were co-treated with or without compounds (1-15) at 80 µM and incubated for 48 h. In addition, 10 µL MTT was added to each well. After 4 h, DMSO was added to dissolve formazan crystals. The absorbance was measured at 490 nm.

Cell Proliferation Assay
MTT assay was used to detect the effect of compounds without toxicity on cell proliferation. Cells were plated into 96-well plates and incubated for 24 h at 37 • C. The cells were divided into 8 groups: NG, 5.6 mmol/L glucose; HG group, 25 mmol/L glucose; HG+EPA (10 µM) group; and HG+compounds at different concentrations (5,10,20,40, and 80 µM) group.

Elisa Assay
Before the experiment, the treated proteins and supernatants were stored at −80 • C. The levels of Nrf2, HO-1, NF-κB, TNF-α, and IL-6 in the cell supernatants and proteins were detected using commercially available Elisa kits (Nanjing Jiancheng Biology Engineering Institute, Nanjing, China) according to the manufacturer s instructions.

Statistical Analysis
All data are presented as the means ± SD from 3 replicates. SPSS 22.0 software was used for one-way ANOVA of multiple groups of data. It was considered a significant difference when the p value was less than 0.05.

Conclusions
In this study, two new diterpenoids and thirteen known triterpenoids were separated from T. sinensis seeds. Compounds 1, 2, 4, 6, and 14 were found to increase the expression of Nrf2/HO-1 and decrease the levels of NF-κB, TNF-α, and IL-6, which indicated that the bioactive terpenoids of T. sinensis seeds could activate the Nrf2/HO-1 pathway and suppress the NF-κB pathway to ameliorate oxidative stress and inflammation, further preventing and reducing the occurrence of DN. This study indicated that the reasonable consumption of T. sinensis seeds might be an effective way to halt DN progression.