Immunosuppressive Sesquiterpene Pyridine Alkaloids from Tripterygium wilfordii Hook. f.

Tripterygium wilfordii Hook. f. is a well-known traditional Chinese medicine used to treat autoimmune diseases. Sesquiterpene pyridine alkaloids (SPAs) are a major class of components found in this herb that have piqued the interest of researchers due to their complex and diverse structures as well as significant biological activities. In this study, ten new SPAs, wilfordatine A–J (1–10), were isolated from the roots of T. wilfordii, along with ten known analogues (11–20). Their structures were primarily elucidated by extensive 1D and 2D NMR spectroscopic analysis. To search for more immunosuppressive ingredients related to the clinical efficacy of T. wilfordii, the total alkaloids (TA) and compounds 4, 5, and 9–16 were tested for their inhibitory effects on nuclear factor-kappa B (NF-κB) pathway in Lipopolysaccharide (LPS) induced HEK293/NF-κB-Luc cells. Among them, TA, compounds 5, 11, and 16 showed potent immunosuppressive activity, with IC50 values of 7.25 μg/mL, 8.75 μM, 0.74 μM, and 15.66 μM, respectively, and no influence on the cell viability at a concentration of 100 μg/mL (TA) or 100 μM (5, 11, and 16). Accordingly, TA, 5, 11, and 16, especially 11, were identified as promising candidates for further investigation into their potential use as immunosuppressive agents.


Introduction
Tripterygium wilfordii Hook. f., a member of the Celastraceae family, has been used medicinally in China for the treatment of rheumatoid arthritis and other autoimmune diseases for centuries [1,2]. Chemical studies on this plant resulted in the isolation of a number of sesquiterpenoid pyridine alkaloids (SPAs) with a variety of biological activities, including anti-inflammatory, insecticidal, anti-HIV, and antitumor activities [3][4][5][6][7][8][9]. SPAs are characterized by a macrocyclic diacetone skeleton composed of a polyoxygenated dihydroβ-agarofuran sesquiterpenoid core and a pyridine dicarboxylic acid moiety. According to the origin of the pyridine dicarboxylic acid, SPAs are mainly classified into four subtypes: wilfordate, evoninate, iso-wilfordate, and iso-evoninate [10]. In addition, the multiple hydroxyl groups of the SPAs are typically esterified by various organic acids, such as acetic, furoic, benzoic, and cinnamic acids. Due to their complex and diverse structures, as well as significant activities, SPAs have attracted continuous attention from researchers.
The total alkaloids of T. wilfordii have been reported to possess good therapeutic effects on collagen-induced arthritis in rats through significant immunosuppressive activity. One of the molecular mechanisms is the inhibition of nuclear factor-kappa B (NF-κB) pathway [11]. To search for more immunosuppressive SPA ingredients related to the clinical efficacy of T. wilfordii, we conducted a systematic study of the chemical constituents
The inhibitory effects of TA, 4, 5, and 9-16 on NF-κB pathway in HEK293/NF-κB-Luc cells induced by LPS were evaluated at a concentration of 100 µM (100 µg/mL for TA). As shown in Table 3, the tested compounds all inhibited NF-κB to varying degrees in the luciferase assay but had no effect on the cell viability in cell counting kit-8 (CCK-8) assay. It is worth noting that the NF-κB inhibitory rates of TA, 5, 11, and 16 were greater than 50%, and their IC 50 values were further determined to be 7.25 µg/mL, 8.75 µM, 0.74 µM, and 15.66 µM, respectively. 74.56 ± 1.83 a TA was tested at a concentration of 100 µg/mL, and compounds 4, 5, and 9-16 were tested at the concentration of 100 µM. b The unit of IC 50 value of TA are expressed as µg/mL. c JSH23 was used as positive control for NF-κB inhibition.
SPAs are common characteristic metabolites of T. wilfordii, of which wilfordate-type are the most abundant. Compared to the majority of reported wilfordate-type SPAs, the new compounds reported herein have different types of ester groups and substitution positions; for example, tigloyloxy groups, which are rare in reported compounds, are first present in the structure of compounds 7 and 8. One the other hand, the absolute configuration of wilfordate-type SPAs, particularly in C-9 position, is a difficult problem for the current study, and only a few reports have solved it, using single-crystal X-ray crystallographic analysis [20,21]. Unfortunately, however, we did not obtained the single crystal using various solvent systems such as methanol-water. Furthermore, we attempted to determine the absolute configuration using the electronic circular dichroism (ECD) method, but due to the macrolide unit, the molecules are more flexible and have many conformations, making the calculation very difficult. Therefore, this issue needs to be further investigated.
Diterpenoids such as triptolide are currently considered as the most active components of T. wilfordii due to their significant immunosuppressive and anti-inflammatory activities, but the high toxicity and low content in the plant limit their further development as drug candidates [22,23]. In contrast, the current study shows that SPAs had almost no cytotoxicity, while some of them, such as compounds 5, 11, and 16, had potent NF-κB inhibitory effects, although the activity may be relatively weak compared to the diterpenoids. Overall, we believe that the above SPAs, particularly 11, are valuable for further investigation of their potential use as immunosuppressive agents.

General Experimental Procedures
Optical rotations were recorded on a Rudolph Research Analytical Autopol III polarimeter. UV spectra were obtained using a Shmadzu UV-2700 UV-visible spectrophotometer, and IR spectra were obtained using a Nicolet iN10 MX spectrometer. NMR experiments were conducted on a Bruker AV-600 spectrometer in CDCl 3 with TMS as the internal standard at 25 • C. In the HMBC experiment, the 1 J C-H was set to 120-170 Hz, and long-range J C-H was set to 8 Hz. In the ROESY experiment, the mixing time was set to 200 ms. HRESIMS spectra were recorded on a Waters Xevo Q-Tof MS spectrometer. Preparative HPLC was conducted on a Waters LC Prep 150 System using various column, such as Waters XBridge Prep OBD C 18 column (30 × 150 mm, 10 µm), Waters XSelected CSH Prep C 18 column (19 × 250 mm, 5 µm), and YMC-Pack Ph column (10 × 250 mm, 5 µm). Neutral alumina (100-200 mesh, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and ODS (50 µm, YMC, Kyoto, Japan) were used for column chromatography.

Plant Material
The roots of Tripterygium wilfordii Hook. f. were collected in August 2020 from Hunan Province, PR China, and identified by Professor Shuai Kang, National Institutes for Food and Drug Control. A voucher specimen (No. 10106900006) has been deposited in the herbarium of Institute for Control of Chinese Traditional Medicine and Ethnic Medicine, National Institutes for Food and Drug Control, Beijing 100050, China.

Extraction and Isolation
The roots of T. wilfordii (50 kg) were powdered and extracted with 95% ethanol (250 L × 2 h × 3) under reflux. The alcohol extract was evaporated under reduced pressure to afford a residue, which was then suspended in water, and partitioned with CHCl 3 . A total of 120 g of the CHCl 3 -soluble extract was dissolved in EtOAc and partitioned three times with a 5% HCl aqueous solution. Then, ammonium hydroxide was added to the HCl aqueous layer to adjust the pH to 8~9. After filtration, the residue was dissolved with EtOAc and chromatographed over a neutral alumina column eluting with EtOAc. After recovering EtOAc by evaporation and drying, 21.36 g of the total alkaloids of T. wilfordii (TA) was obtained.
The TA (12.76 g) was separated by ODS chromatography with a gradient of CH 3