Bioactivities of Steroids and Sesquiterpenes from the Branches and Leaves of Aglaia lawii

Five undescribed steroids and one sesquiterpene, named Aglaians A–F, along with sixteen known analogs, have been isolated from the branches and leaves of Aglaia lawii. Its structure was elucidated by IR, HRESIMS, 1D and 2D NMR, quantum-chemical calculations, electronic circular dichroism (ECD) calculations, and single-crystal X-ray diffraction analysis. The cytotoxic and antibacterial activities of six human tumor cell lines were evaluated (MDA-MB-231, MCF-7, Ln-cap, A549, HeLa, and HepG-2), and four strains of bacteria (Bacterium subtilis, Phytophthora cinnamomic, Acrogenic bacterium, and Ralstonia solanacearum). The bioassay results indicated that compounds 3 and 5 exhibited moderate antitumor activity with IC50 values ranging from 16.72 to 36.14 μM. Furthermore, compounds 3–5 possess antibacterial activities against four bacteria with MIC values of 25–100 μM.


Introduction
Aglaia lawii (Meliaceae), a perennial tree, is mainly distributed in the tropical and subtropical areas of Asia (such as Guangzhou, Guizhou, and Yunnan Provinces, China).The leaves of A. lawii are a traditional folk medicine used as an antibacterial and antitumor [1][2][3][4].Previously, our research group has been devoted to the medicinal plants of the family Meliaceae, from which some limonoids and triterpenoids with novel structures have been found.These compounds exhibit diverse biological activities, such as antitumor, anti-inflammatory, and anti-bacterial [5,6].Currently, existing evidence indicates that A. lawii predominantly comprises sesquiterpenes and triterpenes, conferring antiinflammatory properties.However, fewer studies have reported on A. lawii, and its chemical constituents remain to be systematically isolated and identified, with their biological activities not extensively explored [1][2][3][4].As part of our ongoing work, we extracted structurally novel and biologically active ethanol extract from the branches and leaves of A. lawii and discovered six previous undescribed steroids and sesquiterpene (1-6) (Figure 1), along with sixteen known analogs (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) (Figure 1).Their structures were characterized by comprehensive spectroscopic data analysis, ECD calculations, and X-ray crystallography.Furthermore, their preliminary cytotoxic and antibacterial activities were evaluated through in vitro assay.In the present paper, the isolation, structure elucidation, and biological evaluation of these compounds were presented.
In accordance with the ethnomedical indication of this plant in Chinese folk medicine, all the isolates were tested in vitro for their cytotoxicity against six human cancer cell lines and four antibacterial activities.The novel compounds 3 and 5 displayed moderate cytotoxicity, with IC 50 values ranging from 16.72 to 36.14 µM (Table 3).Compounds 3, 4, and 5 have weak antibacterial activities against four bacteria with MIC values of 25-100 µM (Table 4).

Plant Material
The stem bark of A. lawii was collected in March 2022 from Wangmo Guizhou Province of China, People's Republic of China, and identified by Prof. Chao-yi Deng, Qianxinan Karst Regional Development Institute of Guizhou.A voucher specimen (no.20220301) was deposited at the Natural Products Research Center of Guizhou Province.

Extraction and Isolation
The air-dried branches and leaves (15.0 kg) of A. lawii were powder and extracted with 95% EtOH.The filtrate was evaporated under reduced pressure to give crude extract (1 kg), which was suspended in H 2 O (2 L).Then, the mixture was extracted with petroleum ether (PE) (5 L × 6) and EtOAc (5 L × 5) to afford the PE (530.0 g).Eight fractions (A-H)

Cytotoxicity Assay
The cytotoxic activity of the compounds against human tumor cell lines (231, MCF-7, Ln-Cap, A549, HeLa, and HepG-2) was analyzed through the MTT method [25].Briefly, cells were treated with different concentrations of compounds and incubated for 48 h after being seeded in 96-well plates for 24 h at 37 • C with 5% CO 2 .Untreated cells were incubated in a medium, and the medium served as control and blank, respectively.Meanwhile, doxorubicin was used as a positive control.The supernatant was removed, and the MTT solution (5 mg/mL) was prepared in a medium, which was added to each well until the formazan crystal was fully dissolved.The absorbance of each well was detected by an enzyme-labeled instrument at a wavelength of 490 nm [25].

Antibacterial Assay
The compounds were assessed against four Gram-positive bacteria: B. subtilis, P. cinnamomi, A. bacterium, and R. solanacearum.The minimum inhibitory concentrations (MICs) were determined in 96-well culture plates by a serial dilution of each compound with concentrations ranging from 12.5-100 µm and repeated three times according to the standard microdilution method [26].Each concentration of samples was added to three bacterial culture wells to ensure the repeatability of the experiment.After incubation at 37 • C for 24 h, the lowest concentration of antibiotics without visible bacterial growth were the MICs.Ofloxacin was used as a positive control.

X-Ray Diffraction Analysis
The colorless acicular crystal of Aglaian A (1) was obtained from acetone at a temperature of 4 • C. The X-ray diffraction data were selected and performed on an XtaLAB AFC12 (RINC): Kappa single diffractometer.The crystal was kept at 99.98(11) K, 99.99(10) K, 99.99(10) K, and 100.00(10)K, respectively, during data collection.The structure was solved with the SHELX structure solution program using Intrinsic Phasing, and all nonhydrogen atoms were refined with the SHELX refinement package using least squares minimization.The crystallographic data of the reported structure were deposited in the Cambridge Crystallographic Data Center (CCDC) with a deposition number of 2303496.

ECD Calculations
The conformational search was carried out by the SYBYL-X 2.0 software at the MMFF94s force field.The relative configurations were reoptimized by using the timedependent density functional theory (TDDFT) at the B3LYP/6-31G(d) level in the gas phase via the Gaussian 16 program, and the frequency was calculated at the same level of theory [27].The optimized stable conformers without imaginary frequencies were performed at the B3LYP-SCRF (PCM)/6-31+G(d) level in the PCM MeOH model using the TDDFT method [27].The absolute configurations of compounds 1-6 were analyzed with SpecDis software 1.71 by comparing the experimental ECD spectra.

Figure 5 .
Figure 5.The X-ray ORTEP drawing of 1.The red circle stands for oxygen atom and the blue circle stands for hydrogen atom.

Figure 5 .
Figure 5.The X-ray ORTEP drawing of 1.The red circle stands for oxygen atom and the blue circle stands for hydrogen atom.