Hovendulcisic acid A-D: four novel ceanothane-type triterpenoids from Hovenia dulcis stems with anticancer properties

Sixteen ceanothane-type triterpenoids, including four new compounds—hovendulcisic acids A–D (1–4) —were purified from the stems of Hovenia dulcis Thunb. The structures of 1–4 were confirmed by comprehensive means including ECD and quantum chemical calculations. Putative biosynthetic pathways of 1–16 were proposed, and 3, 5, and 15 exhibited antitumor activity on A549 and MDA-MB-231 cells.

Previous studies revealed that the triterpenoid saponins from H. dulcis inhibited Nrf2 expression (Cai, et al., 2021), indicated potential anti-tumor sensitization activity (Lin et al., 2020).This discovery piqued our interest about the triterpenoid constituents of the Hovenia genus.We found the stems of Hovenia genus to be rich in triterpenoids by LC-MS experimentation.The chemical constituents from the stem of H. dulcis were thus isolated and identified in this study.As a result, four new triterpenoids of hovendulcisic acid A-D (1-4) were isolated from 70% alcohol extract by multicolumn chromatography and HPLC methods.The activity of 1-4 on Nrf2 were measured by luciferase reporter gene assay, and the cell viability of 1-4 on MDA-MB-231 and A549 cells were tested by CCK8 method.This research serves as a reference for further study on the pharmacological effects and drug active substance basis of H. dulcis.

Plant materials
The stems of H. dulcis Thunb.were collected in December 2020 in Zhen-an City, Shanxi Province and identified by pharmacist Ganshu She in the Guangdong Provincial Hospital of Chinese Medicine.The samples were authenticated as voucher number ( 202012001) and stored at the TCM storehouse in the Second Clinical College of the Guangzhou University of Chinese Medicine.

Acid hydrolysis and GC analysis
Acid hydrolysis and GC analysis of the glycoside were carried out according to Cai, et al. (2021).First, Compound 4 (2.1 mg) was hydrolyzed in HCl solution (2 M, 10 mL) in an oven (90 °C, 4 h), and then evaporated to dryness.H 2 O was added to the residue and CHCl 3 was used for extraction twice, then sugar residue was obtained from the H 2 O layer.The sugar fraction was reacted with L-cysteine methyl ester hydrochloride (in pyridine, 1 mL) in an oven (60 °C, 2 h).
After concentration and drying, the residue was reacted with 1-(trimethylsilyl) imidazole (0.2 mL) in an oven (60 °C, 1 h) and then extracted with n-hexane.The n-hexane fraction was acquired for GC analysis.The sugar was identified as D-glucose (t R /min) 25.170, reference D-glucose (t R /min) 25.172.

Computational section
All calculations and processing were conducted using ORCA 5.0.4 and Python 3.10.6.The optimization of the structures used for CD and NMR calculations was performed in pyridine solvents and B3LYP-D3BJ/6-31 g (d, p) levels in the CPCM model, using tight criteria and checking for the absence of virtual frequencies.NMR calculations were performed using the SMD model and at the revTPSS/PCSSEG −1 level (10.1021/ACS.JCTC.1C00604 indicates that this is a good level, and ECD calculations were performed using TDDFT under the SMD model.We calculated 90 excited states at the ωb97x-d4/ def2-TZVP level to cover the excited levels as much as possible, and the rotor intensity and excitation wavelength were expanded using the Gauss function.The FWHM was set at 10 nm to draw the spectrum.

Luciferase reporter gene assay
The cells used in this experiment were MDA-MB-231 stable reporter cell lines transfected with ARE-luciferase plasmid in the previous study (Chen, et al., 2016).The cells were cultured in medium containing puromycin (1.5 mg mL −1 ) in 48 well plates for 24 h.The isolated constituents 1-4 (1-10 μM) were added to the cells for 24 h.The luciferase activities were tested using the manufacturer's protocol after the digestion of the cells.TBHQ was used as positive control.

Cytotoxic activity assay
The CCK8 method was applied in a cell viability assay in human MDA-MB-231 cells and human A549 cells.Compounds 1-4
Compound 4 was isolated as a white amorphous powder with formula C 36  1 and 2).The sugar residue was finally identified as D-glucose according to acid hydrolysis and GC analysis.HMBC correlations between C-28 (δ C 175.7) and H-1′ in Glu (δ H 5.50) indicated that the glycosidic bonds were located at C-28.The HMBC correlations between the aldehyde proton signal H-27 (δ H 10.12) and C-14 (δ C 58.9), C-13 (δ C 39.3), C-15 (δ C 25.4) indicated that the aldehyde group was located at C-27.The position of the isopropenyl group can be determined according The relative configuration of 4 was deduced by the experimental and calculated 13 C NMR method, while the absolute configuration of 4 was approved by experimental and calculated ECD data (Figures 4D, 5D).Therefore, 4 was elucidated as 2α-carboxy-3β-hydroxy-A (1)-norlup-20 (29)-en-27-aldehydo-28-oic acid (hovendulcisic acid B) 28-β-D-glucopyranoside and named "hovendulcisic acid D".

Bioactivity results
The antitumor effects of isolated compounds 1-16 were investigated.The results of the ARE luciferase reporter gene showed that 1-4 showed strong inhibitory effect at low concentrations of 1 to 5 μM (Figure 6).Generally, MDA-MB-231 cells appeared to be more sensitive than A549 cells.Compounds 3, 5, and 15 exhibited significant inhibitory activity against A549 cells (Figure 7A), and 3, 5, 12, and 15 exhibited significant inhibitory activity against MDA-MB-231 cells (Figure 7B).The structure-activity relationships analysis revealed that the acetyl and aldehyde groups played an important role in anti-tumor activity.The above results indicated that 3 suppressed tumor activity by inhibiting Nrf2 expression.
(10-80 μM)were added to the cells for 24 h.The medium was discarded, and CCK8 solution was added and then cultured for 90 min.Absorbance at 450 nm was recorded and used to calculate cell viability.