Chryroxosides A–E: five new triterpene saponins from the leaves of Chrysophyllum roxburghii G.Don. and their cytotoxic activity

Abstract Five undescribed oleanane triterpene glycosides named chryroxosides A–D (1–5), together with five known compounds (6–10) were isolated from the leaves of Chrysophyllum roxburghii G.Don. Their chemical structures were elucidated by extensive spectroscopic data analyses including IR, HR-ESI-MS, 1D and 2D NMR). Compounds 1, 3, and 5 showed cytotoxic effects against KB, HepG2, HL60, P388, HT29, and MCF7 cell lines with the IC50 values ranging from 14.40 to 52.63 μM compared to the positive control compound (ellipticine) with the IC50 values ranging from 1.34 to 1.99 μM. Graphical Abstract

(The World Flora Online).To date, there have been few reports on the chemical composition and biological activity of the genus Chrysophyllum.Pentacyclic triterpenes were found from C. lacourtianum (Talla et al. 2021) and C. cainito (Herrera-España et al. 2022) while alkaloids, saponins, steroids, tannins and volatile oil were defined to be present in C. albidum (Joyce et al. 2014), and some amino acids such as aspartic acid, glutamic acid, proline, and lysine were reported from C. roxburghii fruits (Barthakur and Arnold 1991).In our screening program for biologically active medicinal plants from Vietnamese plants, the methanol extract of C. roxburghii leaves showed significant cytotoxic activity on KB cells with IC 50 value of 3.0 µg/ml.Therefore, this plant was chosen for further study.Herein, we reported ten compounds including five undescribed oleanane triterpene glycosides from C. roxburghii leaves and their cytotoxicity on six cancerous cell lines including KB (human carcinoma in the mouth), HepG2 (human hepatocellular carcinoma), HL60 (human leukemia), P388 (mouse murine leukemia vinorelbine-resistant), HT29 (human colon adenocarcinoma), and MCF7 (human breast carcinoma) by SRB in vitro assay.

Results and discussion
The crude MeOH extract of the C. roxburghii leaves and sub-fractions obtained through partition were screened for their cytotoxic activities against KB, HepG2, HL60, P388, HT29, and MCF7 cancer cell lines.Amongst, only the crude water layer (CHLW) displayed cytotoxic effects against the tested cell lines with the IC 50 values of 12. 6, 18.7, 24.5, 13.2, and 21.9 μg/mL, respectively.Therefore, the CHLW fraction was further fractionated by various chromatograph methods including purification by semipreparative HPLC over C18 silica gel afforded compounds 1-10.The sugar units were identified by TLC after acid hydrolysis of the crude saponin mixture as d-glucose, d-apiose, l-arabinose, l-rhamnose, and d-xylose, after comparison of their optical rotation with those of authentic samples (Eskander et al. 2005;Voutquenne-Nazabadioko et al. 2013).

General
The used characterization equipment is the same as that described in our previous work (Nhung et al. 2022;Tham et al. 2023) (see supporting information).

Plant material
The leaves of Chrysophyllum roxburghii G.Don. (Chrysophyllum lanceolatum (Blume) A.DC.) were collected at Dak Lak province, Vietnam in February 2019 and identified by Dr Do Van Hai at the Institute of Ecology and Biological Resources, VAST.Voucher specimen (HSB2019MM) was deposited at the Institute of Marine Biochemistry, VAST.

4.
Bio-guided fractionation study on the methanol extract of the leaves of C. roxburghii led to the isolation of five undescribed oleanane triterpene glycosides named chryroxosides A-D (1-5), together with five known compounds (6)(7)(8)(9)(10) 5).Compounds 1, 3, and 5 showed cytotoxic effects against KB, HepG2, HL60, P388, HT29, and MCF7 cell lines with the IC 50 values ranging from 14.40 to 52.63 μM compared to the positive control compound (ellipticine) with the IC 50 values ranging from 1.34 to 1.99 μM.Our phytochemical results suggested that glycosides of protobassic acid are the major saponins of C. roxburghii, consistent with the previously published results, as protobassic acid glycosides seem to have been found exclusively in the Sapotaceae family (Gariboldi et al. 1990;Kim et al. 1999).