Phytochemical study of Magonia pubescens A. St.-Hil. and cytotoxicity of branches aqueous extract on breast cancer and leukemia cells

Abstract Magonia pubescens is a natural species from the Brazilian cerrado biome. Its fruits and seeds are used in the treatment of seborrheic dermatitis, a common inflammatory skin disease. In this work, the known compounds lapachol, stigmasterol, maniladiol and scopoletin were isolated from hexane and dichloromethane extracts of M. pubescens branches. The aqueous extract of this material was fractioned through a liquid-liquid partition and the obtained fractions were analyzed by UHPLC-MS/MS. The results obtained were compared with data from three databases, leading to the putative identification of 51 compounds from different classes, including flavonoids, saponins and triterpenes. The cytotoxicity of aqueous fractions was assayed against breast cancer (MDA-MB-231) and leukemia (THP-1 and K562) cells. The best activity was observed for fraction AE3 against MDA-MB-231 cells (IC50 30.72 µg.mL−1). Graphical Abstract

Magonia pubescens is a natural species from the Brazilian cerrado biome.its fruits and seeds are used in the treatment of seborrheic dermatitis, a common inflammatory skin disease. in this work, the known compounds lapachol, stigmasterol, maniladiol and scopoletin were isolated from hexane and dichloromethane extracts of M. pubescens branches.the aqueous extract of this material was fractioned through a liquid-liquid partition and the obtained fractions were analyzed by uHpLC-mS/mS.the results obtained were compared with data from three databases, leading to the putative identification of 51 compounds from different classes, including flavonoids, saponins and triterpenes.the cytotoxicity of aqueous fractions was assayed against breast cancer (mda-mB-231) and leukemia (tHp-1 and K562) cells.the best activity was observed for fraction aE3 against mda-mB-231 cells (iC 50 30.72µg.mL −1 ).

Introduction
Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020 (Ferlay et al. 2020).Leukemia, a type of cancer, accounted for approximately 3.4% of all new cancer cases and 3.8% of all cancer deaths in 2020 Lin et al. 2021.Breast cancer is another disease of concern and is the leading cause of female mortality worldwide.Generally, the treatment of cancers produces severe adverse effects, thereby justifying the search for new chemotherapy drugs that are effective in the treatment of cancer and cause as few side effects as possible.in this context, the cytotoxic activity observed for many plant extracts represents a potential path to the discovery of new anti-cancer drugs (Huang et al. 2019;pathiranage et al. 2021).Some plant extracts from species of Sapindaceae presented cytotoxic activity against breast cancer, for example: Litchi chinensis (Wang et al. 2006), Majidea zanquebarica (Gowri 2020) and Sapindus trifoliatus (Rao and Lakshmi 2012).
Magonia pubescens A. St. Hil., is a Sapindaceae species native to the Brazilian cerrado biome.de mesquita et al. ( 2009) evaluated the cytotoxicity of ethanolic extracts from M. pubescens roots, which had a half maximal inhibitory concentration (iC 50 ) of 7.9 µg.µL −1 against mda-mB-231 breast cancer cell lines.
there are no published cytotoxicity studies involving M. pubescens branch extracts.For this reason, the aqueous extract was fractionated through liquid-liquid partition, and the obtained fractions were tested against breast cancer (mda-mB-231) and leukemia (tHp-1 and K562) cells and analyzed by uHpLC-mS/mS.Furthermore, the hexane and dichloromethane extracts of M. pubescens branches were studied and led to the isolation and characterization of four known compounds.

Results and discussion
to investigate the phytochemical content of M. pubescens branches, two approaches were applied in parallel, the first consisting of classical fractioning through column chromatography and nmR characterization, and the second being performed by uHpLC-mS/mS followed by putative identification of compounds.

Putative identification of compounds by LC-MS
the powdered branches of M. pubescens were submitted to water decoction and the resulting aqueous extract (aE) was fractionated by an acid-base extraction resulting in fractions aE2, aE3 and aE4 (see supplemental material).a total of 18 compounds (B1 to B18) were putatively identified for the aE2 fraction by uHpLC-mS/ mS.using the same technique, aE3 and aE4 lead to the identification of 21 and 19 compounds, respectively.two compounds of aE3 were also present in the aE2 fraction, scopoletin (B2) and glycyrrhetinic acid (B11).For the aE4, three compounds were also detected in the aE2 fraction (lapachol (B8a), quercetin (B5) and afzelin(B6)) and two were identified in the aE3 fraction (cleomiscosin B (B22) and litseakolide L (B30)).

MTT cytotoxic assays
the aE2, aE3 and aE4 fractions of aqueous extract from M. pubescens were tested against cellular proliferation of two leukemia strains (K562 and tHp-1) and one breast cancer strain (mda-mB-231).Healthy lung fibroblasts (Wi-26Va4) were used for the selectivity index calculations.Etoposide, cytarabine and imatinib, used in the treatment of different types of cancers, were used as controls.
all tested fractions showed cytotoxic activity, with iC 50 values of less than 60 µg.mL −1 for either of the cancer cell lines tested (table 1). the best cell growth inhibition was observed for aE3 fraction (iC 50 30.72± 1.66 µg.mL −1 ), against breast cancer cells (mda-mB-231), with a selectivity index (Si 2.48) higher than the etoposide, used as a positive control (Si 0.72).the aE2 fraction showed the best activity against the K562 leukemia cells (iC 50 40.85± 2.25 µg.mL −1 ) and the aE4 fraction was more active against the tHp-1 cell lines (iC 50 32.68 ± 1.62 µg.mL −1 ). the Si values of all samples against the leukemia lines tHp-1 (Si 1.80 to 2.30) and K562 (Si 1.35 to 2.00) were relatively low compared to the control cytarabine (Si 5.97 for tHp-1) and imatinib (Si 7.48 for K562).

Conclusion
the hexane and dichloromethane extracts of M. pubescens branches lead to the isolation of lapachol (1), stigmasterol (2), maniladiol (3) and scopoletin (4), identified for the first time in the branches of this species.the fractions aE2, aE3 and aE4 obtained from the aqueous extract of M. pubenscens branches showed cytotoxic activity against mda-mB-231, breast cancer cells, and tHp-1 and K562, leukemia cell lines.the phytochemical screening of M. pubescens extracts was performed using the uHpLC-mS/ mS technique, showing the presence of triterpenes, saponins, flavonoids, coumarins, and other classes of compounds.through this method, a total of 51 compounds were putatively identified in M. pubescens.