Bioactivity of fractions and constituents of Piper capense fruits towards a broad panel of cancer cells

doi:10.1016/j.jep.2021.113884

Journal of Ethnopharmacology

Volume 271, 10 May 2021, 113884

https://doi.org/10.1016/j.jep.2021.113884 Get rights and content

Abstract

Ethnopharmacological relevance

Piper capense is a medicinal spice whose fruits are traditionally used as aqueous decoction to heal several ailments such as trypanosomiasis, helminthic infections, and cancer.

Aim of the study. (1) To perform phytochemical investigation of the methanol extract of Piper capense; (2) to evaluate the cytotoxicity of botanicals (PCF, fractions PCFa-e), isolated phytochemicals on a broad panel of animal and human cancer cell lines; (3) to evaluate the induction of apoptosis of the most active samples.

Material and methods

Resazurin reduction assay (RRA) was used to determine the cytotoxicity of the studied samples. Cell cycle distribution (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP; JC-1) and reactive oxygen species (ROS; H₂DCFH-DA) were measured by flow cytometry. Column chromatography (CC) was used for the purification of PCF, whilst nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric (MS) analyses were applied for structural elucidation.

Results

The phytochemical investigation of PCF led to the isolation of 11 compounds: licarin B (1), licarin A (2), 7-(1,3-benzodioxol-5-yl)-7,8-dihydro-8-methyl-5-(2-propenyl)-furo[3,2-e]-1,3-benzodioxole (3), nitidine isocyanate (4), 5-hydroxy-7,4′-dimethoxyflavone (5), cardamomin (6), sitosterol (7) and stigmasterol (8), β-sitosterol 3-O-β-_D-glucopyranoside (9), oleanolic acid (10) and lupeol (11). Fraction PCFb, compound 2 and doxorubicin (as positive control drug) revealed cytotoxic effects towards the 18 tested cancer cell lines. The IC₅₀ values ranged from 6.1 μg/mL (against CCRF-CEM cells) to 44.2 μg/mL (against BRAF-V600E homozygous mutant melanoma cells) for PSCb; from 4.3 μM (against CCRF-CEM cells) to 21.8 μM (against HCT116 p53^−/−) for compound 2 and from 0.02 μM (against CCRF-CEM cells) to 123.0 μM (against CEM/ADR5000 cells) for doxorubicin. PCFb and compound 2 induced apoptosis in CCRF-CEM cells mediated by activation of caspase 3/7, 8 and 9, MMP alteration and increased ROS production.

Conclusion

Piper capense is a source of potent cytotoxic botanicals and phytochemicals that could help to fight various types of cancer including multidrug resistance phenotypes. PCFb and compound 2 should further be explored to develop new drugs to fight malignancies.

Graphical abstract

Introduction

Cancer remains among the harsh human killers causing one out of six deaths globally, with a continuing increase of both morbidity and mortality in all parts of the world (IARC, 2018). Chemotherapy, which is one of the major means of treatment of malignancies, is greatly hampered by the development of resistance, or even multi-drug resistance (MDR) of cancer cells to cytotoxic drugs. In effect, cancer cells develop different mechanisms of resistance to any new established cytotoxic molecule, rendering its use obsolete to tackle recalcitrant cancers. In the past decade, scientists have considered this ability of cancer cells to develop drug-resistant cell models to experimentally unravel the underlying mechanisms and to discover new molecules with activity to resistant tumor cells but not normal cells and organs (Efferth et al., 2017, 2019).

Many botanicals and phytochemicals from the African flora have previously shown interesting cytotoxic activities on various models of cancer cell lines expressing MDR phenotypes (Kuete and Efferth, 2015; Mbaveng et al., 2017; Hegazy et al., 2019). Some of most promising botanicals identified earlier include Echinops giganteus, Xylopia aethiopica, Imperata cylindrica and Piper capense (Kuete et al., 2013b), Lawsonia inermis, Trigonella foenum-graecum and Ambrosia maritma (Saeed et al., 2015b), Withania obtusifolia, Jasonia candicans, Centaurea lippii and Pulicaria undulata (Hegazy et al., 2019) as well as Fagara tessmannii (Mbaveng et al., 2019b). Phytochemicals with the ability to hinder the proliferation of MDR cancer cells such as neoambrosin, damsin, 2-acetyl-7-methoxynaphtho[2,3-b]furan-4,9-quinone, 5,7-dihydroxy-4′-methoxy-6,8-diprenylisoflavone, 7,7″-di-O-methylchamaejasmin, ungeremine, aridanin, progenin III and 8,8-bis-(dihydroconiferyl)-diferulate (Saeed et al., 2015a; Kuete et al., 2017; Adem et al., 2019; Mbaveng et al., 2019a; 2020a; 2020b; 2020c) were recently identified in various African medicinal plants. However, the search for new substances capable of hampering the development of refractory cancer should be intensified to increase the arsenal of phytochemicals identified so far. This will increase the probability to establish novel anticancer drugs from African flora in the future.

Therefore, the present investigation was designed to carry out bio-assay guided fractionation of the methanol extract of Piper capense Linn. (Piperaceae), towards a broad panel of animal and human cancer cell lines. The study was extended to the determination of the mode of induction of apoptosis of the most active samples.

Piper capense is a Cameroonian medicinal spice traditionally used in the treatment of cancer as well as other health conditions such as trypanosomiasis or helminthic infections (Kokowaro, 1976; Van Wyk and Gericke, 2000; Kuete et al., 2011). The rationale of the present study come to fact that the fruits Piper capense are used to treat cancer. In effect, the aqueous decoction of fruit is used in Cameroon to treat various types of cancers (Kuete et al., 2011). Besides, infectious diseases, including trypanosomiasis or helminthic infections reflect disease states bearing relevance to cancer or cancer-like symptoms (Cordell et al., 1991; Popoca et al., 1998). The methanol extract of this plant has previously shown good cytotoxic effects towards a panel of cancer cell lines, including CCRF-CEM, HL60 and HL60AR leukemia cells, MDA-MB231 and MDA-MB231/BCRP breast adenocarcinoma cells, HCT116 p53^+/+ and HCT116 p53^−/− colon adenocarcinoma cells, U87.MG and U87.MGΔEGFR glioblastoma cells, and HepG2 hepatocarcinoma cells (Kuete et al., 2013b). In this study, some of the above cancer cell lines were used to ensure the good quality of the newly prepared crude extract from the fruits of Piper capense, and the identification of its active constituents on a broad panel of 19 cancer cell lines was further undertaken.

Section snippets

General procedure

Various spectrometers were used to measure the high resolution mass spectra (HRMS) (Shimadzu hybrid LC-MS-IT-TOF) and NMR Spectra (Agilent DD2 NMR (400 MHz) and Bruker Avance-III (1H-NMR: 600 MHz and 13C-NMR: 151.1 MHz) spectrometer). Chemical shifts are referenced to residual solvent signals and reported in parts per million (ppm) relative to tetramethylsilane (TMS). The silica gel Merck 60 F₂₅₄ [(0.2–0.5 mm) and (0.2–0.063 mm)] (Darmstadt, Germany) was used in column chromatography (CC) while

Phytochemistry

The phytochemical study of P. capense resulted in the isolation of 11 compounds (1–11) with diverse structures (Fig. 1). These structures were determined by exploiting the relative physical characteristics, mass data and NMR spectra resonances of each compound followed by comparison of obtained data with those of similar reported compounds in the literature. The identified compounds included three neolignans: licarin B (1), licarin A (2), and

Discussion

The use of drug-resistant cancer cell lines represents an attractive strategy in the search for new substances to fight refractory tumors. The use of a broad panel of cancer cells also enhances the credibility of data obtained with newly discovered substances. In this work, these two requirements have been considered by using firstly a broad panel of 18 cancer cell lines, and secondly, various models of drug-resistance. The drug-resistant phenotypes included U87.MGΔEGFR glioblastoma multiforme

Conclusion

Piper capense is a source of potent cytotoxic botanicals and phytochemicals acting on various types of cancer cells including MDR phenotypes. The active constituents of Piper capense include licarin B (1), licarin A (2), nitidine isocyanate (4), 5-hydroxy-7,4′-dimethoxyflavone (5) or cardamomin (6). PCFb and neolignan 2 induced apoptosis in CCRF-CEM cells mediated by caspase activation, MMP alteration and increased ROS production. The crude extract from Piper capense, some of its fractions and

Authors’ contribution

ATM, BENW, SBT, AHNL, BCKA and VK carried out the experiments. GTMB, IC and AHNL carried out NMR analyses and contributed to structure elucidation. VK and TE designed the study. ATM, GTMB and VK wrote the manuscript. TE supervised the work, corrected the manuscript and provided the facilities for the study. All authors read and approved the final manuscript.

Declaration of competing interest

There is no conflict of interest.

Acknowledgements

ATM is thankful to Alexander von Humboldt Foundation for an 18 months fellowship in Prof. Dr. Thomas Efferth's laboratory in Mainz, Germany through the ''Georg Foster Research Fellowship for Experienced Researcher'' program. SBT and VK are grateful to the Alexander von Humboldt Foundation for the 6 months further research stay fellowship in Mainz. Authors are also thankful to the Institute of Molecular Biology gGmbH (IMB) (Mainz, Germany), where the flow cytometry measurements of MMP were

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Bioactivity of fractions and constituents of Piper capense fruits towards a broad panel of cancer cells

Abstract

Ethnopharmacological relevance

Material and methods

Results

Conclusion

Graphical abstract

Introduction

Section snippets

General procedure

Phytochemistry

Discussion

Conclusion

Authors’ contribution

Declaration of competing interest

Acknowledgements

Phytomedicine

J. Biol. Chem.

J. Ethnopharmacol.

Phytochemistry

Phytomedicine

J. Ethnopharmacol.

J. Ethnopharmacol.

Phytomedicine

Phytomedicine

Phytochemistry

Phytomedicine

Phytomedicine

J. Ethnopharmacol.

Phytomedicine

Chem. Biol. Interact.

Phytomedicine

Life Sci.

J. Ethnopharmacol.

J. Ethnopharmacol.

Phytochemistry

Bioorg. Med. Chem. Lett

Structures of New Benzphenanthridine Alkaloids from Zanthoxylum Nitidum

Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation

J. Immunol.

Natural Compounds in Cancer Therapy

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