Antioxidant and cytotoxicity activities of δ-tocotrienol from the seeds of Allophylus africanus

Abstract Chemical investigation of Allophylus africanus P. Beauv fruits led to the isolation of a new δ-tocotrienol, 3α-hydroxy-δ-tocotrienol (1) together with eight known compounds (2-9). Compound (1) was allylated (1a) and prenylated (1 b and 1c) to give three new semi-synthesized derivatives which were fully characterized as: 6-O-allyl-3α-hydroxy-δ-tocotrienol (1a), 6-O-prenyl-3α-hydroxy-δ-tocotrienol (1 b) and 6-O,5-C-diprenyl-3α-hydroxy-δ-tocotrienol (1c). The structures of compounds were established using comprehensive spectroscopic analysis including UV, MS, 1 D NMR, 2 D NMR and by comparison with the corresponding literature data. Compound (1) and its semi-synthetic derivatives (1a-c) were tested for their antioxydant activity using DPPH radical scavenging assay and also for their cytotoxicity using human cervix carcinoma KB-3-1 cell lines. The results showed that compound (1) exhibited antioxidant activity with an IC50 value of 0.25 μM compared to the reference control trolox (26 µM); and good cytotoxic activity with IC50 values of 97 μM compared to the reference (+)-griseofulvin (IC50 between17-21 μM). Graphical Abstract


Introduction
Allophylus is one of the largest genera belonging to the Sapindaceae family with about 255 species and are widely distributed in subtropical and tropical regions of Africa, America, Asia, India Archipelago, Australia, Pacific and Madagascar (Piaggio and Delfino 2014;Fatima 2016). Allophylus africanus P. Beauv is one of the most important conserved and valued forest species in Africa due to their multiple uses, high nutritional content (Oladosu et al. 2015;Martin and Martin 2009;Onuminya and Ogundipe 2014) and its medicinal values. The plant is used in the treatment of conjunctivitis, dysentery, arthritic conditions, malaria, skin inflammation and eyes trouble (Burkill 1983;Oladosu et al. 2013;Catarino et al. 2016;Biseko et al. 2019). The plant is also used as antipyretic, anti-diarrhoea, anticancer, anti-headache, anti-migraine and has vermicidal properties (Burkill 1985;Gathirwa et al. 2011;Oladosu et al. 2013;Biseko et al. 2019). Previous phytochemical studies on the whole plant of A. africanus reported the isolation of terpenoids (Oladosu et al. 2015); while in other Allophylus species C-glycosyl flavonoids, sesquiterpenoids, coumarins, vanillic acid derivatives, triterpenoids, as well as ceanothane-type triterpenoids, tocotrienols and fatty acids were reported (Hoffmann-Bohm et al. 1992;David et al. 2004;Ciepichal et al. 2007;Dıaz et al. 2008;Kumar et al. 2010;Zhang et al. 2012;Balogun et al. 2016;Sangsopha et al. 2020). Some exhibited strong antimalarial, antibacterial, anticancer and antioxidant activities (Sofidiya et al. 2007;Oladosu et al. 2014;Balogun et al. 2016). Despite the studies already done on the different parts of A. africanus, no chemical and/or biological study have yet been carried out on the fruits. In our continuing search for bioactive compounds from plants, we investigated the fruits of A. africanus and herein report on the isolation and structure elucidation of a new d-tocotrienol; 3a-hydroxy-d-tocotrienol together with its DPPH antioxidant and cytotoxic assays.
The cytotoxic activities of compound 1, and its semisynthetic derivatives (1a-1c), were also determined in cell-based cytotoxicity assays using human cervix carcinoma KB-3-1 cells line. Only compound 1 exhibited significant cytotoxic activities with IC 50 values of 97 mM, while the semisynthetic derivatives (1a-1c) were inactive, compared to the reference drug griseofulvin (17-21 lM). It's evidence that the free hydroxyl group on the aromatic ring is the main functional group responsible for the activity. These results corroborated some previous data on tocotrienols (Ahsan et al. 2014).

General experiment procedures
The 1 H and 13 C NMR spectra were recorded on Bruker DRX spectrometers at 500 MHz and 125 MHz respectively. HRESIMS spectra were recorded on a Synapt G2Si -Q-IMS-TOF-mass spectrometer. Column chromatography (open column) was performed with silica gel Merck 60 (0.063-0.200 mm) and percolated aluminum backed silica gel 60 F254 sheets were used for TLC. Size exclusion column chromatography was performed Scheme 1. Proposed reaction mechanism for compound 1c using Sephadex LH-20. The TLC spots were visualized under UV light (254 and 365 nm); H 2 SO 4conc. (10%) was used as spraying reagents.

Plant material and identification
The seeds of Allophylus africanus were collected in October 2017 in Bangoua, West Region of Cameroon and identified at the Cameroon National Herbarium, Yaound e, by Victor Nana, with voucher specimen N o 20561/SRF/HNC.

Allylation and prenylation reactions
3.4.1. Allylation of 3a-hydroxy-d-tocotrienol 25.0 mg of 3a-hydroxy-d-tocotrienol were dissolved in 10.0 mL of acetone. Then, 120.0 mg of potassium carbonate (K 2 CO 3 ) and 0,2 mL of allyl bromide were respectively added to this mixture. The reaction medium was refluxed at 70 C for 9 h (Barron et al. 1996). Compounds 1a (yield 55%) was obtained after purification of the reaction product on silica gel CC with petrol ether/CH 2 Cl 2 (95:5) as eluent.

Antioxidant assay
The DPPH (1,1-diphenyl-2-picrylhydrazyl) assay was used to evaluate the free radical scavenging activity of the compounds (Sellem et al. 2016). Briefly, the compounds were dissolved in 10% DMSO and diluted at different concentrations of 250.0 to 1.0 lg/mL. Then, 500.0 mL of a 4% (w/v) solution of DPPH radical in methanol was mixed with 500.0 mL of samples under investigation. The mixture was incubated for 30 min in the dark at room temperature. The scavenging capacity was determined spectrophotometrically by monitoring the decrease in absorbance at 517 nm against a blank. The percentage of antioxidant activity was calculated As antioxidant activity (%) ¼ [(A control -A sample )/A control ] x 100. BHA (Butylhydroxyanisol) and BHT (Butylatedhydroxytoluene) (Sigma, USA) were used as positive controls. A sample was the absorbance of the sample and A control was the absorbance of the blank.

Cytotoxicity assay
Cytotoxic activity screening of the isolates was done as described in previous reports (Sammet et al. 2010;Awantu et al. 2011). The KB-3-1 cell was cultivated as a monolayer in DMEM (Dulbecco's modified Eagle medium) with glucose (4.5 g.L À1 ), L-glutamine, sodium pyruvate and phenol red, supplemented with 10% (KB-3-1) foetal bovine serum (FBS). The cells were maintained at 37 C and 5.3% CO 2 -humidified air. The day before the test, the cells (70% confluence) were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (0.05%; 0.02% PBS) and placed in sterile 96-well plates in a density of 10 000 cells in 100 lL medium per well. The dilution series of the compounds were prepared from stock solutions in DMSO of concentrations of 1 mM or 10.0 mM. The stock solutions were diluted with culture medium 10% FBS [KB-3-1]) at least 50 times. Some culture medium was added to the wells to adjust the volume of the wells to the wanted dilution factor. The dilution prepared from stock solution was added to the wells. Each concentration was tested in six replicates. Dilution series were prepared by pipetting liquid from well to well. The control contained the same concentration of DMSO as the first dilution. After incubation for 72 h at 37 • C and 5.3% CO 2 -humidified air, 30.0 lL of an aqueous resazurin solution (175.0 lM) were added to each well. The cells were incubated at the same conditions for 6 h. Subsequently, the fluorescence was measured. The excitation was affected at a wavelength of 530 nm, whereas the emission was recorded at a wavelength of 588 nm. The IC 50 values were calculated as a sigmoidal dose response curve using GRAPHPAD PRISM 4.03. The IC 50 values equal the drug concentrations, at which vitality is 50%. Griseofulvin was used as reference.

Conclusions
The seeds of A. africanus were investigated for their chemical composition, antioxidant and cytotoxicity activities in this study for the first time. The chemical investigation led to the isolation of nine compounds, including a new tocotrienol namely, 3a-hydroxyd-tocotrienol (1). The allylation and the prenylation of compounds 1 led to the three news semi-synthesis derivative 1a and 1 b-1c respectively namely, 6-O-allyl-3ahydroxy-d-tocotrienol (1a), 6-O-prenyl-3a-hydroxy-d-tocotrienol (1 b) and 6-O-5-Cdiprenyl-3a-hydroxy-d-tocotrienol (1c). DPPH scavenging method and human cervix carcinoma KB-3-1 cells line assays were performed to assess the antioxidant and cytotoxicity of compound 1 and its derivatives. Compound 1 was about a hundred times more active than the reference trolox (26 mM) with IC 50 value of 0.25 mM regarding the antioxidant activity, and exhibited significant cytotoxic activities with IC 50 values of 97 mM compared to the reference drug griseofulvin (17-21 lM), while the semisynthetic derivatives (1a-1c) were inactive. These results corroborate some data on certain d-tocotrienol derivatives, with regard to the antioxidant and the cytotoxicity activities (Beretta et al. 2018;Popova et al. 2020).

Notes
The authors declare no competing financial interest.