Antiproliferative effect of extracts and fractions of the root of Terminalia avicennioides (Combretaceae) Guill and Perr. on HepG2 and Vero cell lines

Gentamicin was from Virbac, Republic of South Africa. Trypsin-EDTA (cat no: BE17-16IF) and L-glutamine (cat no: BE-17-605E) were from Lonza (Verviers, Belgium). Dulbecco’s Modified Eagle’s Medium (DMEM, cat. No: D6546), MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) reagent (cat. no: M5655), phosphate buffered saline (PBS, P4417) and Trypan blue (cat. no: T6146) were from Sigma-Aldrich (Darmstadt, Germany). Dimethyl sulphoxide (DMSO) (cat. no: SAAR1865000LP) was procured from Merck (Darmstadt, Germany). Analytical grade solvents were from Sigma-Aldrich/Merck (Darmstadt, Germany).

The roots of Terminalia avicennioides were collected in the wild, around Zaria (11º 04’ N 7º 42’ E) between the months of October and December (beginning of dry season in northern Nigeria). The plant material comprising the leaves, fruits and seeds were authenticated at the Herbarium, Department of Biological Sciences, Ahmadu Bello University, Zaria. A verification number of VIN 900,239 was given. Sample of T. avicennioides roots collected was washed, allowed to dry in the shade and ground into powder using a laboratory mill. About 4 kg of the powdered material was put in a polythene bag and stored in a cabinet at room temperature. Two hundred and forty (240 g) gram of the powdered root of T. avicennioides divided into 6 parts, 30 g each, was extracted using acetone, absolute ethanol, 30 % ethanol and methanol, hot and cold water by maceration for 24 h. The different solutions were sieved through Whatman No. 1 filter paper. The filtrates were evaporated to obtain solid residues of the different extracts, respectively. Similarly, about 2 kg of the powder was extracted exhaustively using methanol by maceration for 24 h and filtered to obtain the methanol extract (ME). The ME was then concentrated in vacuo using a Rotary evaporator (Büchi® Rotavapor® R II, Vacutec, Switzerland) under lowered pressure to give the solid extract, which was kept for the fractionation process.

The crude ME was serially partitioned using solvents of decreasing polarity starting with butanol, chloroform and hexane. The procedure for each solvent was exhaustively carried out.

The phytochemical composition of extracts was appraised by the methods of Trease and Evans [16].

Two hundred milligram (0.2 g) of extract was placed in 10 mL acid alcohol, boiled and filtered. To 5 mL of the solution, 2 mL of dilute ammonia was added. Chloroform (5 mL) was added and the mixture agitated lightly. To this mixture, 10 mL of acetic acid was added. This was separated into two parts. Draggendorff’s reagent was added to one part. The development of reddish-brown precipitate is positive for alkaloids.

In 0.5 mL filtrate of extract, 5 mL dilute ammonia was added, followed by the addition of 1 mL concentrated sulphuric acid. The existence of flavonoids is identified via yellow colouration of the solution which vanishes on standing.

A tiny amount of extract was liquefied in 2 mL distilled water. Into it was added few drops of 10 % aqueous ferric chloride solution. Production of a blue or green colouration points to the existence of phenols.

The extract was mixed with 5 mL distilled water in a test tube and agitated robustly for about 30 s. The development of a honeycomb-like froth that persists for 10–15 indicates the existence of saponins.

To about 2 mL of the extract, 3–5 drops of ferric chloride (FeCl₃) solution was added. A blue or brownish-blue precipitate indicates the presence of hydrolysable tannins.

One milliliter acetic anhydride was added to about 2 mL of extract, followed by the addition of concentrated sulphuric acid (H₂SO₄₎. A pink to violet colour indicates the presence of terpenoids.

HepG2 human liver cancer (ATCC® HB8065™) and Vero monkey kidney (ATCC® CCL-81™) cells were from Cellonex, South Africa. HepG2 cells were retained in DMEM high glucose (4.5 g/L) encompassing L-glutamine (4 mM) and sodium pyruvate (Hyclone™) supplemented with 10 % foetal calf serum (FCS). Vero cells were sustained in DMEM high glucose (4.5 g/L) having L-glutamine in which 1 % gentamicin and 5 % FCS (Highveld Biological, South Africa) were added. Both cells were kept at 37° C in 175 cm² culture flasks in a 5 % CO₂ incubator (HeraCELL 150^R, Thermo-Electron Corporation). Cells were passaged three times a week and trypsin-EDTA was used to detach the cells. Counting of cells was with a haemocytometer and using trypan blue exclusion to define viable cells. Nunc 96 well microtiter plates were used for seeding cells.

Inhibition of viable cell growth was appraised utilizing the MTT assay [17]. Cells from a sub-confluent flask were collected and centrifuged at 200 x g for 5 min, then suspended in DMEM to 1 × 10⁵ cells/mL. One hundred microlitres (100 µL) of the cell preparation was seeded into wells of columns 2 to 11 of a sterile 96-well microtitre plate. DMEM (200 µL) was placed in wells of columns 1 and 12 to lessen the “edge effect” and sustain moisture. The plates were placed in the incubator overnight to allow for cell attachment and recovery. Test samples (methanol, hot water, cold water, ethylacetate, hexane, chloroform, butanol and residual water extracts) were liquefied in DMSO (making 100 mg/mL). Varying dilutions were made in DMEM starting with 1000 µg/mL. Decreasing concentrations (100 µL each) of the extracts (1000, 750, 500, 250, 100, 50 µg/mL) were pipetted into the matching wells and the plates incubated for 48 h. Cells maintained with growth medium only, representing 100 % viability, functioned as negative control whereas doxorubicin hydrochloride (Pfizer, South Africa) was used as positive control. After 48 h, the cells were washed with 200 µL PBS, before 200 µl fresh medium incorporating 30 µL MTT (5 mg/mL in PBS), was added. Following 4 h incubation, the growth medium was cautiously aspirated, with a suction pump (Integra, USA) and the formed formazan crystals solubilized with 50 µL DMSO. The plate was agitated lightly for 2 min. The MTT reduction was quantified instantly by reading the absorbance using a spectrophotometer (Synergy HT, BioTek^REL808, Winooski, Vermont, USA) at a wavelength of 570 nm and a reference wavelength of 630 nm. Each extract concentration was tested in quadruplicate and the assays repeated twice at weekly intervals.

Percentage cell viability was computed for each extract concentration and the inhibitory concentration 50 (IC₅₀) values were determined with the straight line equation of the plots of viability (%) against log concentration.

Data were presented as mean ± SD (standard deviation). Cell viability as percentage of untreated negative control cells was computed with the equation: