SUPPLEMENTARY MATERIAL

Alkaloid extracts of Ficus species and palm oil-derived tocotrienols

synergistically inhibit proliferation of human cancer cells

Ibrahim Babangida Abubakara, Kuan-Hon Lim

b and Hwei-San Loh

a,c*

a School of Biosciences, Faculty of Science, The University of Nottingham Malaysia

Campus, Jalan Broga 43500 Semenyih, Selangor, Malaysia; b

School of Pharmacy,

Faculty of Science, The University of Nottingham Malaysia Campus, Jalan Broga

43500 Semenyih, Selangor, Malaysia; cBiotechnology Research Centre, The University

of Nottingham Malaysia Campus, Jalan Broga 43500 Semenyih, Selangor, Malaysia

*Corresponding author. Email: Sandy.Loh@nottingham.edu.my

Tocotrienols have been reported to possess anticancer effects other than anti-

inflammatory and antioxidant activities. This study explored the potential synergism of

antiproliferative effects induced by individual alkaloid extracts of Ficus fistulosa, Ficus

hispida and Ficus schwarzii combined with - and γ-tocotrienols against human brain

glioblastoma (U87MG), lung adenocarcinoma (A549) and colorectal adenocarcinoma

(HT-29) cells. Cell viability and morphological results demonstrated that extracts

containing a mixture of alkaloids from the leaves and bark of F. schwarzii inhibited

proliferation of HT-29 cells. Whereas, the alkaloid extracts of F. fistulosa inhibited

proliferation of both U87MG and HT-29 cells and showed synergism in combined

treatments with either - or γ-tocotrienol resulting in 2.2-34.7 folds of reduction in IC50

values of tocotrienols. The observed apoptotic cell characteristics in conjunction with the

synergistic antiproliferative effects of Ficus species derived alkaloids and tocotrienols

assuredly warrant future investigations towards the development of a value-added

chemotherapeutic regimen against cancers.

Keywords: Ficus fistulosa; Ficus hispida; Ficus schwarzii; tocotrienols; antiproliferative;

synergism; cancer cells

Experimental

Tocotrienols, plant collection and preparation of extracts

The palm oil derived - and γ-tocotrienol isomers were supplied in kind by Davos Life

Science Pte Ltd (Singapore). Ficus fistulosa Reinw. ex Blume, Ficus hispida Linn. and

Ficus schwarzii Koord were collected in Selangor, Malaysia and identified by Dr Kien-

Thai Yong (Institute of Biological Sciences, University of Malaya). The plants were

tagged and assigned voucher numbers UNMC 68 (KLU 47988), UNMC 77 (KLU

48172) and UNMC 87 (KLU 48174), respectively and deposited at the Herbarium of

the University of Malaya. The leaves and bark were air-dried, blended into smaller

particles and extracted three times with 95% ethanol with subsequent evaporation to

obtain the ethanol crude extracts. Thereafter, a portion of the ethanol crude extracts

were acidified with 3% tartaric acid and filtered through kieselguhr to remove non-

alkaloidal substances. The filtrate was basified to pH 10 with 28% ammonia and the

liberated alkaloids were extracted with chloroform to furnish the alkaloid crude extracts.

Cell culture and cell viability assay

Plant extracts at the weight of 10 mg were dissolved in 1 ml DMSO. A dose range (0.1,

5, 15, 30, 60 and 120 µg/ml) was prepared from a working concentration of 1 mg/ml.

For tocotrienols, a stock concentration of 25 mg/ml was prepared and thereafter, a dose

range (0.01, 0.2, 2, 6, 12 and 24 µg/ml) was prepared from a working concentration of 1

mg/ml. U87MG, HT-29, A549 and MRC5 cells were maintained under cell culture

conditions as previously described (Lim et al. 2011). Approximately, 5x103

cells were

seeded in 96-well plates and allowed for 24 h of incubation period to facilitate cell

attachment. Thereafter, treatment media or plain media containing equivalent amount of

DMSO (vehicle control) were added and incubated for 72 h. Cell viability was

determined using the neutral red uptake assay as previously described (Repetto et al.

2008; Lim et al. 2013).

For determining synergistic activity, the sub-effective dose, IC20 of the potent

leaf and bark alkaloid crude extracts of F. fistulosa and F. schwarzii were combined

with doses of - and γ-tocotrienols (0.01, 0.2, 2, 6, 12 and 24 µg/ml). The resultant low

dose of tocotrienols responsible for 50% cell growth inhibition in combination with IC20

of the plant extracts was subsequently determined using Graphad prism5 software. The

synergistic index (SI) and dose reduction index (DRI) were determined as described

before (Wali & Sylvester 2007). SI was used to measure the pharmacological

interaction between plant extracts and tocotrienols, while DRI is a representation of fold

decrease of combined treatment that results in 50% cell growth inhibition. An SI value

that is less than or equals to 1 indicates synergistic or additive effect, respectively,

whereas, an SI value above 1 indicates antagonistic effect (Akl et al. 2012). The

strength of synergism or antagonism was deduced accordingly (Chou 2006).

Histochemical staining

Guided by neutral red uptake finding, the alkaloid crude extracts of F. fistulosa and

combinations with - and γ-tocotrienols which had the highest antiproliferative effect

against U87MG cells were thus selected for morphological studies by haematoxylin and

eosin (H&E) staining technique. Approximately 5x103 U87MG cells were seeded in

chamber slides (Nunc Labtek II, Sigma, USA) and treated with IC50 dose of plant

extracts or combination of plant extracts with either - and γ-tocotrienol for 24 h.

Subsequently, cells were fixed and rehydrated with decreasing ethanol concentrations

(100%, 95% and 70%) followed by staining with a mixture of haematoxylin and eosin

dyes (Sigma USA) as previously described (Lim et al. 2011). The stained slides were

mounted and viewed under Nikon 80i microscope (Nikon, Japan) at 40X magnification.

Statistical analysis

The IC50 and IC20 values were determined by the non-linear regression curve fit using

Graphpad Prism (version 5). One way ANOVA using the Dunnet T-test was used to

compare treated and untreated groups. Differences were considered at p < 0.001. The

quantitative cell viability experimental data were expressed as mean value with standard

error of mean (SEM) of the triplicates performed in three separate experiments.

References

Akl MR, Ayoud NM, Sylvester PW. 2012. Mechanisms mediating the synergistic

anticancer effects of combined γ-tocotrienol and sesamin treatment. Planta Med.

17:31-39.

Chou TC. 2006. Theoretical basis, experimental design, and computerized simulation of

synergism and antagonism in drug combination studies. Pharmacol Rev. 58:621-

681.

Lim SW, Loh HS, Ting KN, Bradshaw TD, Zeenathul NA. 2013. Acalypha wilkesiana

ethyl acetate extract enhances the in vitro cytotoxic effects of α-tocopherol in

human brain and lung cancer cells. Int J Biosci Biochem Bioinforma. 3:335-340.

Lim SW, Ting KN, Bradshaw TD, Zeenathul, NA, Wiart C, Khoo TJ, Lim KH, Loh

HS. 2011. Acalypha wilkesiana extracts induce apoptosis by causing single strand

and double strand DNA breaks. J Ethnopharmacol. 138:616-623.

Repetto G, Del Peso A, Zurita JL. 2008. Neutral red uptake assay for the estimation of

cell viability/cytotoxicity. Nat Protoc. 3:1125-1131.

Wali VB, Sylvester PW. 2007. Synergistic antiproliferative effects of gamma-

tocotrienol and statin treatment on mammary tumor cells. Lipids 42:1113-1123.

Table S1: Yield of crude alkaloid extracts of Ficus spp.

Plant Species Plant Part Yield of Alkaloid

Extract (g/kg)

Ficus fistulosa

(FF)

Leaf 0.28

Bark 0.37

Ficus hispida

(FH)

Leaf 0.56

Bark 0.22

Ficus schwarzii

(FS)

Leaf 0.22

Bark 0.07

Table S2: Combinational treatment effects of crude alkaloid extracts with -tocotrienol (-T3) and γ-tocotrienol (-T3) on U87MG and HT-

29 cells.

Cell Line Plant

Species

Plant

Part

Extract

Type

Extract

IC20

(μg/ml)

-T3

IC50

(μg/ml)

Combined

Results:

IC50 (μg/ml)

Synergistic

Index

(SI)

Dose Reduction

Index (DRI) Combined

Results (MRC5):

IC50 (μg/ml) Extract -T3

U87MG FF Leaf Alk 0.24 ± 0.28 3.12 ± 1.26 0.11 ± 0.22 0.29 4.00 28.36 2.23 ± 1.44

Bark Alk 0.53 ± 0.34 3.12 ±1.26 0.09 ± 0.24 0.28 3.96 34.67 4.68 ± 1.17

HT-29

FF Leaf Alk 0.50 ± 0.33 5.71 ± 1.23 2.10 ± 0.16 0.63 3.84 2.72 0.92 ± 0.22

Bark Alk 0.50 ± 0.31 5.71 ± 1.23 2.62 ± 0.12 0.71 4.00 2.17 4.40 ± 1.41

FS Leaf Alk 2.20 ± 0.31 5.71 ± 1.23 5.43 ± 1.29 1.20 4.00 1.05 4.32 ± 1.17

Bark Alk 3.60 ± 0.30 5.71 ± 1.23 4.42 ± 1.26 1.02 4.02 1.29 5.66 ± 1.14

Cell Line Plant

Species

Plant

Part

Extract

Type

Extract

IC20

(μg/ml)

-T3

IC50

(μg/ml)

Combined

Results:

IC50 (μg/ml)

Synergistic

Index

(SI)

Dose Reduction

Index (DRI) Combined

Results (MRC5):

IC50 (μg/ml) Extract -T3

U87MG FF Leaf Alk 0.24 ± 0.28 3.17 ± 1.29 0.11 ± 0.19 0.29 4.00 28.82 1.44 ± 1.48

Bark Alk 0.53 ± 0.34 3.17 ± 1.29 0.12 ± 0.20 0.29 3.96 26.42 3.07 ± 1.12

HT-29

FF Leaf Alk 0.50 ± 0.33 6.14 ± 1.15 2.45 ± 0.18 0.66 3.84 2.51 0.72 ± 0.26

Bark Alk 0.50 ± 0.31 6.14 ± 1.15 2.67 ± 0.19 0.68 4.00 2.30 4.82 ± 1.32

FS Leaf Alk 2.20 ± 0.31 6.14 ± 1.15 8.29 ± 1.17 1.60 4.00 NR 4.67 ± 1.26

Bark Alk 3.60 ± 0.30 6.14 ± 1.15 4.48 ± 1.29 0.98 4.02 1.37 4.31 ± 1.28

Note: Abbreviations, FF - F. fistulosa; FH - F. hispida; FS - Ficus schwarzii; Alk - alkaloid crude, NR - no reduction. Data are presented as

Mean ± SEM (n = 9). The alkaloid extracts of F. fistulosa inhibited proliferation of both U87MG and HT-29 cells and showed synergism in

combined treatments with either - or γ-tocotrienol resulting in 2.2-34.7 folds of reduction in IC50 values of tocotrienols.

Figure S1. Representative cell viability histograms showing the combined growth

inhibitory effects of (a) -tocotrienol + 0.24 µg/ml F. fistulosa leaf (FF-L), (b) -

tocotrienol + 0.53 µg/ml F. fistulosa bark (FF-B), (c) γ-tocotrienol + 0.24 µg/ml F.

fistulosa leaf (FF-L), and (d) γ-tocotrienol + 0.53 µg/ml F. fistulosa bark (FF-B) on

U87MG brain cancer cells after 72 h treatment. Combined treatments have significantly

reduced the cell viability percentage of U87MG as compared to untreated controls.

Results are presented as Mean ± SEM of triplicates in three independent experiments.

*P < 0.001.

Figure S2. Isobologram analysis of combined treatments, i.e. - and γ-tocotrienols with

alkaloid crude extracts of leaves or bark of (a) F. fistulosa on U87MG cells, (b) F.

fistulosa on HT-29 cells, and (c) F. schwarzii on HT-29 cells.

Figure S3. Morphologies of H&E stained U87MG cells receiving single treatments of F.

fistulosa alkaloid crude extracts and combined treatments with either - or γ-tocotrienol.

(a) Untreated, (b) F. fistulosa leaf (0.96 µg/ml), (c) F. fistulosa bark (2.10 µg/ml), (d) F.

fistulosa leaf + -tocotrienol (0.24 + 0.11 µg/ml), (e) F. fistulosa bark + -tocotrienol

(0.53 + 0.09 µg/ml), (f) F. fistulosa leaf + γ-tocotrienol (0.24 + 0.11 µg/ml), (g) F.

fistulosa bark + γ tocotrienol (0.53 + 0.12 µg/ml). Features including vesicle formation

(blue arrow), fragmented nucleus (green arrow), condensed nucleus (red arrow),

apoptotic bodies (red arrow head), cell shrinkage with condensed nucleus and cytoplasm

(blue arrow head) and cytoplasmic extension (black arrow) were evidenced. Scale bar

represents 50 µm.