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Alkaloid extracts of Ficus species and palm oil-derived tocotrienols synergistically inhibit proliferation of human cancer cells a

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Ibrahim Babangida Abubakar , Kuan-Hon Lim & Hwei-San Loh

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Faculty of Science, School of Biosciences, The University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia b

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Faculty of Science, School of Pharmacy, The University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia c

Biotechnology Research Centre, The University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia Published online: 17 Dec 2014.

To cite this article: Ibrahim Babangida Abubakar, Kuan-Hon Lim & Hwei-San Loh (2014): Alkaloid extracts of Ficus species and palm oil-derived tocotrienols synergistically inhibit proliferation of human cancer cells, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.991927 To link to this article: http://dx.doi.org/10.1080/14786419.2014.991927

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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.991927

SHORT COMMUNICATION Alkaloid extracts of Ficus species and palm oil-derived tocotrienols synergistically inhibit proliferation of human cancer cells Ibrahim Babangida Abubakara1, Kuan-Hon Limb2 and Hwei-San Lohac* a


Faculty of Science, School of Biosciences, The University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia; bFaculty of Science, School of Pharmacy, 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 (Received 9 October 2014; final version received 20 November 2014)

Tocotrienols have been reported to possess anticancer effects other than antiinflammatory 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 d- and g-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 the proliferation of HT-29 cells, whereas the alkaloid extracts of F. fistulosa inhibited the proliferation of both U87MG and HT-29 cells and showed synergism in combined treatments with either d- or g-tocotrienol resulting in 2.2– 34.7 fold 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

1. Introduction Plants have been rich sources of bioactive compounds for anticancer drug development and notable examples of anticancer drugs of plant origin include the Vinca alkaloids, vincristine and

*Corresponding author. Email: [email protected] q 2014 Taylor & Francis


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vinblastine (Cragg & Newman 2005). Medicinal application of Ficus species across Asia, Europe, Middle East and Africa dated back to thousands of years and recent ethnopharmacological and phytochemical studies of plants from this genus have resulted in the isolation of secondary metabolites exhibiting various bioactivities such as antioxidant, cytotoxicity and inflammation (Lansky et al. 2008). In addition, tocotrienols have received more recognition for therapeutic properties, primarily anticancer, over the past 14 years and were suggested to be best suited for adjuvant treatment of cancer in combination with other compounds (Constantinou et al. 2008). As part of our continual screening of rainforest plants for potential cytotoxic alkaloids, we investigated the antiproliferative effect of 12 different extracts obtained from three Ficus species parts (Ficus hispida Linn., Ficus fistulosa Reinw. ex Blume and Ficus schwarzii Koord) comprising ethanolic and alkaloid crude extracts of each plant, against three cancer cell lines, i.e., human lung adenocarcinoma (A549), glioblastoma (U87MG) and colon adenocarcinoma (HT-29), as well as the normal lung fibroblast (MRC5) cell line. Subsequently, extracts containing mixture of alkaloids that demonstrated antiproliferative potency were combined with individual d- and g-tocotrienols to allow their potential synergistic activities to be investigated. 2. Results and discussion Chemotherapeutic compounds isolated from plants appear to be a practical approach for cancer prevention and treatment. Development of drug resistance in certain cancers resulting in poor treatment outcome has become a driving force among researchers in searching for new plantderived anticancer agents. In this study, the leaf of F. hispida yielded the highest alkaloid extracts compared with F. fistulosa and F. schwarzii (Supplementary Table S1, online only). Following a 72 h treatment, only four extracts against U87MG cells, five extracts against HT-29 cells and one extract against A549 cells had substantial dose-dependent antiproliferative effects with IC50 values obtained below 20 mg/mL (Table 1, values in bold) which conformed to the required anticancer potency set by the National Cancer Institute standard for the plant extracts (Suffness & Pezzuto 1990). The leaf and bark alkaloid crude extracts of F. fistulosa exhibited the highest potency (Table 1) at concentrations ranging from 0.96 to 2.1 mg/mL against HT-29 and

Table I. Growth inhibitory effects (IC50) of crude alkaloid extracts of Ficus spp. against U87MG, A549, HT-29 and MRC5 cell lines. Plant species

Plant part

Extract type

FF

Leaf

ET Alk ET Alk ET Alk ET Alk ET Alk ET Alk

Bark FH

Leaf Bark

FS

Leaf Bark

U87MG (mg/ml)

A549 (mg/ml)

HT-29 (mg/ml)

MRC5 (mg/ml)

66.02 ^ 1.21 46.95 ^ 1.08 27.73 ^ 1.11 29.32 ^ 1.14 0.96 6 1.12 46.81 ^ 1.25 1.92 6 1.30 12.47 ^ 2.79 21.62 ^ 1.15 49.61 ^ 1.18 32.08 ^ 1.19 30.95 ^ 1.12 2.10 6 1.34 44.35 ^ 1.18 2.00 6 1.23 18.90 ^ 1.36 11.98 6 2.28 49.58 ^ 1.12 43.90 ^ 1.21 18.39 ^ 1.31 17.49 6 1.25 40.82 ^ 1.22 20.71 ^ 1.31 11.27 ^ 1.78 88.01 ^ 1.14 61.41 ^ 1.07 57.09 ^ 1.18 72.20 ^ 1.14 31.24 ^ 1.26 52.98 ^ 1.08 15.14 6 1.21 19.94 ^ 1.16 35.31 ^ 1.31 73.71 ^ 1.09 29.00 ^ 1.19 . 120.00 ^ 2.29 29.39 ^ 1.25 .120.00 ^ 1.15 8.79 6 1.20 63.54 ^ 1.23 84.38 ^ 1.17 19.21 6 1.16 . 120.00 ^ 1.24 61.60 ^ 1.17 107.9 ^ 1.16 44.83 ^ 1.13 14.49 6 1.20 . 120.00 ^ 1.14

Note: FF, F. fistulosa; FH, F. hispida; FS, Ficus schwarzii; ET, ethanol; Alk, alkaloid crude. Data is presented as Mean ^ SEM (n ¼ 9). Plant extracts with IC50 values , 20 mg/ml (bolded) exhibit antiproliferative effects towards the respective cancer cell lines.


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U87MG cell lines. Similar toxicity was also observed in HT-29 cells treated with crude alkaloid extracts from the leaves and bark of F. schwarzii with relatively low toxicity against normal MRC5 cells. Analysis of the cell viability IC50 values enables the potency of the alkaloid crude extracts to be ranked as F. fistulosa leaf . F. fistulosa bark . F. schwarzii leaf . F. schwarzii bark . F. hispida leaf . F. hispida bark, in a descending order. Combinations of low dose d- and g-tocotrienols with alkaloid crude extracts of the leaves and bark of F. fistulosa demonstrated a dose-dependent growth inhibition (Supplementary Figure S1, online only) with strong (SI ¼ 0.28 – 0.29) and moderate synergistic effects (SI ¼ 0.63 –0.71) against U87MG and HT-29 cells, respectively, as shown in the isobologram analysis (Supplementary Figure S2, online only). In contrast, a nearly additive or antagonistic effect (SI ¼ 0.98 –1.60) with high toxicity incurred on normal cells was observed when subeffective doses of d- and g-tocotrienols were combined with alkaloid crude extracts of the leaves and bark of F. schwarzii against HT-29 cells (Supplementary Table S2, online only). In addition to the improved potency of the leaf and bark crude alkaloids of F. fistulosa, the selectivity window between cancer and normal cell lines (calculated by IC50 of MRC5 over that of U87MG cells) was also increased from 9 –13 fold for single treatment to 13– 52 fold for combined treatments with d- and g-tocotrienols. Although the combined treatment IC50 values of F. fistulosa and F. schwarzii alkaloid crude extracts against HT-29 cells have been greatly reduced, it has nonetheless caused significant toxicity to the non-cancerous MRC5 cells. In addition, morphological features including cell shrinkage, condensed and fragmented nuclear chromatin, vesicles and apoptotic bodies resembling apoptotic cell death as previously described (Saraste & Pulkki 2000) were detected by H&E staining technique on U87MG cells treated with the leaf and bark alkaloid crude extracts of F. fistulosa and combined treatments with d- and g-tocotrienols (Supplementary Figure S3, online only). These findings confirmed that the alkaloid crude extracts of F. fistulosa induced cancer cell death by apoptosis contrasting previous studies on the stem-bark of Vietnamese and Singaporean F. fistulosa which yielded non-cytotoxic crude and purified alkaloids with antifungal and antimalarial activities (Zhang et al. 2002; Subramaniam et al. 2009). These differences could be attributed to divergent environmental effects on plants collected in different locations and in fact, the cancer cell lines studied were not the same as ours. Besides, the antiproliferative potential of F. schwarzii which has not been previously reported was explored in this study, which exhibited appreciable activity against HT-29 cells although it was less selective in the combined treatments. 3. Conclusion It has been found out that F. fistulosa and F. schwarzii are potential sources for cytotoxic alkaloids to combat against brain and colon cancers in future. Furthermore, the combined treatment of F. fistulosa crude alkaloids with d- or g-tocotrienol has paved a new insight into developing a value-added chemotherapeutic approach against brain cancer particularly. Supplementary material Experimental details relating to this paper are available online, alongside Tables S1 – S2, Figures S1 –S3. Acknowledgements The authors would like to thank Dr Kien-Thai Yong (Institute of Biological Sciences, University of Malaya) for identifying the plants and Dr Chee-Wai Fong (Davos Life Science Pte Ltd, Singapore) for providing the d- and g-tocotrienols.

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Notes 1. Email: [email protected] 2. Email: [email protected]


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