Ogbole et al. BMC Complementary and Alternative Medicine (2017) 17:494 DOI 10.1186/s12906-017-2005-8 RESEARCH ARTICLE Open Access In vitro cytotoxic activity of medicinal plants from Nigeria ethnomedicine on Rhabdomyosarcoma cancer cell line and HPLC analysis of active extracts Omonike O. Ogbole1* , Peter A. Segun2 and Adekunle J. Adeniji3 Abstract Background: Cancer is a leading cause of death world-wide, with approximately 17.5 million new cases and 8.7 million cancer related deaths in 2015. The problems of poor selectivity and severe side effects of the available anticancer drugs, have demanded the need for the development of safer and more effective chemotherapeutic agents. The present study was aimed at determining the cytotoxicities of 31 medicinal plants extracts, used in Nigerian ethnomedicine for the treatment of cancer. Methods: The plant extracts were screened for cytotoxicity, using the brine shrimp lethality assay (BSLA) and MTT cytotoxicity assay. Rhabdomyosarcoma (RD) cell line, normal Vero cell line and the normal prostate (PNT2) cell line were used for the MTT assay, while Artemia salina nauplii was used for the BSLA. The phytochemical composition of the active plant extracts was determined by high performance liquid chromatography (HPLC) analysis. Results: The extract of Eluesine indica (L.) Gaertn (Poaceae), with a LC50 value of 76.3 μg/mL, had the highest cytotoxicity on the brine shrimp larvae compared to cyclophosphamide (LC50 =101.3μg/mL). Two plants extracts, Macaranga barteri Mull. Arg. (Euphorbiaceae) and Calliandra portoricensis (Jacq.) Benth (Leguminosae) exhibited significant cytotoxic activity against the RD cell line and had comparable lethal activity on the brine shrimps. Further cytotoxic investigation showed that the dichloromethane fraction of Macaranga barteri (DMB) and the ethyl acetate fraction of Calliandra portoricensis (ECP), exhibited approximately 6-fold and 4-fold activity, respectively, compared to cyclophosphamide on RD cell line. Determination of selective index (SI) using Vero and PNT2 cell line indicated that DMB and ECP displayed a high degree of selectivity against the cancer cell under investigation. HPLC analysis showed that 3,5dicaffeoylquinic acid, acteoside, kampferol-7-O-glucoside and bastadin 11 were the major components of DMB while the major components of ECP were neurolenin B, nigrosporolide and trans-geranic acid. Conclusion: The results demonstrate the cytotoxicity of Macaranga barteri and Calliandra portoricensis extracts, which are used in Nigerian folklore for cancer treatment. Keywords: Nigeria, Ethnomedicine, Cancer, MTT assay, Brine shrimp lethality assay, Macaranga Barteri, Calliandra portoricensis * Correspondence: [email protected] 1Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ogbole et al. BMC Complementary and Alternative Medicine (2017) 17:494 Page 2 of 10 Background [13–15]. Although, the cytotoxicity of some of these Cancer remains a leading cause of death worldwide, with plants have been investigated [16, 17], there is yet to be a approximately 17.5 million new cases and 8.7 million plant from Nigerian ethnomedicine that have produced a cancer related deaths in 2015 [1]. With approximately lead in anticancer drug discovery. 20% of the population of Africa and slightly more than In this framework, data on plants used in the treatment half the population of West Africa, Nigeria contributed of cancer by the TMPs in the Ijebu region of southwestern 11% to the estimated 681,000 new cases of cancer that Nigeria were collected, by personal contact with the TMPs occurred in Africa in 2008 [2]. In Nigeria, the common during an ethnobotanical survey conducted between May – cancer types include cancer of the breast, cervix, prostate, September 2015. From the 90 plants that were recorded colorectal, liver cancer and Non Hodgkin Lymphoma [3]. during the survey, bibliographical survey permitted to retain Rhabdomyosarcoma (RD) is the commonest soft tissue 31 indigenous plants for which cytotoxic activity had never sarcoma in children and adolescents below 20 years. In been evaluated or only partially studied. The present study the US, the incidence of RD is approximately five cases investigates the in vitro cytotoxicity of the methanol extract per million children/adolescents per year, and, in >50% of the medicinal plants and the active fractions against of cases, RD occurs during the first decade of life [4]. Artemia salina nauplii and RD, Vero and PNT2 cell line. In Nigeria, rhabdomyosarcoma occurs frequently in the paediatric population, and it is a common cause of Methods mortality in this age group. The most prevalent variant Plant material is the embryonal rhabdomyosarcoma and the commonest The selected plants were collected between September 2015 anatomical sites are the head and neck regions [5]. Several and May 2016 in their natural habitat (Table 1). The plants chemotherapeutic agents are being used for the manage- were identified and authenticated by comparison with appro- ment of cancer but the problem of selective toxicity and priate voucher specimens at Department of Pharmacognosy, severe side effects still exist. Hence, there is an urgent Herbarium, University of Ibadan (DPHUI) by Mr. P. Agwu need to discover new anticancer drug leads. and the Forest Herbarium Ibadan, Forestry Research Natural products have been used since ancient times for Institute of Nigeria (FRIN), by Mr. T.K. Odewo. the treatment of many diseases. Before the 20th century, 80% of all medicines used to treat human and animal Preparation of crude extracts and fractions illness were obtained from the leaves, barks and roots of Plant parts were air-dried at room temperature and milled medicinal plants. During that period, crude botanicals into coarse powder. For each plant material, 300 g of were percolated in readily available fluids like alcohol and material was macerated in methanol, with intermittent the doctor will prescribe tablespoons of the fluid extract stirring, for 72 h at room temperature. The extracts were to be taken for a period. It is noteworthy to mention that filtered and concentrated to dryness using a rotary about 70% of the drugs used today are models of natural evaporator. Dry extracts were stored at 4 °C until ana- products [6]. Between 1981 and 2010, approximately lysis. To obtain fractions of different polarity from the 700 natural products or natural product derived New active extracts, 20 g of the crude extracts (M. barteri Chemical Entities (NCEs) were approved [7]. Biodiversity and C. portoricensis) were re-dissolved in 250 mL of and traditional medical knowledge had provided useful lead distilled water and partitioned (3×) with an equal volume compounds for cancer chemotherapy, as exemplified in the of n-hexane, dichloromethane and ethyl acetate. Fractions discovery of the vinca alkaloids (vincristine and vinblastine), were concentrated under reduced pressure. taxols (paclitaxel and docetaxel), camptothecin and etoposide [8, 9]. Brine shrimp lethality assay (BSLA) According to the World Health Organization (WHO), The BSLA assay is a bench top assay used for screening 80% of people living in developing nations, which includes natural products for the presence of bioactive compounds. Nigeria, depend on herbal medicine to meet their health The experiment was carried out using the method care needs [10]. Nigeria is a country blessed with a vast described by Mclaughlin [18]. Brine shrimp eggs were flora of medicinal importance and the therapeutic potency obtained from the Department of Pharmacognosy, of such plants have been demonstrated by several University of Ibadan. Briefly, Artemia salina cysts (brine researchers [11, 12]. In Nigeria, many people especially shrimp eggs 0.1 g) were allowed to hatch in natural sea those residing in the rural areas, solely depend on the water, containing 3.8 g/L salt, obtained from Bar beach, traditional medical practitioners (TMPs) for the diagno- Ikoyi, Lagos. The larvae (nauplii) were placed in sea water sis and treatment of many diseases including cancer. for 48 h at 25 °C under constant aeration and illumination Interestingly, several studies have documented the indi- to ensure survival and maturity before use. Stock solutions genous knowledge of the TMPs in the treatment of (10 mg/mL) of plant extracts were made and diluted seri- various cancers across the geopolitical zones of Nigeria ally in clean test tubes of 10 mL volume to obtain five final Ogbole et al. BMC Complementary and Alternative Medicine (2017) 17:494 Page 3 of 10 Table 1 Plant species analysed for cytotoxicity S/N Plant Family Local name Part used Voucher number 1 Acanthospermum hispidum D.C. Asteraceae Dagunro gogoro Aerial part FHI 110050 2 Alchornea laxiflora (Benth.) Pax & K.Hoffm. Euphorbiaceae Pepe leaf FHI 110155 3 Boerhavia diffusa