Cytotoxicity of South-African medicinal plants towards sensitive and multidrug-resistant cancer cells

Author’s Accepted Manuscript Cytotoxicity of South-African medicinal plants towards sensitive and multidrug-resistant cancer cells Mohamed E.M. Saeed, Marion Meyer, Ahmed Hussein, Thomas Efferth www.elsevier.com/locate/jep

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S0378-8741(16)30195-7 http://dx.doi.org/10.1016/j.jep.2016.04.005 JEP10082

To appear in: Journal of Ethnopharmacology Received date: 19 January 2016 Revised date: 3 April 2016 Accepted date: 4 April 2016 Cite this article as: Mohamed E.M. Saeed, Marion Meyer, Ahmed Hussein and Thomas Efferth, Cytotoxicity of South-African medicinal plants towards sensitive and multidrug-resistant cancer cells, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cytotoxicity of South-African medicinal plants towards sensitive and multidrug-resistant cancer cells Mohamed E. M. Saeed1, Marion Meyer2, Ahmed Hussein3, Thomas Efferth1* 1

Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes

Gutenberg University, Mainz, Germany. 2 3

Plant Science Department, University of Pretoria, 002 Pretoria, South Africa Chemistry Department, University of Western Cape, Private Bag X17, Belleville 7535, South

Africa *Correspondence address: Prof. Dr. Thomas Efferth, Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany. Telephone: +49-6131-3925751. Fax: 49-6131-23752. E-mail: [email protected]

Running title: Activity of South-African medicinal plants towards multidrug-resistant cancer cells Key words: ABC transporters, Chemotherapy, Multidrug resistance, Natural products, Pharmacogenomics, Phytotherapy

Abbreviations: ABC, ATP-binding cassette IC50, 50% inhibition concentration MDR, multidrug resistance NCI, National Cancer Institute P-gp, P-glycoprotein

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Abstract Ethnopharmacological relevance: Traditional medicine plays a major role for primary health care worldwide. Cancer belongs to the leading disease burden in industrialized and developing countries. Successful cancer therapy is hampered by the development of resistance towards established anticancer drugs. Aim: In the present study, we investigated the cytotoxicity of 29 extracts from 26 medicinal plants of South-Africa against leukemia cell lines, most of which are used traditionally to treat cancer and related symptoms. Material and Methods: We have investigated the plant extracts for their cytotoxic activity towards drug-sensitive parental CCRF-CEM leukemia cells and their multidrug-resistant Pglycoprotein-overexpressing subline, CEM/ADR5000 by means of the resazurin assay. A panel of 60 NCI tumor cell lines have been investigated for correlations between selected phytochemicals from medicinal plants and the expression of resistance-conferring genes (ABC-transporters, oncogenes, tumor suppressor genes). Results: Seven extracts inhibited both cell lines (Acokanthera oppositifolia, Hypoestes aristata, Laurus nobilis, Leonotis leonurus, Plectranthus barbatus, Plectranthus ciliates, Salvia apiana). CEM/ADR5000 cells exhibited a low degree of cross-resistance (3.35-fold) towards the L. leonurus extract, while no cross-resistance was observed to other plant extracts, although CEM/ADR5000 cells were highly resistant to clinically established drugs. The log10IC50 values for two out of 14 selected phytochemicals from these plants (acovenoside A and ouabain) of 60 tumor cell lines were correlated to the expression of ABCtransporters (ABCB1, ABCB5, ABCC1, ABCG2), oncogenes (EGFR, RAS) and tumor suppressors (TP53). Sensitivity or resistance of the cell lines were not statistically associated with the expression of these genes, indicating that multidrug-resistant, refractory tumors expressing these genes may still respond to acovenoside A and ouabain. Conclusion: The bioactivity of South African medicinal plants may represent a basis for the development of strategies to treat multidrug-resistant tumors either by phytotherapeutic approaches with whole plant preparations or by classical drug development with isolated compounds such as acovenoside A or ouabain.

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Introduction One major obstacle in cancer management with established anticancer agents is drug resistance (Mukamoto et al., 2013). The phenomenon of multidrug resistance (MDR) became a point of interest in cancer research after the discovery of P-glycoprotein, which exhibits not only resistance to one but to many drugs (Juliano and Ling, 1976). This phenomenon is defined as cross-resistance of cancer cells to the cytostatic and cytotoxic actions of many anticancer drugs, which are structurally or functionally unrelated and which have different molecular targets (Nakamura et al., 2013). Overexpression of ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp) has been shown to be responsible for MDR in clinical tumors (Efferth and Osieka, 1993). Interestingly, between the years 1981-2006 about a hundred anticancer agents have been developed, and the majority are either pure natural products, natural product derivatives or synthetic compounds with pharmacophores mimicking natural products. Thus, natural products can be considered as a promising source of novel anticancer drugs to combat MDR (Newman and Cragg, 2007). South Africa has a rich flora, including a plethora of medicinal plants, which have not been investigated in detail (Hutchings et al., 1996; Van Wyk et al., 1997; Watt and Breyer-Brandwijk, 1962), although some South-African medicinal plants are known to exhibit cytotoxic activity against breast cancer and leukemia cell lines (Chang et al., 2001; Steenkamp and Gouws, 2006). Here, we performed a study on 26 South-African medicinal plants, which have a rich tradition in the practice of healers for a wide range of diseases and symptoms (Cordell et al., 1991). We tested extracts of these plants for their cytotoxicity towards sensitive and multidrug-resistant, P-gp-overexpressing leukemia cell lines. Interestingly, extracts from seven herbs revealed high cytotoxicity and these seven plants have a documented use in traditional medicine for cancer or cancer-related symptoms. Cancer is frequently not exactly defined in traditional African medicine. Therefore, symptoms and activities related to anticancer and cytotoxic activity should be taken into account to describe the ethnopharmacological use in an appropriate manner. Leonotis leonurus has many traditional applications (Mazimba, 2015). The hottentots used leaf decoctions as purgative and emmenagogue. Colonial settlers employed it against leprosy. Other applications include among many others, influenza, bronchitis, wound healing, asthma tuberculosis, jaundice, muscular cramps, high blood pressure, diabetes, viral hepatitis, 3

dysentery, diarrhea, gynecological disorders, cardiovascular disorders, and epilepsy (Bienvenu et al., 2002; Kuchta et al., 2012; Mazimba, 2015; Scott G et al., 2004). Tea made from the whole plant is used against cancer, arthritis, piles, bladder and kidney disorder, obesity, and rheumatism (Thring and Weitz, 2006). Generally the plant is a general tonic, having reputed dermatological, hypertension, anti-inflammatory, pain and wound healing properties. Plectranthus barbatus has been traditionally used for a wide range of complaints, e.g. stomach ache and as purgative drug (Abdel-Mogib et al., 2002), against hypertension, congestive heart failure, eczema, colic, respiratory disorders, painful urination, insomnia, convulsions, and cancer prevention (Mwitari et al., 2013). In traditional medicine, several species of this genus are used to treat ailments of the digestive tract, respiratory system, nervous system, inflammation, infections, pain and different types of cancers (Lukhoba et al., 2006). The cytotoxic activity of P. barbatus and P. ciliates may be also supported by the contraceptive properties (Yashaswini and Vasundhara, 2011). Hypoestes aristata has been used against eye sores (Hulme, 1954) breast diseases, respiratory infections and malaria (Iwu, 1993; Kokwaro, 1976). According to healers of the Xhosa tribe, the plant is also used to treat complicated and major diseases like cancer, arthritis, tuberculosis, bone fractures etc. (Bhat, 2014). Acokanthera oppositifolia (Vhavenda name: mutsilili; English name: Common poison bush) is traditionally used as highly effective arrow poison in South-Africa (Steyn, 1934; Van Wyk et al., 2002), which indicates his cytotoxic properties. It is also used as anthelminthic against snake-bites and gynecological disorders (Adedapo et al., 2008; Bisi-Johnson MA et al., 2010; Fouche et al., 2008; Steenkamp, 2003). Applied as powdered snuff, it has been traditionally used to treat headaches and as infusions for abdominal pains and convulsions and septicemia (Bhat and Jacobs, 1995; Dold and Cocks, 2001; Watt and Breyer-Brandwijk, 1962). Salvia apiana has been traditionally used for respiratory tract infections and acute vaginitis caused by Candida infection (Foster and Hobbs, 2002; Moore, 1993). It is recommended by herbalists to treat prostate hyperplasia (ttp://oneearthherbs.squarespace.com/diseases/benignprostatic-hyperplasia-bph.html). Laurus nobilis is used among other applications against skin diseases and cancer in the Middle East (Said et al., 2002). This plant is also used in South-Africa. We exemplarily selected two compounds from one of the cytotoxic plants, acovenoside A and ouabain and correlated their 50% inhibition concentrations (IC50) values in a panel of 60 4

tumor cell lines of the National Cancer Institute (NCI, USA) with the expression of genes that are known to confer MDR, i.e. the ABC-transporters ABCB1/MDR1, ABCB5, ABCC1/MRP, ABCG2/BRCP, the oncogene EGFR, and the tumor suppressor gene TP53. Furthermore, we correlated the response of acovenoside A and ouabain with 85 standard anticancer drugs to identify cross-resistance profiles of these two natural compounds with established drugs. The present study may serve as a starting point to further characterize phytochemicals from these plants with the potential to kill MDR cells.

Material and Methods Plant material. Twenty-six plant species were collected from Pretoria (South-Africa) during September 2011 and identified at the H.G.W.J. Schweickerdt Herbarium (PRU) at the University of Pretoria. Traditional uses of the plants against cancer and their voucher numbers were shown in Table 1 and Table 2 respectively. The plant name has been verified with www.theplantlist.org. The phytochemicals of these cytotoxic plants identified in the literature are given in Table 3. Extract preparation. Twenty-nine extracts have been prepared from 26 plant species in this study. Plant material was subjected to the following extraction protocol separately; ~50 g fresh leaves (unless otherwise stated) were homogenized with 150 mL methanol (Merck, Germany). After filtration, the plant material was washed twice with fresh solvent, the combined extract was concentrated under reduced pressure using a vacuum rotary evaporator (Buchi Labortechnik, Flawil, Switzerland). The solvent free extracts were kept at 4°C for further biological evaluation. In case of resin samples, ~ 2.0 g have been extracted. Cell lines. Human drug sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells have been generated as described (Kimmig et al., 1990). The panel of 60 human tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) consisted of leukemia, melanoma, non-small cell lung cancer, colon cancer, renal cancer, ovarian cancer, breast cancer, and prostate carcinoma cells as well as tumor cells of the central nervous system (Alley et al., 1988). Cytotoxicity assay. The resazurin reduction assay is based on reduction of the indicator dye, resazurin, to the highly fluorescent resorufin by viable cells (O'Brien et al., 2000). The protocol used in the present investigation has been previously reported (Kuete et al., 2012). 5

Cells of the National Cancer Institute (NCI, USA) cell line panel were assayed by means of a sulforhodamine B assay (Rubinstein et al., 1990).

Statistical analysis. mRNA microarray data and log10IC50 values of the NCI tumor cell line panel are available (Scherf et al., 2000; Staunton et al., 2001) through the NCI website (http://dtp.nci.nih.gov). Pearson’s correlation test was used to calculate significance values and rank correlation coefficients as a relative measure of the linear dependency of two variables (WinSTAT, Kalmia).

Results Screening of medicinal plants from South-Africa. Screening of 29 extracts from 26 plants at a fixed concentration (10 µg/mL) revealed that seven extracts showed inhibitory activities towards CCRF-CEM and CEM/ADR5000 leukemia cells, i.e. Acokanthera oppositifolia, Salvia apiana, Laurus nobilis, Plectranthus ciliatus, P. barbatus, Hypoestes aristata, Leonotis leonurus. The results are depicted in Figure 1. All active extracts showed growth inhibition of less than 40% except of the Leonotis leonurus extract, which exerted stronger inhibitory effects in multidrug-resistant cells. Dose response curves of active plants. Next, we performed dose response curves in a dose range from 0.1 to 30 µg/mL for all seven active extracts, which were active in the first screening (Figure 1). As shown in Figure 2 and Table 4, the most active extract was Plectranthus ciliatus, which had the lowest IC50 value in sensitive and MDR leukemia cells (1.57±0.09 and 2.3±0.06 µg/mL, respectively). Considerable cytotoxicity towards both cell lines were also observed for Acokanthera oppositifolia (IC50 value: 2.5±0.06 and 2.85±0.08 µg/mL, respectively). The degrees of resistance of sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 were calculated by dividing the IC50 value of the resistant cell line by the corresponding parental sensitive cell line. Interestingly multidrug-resistant leukemia cells showed low levels of cross-resistance towards only one extract (Leonotis leonurus), while the other extracts inhibited sensitive and MDRcells with similar efficacies (Table 4), taking into consideration a two-fold resistance as cutoff value as previously considered (Hall et al., 2009).

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Cytotoxicity of Phytochemicals derived from South-African Medicinal Plants. As a next step, we mined the NCl database for the phytochemicals of the plants as shown in Table 4. Fourteen compounds were identified. The average log10IC50 values of these compounds over the entire NCI panel of 60 cell lines of different tumor types is shown in Figure 3A. The cardiotonic steroids acovenoside A and ouabain were the most cytotoxic phytochemicals. Then, the average log10IC50 values for these two phytochemicals were diversified regarding their tumor type (Figures 3B and C). Renal and lung cancer cell lines were the most sensitive towards both compounds. On the other hand, colon and breast tumor cells were most resistant to acovenoside A and melanoma and colon cancer cell lines to ouabain. Cell lines of all other tumor types were of intermediate responsiveness to these substances. Cross-Resistance Profiles. The log10IC50 values of the NCl cell line panel for acovenoside A and ouabain were compared to those of 85 standard anticancer drugs (Figure 4). First, a significant relationship between both glycosides, acovenoside A and ouabain was found. (R=0.629; P=6.02×10-8). As shown in Figure 4A, the log10IC50 values of acovenoside A significantly correlated with those of 1/3 platin compounds and 1/3 camptothecines, respectively (=33%), as well as 1/4 antibiotics (=25%). Similar results were found for ouabain (Figure 4B). In addition, cross-resistance of 1/4 mTOR inhibitors and 2/14 tyrosine kinase inhibitors (=14%) to ouabain was observed. Cross-resistance to other compounds were not or only marginally visible, indicating that tumor cells resistant to anticancer drugs of these classes were still responsive to acovenoside A and ouabain. For comparison, we determined the cross-resistance profile of doxorubicin as control drug. Figure 4C demonstrates that the cell lines exhibited considerable cross-resistances between doxorubicin and several other drug classes. These data indicate that the cardiotonic steroids acovenoside A and ouabain may still be active, if many established anticancer drugs cannot kill the tumor due to broad spectrum cross-resistances.

Classical mechanisms of drug resistance. To investigate underlying mechanisms of drug resistance, we correlated the log10IC50 values of these two cardiotonic steroids for the NCI cell line panel with different parameters of the ATP-binding cassette (ABC) efflux transporter Pgp/ABCB1/MDR1. We analyzed microarray- or RT-PCR-based mRNA expression of the ABCB1 gene as well as gain of DNA at the chromosomal locus 7p21 (were the ABCB1 gene is located) and the intracellular rhodamine 123 (R123) accumulation rates. R123 is a P-gp substrate and the flow cytometric determination of R123 uptake can serve as functional assay for P-gp activity (Efferth et al., 1989; Tapiero et al., 1984). As shown in Table 5, these 7

parameters did not significantly correlate with the log10IC50 values for acovenoside A or ouabain, indicating that their cellular sensitivity was not related to this ABC-transporter. For comparison, the established anti-cancer drug daunorubicin was used as positive control (Kadioglu and Efferth, 2015). Daunorubicin is a well-known substrate of P-glycoprotein. As expected, the log10IC50 values for daunorubicin significantly correlated with all ABCB1 parameters (Table 5). The microarray data for ABCB1 mRNA expression have been validated by correlation of these mRNA expressions with those of mRNA expression values obtained by RT-PCR. As expected, a statistically significant relationship has been found (Table 5). Similarly, significant relationships have been observed between microarray-based mRNA expression to gain of DNA at chromosomal locus 7q21 and cellular R123 accumulation (Table 5) (Kadioglu and Efferth, 2015). In addition to ABCB1, the correlation of log10IC50 of the two cardiotonic drugs to other ABC-transporters have been analyzed. Both compounds did not significantly correlate to ABCB5 or only with low correlation coefficient (R<0.3) (Table 6). While both compounds did not correlate with expression of BCRP/ABCG2 (Table 8), they did not or inversely correlate with MRP1/ABCC1 expression (Table 7), which does not imply a role of these compounds as substrate of MRP1/ABCC1. By contrast, control drugs known from the literature to be substrates of these transporters revealed significant correlations as can be expected (Tables 5-8). Again, microarray-based mRNA expressions of these ABCtransporters in the cell line panel correlated with RT-PCR-based mRNA expression values, indicating the correctness of microarray hybridizations (Tables 5-8) (Kadioglu and Efferth, 2015). Taken together, these results indicate that neither acovenoside A nor ouabain are substrates of these four MDR-mediating ABC-transporters and these drug efflux transporters do not confer to these two substances. Pleiotropic or broad spectrum drug resistance phenomena are not only known to be caused by ABC-transporters. Oncogenes and tumor suppressor genes also confer drug resistance in addition to their role in carcinogenesis (el-Deiry, 1997, 2003; Shetzer et al., 2014). Therefore, we investigated, whether or not EGFR and RAS oncogenes and the tumor suppressor gene TP53 influence cellular response to ursolic and pomolic acids. As can be seen in Tables 9-10, over-expression or mutation of these genes were not clearly associated with the responsiveness of the cell lines to these two cardiotonic steroids. For comparison, significant

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correlations have been observed for standard anticancer agents, i.e. erlotinib (for EGFR), cisplatin (for TP53), and melphalan (for RAS) (Kadioglu and Efferth, 2015).

Discussion In the present investigation, we found an overall hit rate of 7 out of 29 cytotoxic extracts (=24.1%). In the past, success rates of 10-20% have been reported for plant extracts derived from traditional Chinese medicine (Campbell et al., 2002; Efferth et al., 2008a). According to guidelines of the Developmental Therapeutics Program of the NCI on cytotoxic activity, crude extracts may be considered as being active, if they show IC50 at concentrations below 30 μg/mL after an exposure time of 72 h (Suffness and Pezzuto, 1990). All seven active extracts are below this range, indicating considerable cytotoxicity towards sensitive and MDR leukemia cells. CEM/ADR5000 cells revealed low levels of cross-resistance towards the Leonotis leonurus extract, while no cross-resistance was observed towards six further plant extracts. This is a remarkable result, since CEM/ADR5000 cells exhibit very high degrees of resistance to many standard anticancer agents, including anthracyclines, taxanes, Vinca alkaloids, and epipodophyllotoxins (Efferth et al., 2008b). This indicates that these plant extracts contain phytochemicals, which are not recognized by P-gp as substrates and are thus not expelled out of MDR cells. Hence, these plants or phytochemicals isolated from them may be useful to kill MDR tumors with similar efficacy as sensitive tumors. This is a striking result, since it opens the possibility that phytotherapeutic approaches with the whole plants or isolated phytochemicals from these plants may be suitable to treat drug-resistant tumors. Clinical resistance frequently results in fatal outcome of the disease, since tumor growth cannot be stopped with drugs anymore. Hence, drugs which are still active in progressive cancers are highly valuable. Of the seven plants, whose extracts were cytotoxic in our investigation, Bay leaf (Laurus nobilis) is best described in the literature for its cytotoxic activity towards cancer cell lines in vitro. Extracts of L. nobilis were cytotoxic to various cell lines derived from different tumor types, including breast cancer, brain tumors, colon tumors, leukemia (Al-Kalaldeh et al., 2010; Bennett et al., 2013; Komiya et al., 2004; Pacifico et al., 2013). Essentials oils as well as costunolide dehydrocostuslactone and zaluzanin D from L. nobilis were reported to be 9

cytotoxic against cell lines of various tumor types (Hibasami et al., 2003; Lin et al., 2015; Loizzo et al., 2007; Moteki et al., 2002). Recently, we reported that MDR cells were hypersensitive (collaterally sensitive) toward an extract of L. nobilis compared to their parental drug-sensitive cells (Saab et al., 2015). Dehydrocostus lactone, naturally occurring in L. nobilis, has been reported to down-regulate the expression of ABCB1/MDR1 and ABCG2/BCRP in liposarcoma and synovial sarcoma cells (Kretschmer et al., 2012). Lauroside B, a megastigmane glycoside isolated from L. nobilis, induced apoptosis in human melanoma cell lines by inhibiting NF-κB activation (Panza et al., 2011). Aqueous fractions of L. nobilis induced G1/S phase arrest, generation of reactive oxygen species, DNA double strand breaks, p53-dependent apoptosis (Rodd et al., 2015). Much less is known about the cytotoxicity of the other plants. Acokanthera oppositifolia revealed growth-inhibitory activity towards TK10 renal cancer cells, MCF-7 breast and UACC62 melanoma cells (Fouche et al., 2008). Leonotis leonurus has not been investigated for its activity towards cancer cells as yet, but another spies of this genus, Leonurus japonicus has been intensively been investigated, because it is a well-known plant in traditional Chinese medicine. Approximately 140 chemical compounds have been isolated from L. japonicus. Among them, leonurine and stachydrine are the most widely investigated ones. L. japonicus possesses various pharmacological activities, including effects on the uterus as well as cardioprotective, anti-oxidative, neuroprotective and cytotoxic activities against cancer cells (Shang et al., 2014). Plectranthus barbatus (formerly Coleus forskohlii) contains forskolin, which inhibited lung metastasis of highly metastasizing B16 (F10) murine melanoma cells (Agarwal and Parks, 1983). Another constituent of this plant is coleusin factor, which inhibited osteosarcoma growth by inducing bone morphogenetic protein-2-dependent differentiation (Geng et al., 2014). While acovenoside A from Acokanthera oppositifolia has not been reported as of yet to exert cytotoxic effects towards cancer cells, this is well known for ouabain. Both compounds are cardiotonic steroids. This class of compounds has long been used for the treatment of congestive heart failure and as anti-arrhythmic agents. Ouabain and related compounds inhibit Na+/K+-ATPase leading to strong inotropic effects on the heart (Riganti et al., 2011). Interestingly, cardiotonic steroids may also play a role in the prevention and treatment of 10

cancer. Na+/K+-ATPase it is a key player of cell adhesion and is involved in cancer progression (Chen et al., 2006). Furthermore, Na+/K+-ATPase is not the only target of ouabain and other cardiotonic steroids, as they affect the cell response to hypoxia, modulate several signaling pathways involved in cell death and proliferation. Cardiotonic steroids also inhibit the efflux function of P-glycoprotein and overcome MDR of tumor cells (Zeino et al., 2015a; Zeino et al., 2015b). Their role as novel candidates for cancer therapy deserves more detailed exploration (Newman et al., 2008; Weidemann, 2005). The correlation analysis of log10IC50 values for acovenoside A and ouabain did not reveal any statistically significant associations between cellular responsiveness to the two cardiotonic glycosides and ABCB1 expression and function. The same was true for ABCB5. This is an interesting result as it implies that these compounds may kill otherwise MDR tumors with similar efficacies as drug-sensitive ones. Despite this result is pleasing there is a caveat. Broulliard et al. (2001) reported that ouabain treatment led to the induction of MDR1 expression (Brouillard et al., 2001). It is possible that lethal concentration kill sensitive and MDR tumors with similar efficacies as found by us, but that sub-lethal ouabain concentration rather induce than reduce MDR. This aspect should be investigated in more detail in the future. Furthermore, an association between high ABCC1 expression and resistance to the two glycosides could not be established in the NCI cell line panel, indicating no major role of this ABC-transporter for the unresponsiveness of tumor cells to acovenoside A and ouabain. The literature data are contradictory concerning MRP1 and ouabain. Cells with acquired ouabain resistance overexpressed ABCC1 (Capella et al., 2001). On the other hand, ouabain has been reported to downregulate ABCC1 expression (Valente et al., 2007). Comparable to ABCB1, concentration-dependent effects may play a role whether ABCC1 expression is induced or repressed. Again, this issue has to be clarified in the future. Comparable to the other ABC-transporters, the expression of ABCG2 did also not correlate with the log10IC50 values of acovenoside A or ouabain. Rao et al. (2014) presented an interesting approach that was different from bypassing MDR by non-cross-resistant drugs such as acovenoside A and ouabain (Rao et al., 2014). These authors used a combination treatment of ABCG2-expressing cells with gramicidin D and ouabain, which leads to an increased ATP consumption by this ABC transporter. As a consequence, the intracellular ATP pools of ABCG2-expressing cells were depleted leading to preferential killing and collateral sensitivity compared to non-transfectant parental cells.

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In addition to ABC transporters, oncogenes and tumor suppressor genes also contribute to pleiotropic resistance phenotypes. We have analyzed the relationship of expression or mutational status of EGFR, RAS and TP53 to sensitivity or resistance of the panel of 60 NCI cell lines to acovenoside A and ouabain and did not find consistent correlations, which speak for a role of these three genes for the activity of the two cardiotonic steroids towards tumor cells. This result reflects the published data in the literature. Ouabain has been reported to inhibit EGFR downstream signaling cascades related to RAS, SRC, ERK1/2 and MAPK in some cell lines, whereas it activated them in others (Kometiani et al., 1998; Wolle et al., 2014). The therapeutic implications of these contradictory results have to be further explored. Tumor cells with mutated TP53 are frequently resistant to established anticancer drugs. Therefore, it was pleasing that the activity of acovenoside A and ouabain was not related to the mutational status of this tumor suppressor genes. Wang et al. (2009) reported that ouabain reduced expression of both wild-type and mutant p53 by downregulation of p53 protein expression rather than by p53 protein degradation or down-regulation of mRNA transcription (Wang et al., 2009).

In conclusion, the results of the present investigation demonstrate that South-African plants may be a useful resource for cancer drug development. Future studies should focus on the isolation of bioactive phytochemicals and the elucidation of their molecular modes of action in cancer cells.

Acknowledgement: We declare that there is no conflict of interest. We thank the Sudanese government (National Research Council) for a stipend to M.E.M.S. Conflict of Interest: There is no conflict of interest. Authors’ contribution: M.E.M.S carried out the bioassay experiments, extracted the microarray data and wrote the first draft of the manuscript; M.M and A.H. collected, identified and prepared the extraction of plant materials; T.E. supervised the work, performed the statistical analysis, provided the facilities for the study, and edited the manuscript. All authors read the manuscript and approved the final version.

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References Abdel-Mogib, M., Albar, H.A., Batterjee, S.M., 2002. Chemistry of the Genus Plectranthus. Molecules 7, 271-301. Adedapo, A.A., Jimoh, F.O., Afolayan, A.J., Masika, P.J., 2008. Antioxidant activities and phenolic contents of the methanol extracts of the stems of Acokanthera oppositifolia and Adenia gummifera. BMC Complement Altern Med 8, 54. Agarwal, K.C., Parks, R.E., Jr., 1983. Forskolin: a potential antimetastatic agent. Int J Cancer 32, 801804. Agnihotri, V.K., ElSohly, H.N., Khan, S.I., Smillie, T.J., Khan, I.A., Walker, L.A., 2009. Constituents of Leonotis leonurus Flowering Tops. . Phytochemistry Letters 2, 103–105. Al-Kalaldeh, J.Z., Abu-Dahab, R., Afifi, F.U., 2010. Volatile oil composition and antiproliferative activity of Laurus nobilis, Origanum syriacum, Origanum vulgare, and Salvia triloba against human breast adenocarcinoma cells. Nutr Res 30, 271-278. Al-Rehaily, A.J., Al-Yahya, M.A., Mirza, H.H., Ahmed, B., 2002. Verticillarone: a new seco-fusicoccane diterpenoid ketonepoxide from Hypoestes verticillaris. J Asian Nat Prod Res 4, 117-122. Alipour, G., Dashti, S., Hosseinzadeh, H., 2014. Review of pharmacological effects of Myrtus communis L. and its active constituents. Phytotherapy research : PTR 28, 1125-1136. Alley, M.C., Scudiero, D.A., Monks, A., Hursey, M.L., Czerwinski, M.J., Fine, D.L., Abbott, B.J., Mayo, J.G., Shoemaker, R.H., Boyd, M.R., 1988. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res 48, 589-601. Arora, S., Singh, S., Piazza, G.A., Contreras, C.M., Panyam, J., Singh, A.P., 2012. Honokiol: a novel natural agent for cancer prevention and therapy. Current molecular medicine 12, 1244-1252. Ayensu, E.S., 1981. Medicinal plants of the West Indies. Reference Publications, Algonac, Mich. Bagetti, M., Facco, E.M.P., Piccolo, J., Hirsch, G.E., Rodriguez-Amaya, D., Kobori, C.N., Vizzotto, M., Emanuelli, T., 2011. Physicochemical characterization and antioxidant capacity of pitanga fruits (Eugenia uniflora L.). Ciencia Tecnol Alime 31, 147-154. Barla A, Topcu G, Oksuz S, Tumen G, DGI., K., 2007. Identification of cytotoxic sesquiterpenes from Laurus nobilis L. Food Chem 104, 1478-1484. Bendaoud, H., Romdhane, M., Souchard, J.P., Cazaux, S., Bouajila, J., 2010. Chemical composition and anticancer and antioxidant activities of Schinus molle L. and Schinus terebinthifolius Raddi berries essential oils. Journal of food science 75, C466-472. Bennett, L., Abeywardena, M., Burnard, S., Forsyth, S., Head, R., King, K., Patten, G., Watkins, P., Williams, R., Zabaras, D., Lockett, T., 2013. Molecular size fractions of bay leaf (Laurus nobilis) exhibit differentiated regulation of colorectal cancer cell growth in vitro. Nutr Cancer 65, 746-764. Bhat, R.B., 2014. Medicinal plants and traditional practices of Xhosa people in the Transkei region of Eastern Cape, South Africa. Indian Journal of Traditional Knowledge 13, 292-298. Bhat, R.B., Jacobs, T.V., 1995. Traditional herbal medicine in Transkei. Journal of ethnopharmacology 48, 7-12. Bienvenu, E., Amabeoku, G.J., Eagles, P.K., Scott, G., Springfield, E.P., 2002. Anticonvulsant activity of aqueous extract of Leonotis leonurus. Phytomedicine 9, 217-223. Biggs, M.L., Sorkin, B.C., Nahin, R.L., Kuller, L.H., Fitzpatrick, A.L., 2010. Ginkgo biloba and risk of cancer: secondary analysis of the Ginkgo Evaluation of Memory (GEM) Study. Pharmacoepidemiology and drug safety 19, 694-698. Bisi-Johnson MA, Obi CL, Kambizi L, M., N., 2010. A survey of indigenous herbal diarrhoeal remedies of O.R. Tambo district, eastern Cape Province, South Africa. African Journal of Biotechnology 9, 12451254. Bode, M., Vleggaar, R., Gates, P.J., Schultz, R.A., Naicker, D., Fourie, N., 2009. New compounds for the treatment of cancer, in: Wipo (Ed.), http://www.google.com/patents/WO2009147632A2?cl=en, US. 13

Bozorgi, M., Memariani, Z., Mobli, M., Salehi Surmaghi, M.H., Shams-Ardekani, M.R., Rahimi, R., 2013. Five Pistacia species (P. vera, P. atlantica, P. terebinthus, P. khinjuk, and P. lentiscus): a review of their traditional uses, phytochemistry, and pharmacology. TheScientificWorldJournal 2013, 219815. Breuninger, L.M., Paul, S., Gaughan, K., Miki, T., Chan, A., Aaronson, S.A., Kruh, G.D., 1995. Expression of multidrug resistance-associated protein in NIH/3T3 cells confers multidrug resistance associated with increased drug efflux and altered intracellular drug distribution. Cancer Res 55, 5342-5347. Brouillard, F., Tondelier, D., Edelman, A., Baudouin-Legros, M., 2001. Drug resistance induced by ouabain via the stimulation of MDR1 gene expression in human carcinomatous pulmonary cells. Cancer Res 61, 1693-1698. Bulgaru, A.M., Mani, S., Goel, S., Perez-Soler, R., 2003. Erlotinib (Tarceva): a promising drug targeting epidermal growth factor receptor tyrosine kinase. Expert Rev Anticancer Ther 3, 269-279. Campbell, M.J., Hamilton, B., Shoemaker, M., Tagliaferri, M., Cohen, I., Tripathy, D., 2002. Antiproliferative activity of Chinese medicinal herbs on breast cancer cells in vitro. Anticancer research 22, 3843-3852. Campbell, W.E., Gammon, D.W., Smith, P., Abrahams, M., Purves, T.D., 1997. Composition and antimalarial activity in vitro of the essential oil of Tetradenia riparia. Planta medica 63, 270-272. Capella, L.S., Gefe, M., Silva, E.F., Morales, M.M., Affonso-Mitidieri, O., Lopes, A.G., Rumjanek, V.M., Capella, M.A., 2001. Reduced glutathione protect cells from ouabain toxicity. Biochim Biophys Acta 1526, 293-300. Chan, P.C., Xia, Q., Fu, P.P., 2007. Ginkgo biloba leave extract: biological, medicinal, and toxicological effects. Journal of environmental science and health. Part C, Environmental carcinogenesis & ecotoxicology reviews 25, 211-244. Chang, J.S., Chiang, L.C., Chen, C.C., Liu, L.T., Wang, K.C., Lin, C.C., 2001. Antileukemic activity of Bidens pilosa L. var. minor (Blume) Sherff and Houttuynia cordata Thunb. Am J Chin Med 29, 303312. Chen, H., Xie, C., Wang, H., Jin, D.Q., Li, S., Wang, M., Ren, Q., Xu, J., Ohizumi, Y., Guo, Y., 2014. Sesquiterpenes inhibiting the microglial activation from Laurus nobilis. J Agric Food Chem 62, 47844788. Chen, J.Q., Contreras, R.G., Wang, R., Fernandez, S.V., Shoshani, L., Russo, I.H., Cereijido, M., Russo, J., 2006. Sodium/potassium ATPase (Na+, K+-ATPase) and ouabain/related cardiac glycosides: A new paradigm for development of anti- breast cancer drugs? Breast Cancer Res Treat 96, 1-15. Cole, S.P., Sparks, K.E., Fraser, K., Loe, D.W., Grant, C.E., Wilson, G.M., Deeley, R.G., 1994. Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells. Cancer Research 54, 5902-5910. Cordell, G.A., Beecher, C.W., Pezzuto, J.M., 1991. Can ethnopharmacology contribute to the development of new anticancer drugs? Journal of ethnopharmacology 32, 117-133. da Silva, G., Serrano, R., Silva, O., 2011. Maytenus heterophylla and Maytenus senegalensis, two traditional herbal medicines. Journal of natural science, biology, and medicine 2, 59-65. Dall'Acqua, S., Viola, G., Giorgetti, M., Loi, M.C., Innocenti, G., 2006. Two new sesquiterpene lactones from the leaves of Laurus nobilis. Chem Pharm Bull (Tokyo) 54, 1187-1189. Dalton, W.S., Grogan, T.M., Miller, T.P., 1991. The role of P-glycoprotein in drug-resistant hematologic malignancies. Cancer Treat Res 57, 187-208. de Albuquerque, R., L,, , Kentopff, M.R., Machado, M.I.L., Silva, M.G.V., Matos, F.J.d.A., Morais, S.M., Braz-Filho, R., 2007. Abietane diterpenoids isolated from Plectranthus barbatus Andrews. Quim Nova 30, 1882–1886. De Marino, S., Borbone, N., Zollo, F., Ianaro, A., Di Meglio, P., Iorizzi, M., 2004. Megastigmane and phenolic components from Laurus nobilis L. leaves and their inhibitory effects on nitric oxide production. J Agric Food Chem 52, 7525-7531. de Souza, N.J., Dohadwalla, A.N., Reden, J., 1983. Forskolin: a labdane diterpenoid with antihypertensive, positive inotropic, platelet aggregation inhibitory, and adenylate cyclase activating properties. Med Res Rev 3, 201-219. 14

Deeken, J.F., Robey, R.W., Shukla, S., Steadman, K., Chakraborty, A.R., Poonkuzhali, B., Schuetz, E.G., Holbeck, S., Ambudkar, S.V., Bates, S.E., 2009. Identification of compounds that correlate with ABCG2 transporter function in the National Cancer Institute Anticancer Drug Screen. Mol Pharmacol 76, 946956. DeFeudis, F.V., Papadopoulos, V., Drieu, K., 2003. Ginkgo biloba extracts and cancer: a research area in its infancy. Fundamental & clinical pharmacology 17, 405-417. Dentali, S.J., Hoffmann, J.J., 1990. 16-Hydroxycarnosic acid, a diterpene from Salvia apiana. Phytochemistry 29, 993-994. Dentali, S.J., Hoffmann, J.J., 1992. Potential antiinfective agents from Eriodictyon angustifolium and Salvia apiana. . Inter J Pharmacog 30, 223-231. Dold, A., Cocks, M., 2001. Traditional veterinary medicine in the Alice district of the Eastern Cape Province, South Africa. South African Journal of Science 97, 1-7. Duke, J.A., 1989. Castanospermum and anti-AIDS activity. Journal of ethnopharmacology 25, 227228. Efferth, T., Kahl, S., Paulus, K., Adams, M., Rauh, R., Boechzelt, H., Hao, X., Kaina, B., Bauer, R., 2008a. Phytochemistry and pharmacogenomics of natural products derived from traditional Chinese medicine and Chinese materia medica with activity against tumor cells. Mol Cancer Ther 7, 152-161. Efferth, T., Konkimalla, V.B., Wang, Y.F., Sauerbrey, A., Meinhardt, S., Zintl, F., Mattern, J., Volm, M., 2008b. Prediction of broad spectrum resistance of tumors towards anticancer drugs. Clinical cancer research : an official journal of the American Association for Cancer Research 14, 2405-2412. Efferth, T., Lohrke, H., Volm, M., 1989. Reciprocal correlation between expression of P-glycoprotein and accumulation of rhodamine 123 in human tumors. Anticancer Res 9, 1633-1637. Efferth, T., Osieka, R., 1993. Clinical relevance of the MDR-a gene and it's gene product , Pglycoprotein, for cancer chemotherapy. Tumor Diagn Ther 14, 238-243. el-Deiry, W.S., 1997. Role of oncogenes in resistance and killing by cancer therapeutic agents. Curr Opin Oncol 9, 79-87. El-Deiry, W.S., 2003. The role of p53 in chemosensitivity and radiosensitivity. Oncogene 22, 74867495. Eloff, J.N., Famakin, J.O., Katerere, D.R.P., 2005. Isolation of an antibacterial stilbene from Combretum woodii (Combretaceae) leaves African Journal of Biotechnology 4, 1167-1171. Fajac, A., Da Silva, J., Ahomadegbe, J.C., Rateau, J.G., Bernaudin, J.F., Riou, G., Benard, J., 1996. Cisplatin-induced apoptosis and p53 gene status in a cisplatin-resistant human ovarian carcinoma cell line. Int J Cancer 68, 67-74. Fang, F., Sang, S., Chen, K.Y., Alexander, G.A., Ho, C.-T., Rosen, R.T., 2005. Isolation and identification of cytotoxic compounds from Bay leaf (Laurus nobilis). . Food Chem 93, 497–501. Foster, S., Hobbs, C., 2002. In A Field Guide to Western Medicinal Plants and Herbs. Houghton-Mifflin company, New York, 442. Fouche, G., Cragg, G.M., Pillay, P., Kolesnikova, N., Maharaj, V.J., Senabe, J., 2008. In vitro anticancer screening of South African plants. Journal of ethnopharmacology 119, 455-461. Gabetta, B., Zini, G., Danieli, B., 1989. Minor diterpenoids of Coleus forskohlii. . Phytochemistry 28, 859–862. Gazim, Z.C., Rodrigues, F., Amorin, A.C., de Rezende, C.M., Sokovic, M., Tesevic, V., Vuckovic, I., Krstic, G., Cortez, L.E., Colauto, N.B., Linde, G.A., Cortez, D.A., 2013. New natural diterpene-type abietane from Tetradenia riparia essential oil with cytotoxic and antioxidant activities. Molecules 19, 514-524. Geng, S., Sun, B., Lu, R., Wang, J., 2014. Coleusin factor, a novel anticancer diterpenoid, inhibits osteosarcoma growth by inducing bone morphogenetic protein-2-dependent differentiation. Mol Cancer Ther 13, 1431-1441. Gonzalez, A.G., Aguiar, Z.E., Grillo, T.A., Luis, J.G., 1992. Diterpenes and diterpene quinones from the roots of Salvia apiana. Phytochemistry 31, 1691-1695. Hall, M.D., Handley, M.D., Gottesman, M.M., 2009. Is resistance useless? Multidrug resistance and collateral sensitivity. Trends in pharmacological sciences 30, 546-556. 15

Hauschild-Rogat P, Weiss E, Reichstein, T., 1967. Glycosides and aglycons. CCCI. Cardenolides of Acokanthera oppositifolia 3. Isolation of additional cardenolides and partial structural elucidation. Helv Chim Acta 50, 2299-2321. Hauschild-Rogat P, W., E, Reichstein T, 1967. Glycosides and aglycons. CCCII. Probable structure of opposide. Helv Chim Acta 50, 2322-2330. Hibasami, H., Yamada, Y., Moteki, H., Katsuzaki, H., Imai, K., Yoshioka, K., Komiya, T., 2003. Sesquiterpenes (costunolide and zaluzanin D) isolated from laurel (Laurus nobilis L.) induce cell death and morphological change indicative of apoptotic chromatin condensation in leukemia HL-60 cells. Int J Mol Med 12, 147-151. Hoang, B., Zhu, L., Shi, Y., Frost, P., Yan, H., Sharma, S., Goodglick, L., Dubinett, S., Lichtenstein, A., 2006. Oncogenic RAS mutations in myeloma cells selectively induce cox-2 expression, which participates in enhanced adhesion to fibronectin and chemoresistance. Blood 107, 4484-4490. Hulme, M., 1954. Wild Flowers of Natal. Shuter and Shooter. Pietermaritzburg. Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A., 1996. Zulu Medicinal Plants: An Inventory. University of Natal Press, Pietermaritzburg. Iwu, M.M., 1993. Handbook of African medicinal plants. . CRC Press, Florida. Jin, Q.D., He, B.H., 1998. Minor constituents from Coleus forskohlii. Acta Bot Yunn 20, 469–473. Juliano, R.L., Ling, V., 1976. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochimica et biophysica acta 455, 152-162. Julianti, E., Jang, K.H., Lee, S., Lee, D., Mar, W., Oh, K.B., Shin, J., 2012. Sesquiterpenes from the leaves of Laurus nobilis L. Phytochemistry 80, 70-76. Kadioglu, O., Efferth, T., 2015. Pharmacogenomic Characterization of Cytotoxic Compounds from Salvia officinalis in Cancer Cells. J Nat Prod 78, 762-775. Kaplan, E.R., Rivett, D.E.A., 1968. Structures of compounds X and Y, two labdane diterpenoids, from Leonotis leonurus. J Chem Soc C 262–266. Katti, S.B., Jauhari, P.K., Tandon, J.S., 1979. New diterpenes from Coleus forskohlii: structures of the diterpenes, coleonol-D, coleol and coleonone. Indian J Chem Sect B Org Chem Incl Med Chem 17B, 321–323. Kelecom, A., 1983a. 6-β-Hydroxycarnosol, a newminor diterpene from the false boldo Coleus barbatus Bentham (Labiatae). Quim Nova 6, 117–118. Kelecom, A., 1983b. Isolation, structure determination, and absolute configuration of barbatusol, a new bioactive diterpene with a rearranged abietane skeleton from the labiate Coleus barbatus. Tetrahedron 39, 3603–3608. Kelecom, A., 1984. An abietane diterpene from the labiate Coleus barbatus. . Phytochemistry 23, 1677–1679. Kelecom, A., Dos Santos, T.C., 1985. Cariocal, a new seco-abietane diterpene from the labiate Coleus barbatus. Tetrahedron Lett 26, 3659–3662. Kelecom, A., Dos Santos, T.C., Medeiros, W.L.B., 1987. Secoabietane diterpenes from Coleus barbatus. Phytochemistry 26, 2337–2340. Kim, H.J., Kim, S.H., Yun, J.M., 2012. Fisetin inhibits hyperglycemia-induced proinflammatory cytokine production by epigenetic mechanisms. Evidence-based complementary and alternative medicine : eCAM 2012, 639469. Kimmig, A., Gekeler, V., Neumann, M., Frese, G., Handgretinger, R., Kardos, G., Diddens, H., Niethammer, D., 1990. Susceptibility of multidrug-resistant human leukemia cell lines to human interleukin 2-activated killer cells. Cancer Res 50, 6793-6799. Kokwaro, J.O., 1976. Medicinal plants of East Africa. East African Literature Bureau. Kometiani, P., Li, J., Gnudi, L., Kahn, B.B., Askari, A., Xie, Z., 1998. Multiple signal transduction pathways link Na+/K+-ATPase to growth-related genes in cardiac myocytes. The roles of Ras and mitogen-activated protein kinases. J Biol Chem 273, 15249-15256. Komiya, T., Yamada, Y., Moteki, H., Katsuzaki, H., Imai, K., Hibasami, H., 2004. Hot water soluble sesquiterpenes [anhydroperoxy-costunolide and 3-oxoeudesma-1,4(15),11(13)triene-12,6alpha16

olide] isolated from laurel (Laurus nobilis L.) induce cell death and morphological change indicative of apoptotic chromatin condensation in leukemia cells. Oncol Rep 11, 85-88. Konate, K., Yomalan, K., Sytar, O., Zerbo, P., Brestic, M., Patrick, V.D., Gagniuc, P., Barro, N., 2014. Free Radicals Scavenging Capacity, Antidiabetic and Antihypertensive Activities of Flavonoid-Rich Fractions from Leaves of Trichilia emetica and Opilia amentacea in an Animal Model of Type 2 Diabetes Mellitus. Evidence-based complementary and alternative medicine : eCAM 2014, 867075. Kretschmer, N., Rinner, B., Stuendl, N., Kaltenegger, H., Wolf, E., Kunert, O., Boechzelt, H., Leithner, A., Bauer, R., Lohberger, B., 2012. Effect of costunolide and dehydrocostus lactone on cell cycle, apoptosis, and ABC transporter expression in human soft tissue sarcoma cells. Planta medica 78, 1749-1756. Kuchta, K., Ortwein, J., Rauwald, H.W., 2012. Leonurus japonicus, Leonurus cardiaca, Leonotis leonurus: a novel HPLC study on the occurrence and content of the pharmacologically active guanidino derivative leonurine. Pharmazie 67, 973-979. Kuchta, K., Volk, R.B., Rauwald, H.W., 2013. Stachydrine in Leonurus cardiaca, Leonurus japonicus, Leonotis leonurus: detection and quantification by instrumental HPTLC and 1H-qNMR analyses. Pharmazie 68, 534-540. Kuete, V., Wiench, B., Hegazy, M.E., Mohamed, T.A., Fankam, A.G., Shahat, A.A., Efferth, T., 2012. Antibacterial activity and cytotoxicity of selected Egyptian medicinal plants. Planta Med 78, 193-199. Kumar, D., Sukapaka, M., Babu, G.D., Padwad, Y., 2015. Chemical Composition and In Vitro Cytotoxicity of Essential Oils from Leaves and Flowers of Callistemon citrinus from Western Himalayas. PloS one 10, e0133823. KUMAR, N.N., RAMAKRISHNAIAH, H., KRISHNA, V., RADHIKA, M., 2014. CYTOTOXIC ACTIVITY OF BROUSSONETIA PAPYRIFERA (L.) VENT ON MCF-7, HELA AND HEPG2 CELL LINES International Journal of Pharmacy and Pharmaceutical Sciences 6, 339-342. Kupchan, S.M., Smith, R.M., 1977. Maytoline, maytine, and maytolidine, novel nicotinoyl sesquiterpene alkaloids from Maytenus serrata (Hochst., ex A. Rich.) R. Wilczek. The Journal of organic chemistry 42, 115-118. Laonigro, G., Lanzetta, R., Parrilli, M., Adinolfi, M., Mangoni, L., 1979. The configuration of the diterpene spiro ethers from Marrubium vulgare and from Leonotis leonurus. Gazz Chim Ital 109, 145–150. Li, S., Yang, Q.R., Wang, X.M., Zou, G.A., Liu, Y.W., 2006. Chemical constituents of Coleus forskohlii replanted to Tongcheng. . Zhongcaoyao 37, 824–826. Lin, X., Peng, Z., Su, C., 2015. Potential anti-cancer activities and mechanisms of costunolide and dehydrocostuslactone. Int J Mol Sci 16, 10888-10906. Liu, Y., Wang, X.M., Wu, H., 2007. Main components of Coleus forskohlii extract and relevant extraction method. . Chinese Patent CN 1944384 A. Loizzo, M.R., Tundis, R., Menichini, F., Saab, A.M., Statti, G.A., 2007. Cytotoxic activity of essential oils from labiatae and lauraceae families against in vitro human tumor models. Anticancer Res 27, 32933299. Luis, J.G., Lahlou, E., Andres, L.S., 1996a. Hassananes: C23 terpenoids with a new type of skeleton from Salvia apiana Jeps. Tetrahedron 52, 12309-12312. Luis, J.G., Lahlou, E., Andres, L.S., Sood, G.N., Ripoll, M.M., 1996b. Apiananes: C23 terpenoid with a new type of skeleton from Salvia apiana. Tetrahedron Lett 37, 4213-4216. Lukhoba, C.W., Simmonds, M.S., Paton, A.J., 2006. Plectranthus: a review of ethnobotanical uses. Journal of ethnopharmacology 103, 1-24. Mathela, D.K., Kharkwal, H.B., Mathela, C.S., 1986. Terpenoids of roots of Coleus forskohlii. Fitoterapia 57, 299–301. Matic, S., Stanic, S., Bogojevic, D., Vidakovic, M., Grdovic, N., Arambasic, J., Dinic, S., Uskokovic, A., Poznanovic, G., Solujic, S., Mladenovic, M., Markovic, J., Mihailovic, M., 2011. Extract of the plant Cotinus coggygria Scop. attenuates pyrogallol-induced hepatic oxidative stress in Wistar rats. Canadian journal of physiology and pharmacology 89, 401-411. 17

Mazimba, O., 2015. Leonotis leonurus: a herbal medicine review. . Journal of Pharmacognosy and Phytochemistry 3, 74-82. McKenzie, J.M., Green, I.R., Mugabo, P., 2006. Leonurun, a novel labdane diterpenoid from Leonotis leonurus. S Afr J Chem 59, 114–116. Mirian, M., Behrooeian, M., Ghanadian, M., Dana, N., Sadeghi-Aliabadi, H., 2015. Cytotoxicity and antiangiogenic effects of Rhus coriaria, Pistacia vera and Pistacia khinjuk oleoresin methanol extracts. Research in pharmaceutical sciences 10, 233-240. Moore, M., 1993. Medicinal Plants of the Pacific West. Red Crane Books, Santa Fe, NM, 266-269. Moteki, H., Hibasami, H., Yamada, Y., Katsuzaki, H., Imai, K., Komiya, T., 2002. Specific induction of apoptosis by 1,8-cineole in two human leukemia cell lines, but not a in human stomach cancer cell line. Oncol Rep 9, 757-760. Mukamoto, M., Kimura, R., Hang'ombe, M.B., Kohda, T., Kozaki, S., 2013. Analysis of tryptophan-rich region in Clostridium septicum alpha-toxin involved with binding to glycosylphosphatidylinositolanchored proteins. Microbiology and immunology 57, 163-169. Mukesh, K.S., Bina, G., Anshita, G., Hemant, D., Chanchal, D.K., Pranita, P.K., Tripathi, D.K., 2015. A Review of the Medicinal Plants of Genus Orthosiphon (Lamiaceae). International Journal of Biological Chemistry 9, 318-331. Mwitari, P.G., Ayeka, P.A., Ondicho, J., Matu, E.N., Bii, C.C., 2013. Antimicrobial activity and probable mechanisms of action of medicinal plants of Kenya: Withania somnifera, Warbugia ugandensis, Prunus africana and Plectrunthus barbatus. PLoS One 8, e65619. Naidoo, D., Maharaj, V., Crouch, N.R., Ngwane, A., 2011. New labdane-type diterpenoids from Leonotis leonurus support circumscription of Lamiaceae. . Biochem Syst Ecol 39, 216–219. Nakamura, K., Kohda, T., Seto, Y., Mukamoto, M., Kozaki, S., 2013. Improved detection methods by genetic and immunological techniques for botulinum C/D and D/C mosaic neurotoxins. Veterinary microbiology 162, 881-890. Newman, D.J., Cragg, G.M., 2007. Natural products as sources of new drugs over the last 25 years. J Nat Prod 70, 461-477. Newman, R.A., Yang, P., Pawlus, A.D., Block, K.I., 2008. Cardiac glycosides as novel cancer therapeutic agents. Mol Interv 8, 36-49. O'Brien, J., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European journal of biochemistry / FEBS 267, 5421-5426. Obikeze, K.C., McKenzie, J.M., Green, I.R., P., M., 2008. Characterization and cardiovascular effects of (13S)-9α,13α-epoxylabda-6β(19),15(14)diol dilactone, a diterpenoid isolated from Leonotis leonurus. S Afr J Chem 61, 119–122. Ochwang'i, D.O., Kimwele, C.N., Oduma, J.A., Gathumbi, P.K., Mbaria, J.M., Kiama, S.G., 2014. Medicinal plants used in treatment and management of cancer in Kakamega County, Kenya. Journal of ethnopharmacology 151, 1040-1055. Ogur, R., 2014. Studies with Myrtus communis L.: Anticancer properties. Journal of intercultural ethnopharmacology 3, 135-137. Oyedeji, O.O., Lawal, O.A., Shode, F.O., Oyedeji, A.O., 2009. Chemical composition and antibacterial activity of the essential oils of Callistemon citrinus and Callistemon viminalis from South Africa. Molecules 14, 1990-1998. Pacifico, S., Gallicchio, M., Lorenz, P., Potenza, N., Galasso, S., Marciano, S., Fiorentino, A., Stintzing, F.C., Monaco, P., 2013. Apolar Laurus nobilis leaf extracts induce cytotoxicity and apoptosis towards three nervous system cell lines. Food Chem Toxicol 62, 628-637. Painuly, P., Katti, S.B., Tandon, J.S., 1979. Diterpenes from Coleus forskohlii: structures of coleonol-E and coleonol-F. Indian J Chem Sect B Org Chem Incl Med Chem 18B, 214–216. Pang, S.Q., Wang, G.Q., Lin, J.S., Diao, Y., Xu, R.A., 2014. Cytotoxic activity of the alkaloids from Broussonetia papyrifera fruits. Pharmaceutical biology 52, 1315-1319. Panza, E., Tersigni, M., Iorizzi, M., Zollo, F., De Marino, S., Festa, C., Napolitano, M., Castello, G., Ialenti, A., Ianaro, A., 2011. Lauroside B, a megastigmane glycoside from Laurus nobilis (bay laurel) 18

leaves, induces apoptosis in human melanoma cell lines by inhibiting NF-kappaB activation. J Nat Prod 74, 228-233. Pech B, J., B., 1982. Alcaloïdes Du Laurier Noble, Laurus nobilis. J Nat Prod 45, 560–563. Pettit, G.R., Goswami, A., Cragg, G.M., Schmidt, J.M., Zou, J.C., 1984. Antineoplastic agents, 103. The isolation and structure of hypoestestatins 1 and 2 from the East African Hypoestes verticillaris. J Nat Prod 47, 913-919. Pettit, G.R., Klinger, H., Jorgensen, N.O.N., Occolowitz, J., 1966. Steroids and related natural products. XXVII. Salvia apiana. Phytochemistry 5, 301-309. Peuzzdni, F., L.., Verotta, C.B., Rogers, R., Colombo, B., Pedrotti, G., Balconi, E., M., D.i., 1993. Cell growth inhibitor constituents from Combretum kraussii. Natural Product Letters 1, 273-280. Prakash, O., Roy, R., Tandon, J.S., Dhar, M.M., 1988. Carbon-13 and proton two-dimensional NMR study of diterpenoids of Coleus forskohlii. Magn Reson Chem 26, 117–119. Qin, F.M., Liu, B.L., Zhang, Y., Zhou, G.X., 2015. A new triterpenoid from the fruits of Gardenia jasminoides var. radicans Makino. Natural product research 29, 633-637. Rao, D.K., Liu, H., Ambudkar, S.V., Mayer, M., 2014. A combination of curcumin with either gramicidin or ouabain selectively kills cells that express the multidrug resistance-linked ABCG2 transporter. J Biol Chem 289, 31397-31410. Rattmann, Y.D., de Souza, L.M., Malquevicz-Paiva, S.M., Dartora, N., Sassaki, G.L., Gorin, P.A.J., Iacomini, M., 2012. Analysis of Flavonoids from Eugenia uniflora Leaves and Its Protective Effect against Murine Sepsis. Evid-Based Compl Alt. Rhinehart, B.L., Keith M. R., Chi-Hsin R. K., L., P.S., 1990. Castanospermine-glucosides as selective disaccharidase inhibitors. Biochemical pharmacology 39, 1537-1543. Riganti, C., Campia, I., Kopecka, J., Gazzano, E., Doublier, S., Aldieri, E., Bosia, A., Ghigo, D., 2011. Pleiotropic effects of cardioactive glycosides. Curr Med Chem 18, 872-885. Rodd, A.L., Ververis, K., Sayakkarage, D., Khan, A.W., Rafehi, H., Ziemann, M., Loveridge, S.J., Lazarus, R., Kerr, C., Lockett, T., El-Osta, A., Karagiannis, T.C., Bennett, L.E., 2015. RNA sequencing supports distinct reactive oxygen species-mediated pathways of apoptosis by high and low size mass fractions of Bay leaf (Lauris nobilis) in HT-29 cells. Food Funct. Rotstein, A., Lifshitz, A., Kashman, Y., 1974. Isolation and antibacterial activity of acylphloroglucinols from Myrtus communis. Antimicrobial agents and chemotherapy 6, 539-542. Roy, R., Mishra, A., Varma, N., Tandon, J.S., Saux, M., Carpy, A., 1993. Minor diterpenes from Coleus forskohlii. Phytochemistry 34, 1577–1580. Rubinstein, L.V., Shoemaker, R.H., Paull, K.D., Simon, R.M., Tosini, S., Skehan, P., Scudiero, D.A., Monks, A., Boyd, M.R., 1990. Comparison of in vitro anticancer-drug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines. J Natl Cancer Inst 82, 1113-1118. Ruedi, P., 1986. Novel diterpenoids from leaf glands of Plectranthus barbatus (Labiatae). The absolute configuration of the 2-hydroxypropyl group in coleon E. Helv Chim Acta 69, 972–984. Saab, A.M., Guerrini, A., Zeino, M., Wiench, B., Rossi, D., Gambari, R., Sacchetti, G., Greten, H.J., Efferth, T., 2015. Laurus nobilis L. Seed Extract Reveals Collateral Sensitivity in Multidrug-Resistant PGlycoprotein-Expressing Tumor Cells. Nutr Cancer 67, 664-675. Saeed, M., Kuete, V., Kadioglu, O., Bortzler, J., Khalid, H., Greten, H.J., Efferth, T., 2014. Cytotoxicity of the bisphenolic honokiol from Magnolia officinalis against multiple drug-resistant tumor cells as determined by pharmacogenomics and molecular docking. Phytomedicine : international journal of phytotherapy and phytopharmacology 21, 1525-1533. Said, O., Khalil, K., Fulder, S., Azaizeh, H., 2002. Ethnopharmacological survey of medicinal herbs in Israel, the Golan Heights and the West Bank region. J Ethnopharmacol 83, 251-265. Sashidhara, K.V., Rosaiah, J.N., Kumar, A., Bid, H.K., Konwar, R., Chattopadhyay, N., 2007. Cell growth inhibitory action of an unusual labdane diterpene, 13-epi-sclareol in breast and uterine cancers in vitro. Phytother Res 21, 1105-1108.

19

Savikin, K., Zdunic, G., Jankovic, T., Stanojkovic, T., Juranic, Z., Menkovic, N., 2009. In vitro cytotoxic and antioxidative activity of Cornus mas and Cotinus coggygria. Natural product research 23, 17311739. Schapoval, E.E.S., Silveira, S.M., Miranda, M.L., Alice, C.B., Henriques, A.T., 1994. Evaluation of Some Pharmacological Activities of Eugenia-Uniflora L. Journal of ethnopharmacology 44, 137-142. Scherf, U., Ross, D.T., Waltham, M., Smith, L.H., Lee, J.K., Tanabe, L., Kohn, K.W., Reinhold, W.C., Myers, T.G., Andrews, D.T., Scudiero, D.A., Eisen, M.B., Sausville, E.A., Pommier, Y., Botstein, D., Brown, P.O., Weinstein, J.N., 2000. A gene expression database for the molecular pharmacology of cancer. Nat Genet 24, 236-244. Scott G, Springfield E, N, C., 2004. A pharmacognostical study of 26 South African plant species used as traditional medicines. . Pharm Biol (Lisse, Neth) 42, 186-213. Shan, Y., Wang, X., Zhou, X., Kong, L., Niwa, M., 2007. Two minor diterpene glycosides and an eudesman sesquiterpene from Coleus forskohlii. Chem Pharm Bull (Tokyo) 55, 376-381. Shan, Y., Xu, L., Lu, Y., Wang, X., Zheng, Q., Kong, L., Niwa, M., 2008. Diterpenes from Coleus forskohlii (WILLD.) BRIQ. (Labiatae). Chem Pharm Bull (Tokyo) 56, 52-56. Shan, Y.P., Kong, L.Y., 2006. Isolation and identification of terpenes from Coleus forskohlii. Chin J Nat Med 4, 271–274. Shang, X., Pan, H., Wang, X., He, H., Li, M., 2014. Leonurus japonicus Houtt.: ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine. J Ethnopharmacol 152, 14-32. Shen, Q., Shang, D., Ma, F., Zhang, Z., 1991. [Pharmacological study on anti-hepatitis effect of Cotinus coggygria Scop. Syrup]. Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica 16, 746-749, 763-744. Shetzer, Y., Solomon, H., Koifman, G., Molchadsky, A., Horesh, S., Rotter, V., 2014. The paradigm of mutant p53-expressing cancer stem cells and drug resistance. Carcinogenesis 35, 1196-1208. Shirota, O., Tamemura, T., Morita, H., Takeya, K., Itokawa, H., 1996. Triterpenes from Brazilian medicinal plant ''Chuchuhuasi'' (Maytenus krukovii). Journal of natural products 59, 1072-1075. Singh, T., Gupta, N.A., Xu, S., Prasad, R., Velu, S.E., Katiyar, S.K., 2015. Honokiol inhibits the growth of head and neck squamous cell carcinoma by targeting epidermal growth factor receptor. Oncotarget 6, 21268-21282. Spies, L., Koekemoer, T.C., Sowemimo, A.A., Van De Venter, M., 2011. The anticancer potential of Artemisia afra. . Planta medica 77, PM 101 Stampoulis, P., Tezuka, Y., Banskota, A.H., Tran, K.Q., Saiki, I., Kadota, S., 1999. Staminolactones A and B and norstaminol A: three highly oxygenated staminane-type diterpenes from Orthosiphon stamineus. Organic letters 1, 1367-1370. Staunton, J.E., Slonim, D.K., Coller, H.A., Tamayo, P., Angelo, M.J., Park, J., Scherf, U., Lee, J.K., Reinhold, W.O., Weinstein, J.N., Mesirov, J.P., Lander, E.S., Golub, T.R., 2001. Chemosensitivity prediction by transcriptional profiling. Proc Natl Acad Sci U S A 98, 10787-10792. Steenkamp, V., 2003. Traditional herbal remedies used by South African women for gynaecological complaints. J Ethnopharmacol 86, 97-108. Steenkamp, V., Gouws, M.C., 2006. Cytotoxicity of six South African medicinal plant extracts used in the treatment of cancer. South African Journal of Botany 72, 630-633. Steyn, D., 1934. The toxicology of plants in South Africa. Central News Agency, South Africa, 23-31. Sudhakar, M., Rao, C.V., Rao, A.L., Ramesh, A., Srinivas, N., Raju, D.B., Murthy, B.K., 2006. Antinociceptive and anti-inflammatory effects of the standardized oil of Indian Callistemon lanceolatus leaves in experimental animals. East and Central African Journal of Pharmaceutical Sciences 7, 10-15. Suffness, M., Pezzuto, J.M., 1990. Assays related to cancer drug discovery. In: Hostettmann, K (Ed.), Methods in Plant Biochemistry: Assays for Bioactivity. Academic Press, London 6, 71-133. Sztiller-Sikorska, M., Koprowska, K., Majchrzak, K., Hartman, M., Czyz, M., 2014. Natural compounds' activity against cancer stem-like or fast-cycling melanoma cells. PLoS One 9, e90783. 20

Tandon, J.S., Jauhari, P.K., Singh, R.S., Dhar, M.M., 1978. Structures of three new diterpenes, coleonol B, coleonol C and deoxycoleonol isolated from Coleus forskohlii. Indian J Chem Sect B Org Chem Incl Med Chem 16B, 341–345. Tandon, J.S., Roy, R., Balachandran, S., Vishwakarma, R.A., 1992. Epi-deoxycoleonol, a new antihypertensive labdane diterpenoid from Coleus forskohlii. Bioorg Med Chem Lett 2, 249–254. Tapiero, H., Munck, J.N., Fourcade, A., Lampidis, T.J., 1984. Cross-resistance to rhodamine 123 in Adriamycin- and daunorubicin-resistant Friend leukemia cell variants. Cancer Res 44, 5544-5549. Thring, T.S., Weitz, F.M., 2006. Medicinal plant use in the Bredasdorp/Elim region of the Southern Overberg in the Western Cape Province of South Africa. J Ethnopharmacol 103, 261-275. Traore, M., Zhai, L., Chen, M., Olsen, C.E., Odile, N., Pierre, G.I., Bosco, O.J., Robert, G.T., Christensen, S.B., 2007. Cytotoxic kurubasch aldehyde from Trichilia emetica. Natural product research 21, 13-17. Tretiakova, I., Blaesius, D., Maxia, L., Wesselborg, S., Schulze-Osthoff, K., Cinatl, J., Jr., Michaelis, M., Werz, O., 2008. Myrtucommulone from Myrtus communis induces apoptosis in cancer cells via the mitochondrial pathway involving caspase-9. Apoptosis : an international journal on programmed cell death 13, 119-131. Van Wyk, B., Van Heerden, F., Van Oudtshoon, B., 2002. Poisonous plants of South Africa. Briza Publications, Pretoria, 46-49. Van Wyk, B.E., van Oudtshoorn, B., Gericke, N., 1997. Medicinal Plants of South Africa. Briza Publications, Pretoria. Wang, G., Wang, J., Du, L., Li, F., 2015. Effect and Mechanism of Total Flavonoids Extracted from Cotinus coggygria against Glioblastoma Cancer In Vitro and In Vivo. BioMed research international 2015, 856349. Wang, Z., Zheng, M., Li, Z., Li, R., Jia, L., Xiong, X., Southall, N., Wang, S., Xia, M., Austin, C.P., Zheng, W., Xie, Z., Sun, Y., 2009. Cardiac glycosides inhibit p53 synthesis by a mechanism relieved by Src or MAPK inhibition. Cancer Res 69, 6556-6564. Watt, J., Breyer-Brandwijk, M., 1962. The medicinal and poisonous plants of Southern and Eastern Africa. 2nd edition. Livingstone, London, 15-18. Weidemann, H., 2005. Na/K-ATPase, endogenous digitalis like compounds and cancer development -a hypothesis. Front Biosci 10, 2165-2176. Westenburg, H.E., Lee, K.J., Lee, S.K., Fong, H.H., van Breemen, R.B., Pezzuto, J.M., Kinghorn, A.D., 2000. Activity-guided isolation of antioxidative constituents of Cotinus coggygria. Journal of natural products 63, 1696-1698. Williams, L.A., Conrad, J., Vogler, B., Rosner, H., Porter, R.B., Setzer, W., Barton, E.N., Levy, H.G., Mika, S., Klaiber, I., Nkurunziza, J.P., Kraus, W., 2007. In vitro anti-proliferation/cytotoxic activity of epingaione and its derivatives on the human SH-SY5Y neuroblastoma and TE-671 sarcoma cells. The West Indian medical journal 56, 5-10. Wolle, D., Lee, S.J., Li, Z., Litan, A., Barwe, S.P., Langhans, S.A., 2014. Inhibition of epidermal growth factor signaling by the cardiac glycoside ouabain in medulloblastoma. Cancer Med 3, 1146-1158. Wu, H., Li, J., Fronczek, F.R., Ferreira, D., Burandt, C.L., Jr., Setola, V., Roth, B.L., Zjawiony, J.K., 2013. Labdane diterpenoids from Leonotis leonurus. Phytochemistry 91, 229-235. Xu, L.L., Kong, L.Y., 2004. Isolation and identification of labdane diterpenoids from the roots of Coleus forskohlii. Chin J Nat Med 2, 344–347. Xu, L.L., Kong, L.Y., 2006. Labdane diterpenoids from Coleus forskohlii (Willd.) Briq. J Integr Plant Biol 48, 478–481. Xu, L.L., Lu, J., Li, W.J., Kong, L.Y., 2005. [Studies on the chemical constituents in root of Coleus forskohlii]. Zhongguo Zhong Yao Za Zhi 30, 1753-1755. Yam, M.F., Ang, L.F., Salman, I.M., Ameer, O.Z., Lim, V., Ong, L.M., Ahmad, M., Asmawil, M.Z., Basir, R., 2009. Orthosiphon stamineus leaf extract protects against ethanol-induced gastropathy in rats. Journal of medicinal food 12, 1089-1097. Yashaswini, S., Vasundhara, M., 2011. COLEUS (PLECTRANTHUS BARBATUS) – A MULTIPURPOSE MEDICINAL HERB. . INTERNATIONAL RESEARCH JOURNAL OF PHARMACY 2, 47-58. 21

Ye, B., Aponte, M., Dai, Y., Li, L., Ho, M.C., Vitonis, A., Edwards, D., Huang, T.N., Cramer, D.W., 2007. Ginkgo biloba and ovarian cancer prevention: epidemiological and biological evidence. Cancer letters 251, 43-52. Zeino, M., Brenk, R., Gruber, L., Zehl, M., Urban, E., Kopp, B., Efferth, T., 2015a. Cytotoxicity of cardiotonic steroids in sensitive and multidrug-resistant leukemia cells and the link with Na(+)/K(+)ATPase. J Steroid Biochem Mol Biol 150, 97-111. Zeino, M., Paulsen, M.S., Zehl, M., Urban, E., Kopp, B., Efferth, T., 2015b. Identification of new Pglycoprotein inhibitors derived from cardiotonic steroids. Biochem Pharmacol 93, 11-24. Zelnik, R., Lavie, D., Levy, E.C., Wang, A.H.J., Paul, I., 1977. Barbatusin and cyclobutatusin, two novel diterpenoids from Coleus barbatus Bentham. Tetrahedron 33, 1457–1467.

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Table 1: Traditional uses of South African medicinal plants against cancer.. Family

Acanthaceae

Anacardiacaea

Plant species

Hypoestes aristata Soland. ex Roem & Schult. var. aristata Pistacia vera L.

Part collecte d leaves

(resin),

Traditional uses

According to healers of the Xhosa tribe, the plant is also used to treat complicated and major diseases like cancer, arthritis, tuberculosis, bone fractures. The plant is used for the treatment of eczema, throat infection, renal stone, and asthma. In addition, some Pistacia species have shown various biological activities including anti atherogenic, antioxidant, antifungal and anti-inflammatory. The oleo gum resin extract of Pistacia vera has shown antiangiogenic and cytotoxic effects. The Cotinus coggygria possesses diverse biological activities, including antioxidant, anti-inflammatory, and anticancer effects. Also it is used in the treatment of acute icteric infectious hepatitis.

Cotinus coggygria (Scop)

leaves

Schinus molle L.

leaves and resin

Apocynaceae

Acokanthera oppositifolia (Lam.) Codd,

leaves

Asteraceae

Artemisia afra Jacq. ex Willd.

leaves

It is widely used in South African traditional medicine to treat many different medical conditions, particularly respiratory and inflammatory. It has been reported that A. afra has anticancer potential.

Celastraceae

Maytenus bachmannii (Loes.) Marais.

leaves

Maytenus species are most frequently used in African traditional medicine to treat infectious and inflammatory diseases. In some part of Africa (Kenya) is used traditionally as anticancer.

Combretaceae

Combretum kraussi Hochst.

leaves

C. kraussi has been used traditionally as antibacterial and antiinflamatory. It has been reported that the combretastatins which isolated from the seeds of C. kraussi have shown cytotoxic activity and influenced the tubulin polymerization.

Fabaceae

Castanospermum australe

leaves

The alkaloid, castanospermine, inside C. australe have shown,

Traditionally S. molle is used as antibacterial, topical antiseptic, digestive, for respiratory and urinary infections, analgesic, and central depressant, purgative diuretic. Recently the antioxidant and anticancer activities have been reported. Traditionally used as highly effective arrow poison in SouthAfrica, which indicates his cytotoxic properties.

23

Referenc es (Bhat, 2014) (Bozorgi et al., 2013; Mirian et al., 2015) (Kim et al., 2012; Matic et al., 2011; Savikin et al., 2009; Shen et al., 1991; Wang et al., 2015; Westenbu rg et al., 2000) (Bendaou d et al., 2010) (Steyn, 1934; Van Wyk et al., 2002) (Fouche et al., 2008; Spies et al., 2011). (da Silva et al., 2011; Kupchan and Smith, 1977; Shirota et al., 1996) (Eloff et al., 2005; Peuzzdni et al., 1993) (Duke,

A.Cunn & C.Fraser ex Hook

anti-parasitic anti-HIV and anticancer properties

Ginkgoaceae

Ginkgo biloba L.

(root bark, leaves, fruits),

G. biloba extract has been used as herbal dietary supplements in several European countries and USA because of chemopreventive properties. It may also have effects on angiogenesis, DNA damage, cell proliferation and death, and free radicals scavenging.

Lamiaceae

Leonotis leonurus (L.) R.Br.

Aerial parts, except P. barbatus (roots)

Tea made from the whole plant is used against cancer, arthritis, piles, bladder and kidney disorder, obesity, and rheumatism.

Orthosiphone serratus Schltr

Several Orthosiphon species traditionally used for expectorant, rheumatism indicated antioxidant activity and anticancer activity.

Plectranthus barbatus Andrews

used for a wide range of complaints, e.g. stomach ache and as purgative drug, against hypertension, congestive heart failure, eczema, colic, respiratory disorders, painful urination, insomnia, convulsions, and cancer prevention.

Plectranthus ciliates E. Mey. ex Benth.

Traditionally is used to treat diseases of digestive tract, respiratory system, nervous system, inflammation, infections, pain and different types of cancers It is recommended by herbalists to treat prostate hyperplasia.

Salvia apiana Jeps.

Tetradenia riparia (Hochst.) Codd,

Traditionally it is used in cases of cough, dropsy, diarrhea, fever, headaches, malaria, antimicrobial and toothache. The terpnoids inside the plant have been shown to possess cytotoxic and antioxidant activities.

Lauraceae

Laurus nobilis L.,

leaves

Magnoliaceae

Magnolia x soulangeana

leaves

Used among other applications against skin diseases and cancer in the Middle East. Some of magnolia species are rich of lignans, therefore, are widely used in Chinese and Japanese traditional medicines for their chemo-preventive role and cytotoxic activity.

Meliaceae

Trichilia emetic Sond.

leaves

T. emetic is native to sub-Saharan Africa, it’s possess hypotensive, antioxidant, antibacterial, anti-inflammatory and

24

1989; Rhinehart et al., 1990) (Biggs et al., 2010; Chan et al., 2007; DeFeudis et al., 2003; Ye et al., 2007) (Thring and Weitz, 2006) (Mukesh et al., 2015; Stampoul is et al., 1999; Yam et al., 2009) (AbdelMogib et al., 2002) (Mwitari et al., 2013) (Lukhoba et al., 2006) (http://on eearthher bs.square space.co m/disease s/benignprostatichyperplas iabph.html) . (Campbel l et al., 1997; Gazim et al., 2013) (Said et al., 2002) (Arora et al., 2012; Saeed et al., 2014; Singh et al., 2015) (Konate et al.,

cytotoxic properties

Moraceae

Broussonetia papyrifera L. (Vent.)

leaves

The plant is used traditionally as astringent, diuretic, vulnerary, tonic, ophthalmic, diaphoretic and laxative properties. The cytotoxicity activities against breast and cervical cancer cells have been reported recently.

Myoporaceae

Bontia daphnoides L.,

leaves

Myrtaceae

Myrtus communis L.

leaves

B. daphnoides has been used traditionally for Treating nephritis, hypertension, cough and colds. Its major secondary metabolite, Epingaione, has shown cytotoxic activity against Neuroblastoma and Sarcoma Cells. Inflammation, diarrhea, hemorrhoids, swelling, bleeding, urethritis, conjunctivitis, excessive perspiration, cough, pulmonary and skin diseases, antiseptic, pain, heartburn, antibacterial and anticancer properties.

Callistemon (Curtis) Skeels

citrinus

The leaves oils possess antimicrobial, fungitoxic, antinociceptive, anti-inflammatory and cytotoxic activities.

Eugenia uniflora L.

Rubiaceae

Gardenia H.Mann

Traditionally it is used for treatment of fever, anti-inflammatory, rheumatism, stomach diseases, disorders of the digestive tract, hypertension, antioxidant, yellow fever, and gout

brighamii

leaves

Some Gardenia species are used in African traditional medicine to treat Breast/uterine/skin cancer.

Pachystigma pygmaeum (Schltr.) Robyns

leaves

In South Africa, this plants has been described to cause “Gousiekte” which is described as plant toxicoses of livestock. Novel compound, Pavettamine, was isolated from the plant has been found to possess anticancer effect.

25

2014; Traore et al., 2007) (KUMA R et al., 2014; Pang et al., 2014) (Ayensu, 1981; Williams et al., 2007) (Alipour et al., 2014; Ogur, 2014; Rotstein et al., 1974; Tretiakov a et al., 2008) (Kumar et al., 2015; Oyedeji et al., 2009; Sudhakar et al., 2006) (Bagetti et al., 2011; Rattmann et al., 2012; Schapova l et al., 1994) (Ochwan g'i et al., 2014; Qin et al., 2015) (Bode et al., 2009)

Table 2: Voucher numbers of plant material used for cytotoxicity testing.

Voucher No. SA-CA-0001 SA-CA-0002 SA-CA-0003 SA-CA-0004 SA-CA-0005 SA-CA-0006 SA-CA-0007 SA-CA-0008 SA-CA-0009 SA-CA-0010 SA-CA-0011 SA-CA-0012 SA-CA-0013 SA-CA-0014 SA-CA-0015 SA-CA-0016 SA-CA-0017 SA-CA-0018 SA-CA-0019 SA-CA-0020 SA-CA-0021 SA-CA-0022 SA-CA-0023 SA-CA-0024 SA-CA-0025 SA-CA-0026 SA-CA-0027 SA-CA-0028 SA-CA-0029

Plant Name Artemisia afra Eugenia uniflora Gardenia brighamii Ginkgo biloba (fruits) Ginkgo biloba (leaves) Ginkgo biloba (root bark) Magnolia x soulangeana Maytenus bachmanii Cotinus coggygria Tetradenia riparia Callistemon citrinus Castanospermum australe Combretum kraussi Hypoestes aristata Laurus nobilis Trichilia emetica Acokanthera oppositifolia Orthosiphone serratus Pachystigma pygmaeum Pistacia vera Plectranthus barbatus Plectranthus ciliatus Salvia apiana Schinus molle leaves Schinus molle resin Bontia daphenoides Leonotis leonurus Myrtus communis Broussonetia papyrifera

26

Table 3: Secondary metabolites previously isolated from South-African medicinal plant extracts, which were cytotoxic towards sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. Plant species

Compounds

Acokanthera oppositifolia Hypoestes aristata

Acovenoside A, acofrioside L, acolongifloroside H, acovenoside C, acolongifloroside K, ouabain (Hauschild-Rogat P et al., 1967; Hauschild-Rogat P, 1967). 1(10)seco-fusicocc-3(4)-ene-5,11,14-trione-8 (9),1(7)-diepoxide, 13-hydroxy-7-oxo-labda-8, 14-diene (Al-Rehaily et al., 2002). Hypoestestatin and 14-hydroxy hypoestestatin (Pettit et al., 1984) Laurupene A, and B, ent-germacra-4(15),5,10(14)-trien-1α-ol, 15-acetoxycostuno-lide, 3αacetoxyeudesma-1,4(15),11(13)-trien-12,6α olide, santamarine, reynosin, 1β-hydroxyarbusculin A, zaluzanin D, zaluzanin C (Chen et al., 2014). hydroperoxide-magnolialide,1 -dihydroxy-,H-eudesma-4(15),11(13)-dien-12,6-olide, magnolialide, 3peroxyarmefolin, 11,13-dehydrosantonin, tubiferin, anhydroperoxycostunolide, lucentolide, baynol C, methyl-trihydroxy-H-eudesma-4(15), 11(13)-dien-12-oate, (3aS,5aR,6S,7R,9aR,9bS)-6-hydroxy-7acetoxy-5-methyl-3,9-dimethylidene-3a,4,5,6,7,8,9a,9b -octahydrobenzo[g][1]benzofuran-2-one; (3aS,5aR,6R,7R,9aS,9bS)-6,7-dihydroxy-5,9-dimethyl- 3-methylidene- 4,5,6,7,9a,9b -hexahydro-3-Hbenzo[g][1]benzofuran-2-one, (3aS,5aR,6R,7R,8R,9S,9aS,9bS)-6,7,8,9-diepoxy-5,9-dimethyl-3methylidene-5. 3a,4,5,6,7,8,9a,9b-octahydrobenzo[g][1]benzofuran-2-one, (3aS,5aR,6R,9S,9aS,9bS)6,9-dihydroxy-5,9-dimethyl-3-methylidene-6. 3,4,5,6,7,8,9,9-octahydrobenzo[g][1]benzofuran-2-one, (1S,2S,4aS)-decahydro-1-hydroxy-7-oxo-4,8-dimethyl–methylene-2-naphthaleneacetic acid methylester,1S,2S,4aR,5R,8R,8aS)-decahydro-1,5,8-trihydroxy- 4,8-dimethyl–methylene-2naphthaleneacetic acid methylester, (1S,2S,4aR,5R,6R,7R,8S,8aS)-decahydro-1-hydroxy-5,6,7,8diepoxy-4,8-dimethyl–methylene-2naphthaleneacetic acid methylester, (1S,2S,4aS,7R,8aR)-decahydro -1-hydroxy-7-acetoxy-4-methyl,8-bis(methylene)-2-naphthaleneacetic acid methylester, (1S,2S,4aS,7R,8aR)-decahydro-1,7dihydroxy-4a-methyl-,8-bis(methylene)- 2-naphthaleneacetic acid methylester, (3aS,5aR,6R,9R,9aS,9bS)-6-hydroxy-9-methoxy-5a,9-dimethyl-3- methylidene- 3a,4,5,6,7,8,9a,9boctahydrobenzo[g][1]benzofuran-2-one (Julianti et al., 2012). (+)-Actinodaphinine, (+)-boldine, (+)-reticuline, (+)-neolitsine, (+)-isodomesticine, (+)-cryptodorine, (+)-launobine, (+)-N-Methyl-actinodaphinine, (+)-nandigerine, (+)-nor-isodomesticine, actinodaphnine, reticuline, launobine, andactinodaphnine, (+)-Reticuline, (+)-launobine and(+)-nandigerine (Pech B and J., 1982). Costunolide, gazaniolide, spirafolide, lauroxepine (Barla A et al., 2007). Laurosides A-E, 2-(4-hydroxy-3-methoxyphenyl)-ethyl-O-D-glucopyranoside, benzyl alcohol, xylopyranosyl(1f6)glucopyranoside, lyoniside, 4,5-Dihydroblumenol A, Alangioside A, Ampelopsisionoside, Dendranthemoside A, Icariside B1, Citroside A, Lyoniside, Kaempferol-3-O-L(3,4-di-E-p-coumaroyl) rhamnoside, Kaempferol-3-O-L(2-E-p-coumaroyl) rhamnoside. (De Marino et al., 2004). 5,9-Dimethyl-3-methylene-3,3a,4,5,5a,6,7,8-octahydro-1-oxacyclopenta[c]azulen-2-one, 3chlorodehydrocostuslactone, dehydrocostuslactone, artremorin, costunolide (Dall'Acqua et al., 2006) 10-epigazaniolide (Fang et al., 2005). Marrubin, compounds X and Y (Kaplan and Rivett, 1968). R and S premarrubiin (Laonigro et al., 1979). Leonurun (McKenzie et al., 2006). (13S)-9α,13α-Epoxylabda-6β(19),15(14)- dioldilactone (Obikeze et al., 2008). 9,13-Epoxy-6-hydroxylabdan-16,15-olide, 9,13:15,16-diepoxy-6,16labdanediol, 6-acetoxy-9,13-epoxy-15-methoxy-labdan- 16,15-olide (Naidoo et al., 2011). leonurenones A – C, nepetifolin, luteolin, luteolin7-O-glucoside (He et al., 2012). Leoleorins D-J,16epi-leoleorin F, leoleorin B, leoleorin C (Wu et al., 2013), 1,2,3-Trihydroxy-3,7,11,15tetramethylhexadecan-1-yl-palmitate, succinic acid, uracil, luteolin7-O-glucoside, acteoside, geniposidic acid (Agnihotri et al., 2009). Apigenin 6-C-arabinoside-8-C-glucoside; apigenin 8-Cglucoside, apigenin7-O-glucoside, luteolin7-O-glucoside, luteolin7-O-glucoside-3`-methyl ether; apigenin 7-O-(6``-O-pcoumaroyl)-glucoside; 6-methoxyluteolin-4`-methyl ether, chryseriol; luteolin, apigenin (El-Ansari et al., 2009). Stachydrine (Kuchta et al., 2013).

Laurus nobilis

Leonotis leonurus

Plectranthus barbatus

plectranthone J, (16R)-coleon E, plectrin, coleon F, (16R)-plectrinon A, plectrinon B (Ruedi, 1986) coleon S, coleonT, coleon O, 3β-hydroxy-3-deoxybarbatusin (Zelnik et al., 1977) barbatusin, cyclobutatusin,7β-acetyl-12-deacetoxycyclobutatusin (de Albuquerque et al., 2007) 14-deoxycoleon U, 11-hydroxysugiol (Xu et al., 2005) 6,7-secoabietane diterpene I, 6,7-Secoabietane diterpene II (Kelecom et al., 1987)

27

P. ciliates Salvia apiana

cariocal (Kelecom and Dos Santos, 1985) manoyl oxide, abietatriene (dehydroabietane) (Mathela et al., 1986) ferruginol, barbatusol (Kelecom, 1983b) sugiol (Li et al., 2006) 20-deoxocarnosol (Kelecom, 1984) 6β-hydroxycarnosol (Kelecom, 1983a) coleon C, forskolin (coleonol; colforsin; 1-deacetylforskolin B, 6-deacetylforskolin J), 9deoxyforskolin,1,9-dideoxyforskolin, 1,9 dideoxy-7-deacetylforskolin (de Souza et al., 1983) deacetyl-1-deoxyforskolin, 11-Oxomanoyloxide (Gabetta et al., 1989) 6-acetyl-1-deoxyforskolin, 6-acetyl-1,9-dideoxyforskolin 12-hydroxy-8,13E-labdadien-15-oic acid] (Xu and Kong, 2006) 1,6-Di-O-acetylforskolin (forskolin A; 1,7-diaceylisoforskolin), 1-acetylforskolin (forskolin B), isoforskolin (coleonol B; forskolin C; 1-deacetylforskolin I), 7-deacetylforskolin, 6-deacetylisoforskolin (forskolin D), forskolin E (9-dehydroxyforskolin B), forskolin F (1-deoxyforskolin; 1-deacetoxyforskolin B; coleonol D) (Jin and He, 1998) 1,9-dideoxycoleonol, 1-acetoxy coleosol (Roy et al., 1993) forskolin G (l-deacetyl-6-acetylforskolin E), forskolin H (7-deacetoxy-9-dehydroxyforskolin A; plectromatin C), forskolin I (7-deacetylforskolin A; 1-acetylforskolin C), forskolin J (6-Oacetylforskolin;1-deacetylforskolin A; 7-acetylforskolin C), 1,6-diacetoxy-9-deoxyforskolin (forskolin K; 9-dehydoxyfoskolin A), forskolin L (Xu and Kong, 2004) coleosol (6β,9β-dihydroxy-11-oxomanoyloxide, coleol (Prakash et al., 1988) coleonol E, coleonol F (Painuly et al., 1979) deoxycoleonol, coleonol C (Tandon et al., 1978) 3-hydroxyforskolin, 3-hydroxyisoforskolin (Shan and Kong, 2006) 13-epi-9-deoxycoleonol (Tandon et al., 1992) coleonone (Katti et al., 1979) 13-epi-sclareol (Sashidhara et al., 2007) coleolic acid, coleonic acid (Liu et al., 2007) forskoditerpenoside A, forskoditerpenoside B (Shan et al., 2007) forskoditerpenoside C, forskoditerpenoside D, forskoditerpenoside E, forskoditerpene A (Shan et al., 2008) ------------13,14-dioxo-11-hydroxy-7-methoxy-hassane-8,11,15-trien-(22,6)-olide. (Luis et al., 1996a) 14Hydroxy-7-methoxy-11,16-diketo-apian -8-en-(22,6)-olide; 7-methoxy-11,16-diketoapian-8,14-dien(22,6)-olide (Luis et al., 1996b) 16-hydroxycarnosic (Dentali and Hoffmann, 1992) 6,7-didehydroferruginol; 6,7-didehydrosempervirol; 16-hydroxy-6,7-didehydroferruginol; 11,12,16trihydroxy-20 (10→5)abeo-abieta-1(10),6,8,11,13-pentaene; 16-hydroxyroyleanone; 6-deoxo-5,6didehydrolanugon Q, ferruginol, miltiodiol, cryptotanshinone, lanugon Q; salvicanol (Gonzalez et al., 1992) 16-hydroxycarnosic acid; carnosic acid (salvin) (Dentali and Hoffmann, 1990). α-amyrin; oleanolic; and ursolic acids (Pettit et al., 1966)

28

Table 4: 50% Inhibitory concentration (IC50) values of sensitive CCRF-CEM and multidrugresistant CEM/ADR5000 leukemia cells for cytotoxic active South-African medicinal plants, IC50 values are shown as mean values ± SD of three independent experiments with each six parallel measurements. Scientific name Leonotis leonurus Plectranthus barbatus Hypoestes aristata Salvia apiana Acokanthera oppositifolia Plectranthus ciliatus Laurus nobilis

IC50 (µg/mL) CCRF-CEM CEM/ADR5000 2.57± 0.37 8.6 ± 0.45 5.7 ± 1.29 7.93 ± 0.64 2.28 ± 0.16 3.8 ± 1.5 7.17 ± 0.67 9.91 ± 0.8 2.5 ± 0.06 2.85 ±0.08 1.57 ± 0.09 2.3 ± 0.06 3.47 ± 1.72 5.93 ± 0.9

Degree of resistance 3.35 1.39 1.67 1.38 1.14 1.46 1.71

Table 5: Correlation of log10IC50 values for acovenoside A and ouabain to gain of the chromosomal locus of the ABCB1 gene (7q21), expression of ABCB1 mRNA (by microarray and RT-PCR), and P-glycoprotein function (cellular rhodamine 123 accumulation) in the NCI cell line panel. Daunorubicin was used as positive control, as it is a well-known substrate of P-glycoprotein (Dalton et al., 1991). The analysis was performed by means of Pearson's rank correlation test. The verification of the microarray data by gene copy numbers, RT-PCR and rhodamine 123 accumulation as functional assay for P-glycoprotein as well as the values for the control drug daunorubicin have been reported by us (Kadioglu and Efferth, 2015).

7q21 (Chromosomal

Rvalue Locus of ABCB1 PGene) value ABCB1 Expression Rvalue (Microarray) Pvalue ABCB1 Expression Rvalue (RT-PCR) Pvalue Rhodamine 123 Rvalue Accumulation Pvalue * P < 0.05 and R > 0.3 (or R < -0.3)

ABCB1 Expression (Microarray)

ABCB1 Expression (RT-PCR)

Rhodamine 123 Accumulation

Acovenoside A (log10 IC50, M)

Ouabain

Daunorubicin

(log10 IC50, M)

(log10 IC50, M)

*0.966

0.012

*0.561

-0.038

-0.093

*0.597

*4.24×10-33

0.467

*5.08×10-6

0.392

0.254

*4.82×10-6

*0.799

*0.717

-0.045

-0.117

*0.684

*1.77×10-12

*8.65×10-11

0.368

0.191

*1.57×10-8

*0.564

-0.001

-0.145

*0.579

0.480

0.158

*4.19×10-6

-0.069

-0.117

*0.544

0.305

0.192

*1.51×10-5

-6

*8.30×10

29

Table 6: Correlation of log10IC50 values for acovenoside A and ouabain to expression of ABCB5 mRNA (by microarray analyses and RT-PCR) in the NCI cell line panel. Maytansine was used as positive control, as it is a well-known substrate of ABCB5 (Sztiller-Sikorska et al., 2014). The analysis was performed by means of Pearson's rank correlation test. The verification of the microarray data by RT-PCR as well as the values for the control drug maytansine have been reported by us (Kadioglu and Efferth, 2015). ABCB1 Expression (RT-PCR) ABCB5 Expression (Microarray) ABCB5 Expression (RT-PCR)

R-value P-value R-value P-value

*0.790 *3.24×10-14

Acovenoside A (log10 IC50, M) 0.129 0.166 0.176 0.096

Ouabain

Maytansine

(log10 IC50, M) 0.181 0.085 0.280 0.016

(log10 IC50, M) *0.454 *6.67×10-4 *0.402 *0.0034

* P < 0.05 and R > 0.3 (or R < -0.3)

Table 7: Correlation of log10IC50 values for acovenoside A and ouabain to DNA gene copy number and ABCC1 mRNA expression (by microarray and RT-PCR) in the NCI cell line panel. Vinblastine was used as positive control, as it is a well-known substrate of ABCC1 (Breuninger et al., 1995; Cole et al., 1994). The analysis was performed by means of Pearson's rank correlation test. The verification of the microarray data by gene copy numbers and RT-PCR as well as the values for the control drug vinblastine have been reported by us (Kadioglu and Efferth, 2015). ABCC1 Expression (Microarray)

ABCC1 Expression (RT-PCR)

DNA Gene

R-value

*0.388

0.115

Acovenoside A (log10 IC50, M) 0.140

Copy Number

P-value

*0.0001

0.220

0.145

0.137

*0.001

ABCC1 Expression

R-value

*0.449

-0.216

-0.237

*0.399

(Microarray)

P-value

*8.83×10-4

0.053

0.038

*0.002

ABCC1 Expression

R-value

-0.036

-0.329

0.299

(RT-PCR)

P-value

0.007

0.019

*0.036

* P < 0.05 and R > 0.3 (or R < -0.3)

30

Ouabain

Vinblastine

(log10 IC50, M) -0.145

(log10 IC50, M) *0.429

Table 8: Correlation of log10IC50 values for acovenoside A and ouabain to expression of ABCG2 mRNA (by microarray and RT-PCR) and ABCG2 protein (by Western blot) in the NCI cell line panel. Pancratistatin was used as positive control, as it is a well-known substrate of ABCG2 (Deeken et al., 2009). The analysis was performed by means of Pearson's rank correlation test. The verification of the microarray data by RT-PCR and Western blot as well as the values for the control drug pancratistatin have been reported by us (Kadioglu and Efferth, 2015). ABCG2 Expression ABCG2 Expression (RT-PCR) (Western Blot) ABCG2 Expression

R-value

*0.391 *0.001

*0.905

Acovenoside Ouabain Pancratistatin A (log10 IC50, M) (log10 IC50, M) (log10 IC50, M) -0.047

-0.094

*0.323

*8.59×10

0.364

0.243

*0.006

0.199

0.187

0.071

-23

(Microarray)

P-value

ABCG2 Expression

R-value

*0.370

(RT-PCR)

P-value

*0.002

0.065

0.078

0.295

ABCG2 Expression

R-value

-0.026

-0.074

*0.346

(Western Blot)

P-value

0.423

0.291

*0.004

* P < 0.05 and R > 0.3 (or R < -0.3)

Table 9: Correlation of log10IC50 values for acovenoside A and ouabain to EGFR gene copy number, expression of EGFR mRNA (by microarray and RNAse protection assay) and EGFR protein (by protein array) in the NCI cell line panel. Erlotinib was used as positive control, as it is a well-known tyrosine kinase inhibitor of EGFR (Bulgaru et al., 2003). The analysis was performed by means of Pearson's rank correlation test. The verification of the microarray data by gene copy numbers, RNAse protection assay and protein array as well as the values for the control drug erlotinib have been reported by us (Kadioglu and Efferth, 2015).

EGFR Gene

Rvalue Copy Number Pvalue EGFR Expression Rvalue (Microarray) Pvalue EGFR Expression Rvalue (RNAse Protection) Pvalue EGFR Expression Rvalue

EGFR Expression

EGFR Expression

EGFR Expression (Protein Array)

EGFR Expression (Western Blot)

Ouabain

Erlotinib

0.221

Acovenoside A (log10 IC50, M) -0.229

(Microarray) *0.638

(RNAse Protection) *0.428

(log10 IC50, M) -0.179

(log10 IC50, M) -0.245

*0.455

*2.11×10-8

*4.03×10-04

*1.48×10-4

*0.045

0.041

0.087

*0.029

*0.639

*0.710

*0.628

-0.191

-0.277

*-0.458

*3.46×10-8

*1.51×10-10

*4.01×10-8

0.07

0.017

*1.15×10-4

*0.448

*0.438

-0.035

-0.007

*0.409

*2.35×10-4

*2.97×10-4

0.398

0.478

*7.08×10-4

*0.542

-0.334

-0.210

*-0.376

31

*4.73×10-6

(Protein Array)

Pvalue * P < 0.05 and R > 0.3 (or R < -0.3)

0.005

0.058

Table 10: Correlation of log10IC50 values for acovenoside A and ouabain to mutational status of TP53 and pan-RAS (H-,K-, and N-RAS) in the NCI cell line panel. Cisplatin and melphalan, respectively, were used as positive controls, as TP53 and pan-RAS are cellular determinants for tumors’ response to these drugs (Fajac et al., 1996; Hoang et al., 2006). The analysis was performed by means of Pearson's rank correlation test.

TP53 Mutation pan-RAS Mutation

Acovenoside A (log10 IC50, M)

Ouabain

Cisplatin

(log10 IC50, M)

(log10 IC50, M)

R-value

-0.180

-0.134

*-0.502

P-value

0.097

0.165

*3.50×10-5

N.D.

R-value

-0.228

-0.140

N.D.

0.367

P-value

0.044

0.150

N.D.

0.002

* P < 0.05 and R > 0.3 (or R < -0.3)

32

Melphalan

N.D.

*0.001

Figure 1: Cytotoxicity of 29 extracts from 26 South-African medicinal plants towards CCRF-CEM cells and their P-glycoprotein-expressing, multidrug-resistant subline CEM/ADR5000 at a fixed concentration of 10 µg/mL as determined by the resazurin assay. The bars show mean values ± SD of two independent experiments with each six parallel measurements.

33

Figure 2: Dose response curves of A: Leonotis leonurus, B: Plectranthus barbatus, C: Hypoestes aristata, D: Salvia apiana, E: Acokanthera oppositifolia, F: Plectranthus ciliatus, G: Laurus nobilis towards drug-sensitive parental CCRF-CEM tumor cells and their P-gpexpressing, multidrug-resistant subline, CEM/ADR5000 as determined by the resazurin assay. Mean values and standard deviations of each three independent experiments with each six parallel measurements are shown. 34

Figure 3: Mean log10IC50 values of selected cytotoxic phytochemicals from South-African medicinal plants for tumor cell lines of the NCI drug screening panel. Mean values and standard deviations for the entire tumor cell line panel (A) or grouped according to the tumor origin of the cell lines (B, C). Figure 4: Oncobiogram for ambrosin using the NCI cell line panel. Shown is a correlation profile of different classes of a total number of 85 standard anticancer agents. The log10IC50 values are deposited at the NCI database (http://dtp.nci.nih.gov). The calculations were performed using Pearson correlation test with a cutoff of R≥0.3 and P≤0.05. These drugs were derived from different drug classes:  Alkylanting agents (busulfan, dacarbazine, thiotepa, carmustine, lomustine, semustine, bendamustine, streptozocin, chlorambucil, ifosfamide, mafosfamide, melphalan, azathioprine)  Platin derivatives (cisplatin, carboplatin, oxaliplatin)  Antimetabolites (methotrexate, trimethrexate, pemetrexed, trimetrexate, hydroxyurea, cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, 6-thioguanine, cytarabine, 5-fluorouracil, gemcitabine, tegafur)  Taxanes (paclitaxel, docetaxel)  Vinca alkaloids (vinblastine, vincristine)  Anthracyclines (+ Topo II inhibitors) (doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone)  Antibiotics (bleomycin, dactinomycin, mitomycin C, mithramycin)  Epipodophyllotoxins (+ Topo II inhibitors) (etoposide, teniposide, amsacrine)  Camptothecins (Topo I Inhibitoren) (camptothecin, irinotecan, topotecan)  Antihormones (tamoxifen, fulvestrant, toremifen, anastrozol, exemestane, megostrol, raloxifene)  Tyrosine kinase inhibitors (axitinib, crizotinib, dasotinib, erlotinib, gamitinib G-TPP, gamitinib G4, gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib, vandetanib, vemurafenib)  mTOR inhibitors (everolimus, temsirolimus, sirolimus, sirolimus derivative NSC758664)  Epigenetic inhibitors (azacytidine, 3-azacytidine, decitabine, dihydro-5-azacytidine, vorinostat, vorinostat derivate NSC759852)  Varia (all-trans-retinoic acid, asparaginase, bortezomib, zoledronate, estramustine).

35

-7

-7.2

-7.4

-7.6

-7.8

-8 Colon cancer

Leukemia Lung Cancer Renal cancer

Prostate cancer Lung Cancer Renal cancer

Ovarian cancer Prostate cancer

Leukemia

CNS cancer Ovarian cancer

Melanoma CNS cancer

Breast cancer

Breast cancer

C. Ouabain

Colon cancer

log10IC50 (M)

Limonene Limonene Boldine Marrubin Marrubin Coleonol Forskolin Forskolin Carnosic acid Carnosol Carnosol Apigenin Acolongofloroside Acolongofloroside Ursolic acid Zaluzanin Zaluzanin C Cryptotanshinone Acovenodide AcovenosideAA Ouabain

-9

Melanoma

log10IC50 (M)

log10IC50 (M)

A.Selected phytochemicals -2

-3

-4

-5

-6

-7

-8

B. Acovenoside A

-6.2

-6.4

-6.6

-6.8

-7

-7.2

-7.4

-7.6

36

A. Acavenoside A

Varia (0/5)

Alkylantin agents (0/13) 35

Platin compounds (1/3)

30 25

Antihormones (0/7)

Antimetabolites (1/14)

20 15 10

Epigenetic inhibitors (0/6)

Taxanes (0/2)

5 0 mTOR inhibitors (0/4)

Vinca alkaloids (0/2)

Tyrosine kinase inhibitors (1/14)

Anthracyclines + Topo II inhibitors (0/5)

Epipodophyllotoxines + Topo II inhibitors Antibiotics (1/4) (0/3) Camptothecins (Topo I inhibitors) (1/3)

B. Ouabain

Varia (0/5)

Alkylantin agents (2/13) 35

Platin compounds (1/3)

30 25

Antihormones (0/7)

Antimetabolites (1/14)

20 15 10

Epigenetic inhibitors (0/6)

Taxanes (0/2)

5 0 mTOR inhibitors (1/4)

Vinca alkaloids (0/2)

Tyrosine kinase inhibitors (2/14)

Anthracyclines + Topo II inhibitors (0/5)

Epipodophyllotoxines + Topo II inhibitors Antibiotics (1/4) (0/3) Camptothecins (Topo I inhibitors) (1/3)

C. Doxorubicin

Varia (1/5)

Antihormones (1/7)

Epigenetic inhibitors (1/6)

Alkylantin agents (8/13) 100 Platin compounds (2/3) 90 80 70 Antimetabolites (8/8) 60 50 40 30 Taxanes (2/2) 20 10 0

mTOR inhibitors (1/4)

Vinca alkaloids (2/2)

Tyrosine kinase inhibitors (3/14)

Anthracyclines + Topo II inhibitors (4/5)

Antibiotics (3/4)

Epipodophyllotoxines + Topo II inhibitors (3/3)

Camptothecins (Topo I inhibitors) (3/3)

Graphical abstract

37

Cytotoxcity of South-African plants against MDR tumor cells

38