**3. In vitro anticancer activity of phytochemicals and extracts of medicinal plants**

Cultured cancer-derived cell lines with comparison to normal healthy cell lines are commonly used to assess the anticancer properties of isolated phytochemicals and extracts of medicinal plants (Table 2). The anticancer properties of ethanolic extract of leaves, pulp and seeds of, *Annona glabra* (L.), commonly known as pond apple, were shown, using human drug-sensitive leukemia (CEM) and its multidrug-resistant-derived (CEM/VLB) cell lines [52]. The most potent anticancer activity was shown in the seed extract of *A. glabra* [52]. Both dried rhizome hexane extract and dried fruit hexane extract, partitioned from total methanol extract, of *Aralia nudicaulis* (L.) caused death of cancer cell lines such as human colon cancer cell (WiDr), human leukemia cell (Molt) and human cervix cancer cell (HeLa) at a lower concentration, than that of required for the death of normal cells [53]. Eupatoriopicrin, a sesquiterpene lactone isolated from *Eupatorium cannabinum* (L.) (Bonesets), indicated anticancer properties on FIO 26 (fibrosarcoma) cells with an IC50=1.5 µg/ml [22]. Methanolic extracts of *Hypericum perforatum* (L.) (St. Johns wort) possessed strong antiproliferative activity in the human prostate cell line (PC-3) and the major constituents, hyperforin and hypericin, synergistically contributed to the reduction of the PC-3 cells proliferation [24]. Maslinic acid, (Figure 2(a)) a triterpene from *Olea europaea* (L.) (Olive), has shown to be significantly inhibitory in cell proliferation of the human colorectal adenocarcinoma cell line (HT29) in a dose dependent manner [33]. The major components in the extract were identified to be oleuropein, hydroxytyrosol, hydroxytyrosol acetate, luteolin-7-*O*-glucoside, luteolin-4'-*O*-glucoside and luteolin [34] (Figure 2). All these phytochemicals inhibited the proliferation of cancer and endothelial cells with IC50, at the low micromolar range [37]. Methanolic leaf extract of *Plantago lanceolata* (L.) (Ribwort plantain) inhibited the growth of three different cell lines; human renal adenocarcinoma (TK-10), the human breast adenocarcinoma (MCF-7) and the human melanoma (UACC-62) cell lines and the MCF-7 was totally inhibited [54]. Further, the ethanolic extract of *P. lanceolata* (L.), produced by maceration with ethanol : water, showed significant antiproliferative activity on cervix epithelioid carcinoma (HeLa), breast adenocarcinoma (MCF-7), colon adenocarcinoma (HT-29) and human fetal lung carcinoma (MRC-5) [38]. Several chemical constituents (Figure 3) from *Silybum marianum* (Milkthistle) have been isolated and their cytotoxic and anticancer potential has been investigated, *in vitro*, using both cancer and normal healthy cell lines. Silymarin, isolated from seeds of *S. marianum*, is a mixture of series of flavolignans, major constituents being: silybin A and B, (also known as silibinin), isosilybin A and B, silychristin, and silydianin [55, 56].

differentiation, modulating apoptosis and signal transduction pathways and protecting intercellular communications [10]. In addition, tumor progression can also be inhibited by affecting the hormonal status and the immune system in various ways and by inhibiting tumor

**3. In vitro anticancer activity of phytochemicals and extracts of medicinal**

Cultured cancer-derived cell lines with comparison to normal healthy cell lines are commonly used to assess the anticancer properties of isolated phytochemicals and extracts of medicinal plants (Table 2). The anticancer properties of ethanolic extract of leaves, pulp and seeds of, *Annona glabra* (L.), commonly known as pond apple, were shown, using human drug-sensitive leukemia (CEM) and its multidrug-resistant-derived (CEM/VLB) cell lines [52]. The most potent anticancer activity was shown in the seed extract of *A. glabra* [52]. Both dried rhizome hexane extract and dried fruit hexane extract, partitioned from total methanol extract, of *Aralia nudicaulis* (L.) caused death of cancer cell lines such as human colon cancer cell (WiDr), human leukemia cell (Molt) and human cervix cancer cell (HeLa) at a lower concentration, than that of required for the death of normal cells [53]. Eupatoriopicrin, a sesquiterpene lactone isolated from *Eupatorium cannabinum* (L.) (Bonesets), indicated anticancer properties on FIO 26 (fibrosarcoma) cells with an IC50=1.5 µg/ml [22]. Methanolic extracts of *Hypericum perforatum* (L.) (St. Johns wort) possessed strong antiproliferative activity in the human prostate cell line (PC-3) and the major constituents, hyperforin and hypericin, synergistically contributed to the reduction of the PC-3 cells proliferation [24]. Maslinic acid, (Figure 2(a)) a triterpene from *Olea europaea* (L.) (Olive), has shown to be significantly inhibitory in cell proliferation of the human colorectal adenocarcinoma cell line (HT29) in a dose dependent manner [33]. The major components in the extract were identified to be oleuropein, hydroxytyrosol, hydroxytyrosol acetate, luteolin-7-*O*-glucoside, luteolin-4'-*O*-glucoside and luteolin [34] (Figure 2). All these phytochemicals inhibited the proliferation of cancer and endothelial cells with IC50, at the low micromolar range [37]. Methanolic leaf extract of *Plantago lanceolata* (L.) (Ribwort plantain) inhibited the growth of three different cell lines; human renal adenocarcinoma (TK-10), the human breast adenocarcinoma (MCF-7) and the human melanoma (UACC-62) cell lines and the MCF-7 was totally inhibited [54]. Further, the ethanolic extract of *P. lanceolata* (L.), produced by maceration with ethanol : water, showed significant antiproliferative activity on cervix epithelioid carcinoma (HeLa), breast adenocarcinoma (MCF-7), colon adenocarcinoma (HT-29) and human fetal lung carcinoma (MRC-5) [38]. Several chemical constituents (Figure 3) from *Silybum marianum* (Milkthistle) have been isolated and their cytotoxic and anticancer potential has been investigated, *in vitro*, using both cancer and normal healthy cell lines. Silymarin, isolated from seeds of *S. marianum*, is a mixture of series of flavolignans, major constituents being: silybin A and B, (also known as silibinin), isosilybin A and B, silychristin,

angiogenesis [10].

and silydianin [55, 56].

**plants**

**IC50:** Concentration which inhibited 50% of cell proliferation; **MOA:** Mode of Action; **BBCE:** Bovine Brain Capillary En‐ dothelial cells; **T24:** Human Urinary Bladder Carcinoma cells; **MCF-7:** Human Breast Adenocarcinoma cells; **HL60:** Hu‐ man Promyelocytic Leukaemia cells; **HT29:** Colon Adenocarcinoma cells

(a) Maslinic acid - [(2a, 3b)-2,3-dihydroxyolean-12-en-28-oic acid]: (b) Oleuropein – [(4S,5E,6S)-4-[2-[2-(3,4-dihydroxy‐ phenyl)ethoxy]-2-oxoethyl]- 5-ethylidene-6-[[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)- 2-tetrahydropyran‐ yl]oxy]-4H-pyran-3-carboxylic acid, methyl ester] : (c) Hydroxytyrosol – [4-(2-Hydroxyethyl)-1,2-benzenediol]: (d) Hydroxytyrosol acetate – [2-(3,4-dihydroxy)Phenyl ethyl acetate;4-[2-(Acetyloxy)ethyl]-1,2-benzenediol]: (e) Luteolin-7-O-glucoside - [2-(3,4-dihydroxyphenyl)-5-hydroxy-4-oxo-4H-chromen-7-yl beta-D-glucopyranoside; 4H-1-benzopyr‐ an-4-one, 2-(3,4-dihydroxyphenyl)-7-(beta-D-glucopyranosyloxy)-5-hydroxy-; 2-(3,4-Dihydroxy-phenyl)-5-hydroxy-7- ((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one] (f) Luteolin-4'-Oglucoside – [3',5,7-Trihydroxy-4'-(β-D-glucopyranosyloxy)flavone;2-(4-β-D-Glucopyranosyloxy-3-hydroxyphenyl)-5,7 dihydroxy-4H-1-benzopyran-4-one;2-[3-Hydroxy-4-(β-D-glucopyranosyloxy)phenyl]-5,7-dihydroxy-4H-1 benzopyran-4-one] (g) Luteolin - [2-(3,4-Dihydroxyphenyl)- 5,7-dihydroxy-4-chromenone]

**Figure 2.** Major bio-active compounds present in *Olea europaea* (a,b,c,d,e,f and g) and *Plantago lanceolata* (f and g)

8

Silybin possessed a dose-dependent growth inhibitory effect on parental ovarian cancer cells (OVCA 433), drug-resistant ovarian cancer cells (A2780 WT) and doxorubicin (DOX)-resistant breast cancer cells (MCF-7) [55]. Both L and D diastereoisomers of silybin inhibited A2780 WT cell growth at low IC50 reported with L-diastereoisomer [55]. Furthermore, silybin potentiated the effect of Cisplatin (CDDP, a platinum analog; cis-diamminedichloroplatinum [II]) in inhibiting A2780 WT and CDDP-resistant cell growth. Cisplatin is an inorganic metal com‐ plex which acts as an alkylating agent [57]. Similar results recorded with doxorubicin (DOX) on MCF-7 DOX-resistant cells when silybin associated with doxorubicin. Doxorubicin ((7S,9S)-7- [(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyace tyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione) is an anthracycline antibiotic isolated from *Streptomyces peucetius* var *caesius* [57]. The effect of silybin-CDDP and silybin-DOX combinations resulted in a synergistic action, as assessed by the Berembaum isobole method [55]. Silymarin demonstrated to have marked inhibition of cell proliferation with almost 50% inhibition in a time dependent manner on the human breast cancer cell line (MDA-MB 468), at 25 µg/mI concentration, after five days of treatment. Its potential anticancer activity was dose dependent and showed a complete inhibition of cancer cells at 50 and 75 µg/mI concentra‐ tions at the beginning of Day 2 of exposure [56]. Induction of apoptotic cell death of human prostate cancer (DU145) treated with silibinin is shown to be due to activation of caspase 9 and caspase 3 enzymes [58].
