**5. Mode of action of selected phytochemicals of North American medicinal plants**

Apoptosis (programmed cell death) is the principal mechanism through which unwanted or damaged cells are safely eliminated from the body. This programmed cell death is mediated via either an extrinsic apoptotic pathway or an intrinsic apoptotic pathway [65]. These two apoptosis signaling pathways differ in the origin of their apoptosis signal, but converge upon a common pathway [66].

The extrinsic pathway is initiated by the stimulation of the cell surface 'death receptor' due to the binding of death ligand and the intrinsic pathway is also known as the mitochondrial pathway in which an intracellular apoptotic signal initiates the process [68]. Various natural extracts, obtained from medicinal plants grown in North America, have been found to induce apoptosis pathways at different levels (Figure 4 and Table 04). Leaf extract of *Achyranthes aspera* activated caspase-3 and induced caspase-3 mRNA in tumor cells. It also decreased Akt-1 transcription, as well its phosphorylation. Suppression of pAkt-1 and a corresponding activation of caspase 3 by the leaf extract, induced apoptosis of tumor cells [14]. It was also found that maslinic acid, isolated from *O. europaea,* inhibited considerably the expression of Bcl-2 (B-cell lymphoma 2), whilst increasing that of Bax. Maslinic acid stimulated the release of mitochondrial cytochrome-c and activated caspase-9 and caspase-3 [33]. These results showed the activation of the mitochondrial apoptotic pathway, in response to the treatment

**IC50:** Concentration which inhibited 50% of cell proliferation; **MOA:** Mode of Action; **MCF-7:** Doxorubicin-resistant breast cancer cells; **OVCA 433:** Parental ovarian cancer cells; **A2780:** Drug-resistant ovarian cancer cells; **22Rv1, LAPC4, LNCaP, DU 145:** Human prostate cancer cells

Figure 3 (a) Silybin A – [(2*R*,3*R*)-3,5,7-trihydroxy-2-[(2*R*,3*R*)-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydroben‐ zo[*b*] [1,4]dioxin-6-yl]chroman-4-one]: (b) Silybin B – [(2*R*,3*R*)-3,5,7-trihydroxy-2-[(2*S*,3*S*)-3-(4-hydroxy-3-methoxy‐ phenyl)-2-(hydroxymethyl)-2,3-dihydrobenzo[*b*] [1,4]dioxin-6-yl]chroman-4-one]: (c) Isosilybin A –[(2*R*,3*R*)-3,5,7- Trihydroxy-2-[(2*R*,3*R*)-2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydrobenzo[1,4]dioxin-6-yl]-4-

chromanone]: (d) (c) Isosilybin B –[(2*R*,3*R*)-3,5,7-Trihydroxy-2-[(2*R*,3*R*)-2-(4-hydroxy-3-methoxyphenyl)-3- (hydroxymethyl)-2,3-dihydrobenzo[1,4]dioxin-6-yl]-4-chromanone]: (e) Silychristin – [(2R,3R)-3,5,7-trihydroxy-2-[(2R, 3S)-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1-benzofuran-5-yl]-2,3-dihydrochro‐ men-4-one]:

**Figure 3.** Major bio-active flavonolignans present in *Silybum marianum*

of HT29 colon-cancer cells with maslinic acid. The major flavonoid present in *P. lanceolata,* luteolin-7-*O*-β-glucoside, as well as aglycon luteolin, acted as potent poisons for DNA topoisomerase I on cancer cell lines [54]. Silibinin (major bioactive component from *S. maria‐ num*) markedly activated the DNA-PK-p53 pathway for apoptosis, in response to UVBinduced DNA damage [69]. DNA-PK pull-down assay showed that silibinin pre-treatment strongly increased binding of DNA protein kinase with p53 [69].

20(D)

 */ Apoptosis inductionb*

90-180*<sup>b</sup>*

32*<sup>a</sup>* 20.5*<sup>a</sup>*

**IC50:** Concentration which inhibited 50% of cell proliferation; **MOA:** Mode of Action; **MCF-7:** Doxorubicin-resistant breast cancer cells; **OVCA 433:** Parental ovarian cancer cells; **A2780:** Drug-resistant ovarian cancer cells; **22Rv1,**

(a) Silybin A – [(2*R*,3*R*)-3,5,7-trihydroxy-2-[(2*R*,3*R*)-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydroben‐ zo[*b*] [1,4]dioxin-6-yl]chroman-4-one]: (b) Silybin B – [(2*R*,3*R*)-3,5,7-trihydroxy-2-[(2*S*,3*S*)-3-(4-hydroxy-3-methoxy‐ phenyl)-2-(hydroxymethyl)-2,3-dihydrobenzo[*b*] [1,4]dioxin-6-yl]chroman-4-one]: (c) Isosilybin A –[(2*R*,3*R*)-3,5,7- Trihydroxy-2-[(2*R*,3*R*)-2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydrobenzo[1,4]dioxin-6-yl]-4 chromanone]: (d) (c) Isosilybin B –[(2*R*,3*R*)-3,5,7-Trihydroxy-2-[(2*R*,3*R*)-2-(4-hydroxy-3-methoxyphenyl)-3- (hydroxymethyl)-2,3-dihydrobenzo[1,4]dioxin-6-yl]-4-chromanone]: (e) Silychristin – [(2R,3R)-3,5,7-trihydroxy-2-[(2R, 3S)-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1-benzofuran-5-yl]-2,3-dihydrochro‐

**(e) Silychristin (f) Silydianin** 

*-*

*22Rv1 LAPC4 LNCaP DU 145* 

90-180*<sup>b</sup>*

*-*

*Metabolite* 

*Metabolite IC50(µg/ml)a*

90-180*<sup>b</sup>*

*-*

**LAPC4, LNCaP, DU 145:** Human prostate cancer cells

**Figure 3.** Major bio-active flavonolignans present in *Silybum marianum*

Isosilybin A Isosilybin B

Figure 3

men-4-one]:

Silybin 4.8-24 14(L)

168 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

**(a) Silybin A (b) Silybin B** 

 **(c) Isosilybin A (d) Isosilybin B** 

Increases p53 level

*IC50(µg/ml) MOA Ref MCF7 OVCA 433 A2780* 

25 Induce G1 arrest

Inhibits STAT3 activation Up-regulates DNA-Protein Kinasedependent p53 Activation

 *MOA Ref* 

Activates Akt–NF-kB–AR axis and increases p53 level

56 58 67

68, 69

69



Anticancer Properties of Phytochemicals Present in Medicinal Plants of North America http://dx.doi.org/10.5772/55859 171


**Plant Extraction**

Linum Usitatissimum (Common flax)

Olea europrae (Olive)

Plantago lanceolata (Ribwort plantain)

Rhodiola rosea (Golden root)

Saponaria vaccaria (Cowherb)

**solvent and concentration**

maslinic acid 0– 100 µg/mL

Aqueous extract and methanol artificial mixture

Methanolic extract

Extracted by maceration with ethanol/water during 72 hr at

70% Methanol extract

room temperature

Ethanol extract Breast (MCF-7,

170 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

**Type of cancer cell line**

MDA-MB-231)

Breast (MCF-7), Human urinary bladder (T-24), Bovine brain (BBCE)

Renal (TK-10), breast (MCF-7), melanoma (UACC-62)

cervix epitheloid (HeLa),breast (MCF-7), colon (HT-29), fetal lung

(MRC-5)

(RT4, UMUC-3, T24, 5637, J82)

colon (WiDr), breast (MDA-MB-231), lung (NCI-417), prostate (PC-3),

Not specified Urinary bladder

**IC50 or growth reduction**

Growth reduction of 15.8% in MCF-7 and 11.4% in MDA-MB-231

**Key findings Ref**

reduction of the PC-3 cells

Significantly reduced cell growth and induced apoptotic

dose-dependent manner and causes apoptotic death

Antiproliferative activity of the extracts should mainly be attributed to its identified

Growth of MCF-7 was totally

Selectively inhibit the growth of cancer cell lines with minimal effect on nonmalignant cells

Dose-dependent growth inhibitory and selective apoptosis-inducing activity. Strong in a breast and a prostate cancer cell lines

phytochemicals

inhibited

Showed significant antiproliferative activity

proliferation

cell death

Colon (HT29) 28.8 µg/ml Cell proliferation inhibition in a

72 (MCF-7), 100 (T-24), and 62 (BBCE) for aq. 565 (MCF-7), 135 (T-24), and 42 (BBCE) for methanol µg/ml

"/>250 (TK-10), 47.2 (MCF-7), 50.6 (UACC-62)

μg/mI

172.3 (HeLa), 142.8 (MCF-7), 405.5 (HT-29), 551.7 (MRC-5) µg/ml

264 (RT4),100 (UMUC-3), 71 (T24), 151 (5637), 165 (J82) μg/ml

3.8-9.4 (WiDr), 11.4-19.6 (MDA-MB-231), 12.6-18.4

**.**

31

33

34

54

38

41

43

**Table 2.** Anti-cancer properties of phytochemicals and extracts of medicinal plants revealed from *in vitro* studies using cancer cell lines



**Table 3.** Anti-cancer properties of medicinal plants revealed from *in vivo* studies using experimental animals

**Plant Preparation Animal model**

and residue was dissolved in methanol

Eupatoriopicrin, a sesquiterpene lactone

Lantadene A, pentacyclic triterpenoid

Eupatorium cannabinum (Bonesets)

Hypericum perforatum (Orange root)

Lantana camara (Wild sage)

Silybum marianum (Milkthistle) **used**

172 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

Methanolic extract Human prostatic

Silibinin Lung - Male

Silibinin Prostate - A

Silibinin Skin - SKH-1

Silibinin Athymic (BALB/

Topically applied silibinin in acetone or oral feeding of

silibinin

Syngeneic C57B1 female mice

carcinoma cell

orthotopically implanted athymic male nude mice

Female Swiss albino mice (LACCA)

B6/129- Nos2tm1Lau (iNOS-/-) and B6/129PF2 WT

mice

transgenic adenocarcinoma of mouse prostate (TRAMP)

model

hairless mouse

c,nu/nu) male nude mice

**Table 3.** Anti-cancer properties of medicinal plants revealed from *in vivo* studies using experimental animals

line

i.v. injection of 20 or 40 mg/kg

ip with a dose of 15 mg/kg dissolved in 1% DMSO

50 mg/kg body weight twice a week for 20 weeks

742 mg/kg body weight for 5 d/wk for 18 weeks

Purified diet containing 0% and 1% (w/w) silibinin

acetone/mouse or 1% in diet

200 mg/kg body weight, 5 d/wk for 33 days

1% (w/w) silibinin in diet for

2 weeks

until

Skin – mouse 9 mg in 200 ml

**Dosage Key findings Ref.**

In one treated mouse tumor completely disappeared

22

24

59

60

61

62

63

64

Significantly stronger growth delay of both lung tumours and fibrosarcoma

effects

Inhibited tumor growth by 70% with no observed side

Activity could be linked to the expression of transcriptional factors

Significantly decreases urethane-induced tumor number and size in WT mice.

multiplicity in WT mice, but not in iNOS-/- mice

Decreased the weight of tumor + prostate + seminal vesicle. Significantly decreased tumor angiogenesis and proliferation and increased

silibinin (both topical and oral) strongly inhibited UVBinduced skin tumorigenesis in long-term study

Strong suppression of UVBinduced damage by dietary feeding of silibinin

Significantly inhibits human NSCLC A549 tumor xenograft growth in a time dependent manner

Decreased tumor

apoptosis also.

**Figure 4.** Schematic representation of current knowledge of mode of action of some selected anticancer phytochemi‐ cals in North America (in a hypothetical cancer cell).

Akt (Protein kinase B); Bcl-2 (Protein kinase B); Bax (Bcl-2–associated X protein); Topo-1 (Topoisomerase 1); p53 (tumor protein 53); Ser15 (Serine 15); NF-kB (nuclear factor kappalight-chain-enhancer of activated B cells); AR (Androgen Receptor)



**Table 4.** Mode of action of anticancer activity of phytochemicals present in selected North American medicinal plants

#### **6. Conclusion**

Currently, natural products, especially plant secondary metabolites such as isoprenoids, phenolics and alkaloids, have been demonstrated to be the leading providers of novel anticancer agents. Thiese important groups of phytochemicals represent a vast majority of chemical groups, including alkaloids, flavonoids, flavonols, flavanols, terpenes and terpe‐ noids, phenols, flavonolignans and steroids. Potential anticancer properties of these phyto‐ chemicals have been shown by both cell culture (*in vitro* methods) and animal (*in vivo* methods) studies. However, *in vitro* and *in vivo* findings should be strengthened by valid human clinical trial data before introducing to the medicine cabinet as natural therapeutics or drugs.
