**4. Evidence from animal studies for anticancer activity of North American medicinal plants**

Anticancer and antiproliferative potential of some North American medicinal plants has also been studied in animal studies (Table 3). *In vivo* antitumor activities of *Achyranthes aspera* (L.) (Devil's Horsewhip) on athymic mice, with are subcutaneous xenograft, harboring human pancreatictumorweredemonstrated,usingtheleafextract.Theleafextractsignificantlyreduced both tumor weight and volume in mice treated with leaf extract intraperitonealy [14]. Intrave‐ nous administration of 40 mg/kg body weight eupatoriopicrin, a sesquiterpene lactone present in *E. cannabinum,* significantly delayed the growth of tumor in Lewis lung tumour-bearing syngeneic C57B1 female mice [22]. A 70% inhibition of tumor growth in PC-3 cells, orthopedi‐ cally implanted into the dorsal prostatic lobe in athymic nude mice, was observed, upon their receiving 15 mg/kg intraperitoneal *H. perforatum* methanolic extract [24]. Lantadene A is a pentacyclictriterpenoid,isolatedfromtheweed,*Lantanacamara*(L)[59].FeedingoffemaleSwiss albino mice (LACCA) with a dose of 50 mg/kg body weight of Lantadene A twice a week for 20 weeks, showed potential chemopreventive activity. This chemopreventive activity could be linked to the expression of transcriptional factors and a significant decrease in the mRNA expression of AP-1 and c-fos), NF-kB (p-65) and p53 was observed in Lantadene A treated mice skin tumors [59]. Silibinin decreased tumor multiplicity by 71% (P < 0.01) in wild type mice, but did not show any such considerable effect in iNOS-/- mice upon oral feeding of 742 mg/kg body weight silibinin for 5 days per week for 18 weeks [60]. Lesser effects of silibinin in iNOS-/- mice suggested that most of its chemopreventive and angiopreventive effects were through its inhibition of iNOS expression in lung tumors [60]. Treatment of a purified diet, containing 0.5% to 1.0% silibinin on a transgenic adenocarcinoma of are mouse prostate (TRAMP) model, decreased the weight of the tumor in both the prostate and seminal vesicle, when compared with control mice [61].

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

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

**4. Evidence from animal studies for anticancer activity of North American**

Anticancer and antiproliferative potential of some North American medicinal plants has also been studied in animal studies (Table 3). *In vivo* antitumor activities of *Achyranthes aspera* (L.) (Devil's Horsewhip) on athymic mice, with are subcutaneous xenograft, harboring human pancreatictumorweredemonstrated,usingtheleafextract.Theleafextractsignificantlyreduced both tumor weight and volume in mice treated with leaf extract intraperitonealy [14]. Intrave‐ nous administration of 40 mg/kg body weight eupatoriopicrin, a sesquiterpene lactone present in *E. cannabinum,* significantly delayed the growth of tumor in Lewis lung tumour-bearing syngeneic C57B1 female mice [22]. A 70% inhibition of tumor growth in PC-3 cells, orthopedi‐ cally implanted into the dorsal prostatic lobe in athymic nude mice, was observed, upon their receiving 15 mg/kg intraperitoneal *H. perforatum* methanolic extract [24]. Lantadene A is a pentacyclictriterpenoid,isolatedfromtheweed,*Lantanacamara*(L)[59].FeedingoffemaleSwiss albino mice (LACCA) with a dose of 50 mg/kg body weight of Lantadene A twice a week for 20 weeks, showed potential chemopreventive activity. This chemopreventive activity could be linked to the expression of transcriptional factors and a significant decrease in the mRNA expression of AP-1 and c-fos), NF-kB (p-65) and p53 was observed in Lantadene A treated mice skin tumors [59]. Silibinin decreased tumor multiplicity by 71% (P < 0.01) in wild type mice, but did not show any such considerable effect in iNOS-/- mice upon oral feeding of 742 mg/kg body weight silibinin for 5 days per week for 18 weeks [60]. Lesser effects of silibinin in iNOS-/- mice suggested that most of its chemopreventive and angiopreventive effects were through its inhibition of iNOS expression in lung tumors [60]. Treatment of a purified diet, containing 0.5% to 1.0% silibinin on a transgenic adenocarcinoma of are mouse prostate (TRAMP) model,

caspase 3 enzymes [58].

**medicinal plants**

Treatment of silibinin significantly decreased tumor angiogenesis and proliferation and also there was increased apoptosis in prostate tumor tissue samples in the TRAMP model [61]. The protective effect of silibinin was also demonstrated in mouse skin with tumors caused by acute and chronic UVB-exposure-caused mitogenic and survival signaling and associated biological responses [62]. Mice were treated with silibinin, either topically (9 mg in 200 ml acetone/mouse) or orally (1% of diet), and both administrations strongly inhibited UVB-induced skin tumori‐ genesis in a long-term study [62]. Thymine dimers are formed in DNA, immediately after UVB irradiation, and are considered as an early and important biomarker for UVB induced DNA damage [62]. A noticeable, 71% reduction (P < 0.001) of thymine positive cells was obtained in the mice treated with 1% (w/w) silibinin before the UVB exposure, compared with the UVB alone group [62]. Oral feeding of 200 mg/kg of silibinin for 5 days per week, for 33 days, significantly inhibited human non–small-cell lung cancer cells (NSCLC A549) tumor xenograft growth in nude mice, in a time-dependent manner [63]. This accounted for 58% (P = 0.003) reduction in tumor weight per mouse and intraperitoneal administration of 4 mg/kg doxoru‐ bicin, once a week for four weeks, showed 61% (P = 0.005) reduction in tumor weight. However, interestingly, in silibinin-doxorubicin combination, 76% (P = 0.002, versus control) decrease in tumor weight per mouse was observed, that which was significantly different from either treatment alone, showing enhanced efficacy [63].
