**2.1. Flavonoids and their cytotoxic activities**

Flavonoids and their glycosides are the main phenolic constituents isolate form many natural sources in a reasonable amount. These phenolic compounds are very well-known for their cytotoxic activity and gave very promising results against different types of tumors. The basic structures of flavonoids are based on a C<sup>6</sup> -C<sup>3</sup> -C<sup>6</sup> skeleton derived from 1,3-diphenylpropane. Flavonoids are further divided into three subclasses on the basis of their little structural changes, substitutions and degree of hydroxylation and polymerization, such as, flavonoids (2-phenylchromen-4-one), isoflavonoids (3-phenylchromen-4-one) and neoflavonoids (4-phenylchromen with no hydroxyl group substitution at position 2). More than ten thousand flavonoids have been identified from natural sources and it exhibit a number of human health benefits due to their interactions with different cellular targets including antioxidant, anti-inflammatory, antiviral and anticancer properties. Isoprenylated flavonoids isolated from Moraceae family has many cytotoxic activity in different cancer cell lines including MCF-7 human breast cancer, TK-10 human renal cancer and UACC-62 human melanoma cells, A549 human lung cancer, Hep3B human hepatocellular cancer, HT-29 human colorectal cancer cells.

The cell viability of B16 melanoma cells after 3 days culture with compounds Artocarpin, cudraflavone C, prenylapigenin, kuwanon C, norartocarpin, albanin A and cudraflavone B at different concentrations was determined using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide MTT colorimetric assays. These compounds 1–7 exhibit potent cytotoxicity in a concentration-dependent manner. The IC50 values are 10.3, 9.2, 32.5, 14.2, 7.8, 84.7 and 12.5 μM [3].

Kaempferol is a dietary flavonoid and it has many protective effects for human health, was treated with human breast cancer cell line, MCF-7 and it regulated down the expression of polo-like kinase PLK-1, which has reported to regulate mitotic progression and to be upregulated in several human tumors. This polyphenolic compound significantly reduces cell viabilities of U-2 OS, HOB and 143B cells, but exerts low cytotoxicity on human fetal osteoblast progenitor hFOB cells. It has anticancer effects on Miapaca-2, Panc-1 and SNU-213 human pancreatic cancer cells. In a dose-dependent manner, its decreased viability of pancreatic cancer cells by increasing apoptosis. The anticancer effect of kaempferol mediated by inhibition of EGFR-related Src, ERK1/2 and AKT pathways and it could act as potent pancreatic cancer cells inhibitor [4]. The IC50 value of Dorsmanin F is a prenylated flavonoid and it ranged from 5.34 to 1.94 μM towards leukemia CCRF-CEM cells to 33.30–28.92 μM towards MDA-MB-231- BCRP cells, respectively and from 0.20 to 195.12 μM against CCRF-CEM. This compound induced apoptosis in CCRF-CEM leukemia cells, mediated by MMP disruption and increased reactive oxygen spp (ROS) production [5]. Artocarpesin, cycloartocarpesin (unique flavonoid structures) displayed cytotoxic effect on four cell lines with IC50 values, respectively, below 106, 50 and 25 μM. The IC50 values of these compounds ranged from 23.95 μM for hepatocarcinoma HepG2 cell, 105 μM towards colon carcinoma HCT116 (p53−/−) cells for artocarpesin, from 15.51 μM for leukemia CCRF-CEM cells and 49.83 μM for glioblastoma U87MG.EGFR cells for cycloartocarpesin [6]. 6,7-dimethoxy-4′-hydroxy-8-formylflavon and 4′,7-dihydroxy8-formyl-6-methoxyflavon are formylated flavonoid and showed significant cytotoxicity against PC3 and A549 cell lines with IC50 values of 2.6 and 1.6 μM, respectively [7]. Isoxanthohumol is known as hops and wines flavonoid and it exhibits antiproliferative activity against human breast cancer MCF-7 cell lines, A-2780 ovarian cancer cell lines, prostate cancer PC-3 and DU145 and colon cancer SW620 and HT-29 cells lines. It inhibits the activation of carcinogens: 2-amino-3-methylimidazol-[4, 5-f]quinoline and aflatoxin B1, AFB1 via human cytochrome P450 CYP1A2 [8]. Kushecarpin D is a Chinese traditional herbal medicine and exhibits antiangiogenic activity demonstrated by its effects on migration, adhesion, tube formation of an endothelial cell line. The antiangiogenic activity, together with its antiproliferative effects on endothelial cells, indicates that kushecarpin D is an excellent candidate for development as chemo preventive molecule to be used in combating tumor development [9]. Luteolin is one of the very important flavonoid found in many foods with lot of health benefits and it prevent cancers strongly, it induce cell cycle arrest and apoptosis in various human cancer cells and it synergize antitumor effects of 5-FU on Bel7402 and HepG2 cells, which can related with apoptosis and regulation of 5-FU metabolism. The IC50 value of luteolin on PC<sup>3</sup> and LNCaP cells was 31.44 and 32.05 μM, respectively. It resulted in a marked reduction in cell proliferation in a dose-dependent manner and enhanced the paclitaxel-induced apoptosis in human breast cancer MDA-MB-231 cells by blocking STAT3 [10–12]. Chrysin is found in many flowers and honey and it has preventive effect on cancer induced chemically on xenograft tumor by inducing activity of antioxidant and detoxification enzyme, reducing the activities of cytochrome P450 (CytP450)-dependent monooxygenases, inhibiting cellular proliferation and inducing apoptosis. Chrysin has induced breast cancer resistance protein (BCRP) in Caco-2 cells. Chrysin and many other flavonoids such as fisetin, kaempferol, galangin, myricetin and apigenin reported as potent inhibitors of P-form phenol sulfo transferase mediated sulfation induced carcinogenesis in human hepatoma cell line HepG2. Chrysin kill several histotype cancer cells, including hematological, cervical, liver, colon, lung, breast, nasopharyngeal, glioblastoma, prostate, thyroid and pancreatic cancer [13]. DICO is a nonaromatic B ring flavonoid having potent antitumor activity and inhibited growth of HepG2 cells in different dose/time dependent manners. It induced G2/M cell cycle arrest and apoptosis via a ROS-mediated mitochondrial pathway. It has significant antitumor effect through G2/M cell cycle arrest and apoptosis induction, which suggested DICO has therapeutic potential against tumors [14]. Morusin belongs to the prenylated class of flavonoids and it suppressed signal transducer and activator of transcription 3 STAT3 and nuclear factor-kB/NFkB signaling pathway, which modulate protein expression involved in invasion process. Morusin decreased lung colonization of SK-Hep1 cells in mice. It indicates that morusin possesses antitumor progression potential by suppressing STAT3 and NFkB [15]. Artocarpin showed significant anticancer activities on breast cancer cells (T47D cells) with IC50 value on T47D cells was 12.6 μM on concentration-dependent manner. Anticancer effect of artocarpin in HepG2 and PLC/PRF/5 hepatoma cells is mediated through autophagic cell death mechanism and it showed dose-dependent reduction on cancer cell viability after 24 h of treatment and IC<sup>50</sup> value was calculated to be approximately 15 μM in both cell lines [16, 17]. Genkwanin a methoxy flavonoid, significantly inhibited HT-29 and SW-480 human colorectal cancer cells proliferation and inflammatory cytokine IL-8 secretion. It has a better antitumor activity via enhancing host immunity and decreasing inflammatory cytokine levels [18]. Fisetin is a

**2. Natural phenolics with anticancer properties**


Flavonoids and their glycosides are the main phenolic constituents isolate form many natural sources in a reasonable amount. These phenolic compounds are very well-known for their cytotoxic activity and gave very promising results against different types of tumors. The basic struc-

are further divided into three subclasses on the basis of their little structural changes, substitutions and degree of hydroxylation and polymerization, such as, flavonoids (2-phenylchromen-4-one), isoflavonoids (3-phenylchromen-4-one) and neoflavonoids (4-phenylchromen with no hydroxyl group substitution at position 2). More than ten thousand flavonoids have been identified from natural sources and it exhibit a number of human health benefits due to their interactions with different cellular targets including antioxidant, anti-inflammatory, antiviral and anticancer properties. Isoprenylated flavonoids isolated from Moraceae family has many cytotoxic activity in different cancer cell lines including MCF-7 human breast cancer, TK-10 human renal cancer and UACC-62 human melanoma cells, A549 human lung cancer, Hep3B human hepatocellular cancer,

The cell viability of B16 melanoma cells after 3 days culture with compounds Artocarpin, cudraflavone C, prenylapigenin, kuwanon C, norartocarpin, albanin A and cudraflavone B at different concentrations was determined using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide MTT colorimetric assays. These compounds 1–7 exhibit potent cytotoxicity in a concentration-dependent manner. The IC50 values are 10.3, 9.2, 32.5, 14.2, 7.8, 84.7 and

Kaempferol is a dietary flavonoid and it has many protective effects for human health, was treated with human breast cancer cell line, MCF-7 and it regulated down the expression of polo-like kinase PLK-1, which has reported to regulate mitotic progression and to be upregulated in several human tumors. This polyphenolic compound significantly reduces cell viabilities of U-2 OS, HOB and 143B cells, but exerts low cytotoxicity on human fetal osteoblast progenitor hFOB cells. It has anticancer effects on Miapaca-2, Panc-1 and SNU-213 human pancreatic cancer cells. In a dose-dependent manner, its decreased viability of pancreatic cancer cells by increasing apoptosis. The anticancer effect of kaempferol mediated by inhibition of EGFR-related Src, ERK1/2 and AKT pathways and it could act as potent pancreatic cancer cells inhibitor [4]. The IC50 value of Dorsmanin F is a prenylated flavonoid and it ranged from 5.34 to 1.94 μM towards leukemia CCRF-CEM cells to 33.30–28.92 μM towards MDA-MB-231- BCRP cells, respectively and from 0.20 to 195.12 μM against CCRF-CEM. This compound induced apoptosis in CCRF-CEM leukemia cells, mediated by MMP disruption and increased reactive oxygen spp (ROS) production [5]. Artocarpesin, cycloartocarpesin (unique flavonoid structures) displayed cytotoxic effect on four cell lines with IC50 values, respectively, below 106, 50 and 25 μM. The IC50 values of these compounds ranged from 23.95 μM for hepatocarcinoma HepG2 cell, 105 μM towards colon carcinoma HCT116 (p53−/−) cells for artocarpesin, from 15.51 μM for leukemia CCRF-CEM cells and 49.83 μM for glioblastoma U87MG.EGFR cells for cycloartocarpesin [6]. 6,7-dimethoxy-4′-hydroxy-8-formylflavon and 4′,7-dihydroxy-

skeleton derived from 1,3-diphenylpropane. Flavonoids

**2.1. Flavonoids and their cytotoxic activities**

36 Phenolic Compounds - Natural Sources, Importance and Applications

tures of flavonoids are based on a C<sup>6</sup>

HT-29 human colorectal cancer cells.

12.5 μM [3].

tetrahydroxy flavonoid having many health benefits and it effects on short- and long-term growth of BRAF-mutated A375, SK-MEL-28 and RPMI-7951 melanoma cancer cells. Results of MTT assay demonstrated that fisetin (10–60 μM) treatment significantly decreased the growth of A375 8.64–61.75%, SK-MEL-28, 6.94–59.79% and RPMI-7951, 11.60–64.11% cells in a concentration-dependent manner [19]. Hispidulin is found in many herbs and significantly inhibited HepG2 cell growth in a time and dose-dependent manner. Hispidulin (at 200 μM) inhibited the growth of HepG2 cells by nearly 50, 70 and 90% after 24, 48 and 72 h of treatment, respectively, which suggest that hispidulin promotes HepG2 cell death through apoptosis. It inhibited cell growth in a dose-dependent manner in (HRCC) lines 786-0 and Caki-1 cell lines, whereas Caki-1 cells were found to be more resistant to hispidulin treatment [20, 21]. Quercetin is a dietary flavonoid (berries flavonoid) showed strong cytotoxicity as 1.49-fold in MCF-7 cells and 1.98-fold in MCF-7/dox cells. It suppressed proliferation and survival of HepG2 cancer cells and induced apoptosis by enhancing the expression of p53 and BAX through downregulation of ROS, PKC, PI3K and COX-2 [22, 23]. Hesperetin a citrus flavonoid exhibited inhibition of cell growth in a concentration and time-dependent manner with IC50 at 72 h 200 μM. It could significantly promote apoptosis of Eca109 cells in a dose-/time-dependent manner [24]. Apigenin a trihydroxy flavonoid with potential health benefits exhibited strong growth inhibitory activity in HER2/neu breast cancer cells but was much less effective in inhibiting growth of cells expressing basal levels of HER2/neu. It induces apoptosis in MDA-MB-453 breast cancer cells with involving intrinsic and extrinsic apoptotic pathways. It has shown to downregulate levels of cyclin D1, D3 and cdk4 and increases p27 protein levels in breast cancer cells. Apigenin inhibited human cervical carcinoma HeLa cells growth through an apoptotic pathway. In various human colon carcinoma cells, it resulted cell growth inhibition and G2/M cell cycle arrest. The effects of apigenin on lung cancer cells were evaluated and it inhibited A549 lung cancer cell. It has capability to significantly reduce cell number and induce apoptosis in PWR-1E, LNCaP, PC-3 and DU145 cells [25, 27]. Quercetin-3-*O*-β-dglucopyranoside also known as isoquercetin and found in mangoes in high amount and it is an interesting dietary compound worth further investigation as a cytoprotective agent. Pretreatment of PC12 cells with nontoxic concentrations of this compound protect cells from H2 O2 induced cytotoxicity with decrease in generation of reactive oxygen species (ROS). These observations qualify it as cytoprotective dietary compound [27]. Amplexicaule A is found in many herbs and it increased levels of cleaved caspase-3,-8,-9 and PARP, resulted from suppression of MCL-1 and BCL-2 expression in cells. This compound inactivated the Akt/mTOR pathway of breast cancer cells. It influenced strongly on breast cancer cells, most likely by induction of apoptosis [28]. Triticuside A a dietary flavonoid found in wheat and induced apoptosis accompanied by a significant decrease in Mcl-1 and Bcl-2 proteins and by increase in cleavage of caspases-3, -7, -9 and PARP. It suppressed the level of phospho-Akt and its downstream targets, mTOR and P70 S6 kinase. LY294002, a specific inhibitor of PI3K, significantly enhanced the triticuside A induced apoptosis. It may a potentially useful wheat bran component and can used for treatment of breast cancer [29].

Naringin is a diglycoside flavonoid and found in many citrus fruits and it inhibited cell proliferation and promoted cell apoptosis and G1 cycle arrest, accompanied by increased p21 and decreased surviving. Significant inhibitory effects of naringin on the cell proliferation of TNBC cells were observed. MDA-MB-231 and BT-549 cells treatment with naringin (50, 100 and 200 M) for 48 h, significantly increased apoptosis in breast cancer cells. It exerts significant effects on inhibition of breast cancer cells growth in through mediating-catenin pathway. Treatment of Raji with naringin showed maximum sensitivity towards NK cell lysis and the activity was 2.5-fold with naringin treatment [30–32]. Quercetin-3-*O*-α-l-rhamnopyranosyl- (1→6)-β-d-glucopyranoside induces apoptosis in A549 cancer cells via caspase activation through cytochrome release from mitochondria. It tested against A549 lung cancer cell line, COLO320DM cancer cell line and Vero cell line and it showed prominent cytotoxic activity against A549 lung cancer cell. This compound showed 87.41% activity at dose of 164 μM with IC50 value of 82 μM. COLO320DM cancer cell line was maintained in complete tissue culture medium [32]. Pectolinarin and astragalin isolated from edible plants against H<sup>2</sup> O2 induced cell death of human brain neuroblastoma SK-N-SH cells. These compounds showed protective effects against H<sup>2</sup> O2 -induced cell death and inhibited ROS generation by oxidative stress [33]. Troxerutin showed strong binding with calf thymus DNA in vitro and DNA interaction was confirmed by CD spectropolarimetry. The mode of binding of troxerutin to DNA was assessed by competing with EtBr or DAPI, known DNA intercalator and minor groove binder, respectively. It induced cytotoxicity in radioresistant DU145 and sensitive PC3 prostate cancer cells. When troxerutin was pretreated with DU145 and PC3 cells and it were exposed to gamma-radiation, the cytotoxicity induced in PC3 and DU145 prostate cancer cells and it was monitored by MTT assay. The toxicity induced by troxerutin 5 μM was more than 40% over control at 24 h, but more than 50% increase was observed at 48 h. Radiation per se induced around 18% cell death in PC3 cells [34]. Vicenin gave IC50 values 141.7 μM, 195.7 μM and 369.3 μg mL−1 on treatment with PC-3, DU-145 and LNCaP cells. Curcumin was used as positive control with IC50 17.90 μM. It is an active constituent of the medicinal herbs (Tulsi) and it effectively induced antiproliferative, antiangiogenic and proapoptotic effect in CaP cells (PC-3, DU-145 and LNCaP) [35, 36] (**Figure 1**).
