**5. Selected flavonoids**

Luteolin, naringenin, kaempferol, and baicalein are flavonoids distributed on accessible natural sources such as fruits, beverages, and plants. Like other flavonoids, their biosynthesis occurs via the phenylpropanoid pathway, except that it can be enhanced with heterologous systems. Such natural products' pharmacological properties and mechanisms against human health threats have been documented over the last decade. However, there is a need to expand our knowledge about their capacity to face NSCLC treatment limitations and develop novel active research fields. Therefore, in the following sections, we present the sources, chemical characteristics, biosynthesis, pharmacokinetics, anticancer properties, and anti-NSCLC effects of our four selected flavonoids: luteolin, naringenin, kaempferol, and baicalein.

#### **5.1 Luteolin**

Luteolin (3′,4′,5,7-tetrahydroxyflavone) is a glycosylated flavone present in various plants such as celery, sweet bell peppers, carrots, etc. In addition, it has been isolated from fruits, tea, wine, and seeds. Luteolin has two benzene rings (A, B), an oxygen-containing ring (C), and a 2–3 carbon double bond; the hydroxyl groups are

*Shedding Light on Four Selected Flavonoids with Anti-non-small Cell Lung Cancer Properties DOI: http://dx.doi.org/10.5772/intechopen.105162*

**Figure 3.**

*Chemical structure, sources, and anticancer properties of luteolin.*

placed at carbons 5,7,3′ and 4′; like other members of this category of NP, its condensed form is C6-C3-C6 (**Figure 3**) [41].

In plants, the synthesis of luteolin comprises of two different pathways, depending on the ring synthesis. The ring B is synthesized by the phenylpropanoid pathway by a sequential enzymatic activity, including the conversion of the amino acid phenylalanine into 4-coumaroyl-CoA by phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase, and 4-coumaroyl CoA ligase catalysis [42]. The ring A is then synthesized when a molecule of 4-coumaroyl-CoA is condensed with three molecules of malonyl-CoA by a chalcone synthase [43]. This process generates the intermediate naringenin, which by the activity of a chalcone isomerase, apigenin is transiently synthesized. This last intermediate is converted into luteolin by a flavonoid 3'-hydroxylase activity. This process has been replicated *de novo* in heterologous systems, such as *Streptomyces albus* [44].

Even though flavonoids metabolism and pharmacokinetic parameters have been areas of active research over the last decade, little is known about the bioavailability of flavones. Luteolin, like other flavonoids, is mainly absorbed in the small intestine and is biotransformed by liver and kidney enzymes (e.g., catechol-O-methyltransferase) [45]; its monoglucuronide and aglycone forms have been detected in human plasma [46]. In rat models, luteolin and its metabolites (e.g., luteolin 3′-O--D-glucuronide and luteolin-7-O--D-glucuronide) are distributed in the gastrointestinal tract, liver, kidney and lung, and the biliary excretion is the dominant elimination pathway of conjugated luteolin and its derivatives [47]. Other analogs, such as luteolin-7-Oglucoside, are excreted through urine and feces [48].

Luteolin acts as an anticancer agent against NSCLC by inhibition of proliferation of tumor cells, protection from carcinogenic stimuli, activation of cell cycle arrest, induction of apoptosis through different signaling pathways (e.g., c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), protein kinase (PKC), etc.), induction of the epithelial biomarker E-cadherin expression, reversion of the EMT process, and by modulation of microRNAs (miRNAs) expression [49, 50].

For instance, Jiang and coworkers [51] revealed the tumor-suppressive activity of luteolin on NSCLC cancer cell lines A549 and NCI-H460. In this study, the effect of luteolin was observed in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) of 40 *μ*M [51]. The tumor inhibitory effect of 50, 100, and 200 mg/kg/day luteolin was investigated using the NCI-H460 xenograft nude mice model [51]. Moreover luteolin treatment upregulated the expression of potential tumor suppressors such as miR-34a-5p, and increased the activation of cysteine proteases (i.e., caspase-3 and -9) involved in cell death by apoptosis [51]. In another study, the effect of luteolin on A549 cell migration was assessed. Treatment with 10, 20, and 40 *μ*M of luteolin reduced the number of invading cells across the matrix and transwell membrane in a concentration-dependent manner [52]. In the same concentration range, luteolin reduced the number of filopodia, suppressed the expression of activated focal-adhesion kinase (FAK), and activated proto-oncogene tyrosineprotein kinase (SRC). In another study, treatment with 5, 10, 20, or 40 *μ*M luteolin arrested the cell cycle of NCI-H1975 and NCI-H1650 cells at G1 phase with a concomitant decrease in the expression of cell cycle regulators, such as cyclin D1 and D3 [53].

### **5.2 Naringenin**

Naringenin (2,3-dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) and its derivatives (naringenin-7-rhamnoglucoside and naringenin-7-glucoside) are naturally occurring flavanones found in many edible fruits (e.g., lemon peels, pomelo, grapefruits, oranges, and tomatoes) and vegetables [54–56]. This flavanone is derived from the hydrolysis of its glycone forms naringenin and narirutin (**Figure 4**) [57].

The biosynthesis of naringenin occurs via shikimic acid and acylpolymalonate metabolic pathways. Naringenin possesses 2 aromatic rings joined by a linear 3-carbon chain (C6-C3-C6) with a saturated 3-carbon chain and an oxygen atom at carbon 4. Depending on the arrangement of related functional groups in its structure, naringenin displays a wide array of therapeutic properties such as neuroprotective, antimicrobial, antiviral, antifibrotic, antidiabetic, and anti-inflammatory [58, 59]. In this context, the high reactivity of hydroxyl groups and the presence of carbohydrate moieties confers antioxidant advantages against a variety of radical species [60].

*Chemical structure, sources, and anticancer properties of naringenin.*

*Shedding Light on Four Selected Flavonoids with Anti-non-small Cell Lung Cancer Properties DOI: http://dx.doi.org/10.5772/intechopen.105162*

Regarding its pharmacokinetic properties, the absorption of naringenin takes place through both passive diffusion and active transport. In rats, naringenin and its derivative naringin, have been identified in the lung, trachea, kidney, liver, spleen, heart, muscle, fat, and brain tissues [61]. In order to be metabolized, naringenin undergoes glucuronidation and sulfation with its major metabolites naringenin-o--Dglucuronide, p-hydroxybenzoic acid, p-hydroxyphenylpropionic acid, and p-coumaric acid, are found in liver, kidney, heart, brain, plasma, and urine. Naringenin excretion occurs through the biliary (naringenin 7-glucuronide 4′-sulfate and naringenin 7-glucuronide) and urinary (naringenin 7,4′-disulfate and naringenin 4′-glucuronide) pathways [62].

Naringenin exhibits antidiabetic, anti-inflammatory, anti-atherosclerotic, hepatoprotective, nephroprotective, and neuroprotective properties [63]. Recently, the *O*-ethyl, *O*-dodecyl, *O*-methyl, and *O*-pentyl derivatives of naringenin have been recognized for its anticancer, and antimicrobial properties [64]. The anticancer potential of naringenin has showed that it can induce apoptosis and cell cycle arrest in a variety of cancer cell lines (e.g., breast tumor, glioblastoma, lung tumor) [65], act as an anti-angiogenic chemopreventive agent [66], suppress mutations, modulate oxidative stress, regulate signaling factors involved in the inflammatory response (e.g., interleukin-8, interleukin-6, inducible nitric oxide synthase, etc.), inhibit carcinogenesis, induce major pathways of apoptosis, and promote caspase activation [67, 68]. Similar effects have been observed for other naringenin derivatives such as 5,4′-dihydroxy flavanone-7-yl [69].

In NSCLC, the effect of naringenin has been evaluated in a few models. For example, in A549 cells, the molecular mechanism of the naringenin-induced apoptotic event has been evaluated [70]. In this study, the growth inhibition effect was observed with 100 and 200 μM of naringenin, and an apoptosis activation with a concentration of 800 μM through the activation of caspase-3 and -9 [71]. In another study, the effect of current therapies such as radiation therapy has been improved with naringenin treatment. For example, naringenin promoted radiosensitization of NCI-H23 cells [71]. In such study, the cancer cells' survival was lowered by 50% with a supplementation of 100 μM naringenin before treatment with radiation. After radiation treatment, naringenin acted as a radiosensitizer and radioprotector at 6 Gy. On the other hand, naringenin treatment decreased the expression of protease enzymes such as metalloproteinase-2 (MMP-2) and -9 (MMP-9) and increased the expression of caspase-3 [71]. Similarly, another study reported that 10, 100, and 200 μmol/L naringenin treatment increased 10–140% mRNA expression of caspase-3 and decreased 6–55% MMP-2 and 5–60% MMP-9 expression levels [72].

#### **5.3 Kaempferol**

Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-chromene-4-one) and its derivatives (e.g., kaempferol-3-O-rhamnoside, kaempferol-3-O-arabinofuranoside, kaempferol-3-O-(6-*p*-coumaroyl)-glucoside, kaempferol-3-O-(2,6-di-*p*-coumaroyl) glucoside, etc.) are polyphenols widely distributed in foods (e.g., green chili, asparagus, beans, blueberries, etc.) fruits (e.g., strawberry and gooseberry), vegetables (e.g., cauliflower), beverages (e.g., grapefruit juice, red raspberry juice, teas, etc.) [73], and plants belonging to the families *Aspidiaceae*, *Aspleniaceae*, *Polypodiaceae*, and *Iridaceae* (**Figure 5**) [74].

Structurally, kaempferol is synthesized from diphenyl propane via condensation of three molecules of malonyl-CoA and one molecule of 4-coumaroyl-CoA.

**Figure 5.**

*Chemical structure, sources, and anticancer properties of kaempferol.*

Kaempferol is absorbed from the small intestine by passive diffusion and metabolized into 4-methyl phenol, 4-hydroxyphenyl acetic acid, and phloroglucinol by the normal flora of the colon [75]. Kaempferol metabolites (e.g., kaempferol-3-glucuronide and kaempferol mono- and di-sulfates) are distributed in body tissues, blood, and plasma. Their excretion occurs through feces and urine [76, 77].

Kaempferol exhibits a handful of pharmacological properties, such as anticancer, antidiabetic, anti-obesity, antimicrobial, anti-inflammatory, antiallergic, antiplatelet aggregation, anti-bone disorders, and cardiovascular protection [78]. The anti-cancer potential of kaempferol is primarily achieved by inhibition of the proliferation of cancer cells by triggering apoptosis (e.g., activation of caspases-3, -7, and -9), cell cycle arrest at G2/M phase, downregulation of signaling pathways and expression of EMT-related markers, prevention of the accumulation of reactive oxygen species (ROS), inhibition of angiogenesis [79], sensitization of cancer cell growth to chemotherapeutic pharmaceuticals (e.g., ovarian cancer cells to cisplatin) [80], among other effects.

During the study of therapeutic molecules against NSCLC, kaempferol treatment (50 μM) inhibited the proliferation of A549 (51%) and NCI-H460 (57%) cells, increased the expression of cleaved caspase-3, -9, and Bax, and arrested the cell cycle at the G1 phase [79]. The same dose of kaempferol down-regulated the transcription of major cellular oxidative and inflammatory regulators such as nuclear factor erythroid 2-related factor 2 (Nrf2) [79]. Finally, 25 μM of kaempferol inhibited the key metastatic inducers tumor growth factor 1 (TGF-1) of epithelial-mesenchymal transition (EMT) and herein, interfering with the activation of downstream effectors involved in NSCLC progression [80].

#### **5.4 Baicalein**

Baicalein (5,6,7-trihydroxyflavone) is the major component of the roots of Scutellaria baicalensis Georgi and Scutellaria lateriflora. Its glycone form (5,6-dihydroxy-7-O-glucuronide) is also an important flavone constituent of *S. baicalensis*. Lower concentrations of baicalein can be found in other sources, such as foods

*Shedding Light on Four Selected Flavonoids with Anti-non-small Cell Lung Cancer Properties DOI: http://dx.doi.org/10.5772/intechopen.105162*

#### **Figure 6.**

*Chemical structure, sources, and anticancer properties of baicalein.*

(e.g., citrus fruits and chocolate) and beverages (e.g., wine and tea) [81]. The structure of baicalein lacks a 4′-hydroxyl group on its ring B compared to other flavones (**Figure 6**).

Although baicalein is synthesized by the flavonoid pathway (which is part of phenylpropanoid metabolism), recent evidence suggests that because of the lack of a 4′-hydroxyl group, an alternative path recruits cinnamic acid to form cinnamoylcoenzyme A (CoA) through a CoA ligase. This modification will allow a condensation with malonyl-CoA by a chalcone synthase to form a chalcone, and then isomerized by chalcone isomerase to form pinocembrin. This intermediate could be converted by a flavone synthase to form chrysin, and then modified by hydroxylases, methyltransferases, and glycosyltransferases to form baicalein [82].

Comparably, baicalin is synthesized via the phenylpropanoid pathway by many enzymes (e.g., phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, chalcone synthase, etc.); baicalein can be catalyzed back to baicalin by UDP-glucuronate by the baicalein 7-O-glucuronosyl transferase [83].

Even with the complex metabolic process of most flavones, baicalein was found to be ample in the small and large intestine of mice, especially its glucuronide and glycosylated forms, whereas its dehydroxylated, methylated, and sulfated metabolites have been found in the entire intestine [84, 85].

The safety, tolerability, and pharmacokinetics of oral baicalein tablets (200, 400, and 600 mg) in a phase I single-center study of healthy Chinese subjects were assessed [86]. Results showed that baicalein tablets are safe and well-tolerated with mild adverse events (e.g., proteinuria). The excretion of baicalein and its metabolites (e.g., baicalein-6-O-glucuronide, baicalein-6-O-sulfate, baicalein-7-O-glucuronide, etc.) was determined mainly in the urine.

Baicalein has multiple biological properties, including cardioprotective, neuroprotective, hepatoprotective, attenuates renal dysfunction, increases the cytotoxicity of chemotherapeutic agents (e.g., cisplatin) [87], antibacterial [88], anti-inflammatory, antiviral (e.g., against dengue virus) [89], and antioxidant [90]. Furthermore, in cancer, baicalein has the potential to decrease cancer growth and retard cancerpromoting processes such as angiogenesis, inflammation, and metastasis [91]. Hence, baicalein properties have been studied in distinct cancers like breast cancer, cervical

cancer, gastric cancer, hepatocellular carcinoma, multiple myeloma, pancreatic cancer, prostate cancer, and LC [92].

In another study where 10 or 40 μmol/L baicalein treatment was used, an inhibition of A549 and NCI-H1299 cell invasion and metastasis was observed. Also, a down-regulation of the membrane-cytoskeleton linker proteins (i.e., ezrin) was measured [93].

The combination of baicalein and the chemotherapic docetaxel was investigated for a potential synergism against the proliferation of A549 and LCC cells [94]. Results showed an increase in the percentage of apoptotic cells compared with baicalein or docetaxel individually tested. In addition, the combination of baicalein plus docetaxel arrested the cell cycle of A549 cells at the G1 phase, whereas for LLC cells, it was blocked in the G2/M phase. These events are related to the suppression of cyclin D1, cyclin-dependent kinase 4 (CDK4), cyclin B1, cyclin-dependent kinase 6 (CDK6), and cell division cycle 25C (CDC25C) expression in both cell lines.
