Review
 
Nobiletin and tangeretin (citrus polymethoxyflavones): an overview on their chemistry, pharmacology and cytotoxic activities against breast cancer
Eric Wei Chiang Chan1*, Oi Yoon Michelle Soo1, Yong Hui Tan1, Siu Kuin Wong2, Hung Tuck Chan3
1. Faculty of Applied Sciences, UCSI University, 56000 Cheras, Kuala Lumpur, Malaysia
2. School of Foundation Studies, Xiamen University Malaysia, Bandar Sunsuria, 43900 Sepang, Selangor, Malaysia
3. Secretariat of the International Society for Mangrove Ecosystems (ISME), Faculty of Agriculture, University of the Ryukyus, Okinawa 903-0129, Japan    
 
 
Abstract: The fruit peel of Citrus species (Chenpi), particularly those of mandarin oranges, is a useful source of food and medicine in China. Flavonoids from the citrus fruit peel are mainly polymethoxyflavones (PMFs), of which nobiletin and tangeretin are the most abundant components. In the present review, we summarized the cytotoxic activities of these two PMFs to breast cancer cells. Studies have reported that these two compounds inhibit the growth of breast cancer cells by inducing apoptosis, cytostatic cell death and cell cycle arrest, or by inhibiting cell proliferation, metastasis and tumour angiogenesis, depending on the molecular subtypes of breast cancer. In vitro and in vivo cytotoxic activities involve different molecular targets and signalling pathways. Analyses on the structure-activity relationship (SAR) of nobiletin and tangeretin have shown that the presence of a methoxy group at C8 and a hydroxyl group at C3 or C5 are essential for anti-proliferative activity. Some future perspectives and research needs are suggested. Sources of information are from PubMed, PubMed Central, Science Direct, Google Scholar, J-Stage, PubChem and CNKI using keyword search.                       
Keywords: Polymethoxylated flavones; Nobiletin; Tangeretin; Breast cancer cells; Structure-activity relationship 
CLC number: R284                Document code: A                 Article ID: 10031057(2020)744312 
 
 
1. Introduction
Flavonoids are a major class of polyphenols with a benzo-γ-pyrone structure, and they are ubiquitous to most plants that form an integral part of human and animal diet[1,2]. Having a 15-carbon skeleton consisting of two benzene rings (A and B) linked by a heterocyclic pyrane ring (C), flavonoids are composed of flavones, flavonols, flavanones, flavanonols, flavanols (catechins), anthocyanins and chalcones as sub-groups. Flavonoids from fruits of Citrus species (family Rutaceae) belong to two groups, namely, polymethoxylated flavones (PMFs),such as nobiletin, sinensetin and tangeretin, and flavanone glycosides, such as naringin, hesperidin and neohesperidin[3,4]. PMFs are flavones bearing two or more methoxy (-OCH3) groups with a carbonyl group (keto moiety) at the C4 position of ring C[5,6]. The maximum number of methoxy groups is seven (heptamethoxyflavone), and they can be substituted with hydroxyl (-OH) groups.
Nobiletin and tangeretin are the most abundant PMFs in the fruit peel of citrus, such as mandarin oranges (Fig. 1). From the fruit peel of Citrus reticulata cv. ponkan in Japan, the contents of nobiletin and tangeretin are estimated to be 110 and 124 mg/100 g fresh weight, respectively[7]. From five regions in China, the dried fruit peel of the same variety of C. reticulata ranges from 6.9−8.6 mg/g[8]. The orange fruit peel contains PMFs consisting of tangeretin (19%), heptamethoxyflavone (15%), tetramethoxyflavone (14%), nobiletin (12%), hexamethoxyflavone (11%), and sinensitin (9%)[9]. Nobiletin and tangeretin are also found in the leaves of C. reticulata[10]. 
 
 
 
Figure 1. Fruits (left) and peel (right) of the mandarin orange are rich in PMFs. 
 
 
In China, the peel of Citrus fruits is a useful source of food and medicine. The dried peel (Chenpi) is used as a spice for flavouring food. In traditional Chinese medicine, Chenpi is used to treat disorders, such as nausea, vomiting, indigestion, diarrhoea, cough and expectoration[11]. Chenpi is also a traditional medicine for relieving stomach upset, cough, skin inflammation, muscle pain and ringworm infections, as well as for lowering blood pressure[12].
PMFs have pronounced bioactivities, including anti-allergic, anti-atherosclerosis, anti-cancer, anti-diabetic, anti-inflammatory, antimicrobial, anti-obesity, antioxidant,hypolipidemic, neuroprotective and vasodilatory properties[6,13]. The anti-cancer properties of PMFs involve anti-angiogenesis, anti-metastasis, anti-mutagenic, anti-proliferative, apoptosis, cell cycle arrest, reduction of drug resistance and scavenging of reactive oxygen species (ROS)[6,14]. Types of cancer include those of breast, bone, gastric, leukaemia, liver, lung, melanoma and neuroblastoma. Studies have shown that PMFs possess greater anti-tumour, anti-metastatic and anti-invasive properties compared with hydroxylated flavonoids[15].
Breast cancer is the leading cause of cancer-related death among women worldwide and ranks the second among all cancer death in the world. In 2018, ~2 089 000 new breast cancer cases and ~627 000 deaths are estimated to occur worldwide, representing 11.6% of new cases and 6.6% of deaths[16]. In the United States, the estimated numbers of new cases and deaths due to breast cancer in 2018 are 268 700 and 41 400, respectively[17]. Of these figures, 99% affect females. In terms of the estimated number of new cases and deaths, breast cancer ranks the number one and the number two among females, respectively. In recent years,different molecular subtypes of breast cancer cells have been identified. They include estrogen receptor (ER)+, progesterone receptor (PR)+ and human epidermal growth factor receptor (HER)-2+ subtypes[18]. There is also the triple-negative (TN) breast cancer, defined as tumours that are negative for ER, PR and HER-2, and they represent a minority of breast cancers.
In this review, knowledge on the chemistry, pharmacology and anti-cancer properties of nobiletin and tangeretin was updated. Their cytotoxic activities involve growth inhibition of cancer cells, multiple molecular targets and signalling pathways with some insights on their structure-activity relationships. Focus is on breast cancer because it is most susceptible to nobiletin and tangeretin. The effects and mechanisms of these two PMFs towards the various molecular subtypes of breast cancer cells would be of great interest to oncology.
2. Chemistry
Nobiletin (5,6,7,8,3’,4’-hexamethoxyflavone) is a flavone with molecular formula of C21H22O8 and molecular weight of 402.4 g/mol. Its IUPAC name is 2-(3,4-dimethoxyphenyl)-5,6,7,8-tetramethoxychromen-4-one[19,20]. The molecule has six methoxy groups, four at 5, 6, 7 and 8 positions of ring A, and two at 3’ and 4’ positions at ring B (Fig. 2). The molecule has a double bond between positions 2 and 3, and a ketone moiety in position 4 of ring C. By removing the C8 methoxy group of nobiletin, the molecule becomes sinensetin. 
 
 
Figure 2. Molecular structure of nobiletin. 
 
Tangeretin (4’,5,6,7,8-pentamethoxyflavone) is a PMFwith five methoxy groups, four at 5, 6, 7 and 8 positionsof ring A, and one at 4’ position at ring B (Fig. 3). It has a molecular formula of C20H20O7 and a molecular weight 372.4 g/mol, and its IUPAC nameis5,6,7,8-tetramethoxy-2-(4-methoxyphenyl)chromen-4-one. Like other flavones, tangeretin has two aromatic rings (A and B) joined by ring C with a double bond at C2 and C3positions, and a carbonyl group at the C4 position[20]. 
 
Figure 3. Molecular structure of tangeretin. 
3. Pharmacology
In the pharmacology of drugs, important processes are drugs entering the body (absorption), moving about the body (distribution), changing within the body (metabolism) and leaving the body (excretion)[21]. Recently, the pharmacokinetic, bioavailability and deliverysystems of nobiletin and its derivatives have been reviewed by Goh et al. (2019)[22].
Nobiletin is lipophilic and it can easily permeate the cell membrane[12]. Methoxylated flavonoids display 5 to 8 times higher permeability in the intestinal wall thanunmethoxylated flavonoids[23]. When taken orally, nobiletin undergoes extensive metabolism after being absorbed in the small intestine and in the colon[24]. Nobiletin has a half-life of 1.8 h in rat plasma[25], a half-lifeof 4.6 h and 1.5 h following oral and parenteral (non-oral) administration, respectively[26]. The concentration of nobiletin is rapidly decreased and becomes undetectablein the serum, stomach, intestines, liver and kidney of rat, resulting in less significant adverse effects of nobiletin administration[27]. Nobiletin and tangeretin differ in their bioavailability. A study on rats has shown that nobiletin (with six methoxy groups) has a higher efficacy and bioavailability than tangeretin (with five methoxy groups), suggesting that methoxy groups influence the bioavailability of methoxyflavones[28]. The content of tangeretin in the urine and faecal samples of rats is less than 8%, and this indicates that 92% of tangeretin is either absorbed or excreted in the form of metabolites[29].
4. Cytotoxic activities
4.1. Breast cancer cells
Against MCF-7 breast cancer cells, the cytotoxicity of nobiletin has an IC50 value of 28.2 μM[8]. Stronger cytotoxicity is displayed towards HO8910 ovarian cancer cells (16.8 μM), while the cytotoxicity is weaker towards HL-60 leukaemia cells (31.7 μM) and A549 cells lung adenocarcinoma cells (31.9 μM). Based on IC50 values, the order of cytotoxicity is HO8910 > MCF-7 > HL-60 ~ A549. Nobiletin increases the cytotoxic activity of doxorubicin on MCF-7 cells but not on T47D cells[30]. Against MCF-7 cells, nobiletin exerts significantly stronger IC50 value (44 μM) compared with nobiletin + cytochrome P450 enzyme CYP1A1 inhibitor (69 μM)[31]. The cytotoxicity of nobiletin against MDA-MB-468 breast cancer cells is much stronger than MCF-7 and SK-BR-3 breast cancer cells[32,33]. The IC50 values of nobiletin towards these breast cancer cell lines are 51, 60 and 87 μM after incubation for 3 d, and 20, 40 and 59 μM after incubation for 7 d, respectively[32]. A study on tangeretin has reported that MCF-7 cells (39.3 μM) are much less susceptible than MDA-MB-468 cells (0.25 μM)[34]. In contrast, MCF10A normal breast cells (>100 μM) are unaffected by tangeretin treatment. Recently, tangeretin has been reported to be cytotoxic to MDA-MB-231 breast cancer cells (9.0 μM) and non-cytotoxic to Hs841.T normal breast cells (>100 μM)[35].
From the literature, molecular mechanisms of nobiletinagainst the growth of different breast cancer cells are listed in Table 1. Studies are based on cells of MCF-7, MDA-MB-231, MDA-MB-435, MDA-MB-468 and T47D. Growth inhibition of breast cancer cells by nobiletin involves induction of apoptosis, cytostatic cell death and cell cycle arrest, and inhibition of metastasisand tumour angiogenesis, migration, invasion and sphere formation. Molecular mechanisms include blockingCXC4 and MMP-9 expression, suppressing NF-κB and MAPK signalling pathways, up-regulating CYP1 enzymes, reducing Bcl-xL expression, inhibiting AKT/mTOR activity, regulating Src, FAK and STAT3 signalling through PXN, activating CD36/STAT3/NF-κB signalling, and mediating p38 MAPK, NF-κB, and Nrf2 pathways. 
 
Table 1. Effects and molecular mechanisms of nobiletin towards the growth of different breast cancer cells in vitro.
   
*Molecular subtypes: MCF-7 and T47D are ER+/PR+, SK-BR-3 is (HER)-2+, and MDA-MB-231 and MDA-MB-468 are TN. Abbreviations: Bcl-xL = B-cell lymphomaextra-large, CD = cluster of differentiation, CXC = CXC chemokine receptor, CYP1 = cytochrome P450 enzymes, ER - estrogen receptor, FAK = focal adhesion kinase, HER = human epidermal growth factor receptor, MAPK = mitogen-activated protein kinase, MMP = matrix metalloproteinase, mTOR = mammalian target of rapamycin, NF-κB = nuclear factor-kappa B, Nrf = nuclear factor erythroid 2-related factor, PR = progesterone receptor, PXN = paxillin, Src = steroid receptor coactivator, STAT = signal transducer and activator of transcription, and TN = triple-negative.    
 
From the literature, the effects and molecular mechanisms of tangeretin towards the growth of different breast cancer cells in vitro and in vivo are listed in Table 2. In vitro studies are based on cells of MCF-7, T47D, MDA-MB-231 and MDA-MB-468. Growth inhibition of breast cancer cells by tangeretin involves inhibition of cell growth and proliferation, induction of G1 and G2/M cell cycle arrest, and modulation of cell cycle. Cell death is non-apoptotic but cytostatic[36]. Molecular mechanisms include ERK1/2 phosphorylation, and CYP1A1/B1 enzyme induction and metabolism. Recently, apoptotic cell death has been reported to increase in Bax/Bcl-2 ratio expression and activation of caspases 3, 8 and 9[35]. In vivo studies are based on 7,12-dimethyl benz[α]anthracene (DMBA)-induced breast cancer in rats. DMBA is a polycyclic aromatic hydrocarbon that can efficiently induce breast cancer in rodents[41]. Growth inhibition of breast cancer in rats by tangeretin involves amelioration of oxidative stress in renal tissues, exhibition of chemotherapeutic and anti-tumorigenic effects, exertion of G1/S cell cycle arrest, inhibition of metastasis, amelioration of oxidative stress, reduction of xenobiotic-induced genotoxicity, exertion of chemotherapeuticeffect, and regulation of cellular metabolic energy fluxes. Molecular mechanisms include decline in oxidant and pro-inflammatory cytokine production, decrease in the levels of lipid peroxide, enzymatic antioxidants and non-enzymatic antioxidants, p53/p21 up-regulation, suppressing MMP-2,MMP-9 and VEGF, enhancing Nrf2-mediated antioxidant effect in the liver, reducing the levels of enzymatic and non-enzymatic antioxidants and breast cancer marker CEA, increasing antioxidant activities, and activating glycolytic, TCA cycle and respiratory chain enzyme activities. 
 
 
Table 2. Effects and molecular mechanisms of tangeretin towards the growth of different breast cancer cells in vitro and in vivo.
  
*Molecular subtypes: MCF-7 and T47D are ER+/PR+ and MDA-MB-468 is TN. Abbreviations: Bax = bcl-2-associated X-protein, BC = breast cancer, Bcl-2 = B-cell lymphoma, CEA = carcino-embryonic antigen, CYP1 = cytochrome P450 enzymes, DMBA = 7,12-dimethylbenz(α)anthracene, ER - estrogen receptor, ERK = extracellularsignal-regulated kinases, MMP = matrix metalloproteinase, Nrf = nuclear factor erythroid 2-related factor, PR = progesterone receptor, TCA = tricarboxylic acid, TN = triple-negative, and VEGF = vascular endothelial growth factor. 
 
4.2. Other cancer cells
Besides breast cancer, studies have shown that nobiletin and tangeretin exhibit cytotoxicity against other cancer cells. Nobiletin has been reported to be cytotoxic against colon[36], gastric[50,51], leukaemia[52], lung[53], nasopharyngeal[54], osteosarcoma[55], ovarian[56], prostate[57], and thyroid[58] cancers. Tangeretin exhibits cytotoxicity against colon[36,59], gastric[60−62], glioma[63], leukaemia[64], liver[65], lung[66,67], ovarian[68] and prostate[69] cancers.
4.3. Studies on SAR
There are several studies on the structure-activity relationship (SAR) of PMFs with respect to their cancercytotoxic activities. These studies include hydroxylated or demethylated analogues of nobiletin (Fig. 2) and tangeretin (Fig. 3) as exemplified by natsudaidain 1, 5-demethylnobiletin 2, demethylheptaMF 3, 3-hydroxy-nobiletin 4, sinensetin 5 and 5-demethyltangeretin 6 (Fig. 4). Results have shown that the presence of a methoxy group at C8 and a hydroxyl group at C3 or C5 is essential for their anti-proliferative activity of PMFs[70]. Nobiletin has the C8 methoxy group but not the C3 hydroxyl group. Natsudaidain 1 with both the C8 methoxy and C3 hydroxyl groups displays the strongest anti-proliferative activity towards A549 lung, B16 melanoma 4A5, CCRF-HSB-2 leukaemia and TGBC11TKB gastric cancer cells[70]. IC50 values are 5.2, 3.4, 2.2 and 1.2 μM, respectively. 
 
 
Figure 4. Molecular structures of PMFs and hydroxylated PMFs used in SAR studies. 
 
Studies have shown that hydroxylated PMFs exhibit stronger cytotoxic activity. IC50 values of nobiletin are 1.2 and 2.9 μM against MDA-MB-435 and MCF-7 breast cancer cells, respectively[71]. When the C5 methyl group of nobiletin is hydroxylated(5-demethylnobiletin 2), the cytotoxicity is greatly enhanced by 1.6 and 2.6 times, respectively. Towards HL-60 leukaemia cells, the IC50 value of nobiletin is 41.5 μM[72]. The IC50 values of 5-demethylnobiletin 2 (hydroxylation of C5 methyl group) and 5-demethyl-heptaMF 3 (hydroxylation of C5 methyl group and methylation of C3) are increased to 2.07 and 4.16 μM, respectively. When C3 of nobiletin is hydroxylated (3-hydroxynobiletin4), the cytotoxicity against HL-60 leukaemia cells is totally lost (>100 μM). Sinensetin 5 or nobiletin without the C8 methyl group exhibits weak cytotoxicity (13.3 μM)[72]. Similarly, 5-hydroxy PMFs have also been reported to show much stronger inhibitory effects on HCT116 and HT29 colon cancer cells[73]. Cell viability studies have shown that 5-demethyltangeretin 6 inhibits A549, H460, and H1299 lung cancer cells with IC50 values 79, 57 and 56 times stronger than those of tangeretin, respectively[74].
In a study on PMFs and hydroxylated PMFs as inhibitors of HL-60 leukaemia cells, both the configurationand total number of methoxy groups substantially affect the anti-cancer properties of PMFs[63]. Polymethylation of the hydroxylated groups in PMFs increases the lipophilicity, metabolic stability and membrane transport in the intestine and liver, leading to improved bioavailability[75].
Overall, there is no convincing evidence that nobiletinwith six methyl groups is more cytotoxic than tangeretin with five methyl groups. Against HL-60 leukaemia cells, the IC50 values of nobiletin and tangeretin are 41.5 and >100 μM, respectively[72]. However, against A549 lung, B16 skin, 4A5 melanoma, CCRF-HSB-2 leukaemia and TGBC11TKB gastric cancer cells, the IC50 values are 12, 10, 7.9 and 6.5 μM for tangeretin, and 22, 18, 13 and 8.3 μM for nobiletin, respectively[70]. Against MCF-7 and MDA-MB-435 breast cancer cells, and HT-29 colon cancer cells, the cytotoxicity of tangeretin has been reported to be stronger than nobiletin[36]. IC50 values of tangeretin are 4 μM for all three cell lines, and the IC50 values of nobiletin are 60 μM towards MCF-7 and HT-29 cells, and 100 μM towards MDA-MB-435 cells.
5. Conclusions
Nobiletin and tangeretin are the most abundant PMFs in citrus fruits. Against breast cancer cell lines, these two compounds induce apoptosis, cytostatic cell death, cell cycle arrest, and inhibit cell proliferation, metastasis and tumour angiogenesis, depending on the molecular subtypes of breast cancer. The strong anti-proliferative activities of PMFs suggest their potential as anticancer agents. Both in vitro and in vivo cytotoxic activities involve different molecular targets and signalling pathways. Analyses on the SAR of nobiletin and tangeretin have shown that the presence of a methoxy group at C8 and a hydroxyl group at C3 or C5 is essential for their anti-proliferative activity against cancer cells.Although SAR studies are yielding some interesting findings via transformations, more analyses are needed to test the different molecular subtypes, especially the TN breast cancer cells. Currently, patients with TN breast cancer have poor survival rates. Therefore, there is a real need to develop new therapeutic agents that can improve the survival and quality of life of these patients. Moreover, we should also explore the potential nobiletin and tangeretin as co-chemotherapeutic agents for cancer drugs, such as doxorubicin, in inducing apoptosis and cell cycle arrest of different breast cancer cells. Further studies on the pharmacokinetics, bioavailability and toxicity of these two PMFs are warranted, specifically in targeted cancer treatment using different delivery techniques.
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Dr. Eric Chan, Associate Professor at the Faculty of Applied Sciences, UCSI University, Kuala Lumpur, Malaysia, obtained his PhD (Natural Product Chemistry) from Monash University Malaysia in 2009. To date, Dr. Eric Chan has 88 publications in international refereed journals with 66 (6 in JCPS) as the lead author. His publications have received more than 1450 citations in Scopus and 3000 citations in Google Scholar. He was one of the Top 5 Competitors of the Elsevier Green and Sustainable Chemistry Challenge 2015, out of 500 proposals submitted globally. In April 2016, he presented his proposal at the Green and Sustainable Chemistry Conference in Berlin, Germany. In the same month, he was awarded the Promising Researcher Award by UCSI University.
 
 
 
 
  
 
  
 
 
 
Received: 2020-03-14; Revised: 2020-04-20; Accepted: 2020-05-12.
*Corresponding author. Tel.: +60-3-9101-8880, E-mail: chanwc@ucsiuniversity.edu.my, erchan@yahoo.com       
 
     
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