Diosmetin and tamarixetin (methylated flavonoids): A review on their chemistry, sources, pharmacology, and anticancer properties

This review begins with an introduction to the basic skeleton and classes of flavonoids. Studies on flavonoids have shown that the presence or absence of their functional moieties is associated with enhanced cytotoxicity toward cancer cells. Functional moieties include the C2–C3 double bond, C3 hydroxyl group, and 4-carbonyl group at ring C and the pattern of hydroxylation at ring B. Subsequently, the current knowledge on the chemistry, sources, pharmacology, and anticancer properties of diosmetin (DMT) and tamarixetin (TMT), two lesser-known methylated flavonoids with similar molecular structures, is updated. DMT is a methylated flavone with three hydroxyl groups, while TMT is a methylated flavonol with four hydroxyl groups. Both DMT and TMT display strong cytotoxic effects on cancer cell lines. Studies on the anticancer effects and molecular mechanisms of DMT included leukemia and breast, liver, prostate, lung, melanoma, colon, and renal cancer cells, while those of TMT have only been reported in leukemia and liver cancer cells. These findings suggest that flavones lacking the C3 hydroxyl group at ring C are more cytotoxic than flavonols having the C3 hydroxyl group. The in vitro and in vivo cytotoxic activities of DMT and TMT against cancer cells involve different molecular targets and signaling pathways. From this study, it is clear that little is known about the pharmacology and anticancer properties of DMT and TMT. The potentials for further research into these aspects of the two lesser-known methylated flavonoids are enormous.


INTRODUCTION
Flavonoids represent the largest family of phenolic secondary metabolites from plants with more than 9,000 compounds reported (Wang et al., 2011). They occur in most herbs, fruits, and vegetables (Kopustinskiene et al., 2020;Panche et al., 2016). These polyphenols have a molecular structure consisting of two benzene rings A and B that are joined by a heterocyclic pyran ring C forming the benzopyrone (C6-C3-C6) moiety (Raffa et al., 2017;Singh et al., 2014). Rings A and C are composed of the chroman (C6-C3) nucleus (Kanadaswami et al., 2005). The basic skeleton along with the functional moieties is shown in Figure 1.
Flavonoids are endowed with health-promoting properties including nutraceutical, pharmaceutical, and cosmeceutical applications (Panche et al., 2016). Pharmacological properties include antioxidant, antimicrobial, antiallergic, antiinflammatory, anticarcinogenic, and antidiabetic effects (Guven et al., 2019;Raffa et al., 2017). The medical applications of flavonoids involve protection against cancer and other diseases, such as cardiovascular, rheumatic, obesity, high cholesterol, hypertension, and neurological disorders (Ballard and Junior, 2019;Havsteen, 2002).The anticancer effects of flavonoids operate during the stages of initiation, promotion, and progression of carcinogenesis. In the initiation and promotion stages, flavonoids can inhibit cell proliferation (Abotaleb et al., 2019;Ballard and Junior, 2019). At the stage of progression, flavonoids can inhibit proangiogenesis, regulate metastasis, induce cytotoxicity and apoptosis, promote cell cycle arrest, and reverse multidrug resistance (MDR) or a combination of these mechanisms (Abotaleb et al., 2019;Chahar et al., 2011;Raffa et al., 2017). The antitumor activities of flavonoids include the induction of apoptosis, suppression of protein tyrosine kinase activity, antiproliferation, antimetastasis, anti-invasive effects, and antiangiogenesis (Kanadaswami et al., 2005). Many studies have provided scientific evidence for the anticancer properties of flavonoids in vitro and in vivo (Ren et al., 2013;Wang, 2000). Flavonoids such as quercetin and flavopiridol are now in phase II human clinical trials for different cancers.
When tested against different cancer cells, cytotoxicity of various classes of flavonoids based on IC 50 values was ranked as flavones > flavonols > flavanones > isoflavones ~ flavanols (Kuntz et al., 1999;Li et al., 2008;Plochmann et al., 2007;Sak, 2014). Flavones have the strongest cytotoxicity over the other groups of flavonoids due to the presence of the C2-C3 double bond, compared to the 4-carbonyl (4-oxo or 4-keto) group and the C3-hydroxyl group at ring C ( Fig. 1). Disparity exists as stronger cytotoxicity has been reported in flavonols than flavones, for example, quercetin > kaempferol > apigenin .
The pattern of hydroxylation in ring B influences the degree of cytotoxicity; for example, the ortho-hydroxylated quercetin (3′ and 4′) is three times more cytotoxic than the meta-hydroxylated morin (2′ and 4′). Other factors influencing cytotoxicity are O-methylation and glucuronidation in the A ring which are associated with enhanced cytotoxicity, while a higher number of hydroxyl residues and solubility are inversely correlated with cytotoxicity (Plochmann et al., 2007).
When tested against five different cancer cell lines, flavonoids can be categorized into those with strong and those with weak in vitro cytotoxic effects (Chang et al., 2008). Apigenin, luteolin, and fisetin of the strong category are characterized by having two hydroxyl groups in rings AC, while myricetin and morin of the weak category have three hydroxyl groups in rings AC ( Fig. 1). Both naringenin and apigenin share the same molecular structure. Naringenin without the 2,3-double bond displayed weak cytotoxic effects suggesting the importance of the double bond between C2 and C3 (Chang et al., 2008). Genistein and daidzein are isoflavones in which ring B is attached to ring C at C3 instead of C2.
For polymethylated flavonoids (e.g., natsudaidain), a methoxy group at C8 and a hydroxyl group at C3 are essential for their antiproliferative activity of the flavonoids (Kawaii et al., 1999). Isoflavones (e.g., genistein and daidzein) are flavonoids in which the B ring is linked in position 3 of the C ring (Chang et al., 2008;Lopez-Lazaro, 2002;Lopez-Lazaro et al., 2002). Generally, isoflavones have weaker cytotoxicity than the other flavonoids linked in position 2. In addition, the sugar moiety of flavonoids (e.g., rutin and isoquercetin) reduces their cytotoxic activity (Lopez-Lazaro, 2002;Lopez-Lazaro et al., 2002). In flavonoids, the ring B catechol moiety of flavonoids (e.g., 3′,4′-diOH) and the -OMe group at 5′ are beneficial toward their cytotoxicity, while glycosylation at C5 of ring A has adverse effects on cytotoxicity   .This review begins with an introduction to the basic skeleton and different classes of flavonoids. Subsequently, the current knowledge on the chemistry, sources, pharmacology, and anticancer properties of diosmetin (DMT) and tamarixetin (TMT), two lesser-known methylated flavonoids with similar molecular structure, is updated. Sources of information cited were from Google Scholar, PubMed, PubMed Central, Science Direct, J-Stage, PubChem, and Directory of Open Access Journals.

PHARMACOLOGY Diosmetin
The anti-inflammatory, antioxidant, and hepatoprotective effects of DMT have been reported . Other pharmacological properties of DMT include antimicrobial (Meng Table 1. Anticancer effects and molecular mechanisms of diosmetin (DMT).

Cancer cell line and type
Anticancer effect and molecular mechanism of diosmetin (reference) MDA-MB-468 breast Inhibits cell proliferation, causes G1 cell cycle arrest, and exerts cytostatic effects via CYP1 enzyme-mediated conversion to luteolin (Androutsopoulos et al., 2009a) MCF-7 breast Inhibits cell proliferation and its cytotoxic effects are dependent on CYP1 enzyme conversion to luteolin (Androutsopoulos et al., 2009b) MDA-MB-231 breast Exerts antiproliferative and proapoptotic activities via cell cycle arrest and the mitochondria-mediated intrinsic apoptotic pathway  HepG2 liver Exerts synergistic cytostatic effects and arrest G2/M cell cycle when applied with luteolin via CYP1A-catalyzed metabolism, activation of c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), and P53/P21 upregulation (Androutsopoulos and Spandidos, 2013) HepG2 liver Induces cell apoptosis by upregulating p53 via the transforming growth factorβ (TGF-β) signal pathway (Liu et al., 2016a) SK-HEP-1 liver Inhibits cell metastasis by downregulating the expression levels of MMP-2 and MMP-9 via the protein kinase (PKC)/ mitogen-activated protein kinase (MAPK)/metalloproteinase (MMP) pathways (Liu et al., 2016b) HepG2 liver Inhibits cell proliferation and induces apoptosis by regulating autophagy via the mammalian target of rapamycin (mTOR) pathway (Liu et al., 2016c) HepG2 liver Triggers apoptosis by activation and inactivation of the p53/Bcl-2 pathway and the Notch3/nuclear factor-kappa B (NF-κB) pathway, respectively (Qiao et al., 2016) HepG2 liver Inhibits cell proliferation and promotes cell apoptosis and cell cycle arrest by targeting chk2 (
The anticancer effects and molecular mechanisms of DMT toward different cancer cell lines are listed in Table 1. Against MCF-7 and MDA-MB-468 breast cancer cells, DMT inhibits cell proliferation, arrests G1 cell cycle, and exerts enhanced cytotoxic or cytostatic effects via CYP1 enzyme-mediated conversion to luteolin (Androutsopolous et al., 2009a(Androutsopolous et al., , 2009b. DMT displays antiproliferative and proapoptotic activities against MDA-MB-231 breast cancer cells via cell cycle arrest and the mitochondriamediated intrinsic apoptotic pathway . When used in combination against HepG2 liver cancer cells, DMT and luteolin exhibit cytostatic effects and arrest G2/M cell cycle via CYP1A-catalyzed metabolism, P53/P21 upregulation, and JNK and ERK activation (Androutsopolous and Spandidos, 2013). When tested with HepG2 and SK-HEP-1 liver cancer cells, DMT induces cell apoptosis by upregulating p53 via the TGF-β signal pathway (Liu et al., 2016a); inhibits cell metastasis by downregulating the expression levels of MMP-2 and MMP-9 via the PKC/MAPK/MMP pathways (Liu et al., 2016b); inhibits cell proliferation by inducing apoptosis and by regulating autophagy via the mTOR pathway (Liu et al., 2016c); triggers apoptosis by activation of the p53/Bcl-2 pathway and inactivation of the Notch3/NF-κB pathway (Qiao et al., 2016); suppresses cell proliferation; and enhances cell apoptosis and cell cycle arrest by targeting chk2 (Ma and Zhang, 2020 (Oak et al., 2018); induces apoptosis of [2] by producing ROS and reducing Nrf2 stability via suppression of the PI3K/ Akt/GSK-3β pathway (Chen et al., 2019b); and suppresses tumor progression and metastasis of [3] by inducing cell death and inhibiting angiogenesis (Choi et al., 2019). DMT promotes apoptosis, inhibits cell proliferation, and arrests G2/M cell cycle of [4] mediated by the membrane death receptor (Koosha et al., 2019a); reduces tumor growth of [4] in nude mice via downregulation of Bcl-2 and overexpression of Bax (Koosha et al., 2019b); promotes apoptosis and cytotoxicity of [5] by reducing AKT phosphorylation via p53 upregulation (Qiu et al., 2020); and induces apoptosis of [6] via activation of caspases 8 and 3/7 and the death-inducing cytokine TNFα (Roma et al., 2018).
There are only two studies on the anticancer effects and molecular mechanisms of TMT (Table 2). Against doxorubicin-resistant K562/ADR leukemia cells, TMT inhibits cell proliferation, arrests G2/M cell cycle, and induces apoptosis (Nicolini et al., 2014). In another study, the cytotoxicity of TMT toward HepG2 and PLC/PRF/5 liver cancer cells and nude mice tumor xenograft was reported . In liver cancer cells, TMT suppresses cell viability via apoptosis, lactate dehydrogenase (LDH) release, caspase-3 activation, ROS accumulation, and decreased mitochondrial membrane potential. In liver tumor xenograft, TMT enhances the expression levels of proapoptotic proteins, including Bax and cleaved caspase-3, and inhibits the expression levels of antiapoptotic proteins. Both in vitro and in vivo studies showed that TMT significantly suppressed the phosphorylation of ERK and AKT in liver cancer cells and tumors .

Structure-activity relationship (SAR) studies
There are very few structure-activity relationship (SAR) studies on DMT and TMT related to anticancer activities. In a study of the inhibitory effects of MDR proteins 1 (MRP 1), an important mechanism in MDR during cancer treatment, methylated flavonoids are among the best inhibitors with IC 50 values ranging from 2.7 to 14.3 µM (van Zanden et al., 2005). Inhibition at 25 µM and IC 50 values was 84% and 2.7 µM for DMT and 68% and 7.4 µM for TMT. DMT was the strongest, while TMT ranked third. Values of DMT and TMT were stronger than luteolin and quercetin, suggesting that the 4′-methyl ether moieties of DMT and TMT contribute to their inhibitory effects. In another study on the inhibitory effects of flavonoids on NF-κB signaling in MDA-MB-231 breast cancer cells, DMT (3.7%) displayed stronger inhibition than TMT (2.4%) (Amrutha et al., 2014). Inhibitory values of DMT and TMT were stronger than luteolin (3.0%) and much weaker than quercetin (3.7%), respectively.

CONCLUSION
Flavonoids are the largest family of phenolic secondary metabolites from plants. They have a molecular structure consisting of two benzene rings (A and B) joined by a pyran ring (C) forming a benzo-pyrone (C6-C3-C6) moiety. The majority of the flavonoids have the B ring linked in position 2 to the C ring, and they can be further divided into classes such as flavones, flavonols, flavanones, and flavanols. Studies have shown that the presence or absence of some functional moieties is associated with enhanced cytotoxicity toward cancer cells. They include C2-C3 double bond, a 4-carbonyl group, and a C3 hydroxyl group at ring C and the pattern of hydroxylation (ortho or meta) at ring B.
DMT and TMT are methylated flavonoids. DMT is a methoxyflavone having three hydroxyl groups, while TMT is a methoxyflavonol with four hydroxyl groups. This review on the anticancer properties of DMT and TMT supported the view that flavones without the C3 hydroxyl group are stronger in cytotoxicity against cancer cells than flavonols with the C3 hydroxyl group. However, further investigations are needed to confirm the role of the C3 hydroxyl group in cytotoxicity toward cancer cells.
Further clinical research on DMT and TMT is warranted to evaluate their safety and chemopreventive efficacy when used alone or in combination with other chemotherapy agents. Current knowledge of their pharmacokinetics, bioavailability, and SAR studies is meager. Further research on the structural modifications of DMT and TMT is needed for the synthesis of novel derivatives with enhanced inhibitory effects against different cancer cells and reduced cytotoxicity toward normal cells. For lesser-known bioactive compounds, such as DMT and TMT, their use in purified and standardized extracts containing chemical constituents that have the desired pharmacological activity may be the most practical approach. While Western medicine employs pure and single compounds, Chinese medicine (CM) has long used different combinations of compounds in the form of medicinal herbs to treat, ameliorate, and relieve the symptoms of different diseases. CM may have fewer and less severe side effects than single pure drugs, making them especially attractive to consumers. The development and clinical usage of different formulations of DMT and TMT with synergistic anticancer effects, reduced side effects, and acceptable quality control remain a major challenge. Little is known about the pharmacology and anticancer properties of DMT and TMT. The potentials for further research into these aspects of the two lesser-known methylated flavonoids are enormous. This will generate much research interest among medicinal chemists and researchers who are keen on lesser-known flavonoids. Table 2. Anticancer effects and molecular mechanisms of TMT.

Cancer cell line and type
Anticancer effect and molecular mechanism of TMT (reference) K562/ADR leukemia Inhibits cell proliferation in a concentration-and time-dependent manner, induces apoptosis, and arrests G2/M cell cycle (Nicolini et al., 2014) HepG2 and PLC/PRF/5 liver Suppresses cell viability via enhanced cell apoptosis, LDH release, caspase-3 activation, and ROS accumulation and decreases mitochondrial membrane potential . Phosphorylation of ERK and AKT in liver cancer cells is significantly suppressed Nude mice with HepG2 and PLC/PRF/5 liver xenograft tumor Enhances the expression levels of proapoptotic proteins (including Bax and cleaved caspase-3) and inhibits the expression levels of antiapoptotic proteins after 14-day administration . Phosphorylation of ERK and AKT in xenograft liver tumors is significantly suppressed AKT = protein kinase B; Bax = Bcl-2 associated X protein; Bcl = B-cell lymphoma; ERK = extracellular signal-regulated kinase; LDH = lactate dehydrogenase; ROS = reactive oxygen species.