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. Author manuscript; available in PMC: 2021 Feb 10.
Published in final edited form as: Xenobiotica. 2020 Oct 23;51(2):139–154. doi: 10.1080/00498254.2020.1836433

Liquid chromatography-tandem mass spectrometry analysis of oxidation of 2′-, 3′-, 4′-, and 6-hydroxyflavanones by human cytochrome P450 enzymes

Tsutomu Shimada a, Haruna Nagayoshi b, Norie Murayama c, Shigeo Takenaka d, Jun Katahira a, Vitchan Kim e, Donghak Kim e, Masayuki Komori a, Hiroshi Yamazaki c, F Peter Guengerich f
PMCID: PMC7875482  NIHMSID: NIHMS1665215  PMID: 33047997

Abstract

  1. 2′-Hydroxyflavanone (2′OHFva), 3′OHFva, 4′OHFva, and 6OHFva, the major oxidative products of flavanone by human cytochrome P450 (P450, CYP) enzymes, were studied in regard to further oxidation by human CYP1A1, 1A2, 1B1.1, 1B1.3, and 2A6. The products formed were analyzed with LC-MS/MS and characterized by their positive ion fragmentations on mass spectrometry.

  2. Several di-hydroxylated flavanone (diOHFva) and di-hydroxylated flavone (diOHFvo) products, detected by analyzing parent ions at m/z 257 and 255, respectively, were found following incubation of these four hydroxylated flavanones with P450s. The m/z 257 products were produced at higher levels than the latter with four substrates examined. The structures of the m/z 257 products were characterized by LC-MS/MS product ion spectra, and the results suggest that 3′OHFva and 4′OHFva are further oxidized mainly at B-ring by P450s while 6OHFva oxidation was at A-ring.

  3. Different diOHFvo products (m/z 255) were also characterized by LC-MS/MS, and the results suggested that most of these diOHFvo products were formed through oxidation or desaturation of the diOHFva products (m/z 257) by P450s. Only when 4′OHFva (m/z 241) was used as a substrate, formation of 4′OHFvo (m/z 239) was detected, indicating that diOHFvo might also be formed through oxidation of 4′OHFvo by P450s.

  4. Finally, our results indicated that CYP1 family enzymes were more active than CYP2A6 in catalyzing the oxidation of these four hydroxylated flavanones, and these findings were supported by molecular docking studies of these chemicals with active sites of P450 enzymes.

Keywords: Hydroxy flavanones, LC-MS/MS, oxidation, human, cytochrome P450

Introduction

A variety of plant flavonoids, such as flavones, flavanones, flavonols, flavanols, isoflavonoids, and anthocyanidins, constitute a class of secondary polyphenolic metabolites that show various biological activities (e.g. anti-cancer, anti-oxidative, and anti-inflammatory activities) in laboratory animals and humans (Androutsopoulos et al., 2009; 2010: Arct and Pytkowska, 2008; Bisol 2020: Hostetler et al., 2017; Kale et al., 2008, Zhang et al., 2005). To better understand the basis of mechanisms by which these flavonoids exert their biological activities, studies have been done to examine how these flavonoids are converted to biologically active and/or inert compounds by various enzymes, such as cytochrome P450s (P450s or CYPs) (Androutsopoulos et al., 2009; 2010: Breinholt et al., 2002, Hodek et al., 2002; Kim et al., 2005; Surichan et al., 2012; 2018; Walle et al., 2007; Zhang et al., 2005).

We have recently reported that flavanone is catalyzed by different forms of human P450 enzymes to form several oxidative products, including flavone, 2′-, 3′-. 4′-, and 6-hydroxyflavanone (2OHFva, 3′OHFva, 4′OHFva, and 6OHFva, respectively) and other chemically unidentified products (Kakimoto et al., 2019; Nagayoshi et al., 2019a). It was also found that CYP2A6 is a principal enzyme involved in flavanone 2′-hydroxylation in human liver microsomes (Nagayoshi et al., 2019a). Recent studies have shown that 2′OHFva prevents proliferation of tumor cells in lung, prostate, kidney, stomach, and breast in humans (Awasthi et al., 2018; Ofude et al., 2013; Singhal et al., 2015; 2018; Yue et al., 2020; Zhang et al., 2015) and exerts anticancer activity in dimethylbenz[a]anthracene-induced mammary tumor in SENCAR mice in vivo (Singhal et a., 2019).

The aims of this study were to examine how 2′OHFva and other hydroxylated flavanones 3′OHFva, 4′OHFva, and 6OHFva, the major oxidative products of flavanone catalyzed by human P450 enzymes, were further oxidized to various products by CYP2A6 (highly active in flavanone 2′-hydroxytion) (Nagayoshi et al., 2019a) and by CYP1A1, 1A2, 1B1.1, and 1B1.3 (four active enzymes in the oxidation of several flavonoids including 5-hydroxyflavone, 57diOHFvo, and 2′-. 3′-, and 4′-methoxyflavones) (Nagayoshi et al., 2019b). Our interests are also extended to whether 2′OHFva, 3′OHFva, 4′OHFva, and 6OHFva are also oxidized/desaturated at C-rings of the molecules by P450 enzymes, as in a case of oxidation/desaturation of flavone by P450s (Kagawa et al., 2004; Lam et al., 2014; Mizuno et al., 2016; Kakimoto et al., 2019; Nagayoshi et al., 2019b). LC-MS/MS analysis of formation of di-hydroxylated flavanone (diOHFva) and dihydroxy flavone (diOHFvo) products was done by monitoring parent molecular ions at m/z 257 and m/z 255, respectively, and the formation of other products such as monohydroxy flavones (OHFvo) and trihydroxy flavanone (triOHFva) products were determined by monitoring m/z 239 and m/z 273, respectively. Product ion spectra of these products on LC-MS/MS were characterized to identify chemical structures. Molecular docking analysis of interaction of these hydroxylated flavanones with active sites of P450 enzymes is also reported.

Materials and methods

Chemicals

Flavanone (Fva), flavone (Fvo), 2′-hydroxyflavanone (2′OHFva), 3′-hydroxyflavanone (3′OHFva), 4′-hydroxyflavanone (4′OHFva), and 6-hydroxyflavanone (6OHFva) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Wako Pure Chemicals (Osaka, Japan) (Figure 1). 4′-Dihydroxyflavone (4′OHFvo), 5,7-dihydroxyflavone (57diOHFvo), 3′,4′-dihydroxyflavone (3′4′diOHFvo), 4′,7-dihydroxyflavanone (4′7diOHFva), 4′,5,7-trihydroxyflavone (apigenin, 4′57triOHFvo), and 5,6,7-trihydroxyflavone (baicalein, 567triOHFvo) were also purchased from Sigma-Aldrich and Wako Pure Chemicals. Other chemicals and reagents used in this study were obtained from sources described previously or were of the highest quality commercially available (Shimada et al., 2013; Kakimoto et al., 2019; Nagayoshi et al., 2019a).

Figure 1.

Figure 1.

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Structures of monohydroxy flavanones used in this study

Enzymes

Purified preparations of human CYP1A1, CYP1A2, CYP1B1.1, CYP1B1.3, and CYP2A6 expressed in Escherichia coli were obtained by the methods as described previously (Sandhu et al., 1993; 1994; Parikh 1997; Shimada et al., 2010; Han et al., 2012; Kim et al., 2018). NADPH-P450 reductase and cytochrome b5 (b5) were purified from membranes of recombinant E. coli as described elsewhere (Shen et al., 1989; Yamazaki et al., 2002; Guengerich 2014).

Liver microsomes prepared from human samples HH2 (Cat No., 452002) and HH47 (Cat No., 452047) were obtained from GENTEST-Corning (Woburn, MA) as described previously (Nagayoshi et al., 2019a). The data sheets provided by the manufacture indicated that liver microsomes from a HH2 sample had no detectable coumarin 7-hydroxylation activity, thus indicating that this individual is classified as a poor metabolizer (PM) for CYP2A6 activity.

Oxidation of flavanone (Fva) and 2′-, 3′-, 4′-, and 6OHFva by P450 enzymes

The oxidation of Fva and 2′-, 3′-, 4′-, and 6OHFva by P450 enzymes was determined by the methods described previously (Kakimoto et al, 2019; Nagayoshi et al., 2019a; 2019b, 2020). Briefly, reconstituted monooxygenase systems consisting of each purified P450 (50 pmol), NADPH-P450 reductase (100 pmol), b5 (100 pmol, in the case of CYP2A6), and L-α-1,2 dilaouryl-sn-glycero-3-phosphocholine (DLPC) (50 μg) were incubated (0.25 ml of total volume) with 60 μM Fva or with 2′-, 3′-, 4′-, or 6OHFva [by adding 1.5 μl of 10 mM stock solution in (CH3)2SO] at 37 °C for 20 min, following a pre-incubation of 1 min before adding an NADPH-generating system (0.5 mM NADP+, 5 mM glucose 6-phosphate, and 0.5 unit of yeast glucose 6-phosphate dehydrogenase/ml). Each reaction was terminated by the addition of 0.5 ml of ice-cold CH3CN. The mixture was mixed vigorously (with a vortex device) and centrifuged at 10,000 × g for 5 min, and an aliquot of the upper CH3CN layer was injected and analyzed with LC-MS/MS as described (Kakimoto et al, 2019; Nagayoshi et al., 2019a; 2019b).

LC-MS/MS analyses were performed using an HPLC system (ACQUITY UPLC I-Class system; Waters, Milford, MA) coupled to a tandem quadruple mass spectrometer (XevoTQ-S; Waters) by the methods as described previously (Kakimoto et al, 2019; Nagayoshi et al., 2019a; 2019b). MS/MS analysis was performed in the positive electrospray ionization mode with a capillary voltage of 3000 V and cone voltage of 30 V as described previously (Kakimoto et al, 2019; Nagayoshi et al., 2019a; 2019b).

Other assays

P450 and protein contents were determined by the methods described previously (Omura and Sato, 1964; Brown et al., 1989).

Docking simulations of 2′OHFva, 3′OHFva, 4′OHFva, and 6OHFva with P450 enzymes

Crystal structures of CYP1A1 (Protein Data Bank 418V), 1A2 (2H14), 1B1.1 (3PMO), 2A6 (2FDV) have been reported and were used in this study (Walsh et al., 2013; Sansen et al., 2007; Yano et al., 2006; DeVore et al., 2012). The CYP1B1.3 variant (L432V) model was established by incorporating the mutation into CYP1B1.1 (3PMO) (Wang et al., 2011). Chemical structures of 2′-, 3′-, 4′-, and 6OHFva were used from PubChem (an open chemistry database at the National Institutes of Health) and were optimized in MOE software (ver. 2019.01, Computing Group, Montreal, Canada). Simulations were carried out in the MOE by the methods as described previously (Kakimoto et al., 2019; Nagayoshi et al., 2020). Ligand-interaction energies (U values) were obtained by use of the program ASEdock in MOE.

RESULTS

Oxidation of flavanone (Fva) by human P450s and liver microsomes

We have previously shown that human P450 enzymes (CYP1B1.3 was not examined in that study) and liver microsomes catalyzed oxidation of flavanone to form several hydroxylated flavanones (OHFva) and flavone (Fvo) (Nagayoshi et al., 2019a). Here, we re-examined and compared catalytic activities of oxidation of Fva by CYP1A1, 1A2, 1B1.1, 1B1.3, and 2A6 and liver microsomes of samples HH2 and HH54 (Figure 2 and Supplemental Information Table 1). 2′OHFva, 3′OHFva, 4′OHFva, 6OHFva, and Fvo and other unidentified products, M3, M4, M5, M6, and M7, were detected upon incubation of flavanone with P450s and liver microsomes. Fvo, 6OHFva, and 4′OHFva were found to be the major products catalyzed by these P450s and liver microsomes, except that CYP1A1, 1A2, and 2A6 had lower activities for formation of 4′OHFva, Fvo, and 6OHFva, respectively. As expected, CYP2A6 had the highest activity to catalyze Fva to form 2′OHFva and liver sample HH2 showed very low 2′-hydroxylation activities as compared with the HH47 sample. CYP1B1.1 and 1B1.3 showed similar catalytic activities towards flavanone, except that the latter enzyme produced a hydroxylated product M3 (Figure 2). It was also found that CYP1A1 and 1A2 were active in forming a product M5 (Figures 2A and 2B).

Figure 2.

Figure 2.

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Oxidation of flavanone by human P450 enzymes and human liver microsomes. P450 enzymes used were CYP1A1 (A), 1A2 (B) 1B1.1 (C), 1B1.3 (D), and 2A6 (E), plus liver microsomal samples HH2 (F) and HH47 (G) (the HH2 microsomes were defective in CYP2A6-dependent coumarin oxidation activities) (Nagayoshi et al., 2019a). The products formed were 2′OHFva, 3′OHFva, 4′OHFva, 6OHFva, M3, M4, M5, and M6 (these were detected at m/z 241>121 or 241>131) and flavone (Fvo, detected at m/z 223>129) on LC-MS/MS analysis. The substrate flavanone (Fva) was determined at m/z 225>131.

Specific activities of oxidation of Fva to form Fvo, 2′-, 3′-, 4′-, and 6OHFva by five P450s were determined (Supplemental Information Table S1). CYP2A6 was highly active in producing 2′OHFva, 6OHFva, and Fvo (4.1, 1.8, and 0.67 nmol/min/nmol P450, respectively). CYP1A2, 1B1.1, and 1B1.3 were more active than CYP2A6 in forming 4′OHFva. As expected, liver sample HH2, deficient in CYP2A6 activity, was very low in forming 2′OHFva, as compared with a sample HH47, which was active in catalyzing CYP2A6-dependent coumarin 7-hydroxylation activity.

Oxidation of four hydroxylated flavanones by P450s

We incubated 2′-, 3′-, 4′-, and 6OHFva with CYP1A1, 1A2, 1B1.1, 1B1.3, and 2A6, and the products formed were analyzed with LC-MS/MS by detecting formation of dihydroxylated flavanones (diOHFva) at m/z 257 and di-hydroxylated flavones (diOHFvo) at m/z 255 (Figures 3 and 4). Since we did not have standard materials of these products available, we compared the levels of formation of the products by comparing peak intensities on LC-MS/MS (Figures 3 and 4 and Supplemental Information Tables S2 and S3). In Tables S2 and S3, we included results of formation of the m/z 239 products (monohydroxy flavone, OHFvo) and the m/z 273 products (trihydroxy flavanone, triOHFva) (vide infra).

Figure 3.

Figure 3.

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LC-MS/MS analysis of oxidation of 2′OHFva by CYP1A1 (A and F), CYP1A2 (B and G), CYP1B1.1 (C and H), CYP1B1.3 (D and I), and CYP2A6 (E and J) and LC-MS/MS analysis of oxidation of 3′OHFva by CYP1A1 (K and P), CYP1A2 (L and Q), CYP1B1.1 (M and R), CYP1B1.3 (N and S), and CYP2A6 (O and T). The dihydroxy flavanone products of 2’OHFva (A-E), namely 2′-257a, 2′-257b, and 2′-257c, were detected at m/z 257, and the dihydroxy flavone products of 2’OHFva (F-J), namely 2′-255a and 2′-255b, were detected at m/z 255. Similarly, in the oxidation of 3′OHFva, the dihydroxy flavanone products of 3’OHFva, namely, 3′-257a, 3′-257b, 3′- 257c, and 3′-257d (K-O), were detected at m/z 257 and the dihydroxy flavone products of 3’OHFva (P-T), namely 3′-255a, 3′-255b, 3′-255c, and 3’′-255d, were detected at m/z 255.

Figure 4.

Figure 4.

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LC-MS/MS analysis of oxidation of 4′OHFva by CYP1A1 (A and F), CYP1A2 (B and G), CYP1B1.1 (C and H), CYP1B1.3 (D and I), and CYP2A6 (E and J) and LC-MS/MS analysis of oxidation of 6OHFva by CYP1A1 (K and P), CYP1A2 (L and Q), CYP1B1.1 (M and R), CYP1B1.3 (N and S), and CYP2A6 (O and T). The dihydroxy flavanone products of 4’OHFva (A-E), namely 4′-257a, 4′-257b, and 4′-257c, were detected at m/z 257 and the dihydroxy flavone products of 4’OHFva (F-J), namely 4′-255a and 4′-255b, were detected at m/z 255. Similarly, in the oxidation of 6OHFva, the dihydroxy flavanone products of 6OHFva (K-O), namely 6-257a, 6-257b, 6-257c, were detected at m/z 257 and the dihydroxy flavone products of 6OHFva (P-T), namely 6-255a, and 6-255b, were detected at m/z 255.

P450 enzymes formed three diOHFva products (tentatively termed as 2′-257a, 2′-257b, and 2′-257c; Figures 3A–3E) and four diOHFva products (3′-257a, 3′-257b, 3′-257c, and 3′257d; Figures 3K–3O) from 2′OHFva and 3’′OHFva, respectively. We also detected two diOHFvo products (2′-255a and 2′-255b; Figures 3F–3J) and four diOHFvo products (3′-255a, 3′255b, 3′-255c, and 3′-255d; Figures 3P–3T) from 2′OHFva and 3’′OHFva, respectively. Among the five P450s examined, CYP2A6 was not very active in forming the m/z 257 and m/z 255 products compared with the CYP1 family enzymes, particularly in forming the m/z 255 products (Supplemental Information Table 2). The results also showed that these P450s did not produce the m/z 239 and 273 products under these assay conditions (Supplemental Information Table 2).

The five human P450s were also found to oxidize 4′OHFva and 6OHFva to form three diOHFva products (4′-257a, 4′-257b, and 4′-257c; (Figures 4A–4E) and three diOHFva products (6-257a, 6-257b, and 6-257c; Figures 4K–4O), respectively, and two diOHFvo products (4′-255a and 4′-255b; Figures 4F–4J) and two diOHFvo products (6-255a and 6-255b; Figures 4P–4T), respectively. In addition, CYP1 family enzymes were more active than CYP2A6 in producing the m/z 257 and 255 products (Supplemental Information Table 3). Also, 4′OHFva was converted to 4′OHFvo (m/z 239) and m/z 273 products by P450 enzymes, and these P450s (except CYP2A6) produced m/z 273 from 6OHFva (Table S3).

Fragmentation analysis of the m/z 257 products from 2′-, 3′-, 4′-, and 6-OHFva

LC-MS/MS fragmentation ion spectra of three diOHFva oxidation products (6-257a, 6-257b, and 6-257c) were studied following incubation of 6OHFva with P450 enzymes (Figure 5). Standard 6OHFva and 57diOHFvo were used for comparison (Figures 5A and 5E). The identification of these products was based on the characterization of various fragments obtained with LC-MS/MS analysis after ionization of chemicals; the ionization of flavonoids has been shown to have general rules for the identification of their products (Hedin and Phillips, 1992; Lewars and March, 2007; Burns et al., 2007; van der Hooft et al., 2011; Nagayoshi et al., 2019b). All three products, 6-257a, 6-257b, and 6-257c (products with CYP1A1), had fragment ions of m/z 103 and 153, indicating that these products were oxidized on the A-ring (Figures 5B–5D).

Figure 5.

Figure 5.

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Product ion spectra of m/z 257 products of oxidation of 6OHFva with P450s. The spectra of dihydroxy products, 6-257a (5B, obtained with CYP1A1), 6-257b (5C, obtained with CYP1A1), and 6-257c (5D, obtained with CYP1A1), and those of standards 6OHFva (5A) and 57diOHFvo (5E) are shown. Suggested structures and fragments of these products and standard chemicals are included in the figure.

P450s oxidized 4′OHFva to two types of diOHFva products; one was 4′-257a and 4′-257c which had fragments of m/z 65, 93, 121, and 135 indicating oxidation on the B-ring (Figures 6B and 6D), and the other was 4′-257b which had fragment ions at m/z 81, 120, 137, and 147, indicating oxidation on the A-ring (Figure 6C). The standards 4′OHFva and 4′7diOHFva were included for comparison (Figures 6A and 6E).

Figure 6.

Figure 6.

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Product ion spectra of m/z 257 products of oxidation of 4′OHFva with P450s. The spectra of dihydroxy products, namely 4′-257a (6B, obtained with CYP1A1), 4′-257b (6C, obtained with CYP1B1.1), and 4′-257c (6D, obtained with CYP1B1.3) and those of standards 4′OHFva (6A) and 4′7diOHFva (6E) are shown. Suggested structures and fragments of these products and standard chemicals are shown.

In the case of 3′OHFva, we analyzed fragmentation product ion spectra in the range m/z 0–300 (Figure 7A–7D; scale of the vertical axis was 0–30%) and between m/z 110 and 150 (Figures 7a–7d; the scale was expanded to 0–4%) in order to see differences in 3′OHFva and three m/z 257 products. These 3′-257a, 3′-257b, and 3′-257c products were found to have fragments at m/z 135–136, suggesting that these products were oxidized on the B-ring (Figure 7).

Figure 7.

Figure 7.

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Product ion spectra of m/z 257 oxidation products of 3′OHFva formed with P450s. The spectra of dihydroxy products, namely 3′-257a (7B and 7b, obtained with CYP1B1.3), 3′-257b (7C and 7c, obtained with CYP1B1.1), and 3′-257c (7D and 7d, obtained with CYP1A1) and that of standard 3′OHFva (7A and 7a) are shown. Suggested structures and fragments of these products and standard chemicals are shown. Figures 7A–7C show spectra from m/z 0–300, and Figures 7a–7d are from m/z 110 to 150.

In a case of 2′OHFva, it was not possible to precisely determine the chemical structures of three products (2′-257a, 2′-257b, and 2′-257c) obtained with CYP1A2 (Figure 8). When the product ion spectra were compared for m/z 0–300 (vertical axis set at 0–60%), the spectra of three products (2′-257a, 2′-257b, and 2′-257c) were found to be similar to that of the substrate 2′OHFva (Figures 8A and 8B–8D). However, when comparing the spectra between m/z 91–94 (Figures 8a1 and b1–d1; relative abundance scale 0–20%), between m/z 119–122 (Figures 8a2 and b2–d2; scale 0–20%), and between m/z 125–145 (Figures 8a3 and b3–d3; scale 0–2%), there were differences between 2′OHFva and these three products; some putative fragments of these products are included in the figures.

Figure 8.

Figure 8.

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Product ion spectra of m/z 257 products of oxidation products of 2′OHFva formed by P450s. The spectra of dihydroxy products, namely 2′-257a (8B and 8b, obtained with CYP1A2), 2′-257b (8C and 8c, obtained with CYP1A2), and 2′-257c (8D, obtained with CYP1A2) and that of standard 2′OHFva (8A) are shown. Suggested structures and fragments of these products and standard chemicals are shown. Figures 8A–8D show spectra between m/z 0–300 and Figures 8a1–8d1, Figures 8a2–8d2, and Figures 8a3–8d3 are from m/z 91–94, 119–122, and 125–145, respectively.

Fragmentation analysis of m/z 255 products from 2′-, 3′-, 4′-, and 6-OHFva

Human P450s produced two m/z 255 products (2′-255a and 2′-255b) from 2′OHFva, four (3′-255a 3′-255b, 3′-255c, and 3′-255d) from 3′OHFva, two (4′-255a and 4′-255b) from 4′OHFva, and two (6-255a and 6-255b) from 6OHFva (Figures 3 and 4). The product ion spectra of standard 3′4′diOHFvo (Figure 9A) and 57diOHFvo (Figure 9F) were included in the figure for comparison. Our analysis of product ion spectra suggested that all of these products (2′-255a, 2′-255b, 3′255b, 3′255c, and 4′255a) were dihydroxy flavones oxidized on the B-ring due to the presence of fragments m/z 65, 93, 121, 209, and 224. On the other hand, 6-255a and 6-255b contained fragments m/z 69, 95, 123, 152, and 209, indicating product formation of diOHFvo oxidized on the A-ring (Figure 9I and 9J).

Figure 9.

Figure 9.

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Product ion spectra of m/z 255 products of oxidation of 2′-, 3′-, 4′-, and 6OHFva by P450 enzymes. The spectra of 3′4’diOHFvo (A) and 57diOHF (F) and the di-hydroxylated flavone products including 2′-255a (B), 2′-255b (C), 3′-255c (D), 3′-255b (G), 4′-255a (H), 4′-255b (E), 6-255a (I), and 6-255b (J) are shown. Suggested structures and fragments of these products and standard chemicals are also shown.

Formation of 4′OHFvo (m/z 239) from 4′OHFva (m/z 241) by human P450 enzymes

Only 4′OHFva was converted to a monohydroxy product, 4′OHFvo, by human P450 enzymes, and we found that CYP1B1.1 and 1B1.3 had higher activities in forming 4′OHFvo than CYP1A1, 1A2, and 2A6 (Figure 10B–10F). Product ion spectra showed that 4′-239a (from CYP1B1.1 and 2A6) had a fragmentation pattern similar to that of standard 4′OHFvo (Figure 10G–10I).

Figure 10.

Figure 10.

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LC chromatography of formation of 4′OHFvo upon incubation of 4′OHFva with CYP1A1 (B), 1A2 (C), 1B1.1 (D), 1B1.3 (E), and 2A6 (F) and compared with standard 4′OHFvo (A). Product ion spectra of standard 4′OHFvo (G), 4’-239a on incubation of 4’OHFva with CYP1B1.1 (H) and CYP2A6 (I) are shown.

Formation of trihydroxy flavanones (triOHFva) on incubation of 4′OHFva and 6OHFva with P450 enzymes

Human P450s produced the m/z 273 products (4′-273a and 6-273b) from 4′OHFva and 6OHFva, respectively (Figure 11A and 11B). CYP1B1.1 and 1B1.3 were highly active in forming 4′-273a and 6-273b as compared with other P450s (Figures 11C and 11E). Product ion spectra suggested that 4′-273a was a triOHFva product (Figure 11G) oxidized on the A-ring because it contained a m/z 153 fragment seen in standard 4′57triOHFvo (apigenin, Figure 11F). In addition, 6-273a and 6-273b are suggested to be triOHFva products oxidized on the A-ring (Figure 11H and 11I), having a m/z 169 fragment that was detected with 567triOHFvo (baicalein, Figure 11J).

Figure 11.

Figure 11.

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Formation of m/z 273 products formed on incubation of 4′OHFva (A) and 6OHFva (B) with CYP1B1.3. Formation of 4′-273a (A and C), 6-273a (B and D), and 6-273b (B and E) was analyzed with LC-MS/MS. Product ion spectra of 4′-273a (G), 6-273a (H), and 6-273b (I) were obtained following incubation of 4′OHFva and 6OHFva with CYP1B1.3. The spectra of standards 4′57triOHFvo (F) and 567triOHF (J) are included for comparison. Suggested structures and fragments of these products are shown.

Molecular docking interaction of OHFva in active sites of P450s

The interactions of 2′-, 3′-, 4′-, and 6OHFva with CYP1A1, 1A2, 1B1.1, 1B1.3, and 2A6 were examined by molecular docking analysis (Figure 12). Among several possible cases, we selected reasonable interactions that show lower ligand-interaction energies (U values) between hydroxylated flavanone molecules and the active sites of P450 enzymes (Figure 12). Some interesting findings were the observations that interactions occurred between the B-rings of the flavonoid molecules and active sites of P450s when CYP1 enzymes were used, except that CYP1A1 was interfaced with the A- and C-rings of 4′OHFva (Figure 12K). These results contrast with the results of interactions when CYP2A6 was used, in that there the A-rings of the chemicals were near the active sites of P450 enzymes examined (Figure 12E, J, O, and T). CYP2A6 was relatively slower than other CYP1 family enzymes in oxidizing these hydroxylated flavanones to form the m/z 257 and 255 products (Figures 3 and 4 and Supplemental Information Tables S2 and S3).

Figure 12.

Figure 12.

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Molecular docking analysis of interaction of 2′OHFva (A-E), 3′OHFva (F-J), 4′OHFva (K-O), and 6OHFva (P-T) with CYP1A1 (A, F, K, and P), 1A2 (B, G, L, and Q), 1B1.1 (C, H, M, and R), 1B1.3 (D, I, N, and S), and 2A6 (E, J, O, and T). Ligand-interaction energies (U values) are shown.

As described above, 4′OHFva was oxidized to 4′OHFvo by human P450s, and we examined molecular interactions of 4′OHFvo with five forms of P450s (Figure 13). The results suggest that 4′OHFvo B-ring interacts with the active sites of these P450s, even when CYP2A6 was used (Figure 13E).

Figure 13.

Figure 13.

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Molecular docking analysis of interaction of 4′OHFvo with CYP1A1 (A), 1A2 (B), 1B1.1 (C), 1B1,3 (D), and 2A6 (E). Ligand-interaction energies (U values) are shown.

Discussion

A variety of plants synthesize different types of flavonoids including free, hydroxylated, and methoxylated flavonoids and their C- and O-glycosides (Hostetler et al., 2017; Martens and Mithöfer, 2005; Tanaka et al., 2010; Tanaka and Brugliera, 2013). It has been reported that ionization of flavonoids with LC-MS/MS produces various fragments through dehydration, losses of carbon monoxide, and fission at C-ring bonds, namely retro Diels-Alder cleavage, and that these fragments can be used for identification of chemical structures of these flavonoids and their metabolites (Sasaki et al., 1966; Heiden and Phillips, 1992; Nikolic and van Breeman, 2004; Tsimogiannis et al., 2007).

In this study, we examined the oxidation of four hydroxylated flavanones, i.e. 2′-, 3′-, 4′-, and 6OHFva, by human P450s CYP1A1, 12, 1B1.1, 1B1.3, and 2A6 to form various products by using LC-MS/MS. These four hydroxylated flavanones were further oxidized by P450s mainly to several dihydroxy flavanones with m/z 257, and fragment analysis with LC-MS/MS showed that 6OHFva was oxidized on the A-ring while oxidation of 3′OHFva was on the B-ring of the flavonoid molecule. Two products (4′-257a and 4′257c) from 4′OHFva were oxidized on the B-ring, while one m/z 257 product (4′-257b) was oxidized on the A-ring. We also detected several m/z 255 (diOHFvo) products from four OHFva substrates examined and the LC-MS/MS results suggested that these diOHFvo products were mainly formed through oxidation or desaturation of diOHFva products (m/z 257) by P450s. However, when 4′OHFva (m/z 241) was used as a substrate, 4’OHFvo (m/z 239) was produced by P450s, indicating that diOHFvo might also be catalyzed through oxidation of 4′OHFvo by P450s.

We have previously proposed that there are at least two different mechanisms of formation of flavone from flavanone by human P450 enzymes; one is formation of 2- or 3-hydroxyflavanone by P450s, followed by dehydration to produce flavone, and the other mechanism is suggested by a direct desaturation (Guengerich 2001; Kagawa et al., 2004; Kakimoto et al., 2019). The former hydroxylation reaction begins with hydrogen abstraction from the C2 or C3 carbon of C-ring and the oxygen rebound and gives 2- or 3-hydroxyflavanone. 2-Hydroxynaringenin was reported as a product of naringenin, as well as a (glycosylated) ring-opened product (Du et al., 2010), and 3-hydroxyflavanone was reported as a product of flavanone by Nikolic and van Breeman (2004). An alternate pathway involves a direct desaturation, as has been reported in plants: rice CYP93G1, a flavone synthase II, is able to catalyze direct conversion of flavanones to flavones, such as the desaturation of naringenin and eriodictyol to apigenin and luteolin, respectively (Akashi et al., 1999; Lam et al., 2014; Mizuno et al., 2016; Fliegmann et al., 2010; Martens and Mithöfer 2005).

Recent studies have established that 2′OHFva prevents proliferation of cancer cells in lung, prostate, kidney, stomach, and breast in humans (Awasthi et al., 2018; Ofude et al., 2013; Singhal et al., 2015; 2018; Yue et al., 2020; Zhang et al., 2015). We examined in this study to identify chemical structures of three diOHFva (m/z 257) products and two diOHFvo (m/z 255) products obtained on incubation of 2’OHFva with P450 enzymes (Figure 3). However, in contrast to other three hydroxylated flavanones analyzed (Figures 5–7), we could not precisely characterize structures of these diOHFva and diOHFvo products from 2’OHFva examined in this study (Figure 8).

CYP1 family enzymes were more active than CYP2A6 in oxidizing these four hydroxylated flavanones, and these findings were supported by molecular docking studies of these chemicals with active sites of P450 enzymes. The A-rings of these four hydroxylated flavanones interacted with CYP1A1, 1A2, 1B1.1, and 1B1.3 but not CYP2A6, which was able to interact with the B-rings (Figure 12). However, our results showed that CYP2A6 was also active in forming 4′OHFvo at the low rate of 0.019 min−1, a level was similar to that with CYP1B1.3 (Figure 10). It was also obtained that the B-ring of 4′OHFvo interfaced with CYP2A6 and the other P450s determined, indicating the possibility that diOHFvo products (m/z 255) are formed through OHFvo products (m/z 239) as well as through OHFva (m/z 241).

Pathways for metabolism of 4′OHFva by human P450 enzymes are proposed (Figure 14). Flavanone (Fva) is firstly oxidized to form a 4′OHFva and then this product is oxidized to produce several diOHFva products (m/z 257). These diOHFva products are further oxidized or desaturated to diOHFvo (m/z 255) products by P450s. In addition, we detected small amounts of triOHFva products (m/z 273) that were formed through oxidation of diOHFva products. Alternatively, 4’OHFva is also oxidized or desaturated to form 4′OHFvo (m/z 239) and this metabolite is then converted to diOHFvo (m/z 255) products by P450 enzymes. We propose that other three substrates, 2′OHFva, 3′OHFva, and 6OHFva, might also be oxidized to several products similar to those obtained in a case of 4′OHFva, but the rates of product formation differ with these hydroxylated flavanones examined.

Figure 14.

Figure 14.

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Proposed pathway for oxidation of 4′OHFva by human P450 enzymes.

In conclusion, our study showed that four hydroxylated flavanones, 2′-, 3′-, 4′-, and 6-OHFva, the major oxidative metabolites of flavanone by P450s, were further oxidized by human P450 enzymes to several dihydroxy flavanones (m/z 257) and subsequently oxidized or desaturated to dihydroxy flavones (m/z 255). These oxidative products formed were characterized by positive ion fragmentation and product ion spectra using LC-MS/MS. When 4′OHFva was used as a substrate, we detected formation of 4′OHFvo (m/z 239), indicating that diOHFvo products were also able to be formed through oxidation/desaturation of 4′OHFvo by P450s. Trihydroxy flavanones (m/z 273) were detected on incubation of 4’OHFva and 6-OHFva with P450 enzymes. Although the exact chemical structures of these products formed in this study were not fully defined, the results presented here can be of use for further studies of identification by comparison of synthetic standard materials and NMR spectroscopy and for studies of biological and toxicological significance of these hydroxylated flavanones in plants and mammals.

Supplementary Material

Suppl File

Funding

This study was supported in part by JSPS KAKENHI [16K21710] (to H. N), [15K07770] (to S. T.), [17K08630] (to J. K.), [JP18K0600] (to M. K.), [17K08426] (to N. M.), and [17K08425] (to H. Y.), National Research Foundation of Korea [NRF-2019R1A2C1004722] (to D. K.), and United States Public Health Service grant [R01 GM118122] (to F. P. G.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Abbreviations used

Fva

flavanone

Fvo

flavone

2′OHFva, 3′OHFva, 4′OHFva, 6OHFva

2′-, 3′-, 4′-, and 6-hydroxyflavanone, respectively

diOHFva

dihydroxyflavanone

diOHFvo

dihydroxyflavone

triOHFva

trihydroxyflavanone

4′57triOHFva

4′,5,7-trihydroxyflavanone

57diOHFvo, 78diOHFvo, 457triOHFvo, and 567triOHFvo

5,7-dihydroxyflavone (chrysin), 7,8-dihydroxyflavone, 4′,5,7-trihydroxyflavone (apigenin), and 5,6,7-trihydroxyflavone (baicalein), respectively

P450 or CYP

cytochrome P450

b5

cytochrome b5

DLPC

L-α-1,2 dilaouryl-sn-glycero-3-phosphocholine

Footnotes

Declaration of interest Statement

The authors declare no conflict of interest associated with this manuscript.

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