INTRODUCTION:

Nutritionists, food scientists and customers have been extremely concerned about dietary polyphenols as they play a role in human health. In recent years, research has strongly supported the role of polyphenols in degenerative disease prevention, particularly cancer, cardiovascular disease and neurodegenerative diseases¹. The principal groups of polyphenols are characterized by their carbon skeleton, phenol acids, flavonoids and the less frequent stilbenes and lignans². The phenolic and polyphenolic compounds formed as secondary plant metabolites are known as Flavonoids. Flavonoids, a polyphenol of antioxygen, developed by plant photosynthesis and omnipresent in plants, fruits and grasses, are closely linked to human life³. Published studies show that the typical daily intake of flavonoids in typical Western populations is between 20–190 mg day⁴. Chemically, flavonoids are based on a fifteen-carbon structure consisting of two benzene rings that are bound by a heterocyclic pyrane. They can be divided into groups like flavones, flavanols, flavanones, Flavanonol Isoflavones and Flavan-3-ols based on substitution on heterocyclic ring². At least 10 % of the general population and 30–70 % of individuals with specific disease conditions are consuming herbal product with a growing interest in alternative medicine⁵.

A natural and biologically active compound is flavonoid chrysin (5,7-dihydroxy-2-phenyl-4Hchromen-4-one), found mainly in honey, passion fruit and propolis⁶. Chrysin is one of the natural flavonoids found in plants, with large quantities of honey and propolis and a dietary supplement easily available⁷. Recently, significant attention was given to the antioxidant health effects of honey as it is considered to be a source of both enzymatic and nonenzymatic antioxidants like L-ascorbic acid, flavonoids, and phenolic acids. Chrysin content ranges from 0.10mg/kg (honeydew honeys) to 5.3mg/kg (forest honeys) in various types of honeys⁸. Chrysin has recently been shown to be an effective inhibitor of aromatase and the activation of human immunodeficiency virus in latent infection models. It also displayed antioxidant and anti-inflammatory effects and has shown cancer chemo preventative activity in a variety of human and rat cell types, by inducing apoptosis⁹. Most cancer cells have shown that chrysininduces apoptosis and prevents proliferation, and is more active in cells with leukaemia than other flavonoids that have been studied, with chrysin possibly acting by activating caspases and by disabling cell Akt signalling^10,11.

In a comprehensive study of use of supplementary and alternative drugs, 16% of consumers of prescription medications are reported to use herbal supplements, 2/3 of women used herbs for perimenopausal symptoms, 45% of parents gave herbal treatments to their children and 45% of pregnant women tried herbal remedies⁵. In the meanwhile, some reports indicate that over 60 percent of patients use traditional herbal medicines as normal¹². Although foods have traditionally been thought to be safe, very few food-drug interactions have been reported such as St John's word herb-drug interaction, which can also lead to clearance of CYP1A2 substrates such as theophylline, bergamottin, a major furanocoumarin contained in grapefruit juice¹³, has been reported to increase drug blood concentration by inhibiting hepatic CYP3A activity; thereby toxicity of drugs such as simvastatin, felodipine, and cyclosporine is thus increased¹⁴. Herb interaction mechanisms typically involve the inhibition or induction of cytochrome P450 (CYP) enzymes, UDP glucuronosyltransferase (UGT) and drug transporters⁴. As certain ingredients of herbal products may be substrates, inhibitors and/or CYPs, this could have an effect on the pharmacokinetics of a co-administered CYP-metabolized medication, likely to lead to herbal-drug interactions¹². However, dietary supplements are not classified as drugs and do not require Food and Drug Administration (FDA) approval to be marketed¹⁵. The potential toxicities and drug interactions have not been evaluated thoroughly with herbal products.

Enzymes such as cytochrome P450 (CYP) play an important role in drug interaction because of the fact that many flavonoid compounds are either activated or inhibited these enzymes¹⁶. In addition, chrysin has been documented in a few research papers as modulators of P-gp and medication-metabolizing enzymes such as CYP1A2 and CYP3A4 by in vivo animal models^17,18. To the best of our knowledge, there have been no detailed studies which have evaluated in vitro-assessment of herbal medicinal interactions potentials of chrysin inhibiting effects of chrysin on human liver CYP450 enzymes. The effects of chrysin on the activity of five main human CYPs, such as 1A2, 2C9, 2C19, 2D6, and 3A4 were investigated using a human liver microsome (HLM), in order to determine the potential for herbal medicinal interactions.

MATERIALS AND METHODS:

Chemicals and reagents:

Pooled human liver microsomes (pool of 50 donors; Cat#452165), Human hepatocytes (Cat#DLW454503) and Hepatocryo recovery Hi Viability medium (Cat#DLW454560) from BD Gentest were purchased from Sigma-Aldrich, Bangalore. Krebs Henseleit Buffer (Cat#K3753), DMSO (Cat#D5879), testosterone (Cat#T1500), terfenadine (Cat#T9652), NADPH (Cat#N1630), potassium phosphate monobasic (Cat#P5655), potassium phosphate dibasic (Cat#P2222), Phenacetin (Cat#77440), acetaminophen (Cat#A7302), fluvoxamine (Cat#F2802), bufuralol (Cat#UC168), OH-bufuralol maleate (Cat#UC169), quinidine (Cat#Q3625), S-mephenytoin (Cat#UC-175), 4-OH mephenytoin (Cat#UC126), ticlopidine. HCl (Cat#T6654), sulfaphenazole (Cat#UC166), midazolam (Cat#UC-429), 1-OH midazolam (Cat#UC-430), imipramine hydrochloride (Cat#I0899), Caffeic acid (Cat#C0625), Chrysin (Cat#C80105), Quercetin (Cat#Q4951), Resveratrol (Cat#R5010), Rutin hydrate (Cat#R5143), Gallic acid (Cat#G7384) were purchased from Sigma, Germany. Glipizide (Cat#50402G), 7-OH-Coumarin (Cat#02040m), Diclofenac sodium (Cat#32732D), ketoconazole (Cat#30101K) and glipizide (Cat#50402G) were purchased from Apin Chemicals, Abingdon, UK.96well plates were procured from Axygen, Union City, California.

Metabolic stability of chrysin in human liver microsomes (Phase-I metabolism):

Metabolic stability of chrysin was determined in pooled HLM. In brief, the incubation was done by preincubating microsomes (0.25mg/mL) with 1μM test compound for 5min at 37°C in 0.1M phosphate buffer, pH7.4. The reactions were initiated by adding prewarmed cofactors (2 mM NADPH and 3 mM MgCl₂). After 0, 5, 15, 30 and 60min incubations at 37°C, the reactions were stopped by adding an equal volume of acetonitrile containing 1μM internal standard. The samples were kept in a refrigerator for 30min and then centrifuged at 3000g for 10min. The supernatants were analysed with LC/MS/MS for the amount of parent compound remaining.

Metabolic stability of chrysin in human hepatocytes (Phase-II metabolism):

Cryopreserved hepatocytes were thawed in a water bath at 37°C and transferred to a 50 mL tube containing Hepatocryo recovery Hi Viability medium, further centrifuged at 105g at room temperature for 5min. The supernatant was removed and the hepatocyte pellet was gently resuspended in medium to a final density of 2 x 10⁶cells/mL. Incubations were carried out at a final test compound concentration of 20μM prepared in KHB buffer. Hepatocytes and compound were mixed in 1:1 proportion to get 1x10⁶cells/mL with 10μM compound concentration, these incubations were carried out in 24-well plates at 37°C for 20 min in CO₂ incubator. The viability of hepatocyte was determined by trypan blue staining immediately prior to use and after incubation. The time points for incubations are 0, 3, 5, 10 and 20 min, reactions were terminated by adding 1mL of ice-cold acetonitrile containing internal standard. The samples were kept in a refrigerator for 30 min and then centrifuged at 3000g for 10min. The supernatants were analysed with LC/MS/MS for the amount of parent compound remaining.

Inhibitory potency of chrysin on P450 activities in human liver microsomes:

To evaluate CYP inhibition potential of chrysin, the substrate concentrations selected were less than their Km values reported in FDA White paper. To assess the functionality of CYP inhibition assay Ketoconazole (CYP3A4), Quinidine (CYP2D6), Sulfaphenazole (CYP2C9), Ticlopidine (CYP2C19) and Fluvoxamine (CYP1A2) were run as a positive control. The assay details provided in Table 1. Because of limited literature availability, the concentrations chosen taken for estimating the CYP inhibition values are 100, 30, 10, 1 and 0.1μM. Incubation for CYP inhibition assay were carried out in 96-well plate by addition of diluted microsomes in Kphos buffer followed by addition of respective CYP isoform inhibitor and chrysin. Further aliquots of respective CYP isoform substrate were added and incubated for 5min at 37⁰C, finally reactions were initiated by addition of NADPH and further incubated for 5-10min. Reactions were terminated using 100µL of ice-cold acetonitrile containing internal standard. The plates were centrifuged at 3000g for 15min and 100µL aliquots were submitted for analysis by LC-MS/MS.

Table 1: CYP450 inhibition assay conditions

Protein concentration	0.1 mg/mL → CYP3A4, CYP2D6, CYP2C9 and CYP1A2 0.5 mg/mL → CYP2C19
Inhibitor concentration	100, 30, 10, 1 and 0.1 µM (Chrysin)
Substrate concentration	2.5 µM → Midazolam (CYP3A4), 5 µM→ Bufuralol (CYP2D6), 10 µM → Diclofenac (CYP2C9), 30 µM → S-Mephenytoin (CYP2C19) 35 µM → Phenacetin (CYP1A2)
Metabolite monitored	1`-hydroxymidazolam (CYP3A4), bufuralol-hydroxylation (CYP2D6), 4`-hydroxydiclofenac (CYP2C9), 4`-hydroxymephenytoin (CYP2C19) Acetaminophen (CYP1A2)
NADPH	1 mM

Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis:

The sample analysis procedures were performed using tandem mass spectrometry (LC-MS/MS) in liquid chromatography. In short, an analysis with HPLC ESI-MS / MS was performed on a Shimadzu HPLC system (Columbia, MD, USA) coupled with a triple quadrupole mass spectrometer, API4000 fitted with an electrospray ionization source (Applied Biosystems/MDS Sciex, Concord, Canada). The HPLC system was fitted with two LC-10AD pumps equipped with an in-line CTO-10A controller and DGU-14A solvent degasser and a dual-solvent self-washing machine (Shimadzu US manufacturing Inc., Columbia, MD) auto sampler. For all analyses an injection volume of 10μL has been used. The chromatographic condition consisted of a column Water Symmetry Shield (i.d., 75 × 4.6mm, 3.5μM) with mobile phase of water-formic acid (100:0·1, v/v) (solvent A) and acetonitrile-water-formic acid (95:5:0·1, v/v) (solvent B). Elution conditions were 95 % A to 20 % A in 2.0 min, 20% A to 0.5 min, 20% A to 95% A to 0.5min, and 95% A to re-equilibrium to 2.0 min. The volume of injection was 10μL, and a flow rate of 0.8mL /min.

For chrysin elution conditions were 100% A to 5% A in 2.50 min, 5%A for 3.60min and re-equilibration at 100% for 3.0min using the same column. Generic mass spectrometry parameters of all analytes included declustering potential range (40–80), colliding energy range (20–30), potential collision cell exit range (9–21), curtain gas (10 arbitrary units), collisionally triggered dissociation gas (6, medium), ion spray voltage (5500 V), source temperature (550°C), and ion source gas 1 and gas 2 (55 and 60 arbitrary units e, respectively). Interface heaters were kept on for all analytes. Mass detection was carried out in multiple reactions monitoring (MRM) mode using an electro spray interface operating in positive-ion mode. The parameters of the selected multiple reaction monitoring (MRM) monitoring transitions for the [M+ H]-precursor ion to selected product ion (m/z) were optimized with 152.0/110.0 (acetaminophen), 310.1/265.9 (4-hydroxy diclofenac), 235.0/150.0 (4′-hydroxy- (S)-mephenytoin), 278.0/186.0 (1′-hydroxybufurolol), 342.5/324.4 (1′-hydroxy midazolam), 254.90/69.9 (chrysin) and 446.3/347.0 (glipizide as an internal standard).Applied Biosystems/MDS Sciex Analyst program (version 1.4.2) was used to conduct instrument monitoring, data acquisition, and data evaluation.

Data Analysis:

Calculation of Intrinsic calculation:

The percent compound remaining in metabolic stability in microsomes and hepatocytes were determined by considering peak area ratio in the 0-minute sample as 100%.

Invitro intrinsic clearance (CL’int) (units in mL/min/kg) was calculated using below formula¹⁹.

For liver microsomes, scaling factor used was 45mg microsomal protein per gm liver.

*Indicates liver weight (gm) which varies species wise. The liver weight taken is 20gm for human.

Intrinsic clearance (mL/min/kg) for human hepatocytes was calculated using the formula:

Where K is Elimination rate constant

C cell is number of cell incubated

* Indicates liver weight (gm) which varies species wise. The liver weight taken is 20gm for human.

Half-life calculation:

The Half-life of compounds in microsomes and hepatocytes were calculated by formula:

Half-life (t1/2) (min) = 0.693/k

Where k is gradient of line determined from plot of ln peak area ratio (compound peak area / internal standard peak area) against time.

Calculation of IC₅₀

The IC50 was calculated by using non-linear regression analysis (4PL fit method) with GraphPad Prism with inhibitor drug concentration (log scale) on x-axis and % activity on y-axis using formula:

% Activity = (PA ratios in presence of test compound/ PA ratios of DMSO control) *100

% Inhibition = 100 - % Activity

Where, PA= Metabolite to internal standard ratio for respective CYPs.

RESULTS AND DISCUSSION:

Metabolic stability of chrysin in human liver microsomes (Phase-I metabolism):

To assess the functionality of metabolic stability in human liver microsomes terfenadine was tested along with chrysin, which showed 18% parent compound remaining in 60min incubation period. However, chrysin showed stability in liver microsomes with t_1/2> 60min confirming chrysin is not metabolized by Phase I enzymes (Table 2 and Figure 1).

Table 2: Metabolic stability of Chrysin and Terfenadine in Human liver microsomes

	Chrysin	Terfenadine
% Remaining at 60 minutes (+ NADPH)	98	18
% Remaining at 60 minutes (- NADPH)	95	92
CL_h,int(mL/min/kg)	NC	215.2
half-life (minutes)	> 60	24.3

*NC means not calculated because compound was stable within incubation duration of 60 min

Fig.1: Percent of Terfendine and Chrysin remaining after 60 min incubation in pooled human liver microsomes (0.25 mg/mL) with cofactors (2 mM NADPH and 3 mM MgCl2) at 1μM compound concentration

Metabolic stability of chrysin in human hepatocytes (Phase-II metabolism):

To assess the functionality of cryopreserved human hepatocytes testosterone (Phase I) and 7-OH coumarin (Phase II) was tested along with polyphenols, testosterone showed half-life of 9.8min with 27% parent compound remaining which confirms functionally of phase I enzymes. To access the functionally of phase II enzymes in human hepatocytes 7-OH coumarin was tested which showed half-life of 14.3 min with 40% parent compound remaining. Flavonoid chrysin showed half-life of 28 min with 59% parent compound remaining [Table 3 and Figure 2].

Table 3: Metabolic stability of testosterone, 7-OH coumarin and chrysin in cryopreserved human hepatocytes

Incubation time (min)	% Parent compound remaining
Incubation time (min)	Testosterone	7-OH coumarin	Chrysin
0	100	100	100
3	94	92	82
5	65	76	78
10	36	52	67
20	27	40	59
half-life(min)	9.8	14.3	28
Intrinsic clearance (mL/min/kg)	169.2	115.8	58.2

Fig.2: Percent of Testosterone, 7-OH coumarin and Chrysin after 20 min incubation in human hepatocytes with 1x10⁶cells/mL at 10μM compound concentration

Inhibitory potency of chrysin on P450 activities in human liver microsomes:

To evaluate whether chrysin altered the enzyme activity of CYP450, the probe substrates were incubated with different concentrations of chrysin. The inhibitory potency of chrysin was determined based on the concentration-inhibition curves of the probe substrate, phenacetin (CYP1A2), midazolam (CYP3A4), diclofenac (CYP2C9), S-mephenytoin (CYP2C19) and bufuralol (CYP2D6). To assess the functionality of CYP inhibition assay FDA approved inhibitors for individual CYPs are performed. IC₅₀ values for the inhibition of human hepatic CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 by selective standard inhibitors and test compounds are shown in Table 4. For CYP3A4 inhibitor ketoconazole showed IC₅₀value of 0.1µM, CYP2D6 inhibitor quinidine showed IC₅₀value of 0.1µM, CYP2C9 inhibitor sulfaphenazole showed IC₅₀ value of 3.1µM, CYP2C19 inhibitor ticlopidine showed IC₅₀ value of 6.0µM and CYP1A2 inhibitor fluvoxamine showed IC₅₀value of 0.2µM (Fig.2 (a-b)).Results showed that chrysin had CYP1A2 inhibitory activities in HLMs with IC₅₀ values of 0.6μM. Chrysin showed inhibitory activities of CYP2C9 and CYP2C19 with IC₅₀ values of 28.9 μM and 1.60μM respectively. Chrysin exhibited inhibitory activities of CYP2D6 with IC₅₀ values of 22.3μM and inhibitory activities of CYP3A4 with IC₅₀ values of >100µM(Table 4). These data confirm the potent inhibition of CYP1A2 and CYP2C19 by chrysin in human liver microsomes (Fig.3 (c)).

Table 4: Effect of chrysin on CYP metabolic activity in pooled human liver microsomes (HLMs)

CYP activity	CYP isoform	IC₅₀ (μM) in HLM
CYP activity	CYP isoform	Chrysin	Positive control P450 inhibitor
Phenacetin O-deethylation	CYP1A2	0.6	0.2 (Fluvoxamine)
Diclofenac 4-hydroxylation	CYP2C9	28.9	3.1 (Sulfaphenazole)
S-Mephenytoin 4′-hydroxylation	CYP2C19	1.6	6.0 (Ticlopidine)
Bufuralol 1′-hydroxylation	CYP2D6	22.3	0.1 (Quinidine)
Midazolam 1′-hydroxylation	CYP3A4	>100	0.1 (Ketoconazole)

Fig.3: The inhibitory effects (IC50 value) of positive controls and Chrysin on CYP3A4, CYP2D6, CYP2C9, CYP2C19 and CYP1A2 catalysed reactions in human liver microsomes

CYP2C19, a CYP450 subtype of hepatic cytochrome, metabolizes close to 15% of recognized medicinal products, including drug products commonly used by medicines such as clopidogrel warfarin, and carbamazepine. Carriers with CYP2C19* 2 and* 3²⁰, for instance, have been found to be more likely to have decreased clopidogrel efficacy with clopidogrel and proton pump (CYP2C19 inhibitors). In both normal metabolizers and heterozygous genotype carriers following treatment with fluvoxamine (a CYP2C19 inhibitor) a statistically significant rise for levels of plasma has been seen, with no other clinically significant improvement in bad metabolizers with the highest levels of plasma rabeprazole already seen²¹. Likewise, the CYP1 enzyme family has a small substrate-binding cavity and show the planar nature of substrates and inhibitors to be very specific. CYP1A1, CYP1A2, CYP1B1 contributes respectively 20%, 17% and 11% to the carcinogenic activation reaction. In general, they contribute 7%, 10% and 3% respectively to drug metabolism²². The inhibition can lead to an increase in plasma concentration and toxicity, especially for drug users with narrow therapeutic windows. Vegetarian diets and herbal supplements have become increasingly popular worldwide as they raise awareness of prevention and health safety. In addition, several studies have shown that high intake of fruit and vegetables reduces the incidence of cancer, and polyphenols, in particular flavonoids, contribute to the benefits of those diets²³. Herbals are commonly co-administered with medicinal items, with a high likelihood of clinically relevant activity. Despite this, the data shown by most literature probably happened as normal. These encounters are normal and unreported. In the current study chrysin was selected because little or no data is available in the publications as either a CYP inhibitor of CYP450's major isoforms. Prior to this review, chrysin has been shown to be a potent CY1A2 and CYP2C19 inhibitor.

CONCLUSION:

In conclusion, our data indicated that chrysin is potent inhibitors of human CYP1A2 and CYP2C19 enzymes. Authors suggest the dosage of drugs, which are metabolized by CYP1A2, such as caffeine, melatonin, theophylline and clozapine and drugs metabolised by CYP2C19 such as proton pump inhibitors (PPIs) (e.g., omeprazole, lansoprazole, pantoprazole), antidepressants (e.g., citalopram and amitriptyline), antiplatelet drugs (e.g., clopidogrel), antifungals (e.g., voriconazole), and anticancer compounds (e.g., cyclophosphamide) to be monitored when they are co-administered with dietary supplements and other herbal medicines containing chrysin. These findings provide proof-of-concept for possible clinical drug -drug interactions with of herbal flavonoid chrysin which is consumed by the people daily in form of honey.

ACKNOWLEDGEMENTS:

The authors are highly thankful to Shri Sunil Sharma (Hon’ble Chairperson) and Dr Sudhanshu (Hon’ble Chief Mentor) Suresh Gyan Vihar University, Jaipur for the needful support.

CONFLICT OF INTEREST STATEMENT:

The authors confirm that there are no conflicts of interest.

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LIST OF ABBREVIATIONS

° C	Degree Centigrade
%	Percentage
pH	Potential of hydrogen
µL	Microliter
µM	Micromolar
DMSO	Dimethylsulphoxide
LC-MS/MS	Liquid Chromatography Tandem Mass Spectrometry
mg/mL	Milligram per milliliter
mM	Mill molar
NADPH	β-Nicotinamide Adenine Dinucleotide 2′- Phosphate
IC₅₀	Concentration of an inhibitor required for 50-percent inhibition of an enzyme
Kphos	Potassium phosphate buffer
g	gram
RPM	Revolutions Per Minute
MRM	Multiple Reaction Monitoring
HLM	Human Liver Microsomes
CYP	Cytochromes P450

Received on 14.05.2020 Modified on 23.06.2020

Research J. Pharm. and Tech. 2020; 13(12):6086-6092.

DOI: 10.5958/0974-360X.2020.01061.6