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In vitro effect of important herbal active constituents on human cytochrome P450 1A2 (CYP1A2) activity.

ABSTRACT

This study was designed to investigate eight herbal active constituents (andrographolide, asiaticoside, asiatic acid, madecassic acid, eupatorin, sinensetin, caffeic acid, and rosmarinic acid) on their potential inhibitory effects on human cytochrome P4501A2 (CYP1A2) activity. A fluorescence-based enzyme assay was performed by co-incubating human cDNA-expressed CYP1A2 with its selective probe substrate, 3-cyano-7-ethoxycoumarin (CEC), in the absence or presence of various concentrations of herbal active constituents. The metabolite (cyano-hydroxycoumarin) formed was subsequently measured in order to obtain [IC.sub.50] values. The results indicated that only eupatorin and sinensetin moderately inhibited CYP1A2 with [IC.sub.50] values of 50.8 and 40.2 [micro]M, while the other active compounds did not significantly affect CYP1A2 activity with [IC.sub.50] values more than 100 [micro]M. K, values further determined for eupatorin and sinensetin were 46.4 and 35.2 [micro]M, respectively. Our data indicated that most of the investigated herbal constituents have negligible CYP1A2 inhibitory effect. In vivo studies however may be warranted to ascertain the inhibitory effect of eupatorin and sinensetin on CYP1A2 activity in clinical situations

Keywords:

Herbal active constituents

CYP1A2

Fluorescence-based enzyme assay

Inhibitory effect

Introduction

Andrographolide (Fig. 1A), a diterpenoid lactone, is the most medicinally active phytochemical substance in the plant Andrographis paniculata, which has been traditionally used as folklore herbal remedy in India, China, and European countries for dysentery, cholera, diabetes, consumption, influenza, bronchitis, and gonorrhea (Lim et al. 2012). Many reports revealed that andrographolide exhibited anti-cancer, anti-inflammation and anti-diabetic activities (Lim et al. 2012). Several in vitro and in vivo studies indicated the ability of this compound to modulate the expression and activity of cytochrome P450 (CYP) enzymes including that of CYP1A (Chatuphonprasert et al. 2009; Pekthong et al. 2009; Jaruchotikamol et al. 2007).

Asiaticoside, asiatic acid, and madecassic acid (Fig. 1B, C, and D) are three major active substances derived from herb Centella asiatica, which has been worldwide cultivated as a vegetable or spice in many Asian, African, and Oceanic countries for centuries. Traditionally this herb is used for boosting memory, wound healing, and nowadays it is often used as an active ingredient in tonics, oral slimming formulas, body-beautiful preparations, and anti-aging skin care products (Brinkhaus et al. 2000). Centella asiatica contains a good number of active constituents accounting for its beneficial effects such as preventing age-related cognitive deficits, improving wound healing, anti-inflammation, and anti-cancer action (James and Dubery, 2009). Only limited data are available on the effect of its active ingredients on CYP activities (Pan et al, 2010; Winitthana et al. 2011; Chatchanee, 2003).

Sinensetin (Fig. IE) is one of the major polymethoxyflavones contained in citrus fruits and Orthosiphon stamineus. Its bioactivities include anti-diabetes, anti-cancer, and anti-inflammation (Wei et al. 2013). As a methoxylated flavone, sinensetin is likely to be mainly metabolized by CYP1A subfamily (Walle and Walle, 2007) and studies indicated that CYP1 family enzymes were able to enhance the antiproliferative activity of dietary flavonoids (including sinensetin) on breast cancer cells through bioconversion to more active products (Androutsopoulos et al. 2009). Eupatorin (Fig. 1F) has been reported as an anti-inflammation and anti-cancer agent mainly found in Orthosiphon stamineus (Doleckova et al. 2012). It was reported that eupatorin was selectively concerted to active form by CYP1 family in breast cancer cells, which may account for its anti-proliferative activity (Androutsopoulos et al. 2008).

[FIGURE 1 OMITTED]

Caffeic acid (Fig. 1G), a phenolic acid, and its derivative, rosmarinic acid (Fig. 1H), are both widely distributed in nature and demonstrated anti-cancer and anti-inflammatory activities in numerous in vitro and in vivo studies (Touaibia et al. 2011; Petersen et al. 2009). Recent in vitro study reported that caffeic acid and rosmarinic acid induced CYP1A activities in both HepG2/C3A and MH1C1 cells (Liu et al. 2013). Moreover, rosmarinic acid prevents 1,2-dimethylhydrazine induced activation of CYP2E1 as well as the conversion of chemicals into carcinogens (Venkatachalam et al. 2013).

Human CYP1A2 constitutes around 13% of the total CYP contents in the liver and about 20% of the drugs in clinical use undergo CYPlA2-mediated oxidation before elimination from human body (Wang and Zhou, 2009). Clinically used drugs such as theophylline, clozapine, and tacrine as well as pre-carcinogens including polycyclic aromatic hydrocarbons and imidazoquinoline derivatives are substrates of CYP1A2 (Zhou et al. 2010). Herbal or natural products are of great interest in maintaining and improving good human health and thus they are commonly consumed by the patients together with clinically used drugs without the awareness of doctors. However, serious adverse reactions resulting from drug-herb interactions have received considerable attention in recent decades. St John's wort (Hypericum perforatum), a well-known herb used to treat a variety of conditions, has been reported to cause serious adverse effects by inducing activity of several CYP isoforms (CYP1A2, CYP2C9 and CYP3A4) and thus increasing elimination of co-administrated drugs (Henderson et al. 2002). The present study was designed to evaluate the effects of eight commonly consumed herbal constituents, as mentioned above, on CYP1A2 activity. Evaluation was performed using 3-cyano-7-ethoxycoumarin (CEC) deethylase assay as CYP1A2 activity marker. Inhibition constants were derived from the study and discussed in relation to the propensity of the active constituents concerned to cause drug-herb interaction.

Material and methods

Reagents

Isopropyl [beta]-D-1-thiogalactopyranoside (IPTG), Tris-base, ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), glycerol were purchased from Promega (Madison, WI, USA). Terrific broth media was purchased from Invitrogen Corporation (Carlsbad, CA, USA). All the other chemicals were purchased from Sigma-Aldrich (St. Louis, MI, USA).

Co-expression of CYPIA2 and NADPH-CYP reductase in E. coli

Co-expression of human CYP1A2 protein and NADPH-CYP reductase was induced by IPTG in E. coli DH5[alpha] cells following an established method (Gillam et al. 1994).

3-cyano-7-ethoxycoumarin (CEC) deethylase assay

Incubations with total volume of 200 [micro]l were conducted using 96-well fluorescence microtiter plates based on the method described previously (Crespi et al. 1997). 50 [micro]g cDNA-expressed proteins were added into each reaction well (nine wells in total) together with the buffer mixture (2.6 mM [NADP.sup.+], 6.6 mM glucose-6-phosphate, and 0.8 U/ml glucose-6-phosphate dehydrogenase in 50 mM phosphate buffer pH7.4). The plate was firstly pre-incubated at 37 [degrees]C for 10 min with shaking at 120 rpm, after which 2 [micro]l of 10mM CEC (CYP1A2 probe substrate, dissolved in DMSO) was added into the first well. Subsequently, two times dilution was performed from the first to the eighth well, giving the final CEC concentrations ranging from 0.4 [micro]M to 50 [micro]M. The ninth well contained 50 [micro]M CEC with 50 [micro]g control protein. After incubation at 37 [degrees]C for another 20 min with shaking at 120rpm, reactions were terminated by adding stop reagent (75 [micro]l of 0.5 M Tris base). Generation of fluorescence signal by the metabolite was quantified immediately after termination of reaction using Tecan Infinite[R] 200 Microplate Reader (Tecan AG, Switzerland) with fluorescence filter at wavelength of 430/465 (excitation/emission). Absorbance data were exported to Microsoft Excel spreadsheet for further analysis of pharmacokinetics data ([K.sub.m] and [V.sub.max]).

Inhibition studies with herbal active constituents

In order to assess the inhibitory potential of the eight active herbal active constituents (andrographolide, asiaticoside, asiatic acid, madecassic acid, eupatorin, sinensetin, caffeic acid, and rosmarinic acid), [IC.sub.50] values were determined in the first place. CEC (10 [micro]M, close to Km) was incubated with bacterial expressed enzyme proteins (50 [micro]g per reaction) in the presence of various concentrations (0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 [micro]M) or in the absence (control reaction) of each active constituent following the procedure described above. The CYP1A2 enzyme activities were calculated on the basis of the relative activity of the control reactions (set as 100%). Subsequently, two active constituents (eupatorin and sinensetin) with IC50 value less than 100 [micro]M were further investigated for [K.sub.i] values. For this, four concentrations of CEC (2.5, 5, 10, 15 [micro]M) were incubated with various concentrations of eupatorin (0,12.5,25, 50, 100 [micro]M) and sinensetin (0, 10, 20, 40, 60, 80 [micro]M), respectively.

Data analysis

Non-linear regression analysis using the GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla California USA) was performed to obtain enzyme kinetics ([K.sub.m], [V.sub.max]) for CYP1A2-mediated CEC deethylation. [IC.sub.50] values were estimated by non-linear regression analysis with the help of SigmaPlot[TM] (version 9.0, Systat Software Inc, USA). [K.sub.i] values were determined from the secondary plots constructed using slopes or y-intercepts of Lineweaver-Burk plots. The mechanism of inhibition was decided graphically from the Lineweaver-Burk plots. The initial kinetics estimates from the plots were subsequently used to determine [K.sub.i] values using nonlinear regression analysis by fitting different models of enzyme inhibition to the kinetic data using SigmaPlot[R] Enzyme Kinetics Module.

Results and discussion

Kinetics of CYP1A2-mediated CEC deethylation

Michaelis-Menten plot forCYPlA2-mediated CEC deethylation is shown in Fig. 2. With CEC increasing concentrations ([CEC]), the velocity (V) of the reaction followed a hyperbolic pattern of increment and approached the maximum velocity ([V.sub.max]). [K.sub.m] is the substrate concentration at which the reaction rate is half of its maximum, and it represents the affinity of one enzyme toward a substrate. The [K.sub.m] value determined from this study was 10.5 [+ or -] 1.4 [micro]M (mean [+ or -] standard error), which was within the reported range (3.0-23.7 [micro]M) in the literature (Nayadu et al. 2013; Kong et al. 2011; Donato et al. 2004; Crespi et al. 1997). It indicated that the expressed CYP1A2 enzyme in this study had comparable affinity for CEC and was suitable for the subsequent inhibitory studies. The variation observed in Km values from the literature is likely due to the different sources of CYP1A2 that were employed, such as pooled human liver microsomes and recombinant enzymes, in different studies. [V.sub.max] (1101.8 [+ or -] 53.6 pmol/min/mg protein) value also showed variation from those derived from other studies (64.3-542.0 pmol/min/mg protein) (Nayadu et al. 2013; Kong et al. 2011; Donato et al. 2004; Crespi et al. 1997). This is not uncommon since the [V.sub.max] value is a function of the expression level of enzyme, which varies from one study to another based on enzyme concentration, incubation conditions, different ratios of co-enzymes (NADPH-CYP reductase or cytochrome b5) versus CYP1A2 in incubation mixtures, and different phospholipid composition between liver microsomes and the recombinant enzyme system.

[FIGURE 2 OMITTED]

Effects of herbal active constituents on CYP1A2 activity

As seen in Table 1, after incubation, six out of eight active constituents investigated did not show significant inhibitory effects towards CYP1A2 ([IC.sub.50] values > 100 [micro]M) except sinensetin and eupatorin (with [IC.sub.50] values of 40.2 and 50.8 [micro]M, respectively). Further evaluations of [K.sub.i] values and the inhibitory mechanisms were subsequently carried out for sinensetin and eupatorin. As shown in Fig. 3A, the lines intercepted to the left of the 1/V axis but above the 1/[CEC] axis in the Lineweaver-Burk plot, indicating that the inhibitory effect of sinensetin on CYP1A2 was of mixed type (competitive-non-competitive inhibition). Hence, sinensetin is likely to bind either at the same catalytic site as the substrate CEC or at a different site of CYP1A2 protein. As a result, sinensetin may reduce CYP1A2 activity, either by directly competing for binding site with substrate or by inducing conformational change affecting the catalytic activity from a non-substrate binding site. As mentioned earlier, CYP1 family, CYP1A1, and CYP1A2 in particular, was reported to be involved in the biotransformation of sinensetin (Walle and Walle, 2007; Androutsopoulos et al. 2011). This is supported by our results, which indicate competition for substrate binding and catalysis as one mode of sinensetin inhibition. Our finding further demonstrated that, in addition to direct competition for CEC binding, there is likely an additional site in which sinensetin could bind and influence CYP1A2 catalytic activity either by reducing substrate binding affinity or slowing catalysis step of the enzyme. The [K.sub.i] value derived from secondary plot (Fig. 4A) was 35.2 [micro]M, indicating a moderate inhibitory potential of sinensetin on CYP1A2. As for eupatorin, Lineweaver-Burk plot illustrated in Fig. 3B showed that this compound affected CYP1A2 activity in an uncompetitive manner, as represented by parallel lines in the plot. This suggested that eupatorin bind to CYP1A2-CEC complex at a site away from the catalytic site, and interferes with substrate binding and hampers catalysis in the enzyme-substrate complex. The [K.sub.i] value derived from secondary plot (Fig. 4B) demonstrated that eupatorin was a moderate inhibitor for CYP1A2 with [K.sub.i] of 46.4 [micro]M.

[FIGURE 3 OMITTED]

The fact that sinensetin and eupatorin were more potent than the other herbal constituents in this study appears to be consistent with the CYP1A2 structural findings. Structural studies have shown that CYP1A2 substrates are characterized as planar, neutral, aromatic (2-4 aromatic rings), and lipophilic molecules with at least one putative hydrogen bond donor (Lewis, 2000). This also matches the topology of the active site, which has been described as narrow and flat as characterized using X-ray crystallography (Sansen et al. 2007) and computational models (Ekins et al. 2001). Therefore, a plausible explanation for our findings is that the bulky and hydrophilic (acidic) side chains of asiaticoside, asiatic acid, madecassic acid, caffeic acid, and rosmarinic acid, as well as buckled shape of andrographolide may hinder their interactions with the CYP1A2 enzyme binding site. Sinensetin and eupatorin, on the other hand, possess the structural features (being planar, neutral, and aromatic) that made them fit in well within CYP1A2 cavity.

[FIGURE 4 OMITTED]

Sinensetin and eupatorin are both polymethoxylated flavonoids found as dominant compounds in Orthosiphon stamineus and many other closely related herbs, which are among the most therapeutically important constituents (Laavola et al. 2012). Herbal preparations containing sinensetin and/or eupatorin are commercially available on the market in many forms (tablet, capsule, powder, soft drink etc.), and they are advertised to have diuretic, antioxidant, and antibacterial activity. The use of these preparations in conjunction with prescription drugs is therefore a safety concern from the drug interaction perspective. Prediction of in vivo drug interaction can be made based on in vitro data such as [K.sub.i] value. In general, in vitro inhibition potency values below 1 [micro]M typically generate concern regarding the potential for causing drug interaction (Obach et al. 2005) whereas those greater than 10-30 [micro]M are typically not associated with interaction risk. Based on this cutoff value of 1 [micro]M, it is apparent that both sinensetin and eupatorin would possess minimum potential to cause relevant in vivo CYP-mediated inhibition. However, studies have proven that some inhibitors that did not demonstrate high in vitro potency (i.e. high [K.sub.i] values) yet still had been shown to cause clinically significant drug interactions. These include several CYP3A inhibitors known to cause irreversible inactivation (including clarithromycin, erythromycin and diltiazem), as well as some drugs that attain high concentrations in vivo (such as fluconazole and cimetidine). Thus, whereas in vitro potency can provide some indication of the potential to cause drug interactions in vivo, outlier interactions (i.e. interactions that are beyond expectation and occur outside the norm) do sometimes occur and hence potential for CYP1A2 inhibition by sinensetin and eupatorin cannot be ruled out.

Clinically relevant interactions involving CYP1A2 have been reported in literature. Some CYP1A2 interactions have limited clinical importance; for example, most patients can withstand an elevated caffeine (CYP1A2 substrate) concentration due to concurrent intake of ciprofloxacin (CYP1A2 inhibitor) without significant adverse consequences. Others can however be serious. Historically, the most important CYP1 A2-drug interaction is probably the severe theophylline toxicity due to concurrent use of theophylline with CYP1A2 inhibitors such as ciprofloxacin or fluvoxamine. Whether sinensetin and eupatorin, the two in vitro CYP1A2 inhibitors investigated in the present study, can affect CYP1A2 catalytic activity in clinical situations remains to be investigated. To ascertain this, further investigation of the two compounds using in vivo models such as animal studies or clinical interaction studies may thus be warranted.

In conclusion, our results showed that sinensetin and eupatorin moderately affected CYP1A2 catalytic activities in a mixed type and uncompetitive manner, respectively. Clinical impact of this inhibition however remains to be elucidated in in vivo models. Until such data become available, patients taking traditional medications should probably exercise caution when using herbal remedies containing sinensetin and eupatorin. On the other hand, the inhibitory effect of the remaining herbal constituents (andrographolide, asiaticoside, asiatic acid, madecassic acid, caffeic acid, and rosmarinic acid) was found to be minimum toward CYP1A2. Hence, there is negligible risk of interaction between these constituents with CYP1A2 in vivo.

Abbreviations: CEC, 3-cyano-7-ethoxycoumarin; CYP, cytochrome P450; DMSO, dimethylsulfoxide; [K.sub.m], Michaelis-Menten constant; Vmax, maximum velocity; [IC.sub.50], the half maximal (50%) inhibitory concentration; [K.sub.i], the inhibitor concentration required for a half-maximal inhibition; NADPH, nicotinamide adenine dinucleotide phosphate.

http://dx.doi.org/10.1016/j.phymed.2014.08.003

Acknowledgments

This research was supported by the Malaysian Ministry of Science, Technology and Innovation (Grant: eScienceFund 02-02-09-SF0005).

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ARTICLE INFO

Article history:

Received 9 July 2014

Accepted 16 August 2014

Yan Pan (a), Kai Hung Tiong (b), Badrul Amini Abd-Rashid (c), Zakiah Ismail (c), Rusli Ismail (d), Joon Wah Mak (b), Chin Eng Ong (e), *

(a) Department of Biomedical Science, The University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia

(b) School of Medical Sciences, International Medical University, 126, Jalan 19/155B, Bukit Jalil, 57000 Kuala Lumpur, Malaysia

(c) Herbal Medicine Research Unit, Division of Biochemistry, Institute for Medical Research, Jalan Pahang. 50588 Kuala Lumpur, Malaysia

(d) Centre of Excellence for Research in AIDS (CERiA), Universiti Malaya, Level 17 Wisma REfD, Jalan PantaiBaru, 59990 Kuala Lumpur, Malaysia

(e) School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia

* Corresponding author. Tel.: +6 03 55144918; fax: +6 03 55146323.

E-mail address: ceong98@hotmail.com (C.E. Ong).
Table 1
Summary of the effects of herbal active constituents on CYP1A2
activity.

                             I[C.sub.50]    [K.sub.i]
Herbal active constituents    ([micro]M)    ([micro]M)

Andrographolide                  >100           --
Asiaticoside                     >100           --
Asiatic acid                     >100           --
Madecassic acid                  >100           --
Sinensetin                         40.2        35.2
Eupatorin                          50.8        46.4
Caffeic acid                     >100           --
Rosmarinic acid                  >100           --
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Author:Pan, Yan; Tiong, Kai Hung; Abd-Rashid, Badrul Amini; Ismail, Zakiah; Ismail, Rusli; Mak, Joon Wah; O
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9MALA
Date:Oct 15, 2014
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