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Metabolism mediated interaction of α-asarone and Acorus calamus withCYP3A4 and CYP2D6
Subrata Pandit a, Pulok K. Mukherjee a,⁎, Sivasankaran Ponnusankar a,Murugan Venkatesh a, N. Srikanth b
a School of Natural Product Studies, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, Indiab Central Council for Research in Ayurveda and Siddha (CCRAS), Department of AYUSH, Ministry of Health and Family Welfare, Govt. of India, No.61-65,Institutional Area, Janakpuri, New Delhi, 110058, India
Please cite this article as: Pandit S, et al, MCYP2D6, Fitoterapia (2010), doi:10.1016/
a b s t r a c t
Article history:Received 17 September 2010Accepted in revised form 31 October 2010Available online xxxx
The present study was aimed to investigate the possible interaction of the standardized extract ofAcorus calamus (AC)with Cytochrome P450 enzyme, quantitative determination of theα-asaronein the AC rhizomewas performed byRP-HPLCmethod. In vitro interaction of the plant extractwasevaluatedbyCYP450-carbonmonoxide complex (CYP450-CO)assay. Effect on individual isoformssuchasCYP3A4andCYP2D6 isozymeswereanalyzed throughfluorescenceproduct formation andrespective IC50 valuesweredetermined. CYP450-COassay showedmoderate interaction potential.Extract showed higher IC50 values (46.84±1.83–32.99±2.21 μg/ml) comparing to the standardinhibitors and lower IC50 value than α-asarone (65.16±2.37–42.15±2.45 μg/ml).
Acorus calamus Linn (AC) (Family: Acoraceae) commonlyknown “sweet flag” or “calamus” is used as a flavoring agentin food and also has a very long history of medicinal use inmany herbal traditions with special reference to Ayurveda inIndia [1]. The rhizomes of AC are considered to possessaphrodisiac, diuretic, antispasmodic, rheumatism, eczemaand antihelminthics properties. Roots and rhizomes of thisplant have been used in the Chinese and Indian systems of themedicine for its beneficial role in mental ailments likeepilepsy, memory disorders, improved learning performance,
and its anti-aging effect [2,3]. Reports revealed that alcoholicextract of rhizome of this plant possesses analgesic, sedative,respiratory depressant and moderately hypotensive proper-ties [4]. AC contains several phytoconstituents such asalkaloid, essential oil, methylisoeugenol, caryophyllene, iso-asarone, safrole, acorin etc. While the calamus oil predomi-nantly contain asarone [5,6]. α-asarone, the trans isomers ofthe alkenylbenzene 1-propenyl-2, 4, 5-trimethoxybenzene(Fig. 1), posses neuroprotective and antioxidant activity [7]. Ithas been reported that α-asarone posses hepatocarcinogenicproperty in a pre-weaned mouse model [8].
With the reported incidents and growing awareness onthe effect of the disposition of conventional pharmaceuticalsby herbal remedies and other phytochemicals, the study onherb–drug interactions play a vital role. The majority of theseinteractions involve cytochrome P450 (CYP450). Severalherbs and natural remedies have already been reported toinduce or inhibit CYP3A4, [9–12]. There is now a growingliterature concerning the action of isolated active components,orwhole extracts fromherbals, on the regulation of humanCYPenzyme activities in vitro systems [13]. With the number of
eraction of α-asarone and Acorus calamus with CYP3A4 and
2 S. Pandit et al. / Fitoterapia xxx (2010) xxx–xxx
adverse reactions reporting is increasing year by year, theability to predict the interactions involving CYP enzymes hasbecome key element in the process of drug discovery.
Ayurvedic medicinal plants of Indian origin have beenproved to be safer through their traditional practices; thescientific evaluation of their interaction with drug metabo-lizing enzymes is yet to be explored [14]. An increasingnumber of individuals are adopting complementary andalternative medicine, thus it is essential to identify clinicallyuseful and safe products from medicinal plants. Standardiza-tion of the herbal preparations is essential to get the optimalconcentrations of known active constituents present, and inpreserving their activities [15]. A common analytical methodsuch as high-performance liquid chromatography (HPLC)was used in the present study to quantify the major bioactivemolecule in AC. The present study was targeted to standard-ization of AC and exploring the interaction potential withdrug metabolizing enzymes including CYP3A4 and CYP2D6.
2. Materials and methods
2.1. Chemicals
All chemicals forfluorometric assayswere procured from theBD Gentest™ CYP2D6 and CYP3A4 high throughput inhibitorscreening kit (cat. no. 459200, and cat no459100 BD Biosciences,Woburm, MA, USA). Kit components contain cDNA expressedrecombinant human CYP2D6 (cat. No. : 04-80717) usingbaculovirus (Autographa californica) infected insect calls (BTI-TN-5B1-4) alongwith potassiumphosphate (pH7.4) buffer; Trisbase, NADP+, MgCl2, glucose 6-phosphate, glucose 6-phosphatedehydrogenase, AMMC (3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarine), quinidine and AHMC(3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4-methylcoumar-ine hydrochloride); ketoconazole, BFC (7-Benzyloxy-trifluro-methylcoumarin); HFC (7-Hydroxy-trifluromethylcoumarin).96 well black-microplate (NUNC) were used. Standard α-asarone was purchased from Sigma (Steinheim, Germany). Allthe reagents used for the standardization methods were ofanalytical grade and other chemicals were of laboratory gradeand purchased locally.
2.2. Plant materials
Dried lyophilized hydro-alcoholic extract of A. calamuswas obtained from a research agency of Government of Indiai.e. Central council for Research in Ayurveda and Siddha(CCRAS), New Delhi Government of India. Dried rhizome ofAC was extracted with 70% alcohol by cold macerationmethod. The alcoholic extract were evaporated to dryness
Please cite this article as: Pandit S, et al, Metabolism mediated intCYP2D6, Fitoterapia (2010), doi:10.1016/j.fitote.2010.11.009
in rotary vacuum evaporator, yielding semi-solid residue.This semi-solid residue was lyophilized to fine powder.
2.3. Standardization of the extract using RP-HPLC
Quantitative analysis of α-asarone was performed byReverse-phase high performance liquid chromatographysystem (RP-HPLC) (Shimadzu Prominence, Kyoto, Japan)equipped with two Shimadzu LC-20 AD UFLC reciprocatingpumps, a variable Shimadzu SPD-M20A Prominence PDAdetector and a Rheodyne manual injector with a loop size of20 μl. The peak area was calculated with LC solution software.RP-HPLC analysis was carried out in isocratic conditions usinga C18 reverse phase column with a particle size of 5 μ,250×4.6 mm (Phenomenex-Luna C18, Torrance, CA, USA).Samples were filtered through 0.45 μm ultra membranefilters (Millipore, Germany). Running conditions included :injection volume, 20 μl; mobile phase, methanol: 0.5% aceticacid in water (75: 25 v/v); flow rate, 1 ml/min; and detectionat 210 nm. α-asarone present in AC were identified bycomparing chromatographic peaks with the retention time(Rt) of individual standard. Percentage of α-asarone presentin AC was determined by constructing calibration curve.
2.4. Cytochrome inhibition studies
2.4.1. Preparation of rat liver microsomes (RLM)Microsomes were isolated from the liver of male Swiss
Wistar (N200 g) rats. RLM fractionswere prepared as describedby Iwata et al., 1996 [16]. In brief, rat liverwas quickly perfusedwith 1.15% KCl solution, and homogenized with four volumes(w/v) of ice-cold 1.15% KCl solution. The 20% homogenate (w/v)was centrifuged at9000×g for 20 min (BeckmanCoulter 64RALLEGRA) and the supernatant was collected. Further it wassubjected to ultra centrifugation at 105,000×g for 1 h, 4 °C,(SORVALL RC100) and the microsomal fractions (105,000×gpellet) were collected from the homogenates and it wasresuspended in 1.15% KCl solution and stored at −80 °C forfurther use. Protein concentrations were determined bymodified biuret method using bovine serum albumin asstandard by AMS photoanalyzer PF-2.
CYP450-CO complex assay was done with pooled RLM in96 well microplate, based on the method described byPonnusankar et al. (2010) [17]. Briefly in this method, theRLM was diluted with a phospho glycerol buffer (10 mMpotassium phosphate, pH 7.4, 20% glycerol) and incubatedwith prepared extract (dissolved in ethanol and DMSO). Thereaction of the extract and CYP450 was initiated by additionNADPH generating system (4.20 mg/ml of NADP+ in solutionof 100 mM glucose-6-phosphate, 100 mM MgCl2 and 100 U/ml glucose-6-phosphate dehydrogenase). One plate (P) wassealed with tape and kept in room temperature, whileanother plate (PC) was incubated in the CO chamber for15 min. 0.5 M sodium hydrosulfite was used to reduce thesample. The absorbance was taken using BIORAD microplatereader (Model 680XR) at 450 nm and 490 nm and absorbancedifference was calculated. Ketoconazole used as positivecontrol. Proper solvent controls were used for the study.
eraction of α-asarone and Acorus calamus with CYP3A4 and
3S. Pandit et al. / Fitoterapia xxx (2010) xxx–xxx
Concentration of CYP450 was calculated using the formula;[CYP450] (mM)=(ΔAPC−ΔAP)/91 Where ΔAPC=absorbancedifferenceof the PC sample, andΔAP=absorbance differenceofthe P sample. Percentage inhibition was calculated using thefollowing formula:
2.4.3. Fluorometric assayThe assay was performed based on the method reported
by Ganzera et al. (2006) [18] with modifications. Briefly,
Fig. 2. A. HPLC chromatogram of α-asarone.
Please cite this article as: Pandit S, et al, Metabolism mediated intCYP2D6, Fitoterapia (2010), doi:10.1016/j.fitote.2010.11.009
145 μl NADPH-Co factor mixture (1.3 mM NADP+, 66 mMMgCl2 and 66 mM glucose 6-phosphate) were added to theeach well of first row of black 96 micro-well plates. 100 μlcofactor mixtures were added to remaining all the wellsexcept the blank wells. 5 μl of test extract solution was addedto the first row of each well. Two-fold serial dilution was usedand the plate was incubated for 10 min at 37 °C. Quinidine(25 μM) and ketoconazole (0.25 μM) used as a positiveinhibitor for CYP2D6 and CYP3A4 respectively. The reactionwas initiated by addition of 100 μl enzyme-substrate solutionprepared in phosphate buffer. 10 mM AMMC for CYP2D6(1.9 nmol), and 50 mMBFC for CYP3A4 (1.3 nmol), were used
B. HPLC chromatogram of AC extract.
eraction of α-asarone and Acorus calamus with CYP3A4 and
Fig. 3. A. Concentration dependent inhibition of the AC extract and α-asarone. Values are mean±S.E.M.; n=3. B. Percentage inhibition of ACextract and α-asarone. Bars represent mean±S.E.M. where n=3. a Pb0.001versus positive inhibitor ketoconazole; One-way analysis of variance(ANOVA) followed by Dunnett's multiple comparison Test. (AC HA-ACextract dissolve in hydro alcohol solution; AC DMSO – AC extract dissolve inDMSO; α-asarone ethanol – α-asarone dissolves in ethanol; α-asaroneDMSO – α-asarone dissolves in DMSO).
Table 1IC50 (μg/ml) value of AC extract and α-asarone on the metabolism mediatedby CYP3A4 and CYP2D6. One-way analysis of variance (ANOVA) followed byDunnett's multiple comparison test.
Test sample Solvent IC50 (μg/ml)(CYP3A4)
IC50 (μg/ml)(CYP2D6)
Acorus calamus Ethanol 46.84±1.83a 36.81±3.93b
DMSO 43.96±1.01a 32.99±2.21b
α-asarone Ethanol 65.16±2.37a 55.17±1.62b
DMSO 57.46±3.34a 42.15±2.45b
Ketoconazole Ethanol 5.74±1.24 –
DMSO 4.54±0.70 –
Quinidine Ethanol – 3.52±0.94DMSO – 2.23±0.54
Each value represents the mean±S.E.M (n=3); aPb0.001, b Pb0.001 versuspositive control ketoconazole and quinidine respectively.
4 S. Pandit et al. / Fitoterapia xxx (2010) xxx–xxx
as substrates. After 30 min of incubation, the reaction wasstopped by using 0.5 M tris base. Fluorescence intensity of themetabolites was measured in a Microplate Fluorescencereader (BioTek FLx 800 T, U.S.A.) using an emission andexcitation wavelength of 460 nm, 390 nm for CYP2D6 and530 nm, 409 nm for CYP3A4 respectively. Generation of theproducts was linear over the range used for these assays; theobtained data were analyzed using an Excel spreadsheet.Percentage of inhibition and IC50 was calculated according tothe following formula.
Percentage of inhibition = 100− Signal of well−Blankð Þ × 100Solvent control−Blankð Þ
All data were expressed as mean±S.E.M. The statisticalsignificance was calculated by one-way analysis of variance(ANOVA) using GraphPad InStat Version 5.0. Dunnett'smultiple comparison test were performed by fixing thesignificance level at Pb0.05 and above.
3. Results
3.1. HPLC analysis of α-asarone in AC
The chromatograms obtained from HPLC analyses of theα-asarone and hydroalcoholic extract of AC has been shownin Fig. 2A and B. The peak of α-asarone in the extract wasidentified by comparison with the retention time of referencestandard at 7.659 min. Reference standard exhibited goodlinearity in the range from 1 to 2000 μg/ml in the calibrationcurve. The regression curve for α-asarone was Y=−26040.74+226885.4 X with r2=0.989. These resultswere considered satisfactory and acceptable for subsequentquantitative analysis. The content of α-asarone was found tobe 1.27% (w/w) in the test sample.
3.2. Cytochrome inhibition study
3.2.1. CYP450-CO complex assayThe CYP450 concentration of RLM was found to be
0.417 nmol/mg protein. HA and DMSO solution of the extractand α-asarone showed a concentration dependent inhibitionof CYP450 (Fig. 3A). Results indicated that plant extract andα-asarone showed significantly less inhibition than positiveinhibitor (Fig. 3B). AC extract dissolve in DMSO showedhighest percentage of inhibition (28.56±2.25%) which maybe due to the higher solubility of extract in DMSO than theethanol. Lowest inhibition (15.21±0.89%) was observed inα-asarone prepared in ethanol. This showed that theinteraction of AC extract with pooled CYP450 was morewith the extract rather than its isolated molecule suggestingpossible synergistic effects.
Please cite this article as: Pandit S, et al, Metabolism mediated intCYP2D6, Fitoterapia (2010), doi:10.1016/j.fitote.2010.11.009
3.2.2. Fluorimetric assay and IC50 value determinationAC extract and α-asarone were assayed between concen-
trations ranging from 200 to 1.56 μg/ml. All samples wereassayed in triplicate, and IC50 were determined (Table 1).Concentration dependant percentage inhibitions of the testcompound on both of the isozymes were observed (Fig. 4).The assay showed that the extract has higher IC50 valuescomparing to the standard inhibitors and lower IC50 valuethan α-asarone for both the isozymes. Results indicated that
eraction of α-asarone and Acorus calamus with CYP3A4 and
Fig. 4. Percentage inhibitory effects of AC extracts,α-asarone and positive inhibitors on drug modulating isozymes CYP3A4 and CYP2D6. Values are mean±S.E.M.;n=3. (AC HA-AC extract dissolve in hydro alcohol solution; AC DMSO – AC extracts dissolve in DMSO; α-asarone ethanol – α-asarone dissolves in ethanol; α-asarone DMSO – α-asarone dissolves in DMSO).
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CYP450 inhibition of AC was more comparing to its singlebioactive compound. The higher enzyme inhibition by the ACextract may be related to the synergistic effects due topresence of other constituents in the extract. α-asaroneindicated a good activity against CYP3A4, yet their IC50 valuesseemed high compared to the positive control ketoconazole.
4. Discussion
With the increase in the global use of medicinal plants,several concerns regarding the efficacy and safety of theherbal medicines have also been raised so as to ensure supplyof safe medicinal plant materials with good quality [19].Preclinical biological screening is important not only forestablishing the therapeutic efficacy of the medicinal plantsbut also to validate their historical utilization by traditionalhealers and herbalists. In this study ACwas standardized withrespect to α-asarone the bioactive marker present in it. Druginteractions are not limited to synthetic drugs, naturalproducts may also act as triggers for changes in the CYPactivity; for example CYP-modulating effects are describedfor echinacea, grapefruit juice or St. John's Wort [20,21].
In CYP450-CO method a dose dependent inhibition of theenzyme was observed with the extract and standardcompound. In fluorescence screening assay different concen-trations of extracts and standard showed good linearity ofdose dependant inhibition. IC50 values for positive controlswere in good agreement to literature values [22]. Assay wasrun in endpoint mode in which plate was incubated for thedesired reaction time. Testing ofα-asarone indicate the overall
Please cite this article as: Pandit S, et al, Metabolism mediated intCYP2D6, Fitoterapia (2010), doi:10.1016/j.fitote.2010.11.009
inhibition activitywas explainableby testedcompounds aswellas the other constituents present in the extract. Whileconsidering the selection of the solvent, the DMSO has beenutilized as it renders complete solubility of the compoundseven though it may not be optimal solvent for cytochromeinhibition study because it can inhibit the activity of severalCYP450s (2 C19, 3A4, 2E1, and 2 C8/9) only when used morethan 5% concentration. In this experiment b2% of DMSO andethanol was used as better alternatives as long as the content iskept at a relatively low level. [23]. It is important that the effectsobserved could vary with the experimental conditions such asthe protein content in themicrosomal incubations, particularlyif the inhibition is due to competitive metabolism of thesolvents [24]. So to make sure the maximum solubility of thephytoconstituents, DMSOwas used in a lesser concentration. Itwasobserved that CYP interactionpotentialwasmore in case ofextract dissolve in DMSO rather than in ethanol. The CYP3A4and CYP2D6 are clinically most relevant isoforms. Bindingsites of active constituents from AC with these isozymes arenot known. These results clearly indicate that the AC extractand its major constituents moderately inhibited the CYPenzymes. The procedure could be used for assessment of thepotential risk for metabolic interaction with conventionalpharmaceuticals [22].
5. Conclusion
The purpose of this study was to explore the CYP450interaction potential of AC andα-asarone as one of its bioactivecompound. The higher IC50 values than positive control
eraction of α-asarone and Acorus calamus with CYP3A4 and
6 S. Pandit et al. / Fitoterapia xxx (2010) xxx–xxx
indicated that the test extracts and constituents moderatelyinteracted in drug metabolism. Lower IC50 value of the extractthan pure compound indicated that care should be takenwhenadministering the extract with other CYP450-interactingcompounds, particularly those with low therapeutic indices.
Acknowledgements
The authors are grateful to Central Council for Research inAyurveda and Siddha (CCRAS), Department of AYUSH,Ministryof Health and FamilyWelfare, Government of India, New Delhifor providing financial support through EMR project grant [F.No. 5-29/2006-CCRAS/Tech./Hq./EMR-220] for this work.
References
[1] Mukherjee PK, Rai S, Kumar V, Mukherjee K, Hylands PJ, Hider RC. Plantsof Indian origin in drug discovery. Expert Opin Drug Discov 2007;2:633–57.
[2] Mukherjee PK, Kumar V, Mal M, Houghton PJ. In vitro acetylcholinesterase inhibitory activity of the essential oil from Acoruscalamus and its main constituents. Planta Med 2007;73:283–5.
[3] Mukherjee PK, Kumar V, Mal M, Houghton PJ. Acorus calamus: scientificvalidation of Ayurvedic tradition from natural resources. Pharm Biol2007;45:1–16.
[4] Mehrotra S, Mishra KP, Maurya R, Srimal RC, Yadav VS, Pandeya R, et al.Anticellular and immunosuppressive properties of ethanolic extract ofAcorus calamus rhizome. Int Immunopharmacol 2003;3:53–61.
[5] Lee HS. Fungicidal property of active component derived from Acorusgramineus rhizome against phytopathogenic fungi. Bioresour Technol2007;98:1324–8.
[6] Ahmad I, Beg AZ. Antimicrobial and phytochemical studies on 45 Indianmedicinal plants against multi-drug resistant human pathogens. JEthnopharmacol 2001;74:113–23.
[7] Mukherjee PK, Kumar V, Mal M, Houghton PJ. Acetylcholinesteraseinhibitors from plants. Phytomedicine 2007;14:289–300.
[8] Wiseman RW, Miller EC, Miller JA, Liem A. Structure-activity studies ofthe hepatocarcinogenicities of alkenylbenzene derivatives related toestragole and safrole on administration to pre-weanling maleC57BL/6J×C3H/HeJF1 mice. Cancer Res 1987;47:2275–83.
[9] Tsukamoto S, Aburatani M, Ohta T. Isolation of CYP3A4 inhibitors fromthe black cohosh (Cimicifuga racemosa). Evid Based ComplementAlternat Med 2005;2:223–6.
Please cite this article as: Pandit S, et al, Metabolism mediated intCYP2D6, Fitoterapia (2010), doi:10.1016/j.fitote.2010.11.009
[10] Ito K, Satoh T, Watanabe Y, Ikarashi N, Asano T, Morita T, et al. Effects ofKampo medicines on CYP and P-gp activity in vitro. Biol Pharm Bull2008;31:893–6.
[11] Usia T, Iwata H, Hiratsuka A, Watabe T, Kadota S, Tezuka Y. CYP3A4 andCYP2D6 inhibitory activities of Indonesian medicinal plants. Phytome-dicine 2006;13:67–73.
[12] Foster BC, Vandenhoek S, Tang R, Budzinski JW, Krantis A, Li KY. Effect ofseveral Chinese natural health products of human cytochrome P450metabolism. J Pharm Pharmaceut Sci 2002;5:185–9.
[13] Budzinski JW, Foster BC, Vandenhoek S, Arnason JT. An in vitroevaluation of human cytochrome P450 3A4 inhibition by selectedcommercial herbal extracts and tinctures. Phytomedicine 2000;7:273–82.
[14] Mukherjee PK. Quality control of herbal drugs-an approach toevaluation of botanicals. first ed. New Delhi: Business Horizon; 2002.
[15] Mukherjee PK, Houghton P. Evaluation of herbal medicinal products.first ed. London: Pharmaceutical Press; 2009.
[16] Iwata N, Mukai T, Hara S, Endo T, Tomoda A. Rapid and large-scaleisolation of microsomal fraction of mouse liver by lyophilization andlow speed centrifugation. Tohoku J Exp Med 1996;180:65–71.
[17] Ponnusankar S, Pandit S, Venkatesh M, Bandyopadhyay A, MukherjeePK. Safety evaluation of standardized extract of Terminalia chebula Retzby cytochrome P450 inhibition assay. Phytother Res 2010 doi:10.1002/ptr.2993 [11 July 2010, Electronic publication ahead of print].
[18] Ganzera M, Schneider P, Stuppner H. Inhibitory effects of the essentialoil of chamomile (Matricaria recutita L.) and its major constituents onhuman cytochrome P450 enzymes. Life Sci 2006;78:856–61.
[19] Mukherjee PK, Venkatesh P, Venkatesh M, Ponnusankar S, Khan Y.Strategies of revitalization of traditional medicine. Chin Herb Med2010;2:1–15.
[20] Modarai M, Yang M, Suter A, Kortenkamp A, Heinrich M. Metabolomicprofiling of liquid Echinacea medicinal products inhibiting CytochromeP450 3A4 (CYP3A4). Planta Med 2010;76:378–85.
[21] Heinrich M, Modarai M, Kortenkamp A. Herbal extracts used for upperrespiratory tract infections: Are there clinically relevant interactionswith the Cytochrome P450 enzyme system? Planta Med 2008;74:657–60.
[22] Zou L, HarkeyMR, Henderson GL. Effect of herbal components on cDNA-expressed cytochrome P450 enzyme catalytic activity. Life Sci 2002;71:1579–89.
[23] Easterbrook J, Lu C, Sakai Y, Li AP. Effects of organic solvents on theactivities of cytochrome p450 isoforms, UDP-dependent glucuronyltransferase, and phenol sulfotransferase in human hepatocytes. DrugMetab Dispos 2001;29:141–4.
[24] Chauret N, Gauthier A, Nicoll-Griffith DA. Effect of common organicsolvents on in vitro cytochrome p450-mediated metabolic activities inhuman liver microsomes. Drug Metab Dispos 1998;26:1–4.
eraction of α-asarone and Acorus calamus with CYP3A4 and
