PHYTOTHERAPY RESEARCH Phytother. Res. (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5137
SHORT COMMUNICATION
Effect of Crude Extract of Eugenia jambolana Lam. on Human Cytochrome P450 Enzymes Santhivardhan Chinni,1* Anil Dubala,2 Jayasankar Kosaraju,3 Rizwan Basha Khatwal,2 M. N. Satish Kumar1 and Elango Kannan1 1
Department of Pharmacology, JSS College of Pharmacy, Udhagamandalam, Tamil Nadu, India Department of Pharmaceutical Biotechnology, JSS College of Pharmacy, Udhagamandalam, Tamil Nadu, India 3 Department of Pharmacognosy, JSS College of Pharmacy, Udhagamandalam, Tamil Nadu, India 2
The fruit of Eugenia jambolana Lam. is very popular for its anti-diabetic property. Previous studies on the crude extract of E. jambolana (EJE) have successfully explored the scientific basis for some of its traditional medicinal uses. Considering its wide use and consumption as a seasonal fruit, the present study investigates the ability of E. jambolana to interact with cytochrome P450 enzymes. The standardized EJE was incubated with pooled human liver microsomes to assess the CYP2C9-, CYP2D6-, and CYP3A4-mediated metabolism of diclofenac, dextromethorphan, and testosterone, respectively. The metabolites formed after the enzymatic reactions were quantified by high performance liquid chromatography. EJE showed differential effect on cytochrome P450 activities with an order of inhibitory potential as CYP2C9 > CYP3A4 > CYP2D6 having IC50 of 76.69, 359.02, and 493.05 μg/mL, respectively. The selectivity of EJE for CYP2C9 rather than CYP3A4 and CYP2D6 led to perform the enzyme kinetics to explicate the mechanism underlying the inhibition of CYP2C9-mediated diclofenac 4′-hydroxylation. EJE was notably potent in inhibiting the reaction in a non-competitive manner with Ki of 84.85 ± 5.27 μg/mL. The results revealed the CYP2C9 inhibitory potential of EJE with lower Ki value suggesting that EJE should be examined for its potential pharmacokinetic and pharmacodynamic interactions when concomitantly administered with other drugs. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Eugenia jambolana; Syzygium cumini; cytochrome P450; herb–drug interactions; enzyme kinetics; human liver microsomes.
INTRODUCTION Herbal remedies are an interesting approach for complementary medicine as well as alternative medicine, because their benefits are well documented from a historical perspective in the population (Egede et al., 2002). Herbs have traditionally been used as a first line medicine or as dietary supplements. Nevertheless, herbs may also cause unexpected adverse reactions when consumed concomitantly with other medicines. One aspect of safety is the risk of adverse effects due to pharmacological interactions between herbal medicines and conventional therapies (Kupiec and Raj, 2005). The safety of herbal medicine is compromised because of underestimation, considered safe because of their natural origin, and consumed without consulting a physician because they are self-care products (Rietjens et al., 2008). In contrast to synthetic drugs, therapeutic use of herbal products was materialized from the ancient texts and passed on through generations, without scientific experimental evidence. For this reason, modern research on pharmacology and toxicology of herbal medicine is aimed to validate herbal medicine and to ensure safety and efficacy (Calapai and Caputi, 2007).
* Correspondence to: Santhivardhan Chinni, Research scholar, JSS College of Pharmacy, Udhagamandalam, Tamil Nadu-643001, India. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
Eugenia jambolana Lam. also known as Syzygium cumini (L.) Skeels is a native plant of India, and its seed kernels are known for their anti-diabetic activity (Chatterjee et al., 2012). It is also known for its antiinflammatory, hepatoprotective, cardioprotective, and anti-hyperlipidemic effects. In addition, it is also a principal herb in the anti-diabetic formulations: Dianex, Diabecon, Hyponidd, MTEC, and Dihar, which are currently marketing in India (Baliga et al., 2011). Despite its wide use as a potential treatment, it has never been tested for its metabolic interactions with other medicines. Besides, few constituents of E. jambolana such as gallic acid, ellagic acid, and quercetin are known to interact with cytochrome P450 (CYP450) enzymes to cause potential herb–drug interactions (Ponnusankar et al., 2011; Barch et al., 1994; Kimura et al., 2010). Hence, the present study was aimed to determine the CYP450 interactions of crude extract of E. jambolana (EJE) with conventional medicine.
MATERIALS AND METHODS Chemicals, solvents, and plant extracts. Pooled human liver microsomes were purchased from Xenotech, India (catalog no: H2620). Standardized E. jambolana extract was received as a gift from Natural Remedies Pvt. Ltd., Bangalore, India. High performance liquid chromatography (HPLC) solvents were procured from Merck, Received 29 September 2013 Revised 20 January 2014 Accepted 08 February 2014
S. CHINNI ET AL.
India.NADP, glucose-6-phosphate (G-6-P), G-6-P dehydrogenase, and other chemicals were obtained from SigmaAldrich, Bangalore, India. Plant material and extraction. The fruits of E. jambolana were thoroughly washed with distilled water to separate the pulp from seeds. They were shade dried and coarsely powdered in a mixer grinder. Seed powder (100 g) was extracted with petroleum ether to remove lipids. It was then filtered, and the residue was successively extracted with 50% methanol by soxhlation at a temperature not exceeding 60 °C. The extract was then concentrated using rotavapor under reduced pressure. The yield of crude extract of E. jambolana was 11.1 g/100 g of dried seeds. Standardization of EJE. Quantitative analysis of ellagic acid was performed by HPLC. Shimadzu gradient HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with LC-10 AT-VP solvent delivery system (pump) with UV detection was adopted for analysis. Briefly, sample was injected into a Phenomenex-Luna (Phenomenex Inc., Torrance, CA, USA) C18 (2) column (250 × 4.6mm i.d., 100 Å, 5 μm). Separation was carried out using 25 mM phosphate buffer and acetonitrile (30:70 v/v) as mobile phase under gradient conditions. Detection was performed at 254 nm. The HPLC chromatogram of the standard ellagic acid and the sample are shown in Fig. 1. The amount of ellagic acid in the EJE was found to be 2.3% w/w. The amount of total polyphenols in standardized EJE estimated by Folin–Ciocalteu reagent (Ainsworth and Gillespie, 2007) was found to be 31.5% w/w. Cytochrome P450 inhibition assays. Standardized EJE was solubilized in methanol to produce a final methanol concentration of 0.5% in the reaction mixture. Microsomes alone were used as a control; ketoconazole, sulfaphenazole, and quinidine were used as positive controls for testosterone 6β-hydroxylation, diclofenac 4′-hydroxylation, and dextromethorphan O-demethylation, respectively. P450 activities were measured by pre-incubating EJE (3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000 μg/mL) for 5 min with a mixture of 10 μL of pooled human liver microsomes (20 mg of microsomal protein per milliliter) and corresponding selective substrates: 50 μM diclofenac (CYP2C9), 250 μM testosterone (CYP3A4), and 100 μM dextromethorphan. The reactions were initiated by adding
NADPH generating system (1 mM NADP, 10 mM G-6-P, 2 IU G-6-P dehydrogenase, and 5 mM MgCl2) in 0.1 M phosphate buffer, pH 7.4. The reaction mixtures of CYP2C9 and CYP3A4 were incubated for 10 min, and CYP2D6 was incubated for 30 min at 37 °C (Broly et al., 1989; Waxman et al., 1988). Finally, the reactions were stopped by placing the reaction tubes into an ice bath and then added 100 μL of ice cold acetonitrile, 20 μL of 60% perchlorate, and 200 μL of ice cold methanol for the diclofenac 4′-hydroxylation assay, dextromethorphan O-demethylation assay, and testosterone 6β-hydroxylation assay, respectively (Donato et al., 2004). The mixtures were centrifuged at 12,000 × g for 10 min, and the supernatants were collected. Metabolites obtained from the corresponding reactions, that is, 4′-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6), and 6β-hydroxytestosterone (CYP3A4), were quantified by HPLC. Shimadzu Prominence HPLC system (Shimadzu Corporation, Kyoto, Japan) was utilized to analyze the metabolites. The HPLC system was equipped with series of LC-20AT pump, Rheodyne 7752i injector with a 20-μL loop, and an SPD20A UV/VIS detector. An aliquot of 20 μL from each sample was injected into a reverse phase C18 Phenomenex column (250 mm × 4.6 mm i.d., 5 μm). Assays were performed according to previously reported method (Nagata et al., 2007). Data analysis. The data are represented as mean ± standard deviation. IC50 values were determined by employing non-linear regression analysis in Graphpad (GraphPad Software, Inc., La Jolla, CA, USA) Prism® v. 6.03. The modes of inhibition were initially estimated from a Lineweaver–Burk plot, and the apparent Ki values were estimated with the help of secondary plot of slopes of Lineweaver–Burk plot. The exact Ki values were later estimated by fitting a non-competitive model of enzyme inhibition into non-linear regression analysis using the Graphpad Prism enzyme kinetics module.
RESULTS AND DISCUSSION The inhibitory effect of EJE on CYP2C9, CYP2D6, and CYP3A4 was investigated with their respective probes, diclofenac, dextromethorphan, and testosterone. EJE inhibited the P450 activities distinctly. Among the three inhibition reactions, EJE has shown a higher selectivity
Figure 1. (a) HPLC-UV chromatogram of standard ellagic acid and (b) representative HPLC-UV profile of crude extract of Eugenia jambolana. Chromatogram shows ellagic acid peak at retention time 11.3 min. Copyright © 2014 John Wiley & Sons, Ltd.
Phytother. Res. (2014)
EUGENIA JAMBOLANA INHIBITS CYP2C9
Figure 2. Concentration-dependent CYP450 inhibition of pooled human liver microsomes by EJE. Dotted lines correspond to interpolated IC50 values of respective assays. Data represented as mean ± standard deviation (n = 3).
toward CYP2C9 with an IC50 of 76.69 μg/mL whereas CYP3A4 and CYP2D6 were weakly inhibited with IC50 of 359.02 and 493.05 μg/mL, respectively. A dosedependent inhibition pattern of respective CYP isoforms is depicted in Fig. 2. Diclofenac, an anti-inflammatory drug, is primarily metabolized by CYP2C9, and it has been reported that co-administration with sulfaphenazole inhibits the metabolism (Leemann et al., 1993). Since EJE has also indistinguishable inhibitory effect on the metabolism of diclofenac, it may exert similar kind of effects on the other xenobiotics metabolized by CYP2C9. As a result of its extensive inhibition and selectivity toward CYP2C9 (IC50 < 100 μg/mL), it was further explored in order to reveal the underlying mechanisms via enzyme kinetic studies. On the basis of the IC50, EJE (35–140 μg/mL) was incubated with variable concentrations of diclofenac (5, 10, 20, 40 μM) (Bort et al., 1999). The Lineweaver– Burk plot (Fig. 3) revealed that EJE non-competitively inhibited the enzymatic reaction with a Vmax and Km of 992.5 ± 42.26 pmol/mgCYP/min and 14.14 ± 1.37 μM, respectively. Further, the Ki value estimated by a nonlinear regression model for non-competitive enzyme inhibition of CYP2C9-mediated diclofenac 4′-hydroxylation by EJE was 84.85 ± 5.27 μg/mL. The results of the present study demonstrate the concentration-dependent CYP450 inhibition of EJE using a probe technique. CYP450 is part of a member of metabolic enzymes responsible for the biotransformation of the majority (~75%) of drugs, whereas CYP2C9 handles
Figure 3. Lineweaver–Burk plot for inhibition of CYP2C9-catalyzed diclofenac 4′-hydroxylation by EJE: Inset corresponds to the secondary plot of slopes. Data represented as mean ± standard deviation (n = 3).
~10% of them. It is also important that several drugs with narrow therapeutic indices (i.e., phenytoin and warfarin) are primarily metabolized by CYP2C9 (Pinto and Dolan, 2011). The in vitro enzyme kinetic studies could explain why the EJE possesses moderate inhibitory effects on drugs metabolized by CYP2C9. Considering the need of ascertaining herb–drug interactions, the observed inhibitory effects of EJE on human liver microsomes necessitate the continuation of researching this topic. Currently, our laboratory is conducting in vivo studies and evaluating their clinical relevance.
Acknowledgements The authors are grateful to Dr N. Muruganantham, Senior Research Officer, Natural Remedies Pvt. Ltd. for his support in standardizing the herbal extracts. The authors are also thankful to Dr Rajkumar Mandraju, Aaron Diamond AIDS Research Center, Rockefeller University, NY, USA, for his valuable inputs during manuscript revision. This work was financially supported by All India Council for Technical Education, New Delhi, with a MODROBS grant (8024/RIFD/MOD124(Pvt.)/Policy-III/2011-2012). S. C. is a recipient of senior research fellowship from Indian Council of Medical Research, New Delhi.
Conflict of Interest The authors have no conflicts of interest to disclose.
REFERENCES Ainsworth EA, Gillespie KM. 2007. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nat Protoc 2: 875–877. Baliga MS, Bhat HP, Baliga BR, Wilson R, Palatty PL. 2011. Phytochemistry, traditional uses and pharmacology of Eugenia jambolana Lam. (black plum): a review. Food Res Int 44: 1776–1789. Barch DH, Rundhaugen LM, Thomas PE, Kardos P, Pillay NS. 1994. Dietary ellagic acid inhibits the enzymatic activity of CYP1A1 without altering hepatic concentrations of CYP1A1 or CYP1A1 mRNA. Biochem Biophys Res Commun 30: 1477–82. Copyright © 2014 John Wiley & Sons, Ltd.
Bort R, Mace K, Boobis A, Gomez-Lechon M, Pfeifer A, Castell J. 1999. Hepatic metabolism of diclofenac: role of human CYP in the minor oxidative pathways. Biochem Pharmacol 58: 787–796. Broly F, Libersa C, Lhermitte M, Bechtel P, Dupuis B. 1989. Effect of quinidine on the dextromethorphan O-demethylase activity of microsomal fractions from human liver. Br J Clin Pharmacol 28: 29–36. Calapai G, Caputi AP. 2007. Herbal medicines: can we do without pharmacologist. Evid Based Complement Alternat Med 4: 41–43. Phytother. Res. (2014)
S. CHINNI ET AL. Chatterjee K, Ali KM, De D, Panda DK, Ghosh D. 2012. Antidiabetic and antioxidative activity of ethyl acetate fraction of hydromethanolic extract of seed of Eugenia jambolana Linn through in-vivo and in-vitro study and its chromatographic purification. Free Rad Antiox 2: 21–30. Donato MT, Jimenez N, Castell JV. 2004. Gomez-Lechon MJ. Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metab Dispos 32: 699–706. Egede LE, Zheng D, Ye X, Silverstein MD. 2002. The prevalence and pattern of complementary and alternative medicine use in individuals with diabetes. Diabetes care 25: 324–329. Kimura Y, Ito H, Ohnishi R, Hatano T. 2010. Inhibitory effects of polyphenols on human cytochrome P450 3A4 and 2C9 activity. Food Chem Toxicol 48: 429–435. Kupiec T, Raj V. 2005. Fatal seizures due to potential herb-drug interactions with Ginkgo biloba. J Anal Toxicol 29: 755–758. Leemann T, Transon C, Dayer P. 1993. Cytochrome P450TB (CYP2C): a major monooxygenase catalyzing diclofenac 4′hydroxylation in human liver. Life Sci 52: 29–34.
Copyright © 2014 John Wiley & Sons, Ltd.
Nagata M, Hidaka M, Sekiya H, et al. 2007. Effects of pomegranate juice on human cytochrome P450 2C9 and tolbutamide pharmacokinetics in rats. Drug Metab Dispos 35: 302–305. Pinto N, Dolan ME. 2011. Clinically relevant genetic variations in drug metabolizing enzymes. Curr Drug Metab 12: 487–497. Ponnusankar S, Pandit S, Babu R, Bandyopadhyay A, Mukherjee PK. 2011. Cytochrome P450 inhibitory potential of Triphala—a Rasayana from Ayurveda. J Ethnopharmacol 133: 120–125. Rietjens IM, Slob W, Galli C, Silano V. 2008. Risk assessment of botanical preparations intended for use in food and food supplements: emerging issues. Toxicol Lett 180: 131–136. Waxman DJ, Attisano C, Guengerich FP, Lapenson DP. 1988. Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6β-hydroxylase cytochrome P-450 Enzyme. Arch Biochem Biophys 263: 424–436.
Phytother. Res. (2014)
SHORT COMMUNICATION
Effect of Crude Extract of Eugenia jambolana Lam. on Human Cytochrome P450 Enzymes Santhivardhan Chinni,1* Anil Dubala,2 Jayasankar Kosaraju,3 Rizwan Basha Khatwal,2 M. N. Satish Kumar1 and Elango Kannan1 1
Department of Pharmacology, JSS College of Pharmacy, Udhagamandalam, Tamil Nadu, India Department of Pharmaceutical Biotechnology, JSS College of Pharmacy, Udhagamandalam, Tamil Nadu, India 3 Department of Pharmacognosy, JSS College of Pharmacy, Udhagamandalam, Tamil Nadu, India 2
The fruit of Eugenia jambolana Lam. is very popular for its anti-diabetic property. Previous studies on the crude extract of E. jambolana (EJE) have successfully explored the scientific basis for some of its traditional medicinal uses. Considering its wide use and consumption as a seasonal fruit, the present study investigates the ability of E. jambolana to interact with cytochrome P450 enzymes. The standardized EJE was incubated with pooled human liver microsomes to assess the CYP2C9-, CYP2D6-, and CYP3A4-mediated metabolism of diclofenac, dextromethorphan, and testosterone, respectively. The metabolites formed after the enzymatic reactions were quantified by high performance liquid chromatography. EJE showed differential effect on cytochrome P450 activities with an order of inhibitory potential as CYP2C9 > CYP3A4 > CYP2D6 having IC50 of 76.69, 359.02, and 493.05 μg/mL, respectively. The selectivity of EJE for CYP2C9 rather than CYP3A4 and CYP2D6 led to perform the enzyme kinetics to explicate the mechanism underlying the inhibition of CYP2C9-mediated diclofenac 4′-hydroxylation. EJE was notably potent in inhibiting the reaction in a non-competitive manner with Ki of 84.85 ± 5.27 μg/mL. The results revealed the CYP2C9 inhibitory potential of EJE with lower Ki value suggesting that EJE should be examined for its potential pharmacokinetic and pharmacodynamic interactions when concomitantly administered with other drugs. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Eugenia jambolana; Syzygium cumini; cytochrome P450; herb–drug interactions; enzyme kinetics; human liver microsomes.
INTRODUCTION Herbal remedies are an interesting approach for complementary medicine as well as alternative medicine, because their benefits are well documented from a historical perspective in the population (Egede et al., 2002). Herbs have traditionally been used as a first line medicine or as dietary supplements. Nevertheless, herbs may also cause unexpected adverse reactions when consumed concomitantly with other medicines. One aspect of safety is the risk of adverse effects due to pharmacological interactions between herbal medicines and conventional therapies (Kupiec and Raj, 2005). The safety of herbal medicine is compromised because of underestimation, considered safe because of their natural origin, and consumed without consulting a physician because they are self-care products (Rietjens et al., 2008). In contrast to synthetic drugs, therapeutic use of herbal products was materialized from the ancient texts and passed on through generations, without scientific experimental evidence. For this reason, modern research on pharmacology and toxicology of herbal medicine is aimed to validate herbal medicine and to ensure safety and efficacy (Calapai and Caputi, 2007).
* Correspondence to: Santhivardhan Chinni, Research scholar, JSS College of Pharmacy, Udhagamandalam, Tamil Nadu-643001, India. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
Eugenia jambolana Lam. also known as Syzygium cumini (L.) Skeels is a native plant of India, and its seed kernels are known for their anti-diabetic activity (Chatterjee et al., 2012). It is also known for its antiinflammatory, hepatoprotective, cardioprotective, and anti-hyperlipidemic effects. In addition, it is also a principal herb in the anti-diabetic formulations: Dianex, Diabecon, Hyponidd, MTEC, and Dihar, which are currently marketing in India (Baliga et al., 2011). Despite its wide use as a potential treatment, it has never been tested for its metabolic interactions with other medicines. Besides, few constituents of E. jambolana such as gallic acid, ellagic acid, and quercetin are known to interact with cytochrome P450 (CYP450) enzymes to cause potential herb–drug interactions (Ponnusankar et al., 2011; Barch et al., 1994; Kimura et al., 2010). Hence, the present study was aimed to determine the CYP450 interactions of crude extract of E. jambolana (EJE) with conventional medicine.
MATERIALS AND METHODS Chemicals, solvents, and plant extracts. Pooled human liver microsomes were purchased from Xenotech, India (catalog no: H2620). Standardized E. jambolana extract was received as a gift from Natural Remedies Pvt. Ltd., Bangalore, India. High performance liquid chromatography (HPLC) solvents were procured from Merck, Received 29 September 2013 Revised 20 January 2014 Accepted 08 February 2014
S. CHINNI ET AL.
India.NADP, glucose-6-phosphate (G-6-P), G-6-P dehydrogenase, and other chemicals were obtained from SigmaAldrich, Bangalore, India. Plant material and extraction. The fruits of E. jambolana were thoroughly washed with distilled water to separate the pulp from seeds. They were shade dried and coarsely powdered in a mixer grinder. Seed powder (100 g) was extracted with petroleum ether to remove lipids. It was then filtered, and the residue was successively extracted with 50% methanol by soxhlation at a temperature not exceeding 60 °C. The extract was then concentrated using rotavapor under reduced pressure. The yield of crude extract of E. jambolana was 11.1 g/100 g of dried seeds. Standardization of EJE. Quantitative analysis of ellagic acid was performed by HPLC. Shimadzu gradient HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with LC-10 AT-VP solvent delivery system (pump) with UV detection was adopted for analysis. Briefly, sample was injected into a Phenomenex-Luna (Phenomenex Inc., Torrance, CA, USA) C18 (2) column (250 × 4.6mm i.d., 100 Å, 5 μm). Separation was carried out using 25 mM phosphate buffer and acetonitrile (30:70 v/v) as mobile phase under gradient conditions. Detection was performed at 254 nm. The HPLC chromatogram of the standard ellagic acid and the sample are shown in Fig. 1. The amount of ellagic acid in the EJE was found to be 2.3% w/w. The amount of total polyphenols in standardized EJE estimated by Folin–Ciocalteu reagent (Ainsworth and Gillespie, 2007) was found to be 31.5% w/w. Cytochrome P450 inhibition assays. Standardized EJE was solubilized in methanol to produce a final methanol concentration of 0.5% in the reaction mixture. Microsomes alone were used as a control; ketoconazole, sulfaphenazole, and quinidine were used as positive controls for testosterone 6β-hydroxylation, diclofenac 4′-hydroxylation, and dextromethorphan O-demethylation, respectively. P450 activities were measured by pre-incubating EJE (3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000 μg/mL) for 5 min with a mixture of 10 μL of pooled human liver microsomes (20 mg of microsomal protein per milliliter) and corresponding selective substrates: 50 μM diclofenac (CYP2C9), 250 μM testosterone (CYP3A4), and 100 μM dextromethorphan. The reactions were initiated by adding
NADPH generating system (1 mM NADP, 10 mM G-6-P, 2 IU G-6-P dehydrogenase, and 5 mM MgCl2) in 0.1 M phosphate buffer, pH 7.4. The reaction mixtures of CYP2C9 and CYP3A4 were incubated for 10 min, and CYP2D6 was incubated for 30 min at 37 °C (Broly et al., 1989; Waxman et al., 1988). Finally, the reactions were stopped by placing the reaction tubes into an ice bath and then added 100 μL of ice cold acetonitrile, 20 μL of 60% perchlorate, and 200 μL of ice cold methanol for the diclofenac 4′-hydroxylation assay, dextromethorphan O-demethylation assay, and testosterone 6β-hydroxylation assay, respectively (Donato et al., 2004). The mixtures were centrifuged at 12,000 × g for 10 min, and the supernatants were collected. Metabolites obtained from the corresponding reactions, that is, 4′-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6), and 6β-hydroxytestosterone (CYP3A4), were quantified by HPLC. Shimadzu Prominence HPLC system (Shimadzu Corporation, Kyoto, Japan) was utilized to analyze the metabolites. The HPLC system was equipped with series of LC-20AT pump, Rheodyne 7752i injector with a 20-μL loop, and an SPD20A UV/VIS detector. An aliquot of 20 μL from each sample was injected into a reverse phase C18 Phenomenex column (250 mm × 4.6 mm i.d., 5 μm). Assays were performed according to previously reported method (Nagata et al., 2007). Data analysis. The data are represented as mean ± standard deviation. IC50 values were determined by employing non-linear regression analysis in Graphpad (GraphPad Software, Inc., La Jolla, CA, USA) Prism® v. 6.03. The modes of inhibition were initially estimated from a Lineweaver–Burk plot, and the apparent Ki values were estimated with the help of secondary plot of slopes of Lineweaver–Burk plot. The exact Ki values were later estimated by fitting a non-competitive model of enzyme inhibition into non-linear regression analysis using the Graphpad Prism enzyme kinetics module.
RESULTS AND DISCUSSION The inhibitory effect of EJE on CYP2C9, CYP2D6, and CYP3A4 was investigated with their respective probes, diclofenac, dextromethorphan, and testosterone. EJE inhibited the P450 activities distinctly. Among the three inhibition reactions, EJE has shown a higher selectivity
Figure 1. (a) HPLC-UV chromatogram of standard ellagic acid and (b) representative HPLC-UV profile of crude extract of Eugenia jambolana. Chromatogram shows ellagic acid peak at retention time 11.3 min. Copyright © 2014 John Wiley & Sons, Ltd.
Phytother. Res. (2014)
EUGENIA JAMBOLANA INHIBITS CYP2C9
Figure 2. Concentration-dependent CYP450 inhibition of pooled human liver microsomes by EJE. Dotted lines correspond to interpolated IC50 values of respective assays. Data represented as mean ± standard deviation (n = 3).
toward CYP2C9 with an IC50 of 76.69 μg/mL whereas CYP3A4 and CYP2D6 were weakly inhibited with IC50 of 359.02 and 493.05 μg/mL, respectively. A dosedependent inhibition pattern of respective CYP isoforms is depicted in Fig. 2. Diclofenac, an anti-inflammatory drug, is primarily metabolized by CYP2C9, and it has been reported that co-administration with sulfaphenazole inhibits the metabolism (Leemann et al., 1993). Since EJE has also indistinguishable inhibitory effect on the metabolism of diclofenac, it may exert similar kind of effects on the other xenobiotics metabolized by CYP2C9. As a result of its extensive inhibition and selectivity toward CYP2C9 (IC50 < 100 μg/mL), it was further explored in order to reveal the underlying mechanisms via enzyme kinetic studies. On the basis of the IC50, EJE (35–140 μg/mL) was incubated with variable concentrations of diclofenac (5, 10, 20, 40 μM) (Bort et al., 1999). The Lineweaver– Burk plot (Fig. 3) revealed that EJE non-competitively inhibited the enzymatic reaction with a Vmax and Km of 992.5 ± 42.26 pmol/mgCYP/min and 14.14 ± 1.37 μM, respectively. Further, the Ki value estimated by a nonlinear regression model for non-competitive enzyme inhibition of CYP2C9-mediated diclofenac 4′-hydroxylation by EJE was 84.85 ± 5.27 μg/mL. The results of the present study demonstrate the concentration-dependent CYP450 inhibition of EJE using a probe technique. CYP450 is part of a member of metabolic enzymes responsible for the biotransformation of the majority (~75%) of drugs, whereas CYP2C9 handles
Figure 3. Lineweaver–Burk plot for inhibition of CYP2C9-catalyzed diclofenac 4′-hydroxylation by EJE: Inset corresponds to the secondary plot of slopes. Data represented as mean ± standard deviation (n = 3).
~10% of them. It is also important that several drugs with narrow therapeutic indices (i.e., phenytoin and warfarin) are primarily metabolized by CYP2C9 (Pinto and Dolan, 2011). The in vitro enzyme kinetic studies could explain why the EJE possesses moderate inhibitory effects on drugs metabolized by CYP2C9. Considering the need of ascertaining herb–drug interactions, the observed inhibitory effects of EJE on human liver microsomes necessitate the continuation of researching this topic. Currently, our laboratory is conducting in vivo studies and evaluating their clinical relevance.
Acknowledgements The authors are grateful to Dr N. Muruganantham, Senior Research Officer, Natural Remedies Pvt. Ltd. for his support in standardizing the herbal extracts. The authors are also thankful to Dr Rajkumar Mandraju, Aaron Diamond AIDS Research Center, Rockefeller University, NY, USA, for his valuable inputs during manuscript revision. This work was financially supported by All India Council for Technical Education, New Delhi, with a MODROBS grant (8024/RIFD/MOD124(Pvt.)/Policy-III/2011-2012). S. C. is a recipient of senior research fellowship from Indian Council of Medical Research, New Delhi.
Conflict of Interest The authors have no conflicts of interest to disclose.
REFERENCES Ainsworth EA, Gillespie KM. 2007. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nat Protoc 2: 875–877. Baliga MS, Bhat HP, Baliga BR, Wilson R, Palatty PL. 2011. Phytochemistry, traditional uses and pharmacology of Eugenia jambolana Lam. (black plum): a review. Food Res Int 44: 1776–1789. Barch DH, Rundhaugen LM, Thomas PE, Kardos P, Pillay NS. 1994. Dietary ellagic acid inhibits the enzymatic activity of CYP1A1 without altering hepatic concentrations of CYP1A1 or CYP1A1 mRNA. Biochem Biophys Res Commun 30: 1477–82. Copyright © 2014 John Wiley & Sons, Ltd.
Bort R, Mace K, Boobis A, Gomez-Lechon M, Pfeifer A, Castell J. 1999. Hepatic metabolism of diclofenac: role of human CYP in the minor oxidative pathways. Biochem Pharmacol 58: 787–796. Broly F, Libersa C, Lhermitte M, Bechtel P, Dupuis B. 1989. Effect of quinidine on the dextromethorphan O-demethylase activity of microsomal fractions from human liver. Br J Clin Pharmacol 28: 29–36. Calapai G, Caputi AP. 2007. Herbal medicines: can we do without pharmacologist. Evid Based Complement Alternat Med 4: 41–43. Phytother. Res. (2014)
S. CHINNI ET AL. Chatterjee K, Ali KM, De D, Panda DK, Ghosh D. 2012. Antidiabetic and antioxidative activity of ethyl acetate fraction of hydromethanolic extract of seed of Eugenia jambolana Linn through in-vivo and in-vitro study and its chromatographic purification. Free Rad Antiox 2: 21–30. Donato MT, Jimenez N, Castell JV. 2004. Gomez-Lechon MJ. Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metab Dispos 32: 699–706. Egede LE, Zheng D, Ye X, Silverstein MD. 2002. The prevalence and pattern of complementary and alternative medicine use in individuals with diabetes. Diabetes care 25: 324–329. Kimura Y, Ito H, Ohnishi R, Hatano T. 2010. Inhibitory effects of polyphenols on human cytochrome P450 3A4 and 2C9 activity. Food Chem Toxicol 48: 429–435. Kupiec T, Raj V. 2005. Fatal seizures due to potential herb-drug interactions with Ginkgo biloba. J Anal Toxicol 29: 755–758. Leemann T, Transon C, Dayer P. 1993. Cytochrome P450TB (CYP2C): a major monooxygenase catalyzing diclofenac 4′hydroxylation in human liver. Life Sci 52: 29–34.
Copyright © 2014 John Wiley & Sons, Ltd.
Nagata M, Hidaka M, Sekiya H, et al. 2007. Effects of pomegranate juice on human cytochrome P450 2C9 and tolbutamide pharmacokinetics in rats. Drug Metab Dispos 35: 302–305. Pinto N, Dolan ME. 2011. Clinically relevant genetic variations in drug metabolizing enzymes. Curr Drug Metab 12: 487–497. Ponnusankar S, Pandit S, Babu R, Bandyopadhyay A, Mukherjee PK. 2011. Cytochrome P450 inhibitory potential of Triphala—a Rasayana from Ayurveda. J Ethnopharmacol 133: 120–125. Rietjens IM, Slob W, Galli C, Silano V. 2008. Risk assessment of botanical preparations intended for use in food and food supplements: emerging issues. Toxicol Lett 180: 131–136. Waxman DJ, Attisano C, Guengerich FP, Lapenson DP. 1988. Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6β-hydroxylase cytochrome P-450 Enzyme. Arch Biochem Biophys 263: 424–436.
Phytother. Res. (2014)
