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Arti Nayak et al. / Journal of Pharmacy Research 2012,5(4),2244-2246 Research Article Available online through ISSN: 0974-6943 www.jpronline.info Synergetic inhibitory activity of and piperlongumine on CYP3A4

Arti Nayak1, Dr.Tushar Nayak2, Chaitanya Bhatt3 1APMC College of Pharmaceutical Education and Research, Himatnagar- 383001, Gujarat-India. 2. B. J. Medical College, Ahmedabab, Gujarat.India. 3. K.B. Institute of Pharmaceutical Education and Research, Sector-23, GH-6, Gandhinagar 382023, Gujarat. India. Received on:16-01-2012; Revised on: 11-02-2012; Accepted on:26-04-2012

ABSTRACT CytochromeP450 (CYP450) enzymes play a key role in the metabolism of many exogenous and endogenous substances, with CYP3A4 the major hepatic and intestinal CYP subfamilies in humans, metabolizes more than 55% drugs. We here investigated inhibitory activity of piperine and piperlongumine because it has been reported that “Trikatu churna” an Ayurvedic formulation, is widely used to enhance the of drugs, contains powder of fruits of longum as one of the three ingredients, in which both piperine and piperlongumine are present, so the bioavailability enhancing property of “Trikatu churna” may be not only due to piperine but piperlongumine might also be contributing for the same. In vitro CYP3A4 activity was done by N- demethylation assay. Piperine (20µM), Piperlongumine (10µM) and (20µM) showed good inhibition and its combination showed good synergetic inhibitory activity on CYP3A4 isozymes.

Key words: Piperine, Trikatu churna, Piperlongumine, erythromycin N- demethylation, CYP3A4.

INTRODUCTION CYP450 enzymes play a key role in the metabolism of many exogenous and shown in clinical trials to increase the Cmax and AUC of , propra- endogenous substances, with CYP3A4 being one of the most important CYP nolol, and theophylline. Metabolic inhibition may be the mechanism for subfamilies for . CYP3A4, the major hepatic and intestinal these drug interactions [13-17]. The most widely studied natural product is CYP in humans, metabolizes more than 50% of clinically used drugs such as grapefruit juice, which has been found to increase the bioavailability and/or to cyclosporine A, dihydropyridines, , midazolam, terfenadine, prolong the metabolic elimination of many drugs such as dihydropyridine- and triazolam[1]. Recently, several reports have demonstrated that natural type channel blockers, histamine-1 receptor antagonists (e.g., compounds and herbal products may cause pharmacokinetic interaction with terfenadine), quinidine, benzodiazepines (e.g., midazolam), 17b-, and western drugs used clinically when they are simultaneously administrated. caffeine[18-20]. The use of medicinal herbs has particularly increased over the past few years among specific patient populations including HIV-infected patients. In vitro CYP3A4 is induced by a wide variety of ligands. These ligands bind to the studies shows that St. John’s wort extract was a potent inducer of CYP3A4, (PXR). The activated PXR complex forms a heterodimer and the responsible component was . St. John’s wort altered with the Retinoid X Receptor (RXR) which binds to the XREM region of the of the HIV protease inhibitor, in individuals on CYP3A gene. XREM is a regulatory region of the CYP3A gene, and binding retroviral therapy. Indeed, plasma concentrations of the HIV protease inhibi- causes a cooperative interaction with proximal promoter regions of the gene, tor, saquinavir, have been decreased in individuals exposed long-term to resulting in increased transcription and expression of CYP3A [21-22]. supplements. The increased clearance of saquinavir may be due to induction of hepatic and/or intestinal CYP3A4.Clinical studies have docu- MATERIALS AND METHODS mented that St. John’s wort reduced the plasma concentrations of cyclosporine, amitriptyline, digoxin, indinavir and oral contraceptives [2-12]. Plant material Crude fruits of Piper longum were procured from local drug supplier in It has been reported that “Trikatu churna” an Ayurvedic formulation, is Ahmedabad. widely used to enhance the bioavailability of drugs. “Trikatu churna” con- tains powder of fruits of Piper longum as one of the three ingredients. In Isolation of piperine and piperlongumine from piper longum Piper longum, both piperine and piperlongumine are present, so the Dried fruits were ground in a mill and powder(200gm)was taken in flask and bioavailability enhancing property of “Trikatu churna” may be not only due refluxed with hexane(500 ml) for 8 hours, filtered the solvent and concen- to piperine but piperlongumine might also be contributing for the same. trated the filtrate, Then hot Petroleum was added and cooled the Piperine, an active , isolated from piper species, inhibit CYP3A4 filtrate. Upon cooling got needle shape (Fig. 1A), and sunflower shape crys- activity using cDNA-expressed human microsomes. At molecular level pip- tals (Fig. 1B). Piperine and piperlongumine was separated with solvent ether. erine acts by suppressing activity of p-glycoprotein and CYP450 enzyme Then dried and collected in air tight bottle. which enhance the metabolism of drug. The levels of drug combining with piperine were required in reduced dose of drug. Piperine also decreases the intestinal production of glucuronic acid which allows more substance to enter in the body in active form. This wonder compounds allows many drugs to enter and remain within target cells for longer duration. Piperine has been *Corresponding author. Arti Nayak, Assistant Professor Department of Pharmacognosy) APMC College of Pharmaceutical Education and Research, Himatnagar- 383001, Gujarat-India. (A) (B) Fig.1: Piperine (A) and piperlongumine (B) Journal of Pharmacy Research Vol.5 Issue 4.April 2012 2244-2246 Arti Nayak et al. / Journal of Pharmacy Research 2012,5(4),2244-2246

Preparation of Microsomes The study was approved by Institutional Animal Ethics Committee (Ap- proval No. KB/10/210, date 23/02/2011). Five male wistar albino rats (230– 270 g) were taken. Inducing agent, (CYP3A inducer) was sus- pended in methyl cellulose (1%) and administered by single daily gavage for 3 days [23]. Dose of 100 mg/kg/day used for clotrimazole. were excised 48 hrs after the last dose of clotrimazole. The liver microsomes was isolated from rats was mince and homogenize in appropriate amount of 0.25 M containing 10mM Tris–HCl (pH 7.4), and then centrifuged at 600 × g for 5 min followed by 12,000 × g for 10 min. The post-mitochondrial supernatant was separated, mixed with solid CaCl2 so that its concentration in the given volume of supernatant was 8.0 mM and then centrifuged at 20,000 × g for 20 min. The pellet was re-suspended in mixture of 150 mM KCl–10mM Tris–HCl, and centrifuged at 20,000 × g for 20 min to obtain pinkish microsomal pellet, which was suspended in 0.5 ml of 0.1 M potas- sium phosphate buffer containing 20% glycerol and store at -80 ?C until needed [24]. Protein concentration of samples was determined by end point assay (Modified Biuret method) [25].

Erythromycin-N-demethylation assay The mixture of microsomal suspension (0.1 ml, 25%), erythromycin (0.1 ml, 10 mM)and potassium phosphate (0.6 ml, 100 mM, pH7.4) was incubated (A) at 37°C along with drug piperine and piperlongumine at a different concen- tration. The reaction between these agents was initiated by adding NADPH (0.1 ml, 10mM) solution for 10 min. It was centrifuged (2000×g; 10min) to remove proteins. To 1.0 ml of this supernatant, 1.0 ml of Nash reagent (2 M ammonium acetate, 0.05 M glacial , 0.02 macetylacetone) was added [27], and heated in a water bath at 50°C for 30 min. After cooling, the absorbance was measured at 412 nm. The activity was calculated from stan- dards (1–100 mM ) prepared by substituting sample with standard solution which were run in parallel .The CYP3A4 activity was expressed as nM of formaldehyde obtained per milligram of protein per hour [26]. Assay was carried out as “control,” “positive control,” “test.” The control represented the methanol vehicle control, whereas the (10 µM) represented the positive control.

RESULTS In vitro CYP3A4 activity of piperine and piperlongumine was determined by Erythromycin-N-demethylation assay. Assessment of in vitro CYP3A4 activity was done by preparing the standard Calibration curve of formalde- hyde (Fig.3) and Protein concentration in liver microsomes was 85.023 mg/ ml. The CYP3A4 activity was expressed as nM of formaldehyde obtained per milligram of protein per hour (Fig.4, Tab.1) whereas “control” repre- sented the methanol vehicle control and the ketoconazole (10 µM) repre- (B) sented “positive control”. Piperine (20µM), piperlongumine (10µM) and (20µM) inhibitory effect on CYP3A4 was found to be 59.83%, 73.9% and 64.5% respectively. In combination of piperine (10µM) and piperlongumine (10µM) showed inhibition 71.4%.

(C) Fig.2: HPTLC finger prints of Piperine and piperlongumine. Fig. 3: Calibration curve of formaldehyde.

Journal of Pharmacy Research Vol.5 Issue 4.April 2012 2244-2246 Arti Nayak et al. / Journal of Pharmacy Research 2012,5(4),2244-2246 Tab .1 Assessment of CYP3A4 activity. activity. Clin Pharmacol Ther 2001;70:317– Drugs nM/mg protein % Inhibition of 326 per min CYP3A4 activity 8 Markowitz J S, DeVane CL, Boulton DW, Carson SW, Nahas Z, Risch (Mean ± STD) (n=3) SC. Effect of St. John’s wort () on cyto- chrome P-450 2D6 and 3A4 activity in healthy volunteers. Life Sci Control 998.7±11.231 - 2000;66:133–139 Ketoconazole 252.41±5.67 74.7 9 Johne A, Brockmoller J, Bauer S, Maurer A, Langheinrich M. Phar- Piperine (20µM) 401.08±5.34 59.83 Piperlongumine (10µM) 259.93±9.75 73.9 macokinetic interaction of digoxin with an herbal extract from St. Piperlongumine (20µM) 354.03±12.32 64.5 John’s wort (Hypericum perforatum). Clin Pharmacol Ther Piperine (10µM) + P.Longumine(10µM) 285.81±11.339 71.4 1999;66:338–345 10 Johne A, Schmider J, Brockmoller J, Stadelmann AM, Stormer E, Bauer S, Scholler G, Langheinrich M. Decreased plasma levels of amitriptyline and its metabolites on comedication with an extract from St. John’s Wort (Hypericum perforatum). J Clin Psychopharmacol 2002;22:46–54 11 Piscitelli SC, Burstein AH, Chaitt D, Alfaro RM, Falloon J. Indinavir concentrations and St. John’s wort. Lancet 2000;355:547–548 12 Yue QY, Bergquist C, Gerden B. Safety of St John’s wort (Hyperi- cum perforatum). Lancet 2000;355:548–549 13 Tsukamoto S, Cha BC, Ohta T.Dipiperamides A, B, and C: bisalkaloids from the white pepper Piper nigrum inhibiting CYP3A4 activity. Tetrahedron 2002;58:1667–1671 14 Volak LP, Ghirmai S, Cashman JR, Court MH. Curcuminoids inhibit multiple human cytochromes P450 (CYP), UDP glucuronosyltransferase (UGT), and sulfotransferase (SULT) en- zymes, while piperine is a relatively selective CYP3A4 inhibitor. American Society for Pharmacology and Experimental Therapeu- tics 2008:1-43 15 Singh J, Reen RK. Modulation of constitutive, benzaanthracene and -inducible cytochromes-P450 activities in rat Fig .4: In vitro CYP3A4 Activity. hepatoma H4IIEC3/G- cells by piperine. Curr Sci 1994;66:365– 369 DISCUSSION 16 Bano G, Amla V, Raina RK, Zutshi U, Chopra CL. The effect of CYP3A4 is one of the major CYP subfamilies involved in drug metabolism in piperine on pharmacokinetics of phenytoin in healthy volunteers. humans accounting for the metabolism of more than 55% of current pharma- Planta Med 1987; 53:568–569 ceuticals. Ketoconazole and erythromycin are frequently used as a selective 17 Bano G, Raina RK, Zutshi U, Bedi KL, Johri RK, Sharma SC. Effect CYP3A4 inhibitor. 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