Curcuma xanthorrhiza is a native Indonesian plant and traditionally utilized for a range of illness including liver damage, hypertension, diabetes, and cancer.
The study determined the effects of C. xanthorrhiza extracts (ethanol and aqueous) and their constituents (curcumene and xanthorrhizol) on UDP-glucuronosyltransferase (UGT) and glutathione transferase (GST) activities.
The inhibition studies were evaluated both in rat liver microsomes and in human recombinant UGT1A1 and UGT2B7 enzymes. p-nitrophenol and beetle luciferin were used as the probe substrates for UGT assay while 1-chloro-2,4-dinitrobenzene as the probe for GST assay. The concentrations of extracts studied ranged from 0.1 to 1000 μg/mL while for constituents ranged from 0.01 to 500 μM.
In rat liver microsomes, UGT activity was inhibited by the ethanol extract (IC50 =279.74 ± 16.33 μg/mL). Both UGT1A1 and UGT2B7 were inhibited by the ethanol and aqueous extracts with IC50 values ranging between 9.59–22.76 μg/mL and 110.71–526.65 μg/Ml, respectively. Rat liver GST and human GST Pi-1 were inhibited by ethanol and aqueous extracts, respectively (IC50 =255.00 ± 13.06 μg/mL and 580.80 ± 18.56 μg/mL). Xanthorrhizol was the better inhibitor of UGT1A1 (IC50 11.30 ± 0.27 μM) as compared to UGT2B7 while curcumene did not show any inhibition. For GST, both constituents did not show any inhibition.
These findings suggest that C. xanthorrhiza have the potential to cause herb-drug interaction with drugs that are primarily metabolized by UGT and GST enzymes.
Findings from this study would suggest which of Curcuma xanthorrhiza extracts and constituents that would have potential interactions with drugs which are highly metabolized by UGT and GST enzymes. Further clinical studies can then be designed if needed to evaluate the in vivo pharmacokinetic relevance of these interactions
Abbreviations Used: BSA: Bovine serum albumin, CAM: Complementary and alternative medicine, cDNA: Complementary deoxyribonucleic acid, CDNB: 1-Chloro-2,4-dinitrobenzene, CuSO4.5H2O: Copper(II) sulfate pentahydrate, CXEE: Curcuma xanthorrhiza ethanol extract, CXAE: Curcuma xanthorrhiza aqueous extract, GC-MS: Gas chromatography-mass spectroscopy, GSH: Glutathione, GST: Glutathione S-transferase, KCl: Potassium chloride, min: Minutes, MgCl2: Magnesium chloride, mg/mL: Concentration (weight of test substance in milligrams per volume of test concentration), mM: Milimolar, Na2CO3: Sodium carbonate, NaOH: Sodium hydroxide, nmol: nanomol, NSAIDs: Non-steroidal antiinflammatory drug, p-NP: para-nitrophenol, RLU: Relative light unit, SEM: Standard error of mean, UDPGA: UDP-glucuronic acid, UGT: UDP-glucuronosyltransferase.
Curcuma xanthorrhiza or commonly known as “temulawak” or Javanese turmeric is a member of the ginger family (Zingeberaceae), which is a native Indonesian plant. It is planted in Thailand, Philippines, Sri Lanka, and Malaysia. C. xanthorrhiza is a low-growing plant with a root (rhizome) that looks like ginger.Traditionally, this plant is used as an ingredient in health supplements known as “jamu” or to cure certain health problems including hepatitis, liver complaints, diabetes, rheumatism, anticancer, hypertension, and heart disorders.
C. xanthorrhiza has also shown antidiuretic, anti-inflammatory, antioxidant, antihypertensive, antihepatotoxic, antibacterial, and antifungal effects. Different phytochemicals found in the herbs have the potential to modulate drug-metabolizing enzymes activities, thus resulting in herb-drug interaction The traditional benefits of C. xanthorrhiza were further confirmed by the isolation and identification of several constituents, including xanthorrhizol and curcumene, and a few volatile substances. Xanthorrhizol is the major component of the essential oil of C. xanthorrhiza, which is a bisabolene-type sesquiterpenoid obtained through hydrodistillation. The presence of xanthorrhizol in C. xanthorrhiza differentiates this plant from other Curcuma species. Xanthorrhizol has been reported to encompass a wide range of biological activities such as antibacterial, antiseptic, and antibiotic.[Curcumene, which is also a bisabolene-type sesquiterpenoid, is one of the constituents found in the essential oil of C. xanthorrhiza with content ranging from 2.6% to 13.6%.
Phase II enzymes are becoming increasingly crucial in drug discovery and drug development. UDP-glucuronosyltransferase (UGT) and glutathione transferase (GST) are the major Phase II enzymes catalyzing the glucuronidation and glutathione (GSH) conjugation, respectively. Glucuronidation of xenobiotics is an important pathway for many diverse organisms to protect themselves against toxins, and the enzymes that detoxify xenobiotics by glucuronidation are the UGTs. GSH conjugation, typically regarded as a detoxification reaction which is catalyzed by GST, is an important host defense mechanism, serving as a scavenger of electrophilic xenobiotics and their re-metabolites.
Herb-drug interaction is becoming an important area of research due to increased concomitant use of herb and conventional drugs. The interaction may involve having a herb component causing an increase or decrease in the amount of drug in the blood through induction or inhibition of drug-metabolizing enzymes. Alteration of drug metabolism by interference with herbal medicines is significantly important since most of the reported clinical adverse drug interactions were due to the modulation of drug-metabolizing enzymes activity. Furthermore, the rapid increase in the consumption of herbal products among herbal practitioners, and although health benefits may be derived from the use of C. xanthorrhiza, this herbal plant extracts and its constituents may have the potential to interact with coadministered drugs. Most of the studies of C. xanthorrhiza focused on its pharmacological activity, and up to date, no report of herb-drug interactions involving C. xanthorrhiza and Phase II drug-metabolizing enzymes has been published. However, previous studies have reported that this herbal plant did show a very low inhibition toward cytochrome P450 (CYP450) enzymes.This situation has triggered the investigation on the effects of C. xanthorrhiza extracts and its constituents on Phase II drug-metabolizing enzymes activity in vitro.
Bovine serum albumin, potassium sodium tartrate tetrahydrate (KNaC4H4O6.4H2O), copper (II) sulfate pentahydrate (CuSO4.5H2O), magnesium chloride (MgCl2), Triton X-100, p-nitrophenol (p NP), tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), UDP-glucuronic acid (UDPGA) (trisodium salt), and diclofenac sodium salt were purchased from Sigma-Aldrich Co.(St. Louis, MO, USA). Ethanol (96%) was purchased from Merck (Darmstadt, Germany). Glycerol was purchased from BDH Chemicals LTD (Poole, UK). Potassium dihydrogen orthophosphate (KH2 PO4) was purchased from AJAX Chemicals (New South Wales, Australia). Sodium nitrate (NaNO3) and potassium chloride (KCl) were purchased from Bendosen Laboratory Chemicals (Selangor, Malaysia). Sodium carbonate (Na2 CO3) and sodium hydroxide (NaOH) were purchased from Systerm® (Shah Alam, Malaysia). Folin and Ciocalteu's phenol reagent were purchased from R and M Chemicals (Canada). Dipotassium hydrogen phosphate (KH2 PO4) was purchased from Riedel-de Häen, Seelze (Germany). Tannic acid was purchased from HmbG Chemicals (Germany). UGT-Glo™ Screening System was purchased from Promega, USA. Purified recombinant human GSTP1 was a gift from Associate Professor Dr. Mohd Nizam Mordi and Mr. Mohammed Nooraldeen Mahmod (Centre for Drug Research, Universiti Sains Malaysia).
Fresh rhizomes of C. xanthorrhiza were purchased from Temerloh, Pahang, Malaysia, in February 2010. The plant was authenticated by Mr. Shanmugam and deposited at the Herbarium Unit of the School of Biological Sciences, USM. The voucher specimen number of the plant is USM11022. The rhizomes were air-dried, ground into fine powder, and stored prior usage.
C. xanthorrhiza extracts were standardized in reference to the content of its constituents, xanthorrhizol, and curcumene. Both xanthorrhizol and curcumene were measured using gas chromatography mass spectrometry as reported by Ab-Halim et al.Compared to curcumene, xanthorrhizol was found to be higher in both ethanol and aqueous extracts (ethanol: 22.89%, aqueous: 3.41%), followed by curcumene (ethanol extract: 13.61%, aqueous extract: 2.11%).
Male Sprague Dawley rats (150–200 g) were obtained from the Animal House of USM. The rats were maintained under controlled temperature (25°C ± 2°C), 12 h light/12 h dark conditions for 1 week before the start of the experiments. They were provided with water and food ad libitium. Animals were maintained and handled according to the recommendations of the USM Ethical Committee which approved the design of the animal experiments with the reference number USM/Animal Ethics Approval/2011/(72) (340).
Rat livers were removed immediately after sacrifice and were rinsed with ice-cooled distilled water followed by ice-cooled 67 mM potassium phosphate buffer (pH 7.4), blotted dry, and weighed. Isolated rats liver samples were homogenized in 67 mM potassium phosphate buffer (pH 7.4) containing 1.15% (w/v) KCl where the volume of buffer is 3 times the weight of the liver samples using a Potter-Elvehjem homogenizer. After centrifugation of the homogenate fraction at 12,500 ×g for 20 min at 4°C, the resultant supernatant was decanted to ultracentrifuge tubes (Optiseal™) and centrifuged at 100,000 ×g for 60 min in Optima™ TLX refrigerated ultracentrifuge (Beckman Coulter, Inc., USA). The supernatant obtained was the cytosolic fraction. The microsomal pellet obtained was scrapped and each was resuspended in 300 µL 67 mM potassium phosphate buffer with 1.15% KCl and 20% (v/v) glycerol. The pooled microsomes were homogenated again to mix the solution properly. The cytosolic fractions and microsomes were aliquoted into several microfuge tubes and were stored at −80°C until used. Protein content of microsomes and cytosolic fractions were determined by a method by Lowry et al.
The inhibition studies with rat liver microsome as the UGT source was carried out according to a previous method by Embola et al., with slight modification. The typical 200 µL incubation mixture contained 0.01% (v/v) Triton X-100, 0.5 mg/mL of UGT enzymes (rat liver microsomes), different concentrations of C. xanthorrhiza, 0.1 M Tris HCl buffer, 0.005 MgCl2, and 0.5 mM p NP in polypropylene microfuge tubes. Microsomes were preincubated with Triton X-100 on ice for 5 min to activate UGT enzymes. The range of C. xanthorrhiza extracts determined was 0.1–1000 µg/mL while the concentration range for the positive inhibitor, diclofenac ranged from 0.1 to 1000 µM. The concentration range of C. xanthorrhiza constituents determined was from 0.02 to 200 µM. The final concentration of ethanol and dimethyl sulfoxide used in this study was <2% (v/v). Controls were prepared as described above except that no extract or constituent was added. Blank for control, xanthorrizol, and ar-curcumene contained the same as described above except without addition of Triton X-100, microsomes, constituents, and UDPGA. However, blank for each extract differ from the others since each concentration determined have their own blank. This is due to the presence of color in the extracts that may interfere with the absorbance reading. The reaction was started by addition of 30 mM UDPGA which was dissolved in distilled water. All tubes were vortexed and incubated in a shaking water bath at 37°C for 30 min. At the end of each incubation time, the reaction was stopped by adding 200 µL of 20% (w/v) ice-cold trichloroacetic acid. All tubes were vortexed for at least 2 min and incubated in ice for at least 10 min. Then, all tubes were centrifuged at 2000 rpm for 10 min. After centrifugation, 200 µL of supernatant from each tube was transferred into new polypropylene microfuge tubes and 800 µL of 0.5 M NaOH was added. Tubes were vortexed and incubated at room temperature for 10 min. After 10 min incubation, 200 µL of mixture was transferred to a 96 microtiter well plate. The absorbance at 405 nm was measured using a Plate CHAMELEON™ multitechnology plate reader, 425-106 (Hidex Oy, Finland).
The luminescent assay was conducted as described in the UGT-Glo™ Screening System protocol. For each inhibition study, the concentrations of extracts and positive control ranged from 0.1–1000 µg/mL to 0.1–1000 µM whereas the concentrations for constituents ranged from 0.02 to 200 µM. The highest concentration determined for constituents was only at 200 µM since maximum inhibition was observed at this concentration. Reactions were conducted in white 96-well luminometer plate using a 37°C incubator. The final reaction volume (40 µL per 96-well) consisted of UGT reaction buffer, 4 mM UDPGA or an equivalent volume of water, and 20 µM UGT multienzyme substrate with 0.1 mg/mL UGT1A1 or UGT2B7 supersomes. Negative control reactions contained the same components as UGT reactions, except the UGT supersomes were replaced with an equivalent amount of control microsomes, devoid of UGT activity. The amount of incubation time for the UGT1A1 reaction was 90 min while it was 60 min for the UGT2B7 reaction. The reaction was stopped by addition of 40 µL reconstituted luciferin detection reagent (LDR) and D-cysteine to all determined wells and further incubated at room temperature for 20 min to stabilize the luminescence signal. UGT activity was detected by the addition of LDR and luminescence signal was recorded as relative light units.
The inhibition studies using rat liver GST and human GST Pi-1 (hGSTP1) were carried out according to a previous method with slight modifications using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Incubation mixtures (300 μL for rat liver GST, 200 μL for hGSTP1) contained 0.1 M potassium phosphate buffer (pH 6.5) and 0.001 M of GSH. This was followed by addition of GST enzyme (0.125 mg/mL of rat liver GST, 0.003 mg/mL of hGSTP1) and different concentrations of extracts and compounds. The control sample contained the same as mentioned except that no extracts or compounds were added. The mixture for blank sample for each concentration of extracts and compounds also contained the same as mentioned except that the rat liver cytosolic fraction was denatured at 60°C for 20 min while blank sample for hGSTP1 was replaced with 0.1 M of potassium phosphate buffer (pH 6.5). The concentrations of plant extracts and tannic acid used ranged from 0.01 to 1000 µg/mL. The concentrations of pure compounds used in this study ranged from 0.01 to 500 µM. The reason for not determining the activity above 1000 µg/mL and 500 µM was because maximum absorbance was reached at the highest concentration determined. GST-mediated conjugation of CDNB to GSH was measured using a Plate CHAMELEON™ multitechnology plate reader, 425-106 (Hidex Oy, Finland) at wavelength of 340 nm for 5 min for rat liver GST while 20 min for hGSTP1. All experiments were carried out in 5 replicates.
All results are presented as mean ± standard error of mean of five determinations except for UGT luminescence assay (three determinations from two independent experiments). The IC50 values, concentration of inhibitor causing 50% inhibition of enzyme activity, were calculated by the nonlinear regression analysis of percentage of remaining enzyme activity (expressed as percent of control) versus the logarithm of inhibitor concentration. ANOVA followed by Dunnett test was used to evaluate the significant differences of the results obtained. P < 0.05 was considered statistically significant. All computations were performed using GraphPad Prism® 5 for Windows software (Version 5.01, GraphPad Software, Inc., USA).
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