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Year : 2020  |  Volume : 11  |  Issue : 2  |  Page : 100-104  

Enhanced oral bioavailability of diltiazem by resveratrol in healthy human subjects: An open-label, two-period, sequential study

Department of Pharmacology, DMPK and Clinical Pharmacology Division, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana, India

Date of Submission19-Jul-2019
Date of Decision28-Oct-2019
Date of Acceptance01-May-2020
Date of Web Publication22-Jul-2020

Correspondence Address:
Prasad Neerati
Department of Pharmacology, DMPK and Clinical Pharmacology Division, University College of Pharmaceutical Sciences, Kakatiya University, Warangal - 506 009, Telangana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jnsbm.JNSBM_163_19

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Background: The purpose of this study was to investigate the bioavailability enhancing potential of resveratrol (RSV) on diltiazem (DLT) treatment in healthy human volunteers. Materials and Methods: An open-label, two-period, sequential study was conducted in 12 healthy human male volunteers. A single dose of RSV 500 mg was administered daily for 10 days during the treatment phase. A single dose of DLT 30 mg was administered during the control phase and after treatment phases under fasting conditions. The blood samples were collected at predetermined time intervals after DLT dosing and analyzed using high-performance liquid chromatography. Results: Treatment with RSV significantly enhanced maximum plasma concentration, area under the curve, whereas the volume of distribution and apparent clearance (CL/F) of DLT was significantly decreased when compared to control. Conclusions: The results suggested that enhanced bioavailability of DLT might be attributed by bio enhancing the potential of RSV resulted by inhibition of P-gp and CYP3A4. Further, the dosage of DLT should be readjusted when it is used concomitantly with RSV supplements or food containing RSV.

Keywords: CYP3A4, diltiazem, high-pressure liquid chromatography, P-gp, resveratrol

How to cite this article:
Athukuri BL, Neerati P. Enhanced oral bioavailability of diltiazem by resveratrol in healthy human subjects: An open-label, two-period, sequential study. J Nat Sc Biol Med 2020;11:100-4

How to cite this URL:
Athukuri BL, Neerati P. Enhanced oral bioavailability of diltiazem by resveratrol in healthy human subjects: An open-label, two-period, sequential study. J Nat Sc Biol Med [serial online] 2020 [cited 2021 Jan 19];11:100-4. Available from:

   Introduction Top

Diltiazem (DLT), a calcium channel blocker, indicated for the treatment of angina, supraventricular arrhythmias, and hypertension.[1],[2] DLT undergoes an extensive hepatic first-pass metabolism, and its oral bioavailability is around 38 ± 11% in humans.[3] In humans, DLT is rapidly and almost completely metabolized in intestine and liver through deacetylation, N-demethylation, and O-demethylation to produce a number of active and inactive metabolites.[4] The cytochrome P4503A (CYP4503A) is the main enzyme responsible for the biotransformation of DLT in humans.[4],[5] Desacetyl DLT is one of the main active metabolites of DLT, has been shown to have coronary vasodilatory potency one half to DLT and inhibition of thrombocyte aggregation about three-fold greater than that of DLT.[6],[7]

CYP3A is one of the most important CYP isoforms responsible for drug metabolism by humans and found in various tissues such as the enterocytes and liver. In addition, it is involved in the oxidative metabolism of numerous clinically useful therapeutic drugs.[8] CYP3A4 in the gastrointestinal tract is, particularly, account for drug interactions characteristic of CYP3A4 substrates, and it suggests a strategy for controlling entry into the body of a major class of drugs. Since herbal products can also competitively inhibit CYP enzymes, simultaneous intake of these phytochemicals may increase plasma levels of therapeutic agents, thus exposing patients to a greater risk of serious adverse effects.[9]

P-glycoprotein (P-gp), an intestinal drug efflux transporter, is a member of the superfamily of transporters known as ATP-binding cassette transporters or ABC transporter. P-gp is well identified as a major source for the low or variable oral bioavailability of several structurally unrelated therapeutic agents.[10] Both CYP3A4 and P-gp have different substrate specificities, and tissue localization and P-gp regulates the access of the drug to the CYP enzyme, which increases the metabolism due to prolonged exposure to the enzyme through repeated cycles of drug absorption and efflux. Thus, these two proteins work together to protect the body from the absorption of toxic drug substances.[11],[12]

Resveratrol (RSV) (trans-3, 4', 5-trihydroxystilbene) is a naturally occurring phytopharmaceutical found in grapes and is often used as a food supplement. Many positive health benefits, including cardioprotection, tumor suppression, and immune modulation, are associated with the regular intake of RSV and are well tolerated in healthy volunteers without any co-medication. It has been reported that RSV mimics calorie-restriction effects in obese humans,[12] and also reported to block the transcription of various cytochrome P450 metabolic enzymes (CYPs) through antagonism of the nuclear aryl hydrocarbon receptor. These mechanisms are expected to reduce the cellular load of chemically reactive and, therefore, potentially toxic drug metabolites. On the other hand, inhibition of CYP activity by RSV could lead to safety problems by changing the pharmacokinetics of coadministered drugs. RSV has been reported to show inhibitory effects substrates of CYP3A4, CYP2D6, CYP2C9, and CYP2C19. The study was carried out to see whether the resveratrol can enhance the bioavailability of diltiazem in healthy volunteers.

   Materials and Methods Top


DLT and buspirone (BSP) were obtained from AET Labs Pvt Ltd (Hyderabad, India). RSV 500 mg capsules were purchased from Medizen Labs Pvt Ltd (Bangalore, India). DLT 30 mg tablets purchased from Torrent pharmaceuticals Ltd (Gangtok, India). Solvents used for quantitative analysis (Merck, India) and all other chemicals, reagents that were used in the study are of analytical grade.


Protocol for human volunteer study

Twelve healthy male volunteers with a mean age of 26.33 ± 2.15 years (range 25 to 30 years), mean height of 5.75 ± 2.23 inch (range 5' 4” to 5' 8”) and mean body weight of 62.25 ± 4.99 kg (55 to 69 kg) participated in the study after undergoing a thorough physical examination. The volunteers were briefed about the study, and written informed consent was obtained from all of them. The Institutional Human Ethical Committee of University College of Pharmaceutical Sciences (UCPSC/KU/BA/2016-4), Kakatiya University approved the study protocol, and the study was followed the principles of Helsinki with Good Clinical Practice guidelines. The volunteers had no history of any ill health during the preceding 6 months and none had taken any medication for at least 15 days before the administration of DLT in the study. They had to avoid nicotine, alcohol, caffeine, and citrus fruit products for one week before and throughout the study period. Volunteers were excluded from the study if they had food allergies or were allergic to DLT or RSV. Study drugs were taken in the morning with 100 mL of drinking water just after voiding, and a common standard fixed quantity of diet for all the volunteers was followed.

The first part of the study included oral administration of 30 mg of DLT tablet (B. No: 2D61C006, Torrent Pharmaceuticals Ltd, Gangtok, India) alone, and blood was sampled from the antecubital vein. Blood samples were obtained at intervals of 0, 1, 3, 3.5, 4, 6, 8, 12, and 24 h after the administration of DLT. The second part of the study was conducted after the washout period of 2 weeks. 500 mg of RSV (RSV, Medizen Labs Pvt Ltd, Bangalore, India) was administered daily ones for 10 days. On day 10, single tablet, each of 30 mg of DLT and 500 mg of RSV, were administered concomitantly. Blood samples were collected as described above and centrifuged at 3000 rpm for 15 min, and the supernatant serum was separated and stored at −20°C until the analysis.

Serum samples analysis

DLT in the serum samples was estimated using the reverse phase high-pressure liquid chromatography method.[13]

Method of analysis of diltiazem by high-performance liquid chromatography

A Shimadzu high-performance liquid chromatography (HPLC) system equipped with a LC- 20AT pump and SPD 20 AVP UV visible detector and RP C18 column (250 mm × 4.6 mm ID, particle size 5 μm, (Phenomenex, Kondapur, and Hyderabad, India) was used for the HPLC analysis of serum samples. The mobile phase used was 25 mM potassium dihydrogen orthophosphate (pH- 4.6): acetonitrile: 65:35, and the elution was monitored at 238 nm with a flow rate of 1.0 mL/min. The retention time of DLT was 11 min, and the internal standard used was BSP with a retention time of 9.2 min. Partial validation of the bioanalytical method has been done to measure the accuracy, precision, limit of detection, and limit of quantification of the analyte.

Extraction procedure for serum samples

To 500 μL serum, 100 μL of internal standard (50 μg/mL) and 500 μL of 0.1M KH2 PO4 were added. This mixture was vortexed for 2 min, and extraction was performed by adding 5 mL of diethyl ether and centrifuged at 1000 g for 10 min. The organic layer was separated and to this 100 μL of a 0.1 M H3 PO4 solution was added. The mixture was vortexed and centrifuged at 1000 g for 10 min. The phosphoric layer was then separated, and a 20 μL aliquot was injected into the HPLC system.

Analysis of pharmacokinetic parameters

All the pharmacokinetic parameters were analyzed using Phoenix WinNonlin version 6.2 software (Certara, Pharsight Corporation, L.P, USA). The statistical analysis was performed using paired t-test (two-tailed) using Graph Pad Prism version (5.0) (Graph Pad Software Inc., San Diego, CA, USA) at the significance level of P < 0.05.

   Results Top

All the human volunteers tolerated the treatments well and there were no cases of severe adverse affects during the study period. The mean serum concentration-time plots of DLT alone and after pretreatment with RSV for 10 days is shown in [Figure 1]. The pharmacokinetic parameters of DLT in both control and treatment phases are shown in [Table 1]. The serum DLT concentrations were increased after RSV treatment when compared to the control phase. After pretreatment with RSV maximum plasma concentration (Cmax) was increased from 38.5 ± 4.14 to 113.14 ± 11.58 ng/mL, area under the curve (AUC)0-t increased from 219.4 ± 28.84 to 784.99 ± 85.47 ng/hr/mL and AUC0- increased from 295.5 ± 96.68 to 920.66 ± 102.18 ng/hr/mL. A statistically significant difference in the pharmacokinetic parameters, Cmax, AUC0-∞, AUC0-t, Vd and Clearance, was observed. No statistically significant difference was observed in the pharmacokinetic parameter, Tmax and Kel. RSV pretreatment increased the Cmax, AUC0-∞, AUC0-t of DLT 2.9-, 3.12-, and 2.66-fold, respectively, compared to the control phase.
Figure 1: Serum concentration-time profiles of diltiazem in human volunteers after the administration of 30 mg of diltiazem during the control phase and after treatment with 500 mg resveratrol once daily for 10 days

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Table 1: Pharmacokinetic parameters of diltiazem in human volunteers after the administration of 30 mg of diltiazem during control and treatment phase with 500 mg resveratrol once daily for 10 days

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   Discussion Top

Dietary supplements and foods, including fruits, vegetables, herbs, spices, and teas that contain complex mixtures of active constituents, have the greater potential to inhibit or induce the expression and activity of drug-metabolizing enzymes and/or drug-efflux transporters. There is a general opinion among the people that herbal products are “good for you” since they are “all-natural.”[14] Herbal–drug interaction is a major safety concern particularly for drugs with narrow therapeutic indices, and may lead to severe adverse effects. CYPs and P-gp may be particularly vulnerable to modulation by the multiple phytochemicals of foods, including dietary supplements. CYP3A4 is known to be involved in the most common herbal-drug interactions, as demonstrated by reports of clinically important interactions involving orally administered drugs that are substrates of this enzyme.

RSV has been reported to show an inhibitory effect on CYP3A4in vitro and in vivo. A high intake of RSV could enhance the oral bioavailability of drugs that undergo extensive first-pass metabolism by CYP3A4. RSV has been reported to inhibit human CYP3A4-dependent biotransformation of cyclosporine and also hydroxylation of testosterone by CYP3A4.[15] Chow et al., reported that an increased area under the plasma concentration versus time curve (AUC) of BSP was observed after administrating RSV for 4 weeks.[16] For the CYP3A4 substrate nicardipine, which has poor oral bioavailability, RSV has been shown to increase the AUC and Cmax by a factor of 2.3 and 2.2, respectively. Human microsomes observed to be more sensitive to inhibition because the IC50 value for CYP3A-mediated testosterone 6β-hydroxylation in rat microsomes was observed to be much higher 20 versus 4 μM.[17]

Nabekura et al. reported that the accumulation of rhodamine 123 in KB-C2 cells was increased, and the efflux of rhodamine 123 from KB-C2 cells was decreased by RSV through inhibitory effects on P-glycoprotein.[18] RSV has been reported to reverse the multidrug resistance in KBv200 cells by inhibiting the multidrug-resistant gene expression.[19] Furthermore,in vivo studies in rats demonstrated that RSV was able to increase the bioavailability of P-gp substrates through the inhibition of P-gp-mediated drug efflux.[20],[21] RSV has been shown to potentiate the cytotoxic activity of docetaxel and doxorubicin (P-gp substrates) by enhancing intracellular drug accumulation through inhibition of P-gp and downregulation of the mdr1 gene.[22]

It has been reported that RSV enhanced the oral bioavailability of DLT by inhibiting P-gp-mediated drug efflux and CYP3A mediated drug metabolism in rat liver and intestine.[21],[22] In addition, RSV and its metabolite RSV-3-sulfate have been reported as inhibitors of CYP3A.[23] Previously it has been reported that RSV exerted an inhibitory effect on the metabolism of carbamazepine[24] and nicardipine.[20]

In the study, we assessed the effect of RSV on the pharmacokinetics of DLT, a substrate of both P-gp and CYP3A4, in human volunteers and found that RSV affected the pharmacokinetics of DLT and increased its oral bioavailability. RSV treatment significantly enhanced the mean Cmax and AUC of DLT compared to the control phase. There is no significant change in Tmax of DLT was observed upon RSV treatment compared to control. On the other hand, mean clearance and Vd of DLT were significantly decreased compared to control phase. These results suggested that RSV could inhibit P-gp, an efflux transporter, and the first-pass metabolism during intestinal absorption. These results are consistent with the previous reports stating that the extraction ratios of DLT in the small intestine and liver after an oral administration to rats were about 85% and 63%, respectively, suggesting that DLT is highly extracted in the small intestine as well as liver.[25],[26],[27] Therefore, the decrease of intestinal extraction by the concomitant use of RSV resulted in the enhanced oral bioavailability of DLT. These results are similar to Choi and Han, who reported that morin, an inhibitor of P-gp and CYP 3A4, increased the bioavailability of DLT in rats, and consistent with those of.[17],[18] These results confirmed the possibility that the increased bioavailability of DLT in the presence of RSV might be associated with the inhibition of P-gp and CYP activity. As a result, RSV may become a dual inhibitor of CYP 3A4[28],[29] and P-gp; therefore, RSV resulted in significantly enhancing the bioavailability of DLT.

   Conclusions Top

This study demonstrates that RSV increases the bioavailability of oral DLT due to the inhibition of P-gp and CYP 3A4. Thus, there is a potential pharmacokinetic interaction between RSV and DLT has been observed. Accordingly, caution should be taken when RSV supplements are used in combination with therapeutic drugs, which are the substrates of P-gp and/or CYP3A4. Dose adjustment of these substrates is necessary while taking concomitant therapy with RSV supplements.


This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. University College of pharmaceutical sciences, Kakatiya University, support for the routine reagents, and permission to animal holding for this research.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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