Abstract
Objectives:
Resveratrol (3,4,5-trihydroxystilbene) is a naturally occurring phytoalexin with purported health-promoting effects, but with limited oral bioavailability. Our prior murine modeling research observed enhanced resveratrol bioavailability with piperine co-administration. In this study, single-dose pharmacokinetics of resveratrol with or without piperine and the associated toxicities were studied on a cohort of healthy volunteers.
Methods:
We performed a double-blind, randomized, three arm pilot study. Participants were randomized to receive a single dose of resveratrol 2.5 grams, with piperine in 0 mg, 5 mg or 25 mg dose. An improved LC/MS/MS assay was used to determine serum levels of resveratrol and resveratrol glucuronide. Baseline through 24 hours post study-drug serum analyses were performed and adverse events were followed for 30 days.
Results:
Twenty-four participants were enrolled. No significant relationship between dose and pharmacokinetic values were found. In the gender stratified analysis, Cmax for resveratrol in females showed a trend (p = 0.057) toward an increase with piperine. Pharmacokinetic values for resveratrol were: Cmax – 18.5±16 ng/ml Resveratrol alone, 29±29 Resveratrol + 5 mg piperine, 16±13 Resveratrol + 25 mg piperine; AUC - 1270±852 ng/h/ml Resveratrol alone, 2083±2284 Resveratrol + 5 mg piperine, 1132±222 Resveratrol + 25 mg piperine. All subjects tolerated their protocol therapy with minimal to no toxicity and no evidence of differences between the 3 groups.
Conclusion:
The coadministration of resveratrol with piperine at 5 and 25 mg doses did not sufficiently alter the pharmacokinetics of resveratrol or resveratrol glucuronide to demonstrate the significant enhancement observed in murine modeling.
Keywords: Piperine, Resveratrol, Bioavailability, Bioenhancer
INTRODUCTION
Resveratrol (3,4,5-trihydroxystilbene) is a naturally occurring phytoalexin known to possess a multitude of health-promoting properties (Bhat and Pezzuto, 2002; Aziz et al., 2005; Baur and Sinclair, 2006). The multitude of observed in vitro and in vivo effects have led to study of resveratrol in diseases and disciplines ranging from cancer prevention and therapy, improved metabolic and cardiovascular health, and neurodegenerative diseases (Chen et al., 2009; Crescente et al., 2009; Kang et al., 2010; Palsamy and Subramanian, 2010; Subramanian et al., 2010; De Amicis et al., 2011; De Leo et al., 2011; Zhu et al., 2012; Osmond et al., 2012; Zhang et al., 2013; Tomayko et al., 2014; Wightman et al., 2014; Turner et al., 2015; Yiu et al., 2015; Bhullar and Udenigwe, 2016; Feng et al., 2016). There have been a few well-controlled, short duration studies exploring dosing, pharmacokinetics and some biomarker analysis in humans and numerous observational reports of longer-term exposure and its effects (Patel et al., 2011). Well-controlled studies have employed doses of a few mg to ≥ 1 gm per day for days to weeks with little to no reports of toxicity with doses less than 500 to 1000 mg (Patel et al., 2011). An issue with human studies of resveratrol has been relatively low concentrations of free resveratrol in serum when compared to the concentrations tested in vitro (Mukherjee, Dudley and Das, 2010; Heebøll et al., 2016; Thazhath et al., 2016; Zortea et al., 2016), or in animal studies (Asensi et al., 2002). Pharmacokinetic studies in animals and humans have identified two attributes of resveratrol. First, the majority of resveratrol metabolites were found to be derived by glucuronidation or sulfation, with at least seven different identified forms (Burkon and Somoza, 2008). Second, the metabolism of resveratrol has been shown to be very rapid with the majority of biotransformation occurring within the first hour following oral administration. Resveratrol metabolites have very short half-life (8 to 14 minutes) and are subjected to rapid urinary elimination (Wenzel and Somoza, 2005; Kinoshita et al., 2006; Wang, Xu and Liu, 2008). A few clinical studies have suggested that the time to maximum serum concentration (Tmax) of resveratrol could be impacted and delayed by certain types of foods (Vaz-da-Silva et al., 2008; Almeida et al., 2009). However, combining resveratrol with these foods did not significantly increase the absorption of resveratrol. This rapid metabolism of resveratrol may be a major obstacle in translating its observed therapeutic effect in pre-clinical studies to controlled clinical settings in humans (Ndiaye, Kumar and Ahmad, 2011).
The relative poor bioavailability or rapid metabolism of orally administered natural products have led to researchers seeking dietary-based maneuvers to increase systemic exposure of key nutrient constituents (Johnson et al., 2011). An example of this is the co-administration of piperine, an alkaloid derived from black pepper (Piper spp.), with natural products (Reen et al., 1993; Shoba et al., 1998; Lambert et al., 2004). Multiple researchers have observed piperine to inhibit the glucuronidation of natural products like curcumin or epigallocatechin-3-gallate (Atal, Dubey and Singh, 1985; Srinivasan, 2007; Johnson, Bailey and Mukhtar, 2010). Consistent with these findings, we observed piperine co-administration with resveratrol in a mouse model to significantly increase peak serum resveratrol concentration (Cmax) by 1,544%, the Cmax of resveratrol-O-β-D-glucuronide by 184% and delay Tmax from 0.25 h to 0.5 h as compared to resveratrol alone (Johnson et al., 2011). However, pharmacokinetic parameters of co-administration of piperine in humans are largely unknown, as is the optimal dosing strategy. Given these data we performed a randomized, placebo-controlled, double-blind clinical trial of resveratrol with and without piperine in healthy adult volunteers. The aim of this trial was to characterize the single-dose pharmacokinetics of resveratrol with or without piperine. The secondary objectives were to determine the pharmacodynamics and toxicities of single dose resveratrol with or without piperine.
METHODS
Study Design
This study was designed as a single-center, randomized, double-blind, dose-ranging trial to determine the effect of co-administration of piperine on resveratrol bioavailability. Participants were randomized using a permuted block of size six on a 1:1:1 basis stratified by gender (4 men and 4 women in each group) to receive a single dose of either resveratrol 2.5 gm alone, resveratrol 2.5 gm and piperine 5 mg, or resveratrol 2.5 gm and piperine 25 mg. Participants met the inclusion criteria: a Karnofsky performance status >70%, capable of providing informed consent for the study, 18–65 years of age, normal screening blood tests (heamoglobin >10 gm/dl, platelets >100,000/mm3, white blood cell count >3.000/mm3, bilirubin < 1.4 mg/dL; aminoaspartate transferase < 1.5 x upper limit of normal, creatinine 0.6–1.3 mg/dL, and within normal limits for serum sodium, potassium, chloride and CO2). Participants could not be taking any scheduled medications (exception fororal contraceptives), had to refrain from alcohol ingestion or consuming supplements or dietary ingredients known to contain resveratrol during study participation. All participants underwent study treatment and pharmacokinetic sampling as inpatients in the University of Wisconsin Clinical Research Unit (UW CRU). Participants were seen on two separate visits and followed for adverse events for 30 days post study drug. The pre-study visit was to determine eligibility. On day 1 of the study, normal volunteers ingested a single dose of resveratrol with or without piperine in the morning, following fasting after midnight the evening prior to starting drug. This was followed by a 24 hour stay. On days 8 and 30, the participants were contacted via telephone to assess any toxicities.
Ethical statement
The study underwent scientific and protocol review by the University of Wisconsin Carbone Cancer Center prior to review and approval by Institutional Review Board for protection of Human Subjects at the University of Wisconsin.
Formulation and Dose of Investigational Agent
Trans-resveratrol (>95%) and piperine powder was prepared according to Good Manufacturing Practice and provided to the University of Wisconsin Pharmaceutical Research Center by Sabinsa Corporation (East Windsor, NJ, USA) and dispensed according to a randomization schedule prepared by a study statistician.
Sample Collection and Processing
Baseline blood samples were drawn before the participants were allowed to eat, which was not until 2 hours following ingestions of the study drug. After the single dose, whole peripheral blood samples (3 ml) for pharmacokinetic analysis were collected into heparinized tubes at the following time points: 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours. All samples were processed and frozen within 30 minutes of sampling. The same laboratory tests that were done for eligibility were repeated at the 24-hour time point to monitor the participant’s safety.
All samples were processed at the UW CRU. The samples were centrifuged at 2000 rpm for 10 minutes to obtain plasma, which was then stored at −70 °C until extraction. All extractions were carried out swiftly and kept away from direct light. Samples were thawed at room temperature or in the hand, kept in the dark, and thoroughly mixed. Plasma was acidified with 17.5 μL of concentrated HCl per 1 mL of plasma. Plasma (250 μL) was pipetted into a clean 1.5 mL tube and 250 μL of methanol added. The sample was then vortex-mixed for 1 min and placed at −20 °C to precipitate the protein. The samples were spun at 13,000 × G in a microcentrifuge at 4 °C for 15 min to discard denatured proteins. The supernatant was pipetted off into a clean microtube and dried down at room temperature in the dark under nitrogen. Once dry, the samples were reconstituted in 200 μL of 50:50 MeOH:H2O and thoroughly mixed. A final 13,000 × G centrifugation at 4 °C was carried out prior to pipetting into the appropriate analysis vials, and if not injected immediately, kept in the dark at 4 °C in the autosampler.
Bioanalytical Methodology
Initial analysis of resveratrol and the resveratrol-glucuronide was done using the previously described method (Johnson et al., 2011). We observed an increase in photo-activated isomerization of resveratrol during sample preparation and a consequent reduction in lower limit of quantification and limit of detection presumably due to larger numbers of samples, sampling volumes and increased light exposure. Furthermore, the inter-day variation with this method was high, in some cases exceeding 15%. With this in mind, we developed a solid-phase extraction (SPE) preparation that involved less sample handling and would remove common serum components known to interfere with liquid chromatography/mass spectrometry (LC/MS/MS) analysis. We achieved typical inter-day variation <5% relative standard deviation (RSD) as compared to our previous method which had >12% RSD variations. Detailed protocol is described in supplemental digital content (see Text document, Supplemental Digital Content 1).
LC/MS Data Analysis
Data were analyzed using the Multi-Quant software (Sciex, Framingham, MA, USA). Chromatograms for all transitions of the analytes and internal standard were created as well as a “total” chromatogram which summed the intensities of 3 transitions. Quadratic modeling with 1/x weighting of the analyte area under the curve relative to the internal standard area was used to determine concentrations of resveratrol and resveratrol-3-O-β-D-glucuronide in subject samples. Analyte peaks typically had >15 data points/peak. Limits of detection were defined by analyte signals with a signal-to-noise ratio of 3. The limit of quantitation was defined as an analyte signal with a signal-to-noise ratio of 10. The percent recovery from plasma samples was calculated to be 63% for resveratrol-3-O-β-D-glucuronide and 76% for resveratrol comparing the intensity of a quality control mid sample to that of blank plasma sample spiked with analytes after processing using the Ostro matrix (Waters Corporation, Milford, MA, USA).
Statistical Considerations
The study sample size of 24 was estimated to detect a 80% change in Cmax (similar to the observed change in murine modeling (Johnson et al. 2011)) the primary pharmacokinetic parameter of interest, between the control group (resveratrol alone) and the experimental group (resveratrol with two piperine dose groups combined) with power 0.85 according to a two-tailed two-sample t-test with significance level 0.05. If the comparison between the control group and the experimental group were statistically significant at significance level 0.05, pairwise comparisons were to be made among three groups. Pharmacokinetic parameters, Cmax, Tmax, AUC0-last, AUC0−∞, T1/2, volume (V) and clearance (CL), were summarized using descriptive statistics and compared between treatment groups using t-test, with normalizing transformation such as logarithm, or Wilcoxon rank-sum or Kruskal-Wallis test.
RESULTS
Participants
Twenty-four participants were registered into the study and randomized to three treatment groups. The mean age at enrollment was 31.8 years (standard deviation 11.5) with gender evenly distributed within the groups (Table 1). Twenty - three patients (96%) were white and 1 patient (4%) was African American. Of white patients, 3 (12%) were Hispanic.
Table 1.
Baseline Patient Characteristics
| Characteristic* | Resveratrol 2.5 gm (n=8) |
Resveratrol 2.5 gm + Piperine 5 mg (n=8) |
Resveratrol 2.5 gm + Piperine 25 mg (n=8) |
All Combined (n=24) |
||||
|---|---|---|---|---|---|---|---|---|
| Age, years | 31.0 | ±10.5 | 32.9 | ±15.1 | 31.6 | ±9.7 | 31.8 | ±11.5 |
| Gender (Male:Female) | 4:4 | 4:4 | 4:4 | 12:12 | ||||
| Body Mass, kg/m2 | 25.1 | ±3.4 | 25.0 | ±3.7 | 30.1 | ±8.2 | 26.7 | ±5.8 |
| ECOG Status 0 | 8 | (100) | 8 | (100) | 8 | (100) | 24 | (100) |
| Ethnicity | ||||||||
| Not Hispanic or Latino | 7 | (88) | 8 | (100) | 6 | (75) | 23 | (96) |
| Hispanic or Latino | 1 | (12) | 0 | (0) | 2 | (25) | 1 | (4) |
| Race | ||||||||
| White | 8 | (100) | 8 | (100) | 7 | (88) | 23 | (88) |
| Asian | 0 | (0) | 0 | (0) | 1 | (12) | 1 | (12) |
| Weight, kg | 70.2 | ±10.2 | 75.3 | ±15.6 | 89.1 | ±29.2 | 78.2 | ±20.8 |
Presented as number (percentage) or mean ± SD
ECOG - Eastern Cooperative Oncology Group
All participants tolerated their protocol therapy without significant sequelae. Seventeen (71%) of the patients had at least one adverse event; ten patients (42%) report having multiple adverse events. Across the three treatment groups, approximately 50–75% of participants reported or were observed to have grade 1 to 2 toxicity. The most common adverse events were transient grade 1 nausea or stomach “queasiness” in the 24 hours post-dose reported by 1–2 participants in each group, or grade 1 headache after study drug dosing. Most participants thought the headache may have been due to caffeine withdrawal during their inpatient stay on day 1 (no caffeine was allowed prior to and for 24 hours post study drug). Examination of adverse events by grade, attribution or frequency revealed no differences among the treatment arms. Examination of safety lab values (complete blood count, INR, electrolytes, kidney and liver function tests) from baseline to 24 hours post-dose revealed no significant changes within patients or across treatment groups (data not shown).
Pharmacokinetics analysis
The pharmacokinetics parameters of resveratrol and resveratrol glucuronide are summarized in Table 2. Peak concentrations (Cmax) of resveratrol were 18.5 ±16 ng/mL for resveratrol alone, 29±29 for resveratrol + 5 mg piperine and 16±13 for resveratrol + 25 mg piperine. Area under the concentration x time curve (AUC) was 1,270±852 ng/h/mL for resveratrol alone, 2,083±2,284 for resveratrol + 5 mg piperine and 1,132±222 for resveratrol + 25 mg piperine. The measured half-life (t1/2) was 30 min for resveratrol alone, 25 for resveratrol + 5 mg piperine and 20 for resveratrol + 25 mg piperine. The assessment of resveratrol glucuronide revealed a Cmax of 343±295 ng/mL for resveratrol alone, 388±345 for resveratrol + 5 mg piperine and 370±439 for resveratrol + 25 mg piperine. Exposure as measured by AUC was 31,671 ng/h/mL for resveratrol alone, 49,711 for resveratrol + 5 mg piperine and 34,477 for resveratrol + 25 mg piperine. Half-life values for this metabolite were 7.4 min for resveratrol alone, 13.6 for resveratrol + 5 mg piperine and 10 for resveratrol + 25 mg piperine. No relationship between piperine dose and resveratrol or resveratrol glucuronide pharmacokinetic values were found with Jonkheere-Terpstra test for the study data as a whole. However, for females, resveratrol Cmax showed a trend (p = 0.057) according to Jonckheere-Terpstra test with piperine dose. For the cohort of males, there was no evidence of a similar relationship with piperine dose.
Table 2.
Pharmacokinetic parameters of Resveratrol and Resveratrol-Glucuronide.
| Table 2A. All subjects | ||||||||
|---|---|---|---|---|---|---|---|---|
| Characteristic | Resveratrol 2.5 gm (n=8) |
Resveratrol 2.5gm + Piperine 5mg (n=8) |
Resveratrol 2.5gm + Piperine 25mg (n=8) |
All Combined (n=24) |
||||
| Resveratrol | ||||||||
| Cmax, ng/mL | 220 | (83.6–1120) | 289 | (124–653) | 321 | (153–693) | 279 | (83.6–1120) |
| AUC0−∞, ng/mL | 1010 | (524–3000) | 1370 | (500–7540) | 1060 | (934–1540) | 1160 | (500–7540) |
| Tmax, h | 1.00 | (0.500–2.00) | 1.00 | (0.750–2.00) | 1.00 | (0.500–2.00) | 1.00 | (0.500–2.00) |
| T1/2, h | 6.06 | (3.87–28.7) | 6.52 | (2.18–45.1) | 5.59 | (2.72–17.1) | 5.91 | (2.18–45.1) |
| CL, L/h | 2.61 | (0.832–4.77) | 1.83 | (0.332–5.00) | 2.36 | (1.62–2.68) | 2.16 | (0.332–5.00) |
| Resveratrol-Glucuronide | ||||||||
| Cmax, ng/mL | 5020 | (2440–0300) | 9440 | (3260–8100) | 5870 | (1440–1700) | 6530 | (1440–20300) |
| AUC0−∞, ng/mL | 30100 | (11900–69800) | 50500 | (13200–85300) | 30900 | (11000–94500) | 35700 | (11000–94500) |
| Tmax, h | 2.00 | (1.00–4.00) | 1.50 | (1.00–2.00) | 1.50 | (1.00–4.00) | 1.50 | (1.00–4.00) |
| T1/2, h | 6.12 | (2.46–18.0) | 6.80 | (3.42–45.3) | 5.31 | (3.70–31.5) | 5.62 | (2.46–45.3) |
| CL, L/h | 0.0850 | (0.0360–0.211) | 0.0495 | (0.0290–0.190) | 0.0820 | (0.0270–0.228) | 0.0700 | (0.0270–0.228) |
| Table 2B. Female subjects | ||||||||
| Characteristic | Resveratrol 2.5 gm (n=4) |
Resveratrol 2.5gm + Piperine 5mg (n=4) |
Resveratrol 2.5gm + Piperine 25mg (n=4) |
All Combined (n=12) |
||||
| Resveratrol | ||||||||
| Cmax, ng/mL | 128 | (83.6–236) | 273 | (124–653) | 321 | (165–546) | 211 | (83.6–653) |
| AUC0−∞, ng h/mL | 705 | (524–1930) | 1590 | (1330–2360) | 1100 | (961–1540) | 1250 | (524–2360) |
| Tmax, h | 0.875 | (0.750–2.00) | 1.00 | (0.750–2.00) | 1.50 | (0.750–2.00) | 1.00 | (0.750–2.00) |
| T1/2, h | 4.88 | (3.87–11.5) | 9.56 | (2.18–26.8) | 5.55 | (2.72–17.1) | 6.70 | (2.18–26.8) |
| CL, L/h | 3.58 | (1.30–4.77) | 1.59 | (1.06–1.88) | 2.28 | (1.62–2.60) | 2.01 | (1.06–4.77) |
| Resveratrol-Glucuronide | ||||||||
| Cmax, ng/mL* | 3830 | (2440–5240) | 10500 | (8560–18100) | 6170 | (2030–11200) | 6740 | (2030–18100) |
| AUC0−∞, ng h/mL | 24400 | (11900–37700) | 50500 | (36800–71500) | 38200 | (11000–94500) | 37200 | (11000–94500) |
| Tmax, h | 2.00 | (2.00–4.00) | 1.75 | (1.50–2.00) | 1.50 | (1.00–4.00) | 2.00 | (1.00–4.00) |
| T1/2, h | 7.70 | (2.46–18.0) | 4.81 | (3.42–14.4) | 5.67 | (3.70–31.5) | 5.67 | (2.46–31.5) |
| CL, L/h | 0.103 | (0.0660–0.211) | 0.0495 | (0.0350–0.0680) | 0.0655 | (0.0270–0.228) | 0.0670 | (0.0270–0.228) |
| Table 2C. Male subjects | ||||||||
| Characteristic | Resveratrol 2.5 gm (n=4) |
Resveratrol 2.5gm + Piperine 5mg (n=4) |
Resveratrol 2.5gm + Piperine 25mg (n=4) |
All Combined (n=12) |
||||
| Resveratrol | ||||||||
| Cmax, ng/mL | 362 | (205–1120) | 289 | (140–316) | 318 | (153–693) | 304 | (140–1120) |
| AUC0−∞, ng h/mL | 1370 | (640–3000) | 880 | (500–7540) | 1020 | (934–1390) | 1110 | (500–7540) |
| Tmax, h | 1 | (0.500–2.00) | 1 | (0.750–1.00) | 0.875 | (0.500–1.00) | 1 | (0.500–2.00) |
| T1/2, h | 7.2 | (5.88–28.7) | 4.66 | (3.92–45.1) | 5.59 | (3.16–6.54) | 5.91 | (3.16–45.1) |
| CL, L/h | 1.84 | (0.832–3.91) | 3.13 | (0.332–5.00) | 2.47 | (1.80–2.68) | 2.25 | (0.332–5.00) |
| Resveratrol-Glucuronide | ||||||||
| Cmax, ng/mL | 9100 | (3420–20300) | 5280 | (3260–14400) | 5320 | (1440–11700) | 6530 | (1440–20300) |
| AUC0−∞, ng h/mL | 36300 | (12600–69800) | 45000 | (13200–85300) | 22800 | (13900–34600) | 34600 | (12600–85300) |
| Tmax, h | 1.25 | (1.00–2.00) | 1.25 | (1.00–2.00) | 1.5 | (1.00–1.50) | 1.5 | (1.00–2.00) |
| T1/2, h | 5.58 | (5.15–7.01) | 15.9 | (3.52–45.3) | 5.26 | (5.17–16.6) | 5.62 | (3.52–45.3) |
| CL, L/h | 0.069 | (0.0360–0.199) | 0.058 | (0.0290–0.190) | 0.114 | (0.0720–0.180) | 0.072 | (0.0290–0.199) |
Presented as median (range)
P-Value (resveratrol 2.5 gm vs resveratrol + piperine 5 mg) < 0.05
P-Value (resveratrol 2.5 gm + piperine 5 mg vs resveratrol + piperine 25 mg) < 0.05
DISCUSSION
The present study aimed to provide an initial clinical evaluation of the bioenhancing effects of piperine on resveratrol pharmacokinetics in humans. Despite our observation of piperine coadministration significantly increasing Cmax and AUC of resveratrol in a murine model, we did not observe a significant increase in resveratrol or decrease in resveratrol glucuronide Cmax or AUC in healthy adults taking 2.5 gm of resveratrol with piperine (5 or 25 mg) as compared to resveratrol alone.
In our earlier study in a murine model, 48 C57BL mice (24 in each arm) were given a single dose (by oral gavage) of 100 mg/kg of trans-resveratrol with or without 10 mg/kg of piperine while in the fasting state, with frequent blood sampling for determination of serum concentrations of resveratrol and resveratrol-3-O-β-D-glucuronide (Johnson et al., 2011). Primarily through a profound increase in peak concentration of resveratrol (more than 15-fold increase in Cmax), piperine co-administration significantly enhanced resveratrol AUC (229% increase). While mice receiving resveratrol + piperine also had an increase in resveratrol-3-O-β-D-glucuronide peak concentrations, the metabolite’s AUC was decreased by 81% compared to resveratrol alone. These data implied that piperine could increase the AUC of resveratrol through primarily inhibiting glucuronidation and potentially via effects on enhanced absorption as well (Alcala, Lieska and Maisel, 1975). Similarly, a recent study of oxyresveratrol (a compound with similar chemical structure to resveratrol) combined with piperine in a rat model showed combination with piperine could increase the oral bioavailability of oxyresveratrol approximately 2-fold, further supporting the hypothesis that piperine reduces the glucuronide reaction of oxyresveratrol. (Junsaeng et al., 2019). In addition, preclinical and clinical data with piperine co-administration with other agents (e.g. curcumin) also support this premise (Atal, Dubey and Singh, 1985; Reen et al., 1993; Shoba et al., 1998; Lambert et al., 2004; Srinivasan, 2007).
Why our results are different than our own animal data and the data of others is uncertain. The usual interpatient variability in resveratrol pharmacokinetics was evident in our data which would limit our sensitivity to detect a modest effect by piperine. But our animal data observed a large effect, so we clearly are not duplicating this effect in humans, but we acknowledge the possibility of not detecting a small piperine effect (e.g ≈ 20% increase). Note that the study was designed to detect an 80% increase with power 0.85 at a two-sided 0.05 level test. Examination of the data by gender did show a trend toward improved Cmax, AUC and decreased clearance of resveratrol in women supporting the potential of a modest enhancement of resveratrol pharmacokinetics whether gender specific or not. If a piperine effect truly exists and is not detected, another possibility would be incorrect study dosage of piperine. Since we used the same supplier of piperine for our animal studies and formulated our own capsules for the human study, we are assuming our delivered piperine human doses were accurate.
To the best of our knowledge, the favorable effects of piperine co-administration on resveratrol bioavailability observed in in vitro and murine models have not yet been demonstrated in any clinical trials in humans. In the study by Wightman et al., 20mg of piperine was co-administered with 250mg of resveratrol to observe the bioefficacy of resveratrol on cerebral blood flow and cognitive function. In their bioavailability analysis, the authors found no significant difference in any of the average concentrations of resveratrol metabolites compared to resveratrol alone, despite observing an improvement in bioefficacy (Wightman et al., 2014). A variety of other methods to improve resveratrol bioavailabilty have been proposed (de Vries, Strydom and Steenkamp, 2018), but none of them have been validated in clinical trials.
During the course of this study, a previously designed protocol for analyzing serum resveratrol and its metabolites concentrations in mice has been altered to minimize errors arising from larger volumes and number of samples, and higher light exposure. We first adapted a method for SPE preparation of resveratrol and its glucuronide using a commercially available stationary phase that specifically targets removal of phospholipids. With this modified protocol, we achieved inter-day variation 5% for resveratrol and 6% for resveratrol glucuronide, compared to 15% and higher using the previously described method (Johnson et al., 2011).
In conclusion, we performed an initial clinical evaluation of the bioenhancing effects of piperine on resveratrol pharmacokinetics in humans. Our data shows co-administration of piperine with 2500mg of resveratrol was well tolerated with no signs of significant adverse events. Piperine at doses 5mg and 25mg did not significantly alter the pharmacokinetics of resveratrol and its main glucorunidated species for us to detect an increase in Cmax of 80% or higher. The observed improvement of bioavailabilty of resveratrol with piperine in murine models was therefore not demonstrated in our patient cohort under the experimental conditions used. Further detailed studies at additional dosage as well as timing of blood draw post-administration may be useful. Similarly, instead of single dose, chronic administration of these agents at low dosage could provide additional information regarding the potential usefulness of resveratrol piperine combination.
Supplementary Material
Source of funding:
This project was supported in part by the National Institutes of Health grant R21 CA149560 to Dr. Ahmad and by the National Cancer Institute Cancer Center Support Grant P30 CA014520.
Footnotes
Statement of conflicts of interests: None declared
REFERENCES
- Alcala J, Lieska N and Maisel H (1975) ‘Protein composition of bovine lens cortical fiber cell membranes’, Exp. Eye Res, 21(6), pp. 581–595. doi: 10.1016/0014-4835(75)90040-8. [DOI] [PubMed] [Google Scholar]
- Almeida L et al. (2009) ‘Pharmacokinetic and safety profile of trans-resveratrol in a rising multiple-dose study in healthy volunteers’, Mol. Nutr. Food Res, 53(SUPPL. 1). doi: 10.1002/mnfr.200800177. [DOI] [PubMed] [Google Scholar]
- De Amicis F et al. (2011) ‘ Resveratrol, through NF-Y/p53/Sin3/HDAC1 complex phosphorylation, inhibits estrogen receptor α gene expression via p38 MAPK /CK2 signaling in human breast cancer cells ‘, FASEB J. doi: 10.1096/fj.10-178871. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Asensi M et al. (2002) ‘Inhibition of cancer growth by resveratrol is related to its low bioavailability’, Free Radic. Biol. Med doi: 10.1016/S0891-5849(02)00911-5. [DOI] [PubMed] [Google Scholar]
- Atal CK, Dubey RK and Singh J (1985) ‘Biochemical basis of enhanced drug bioavailability by piperine: Evidence that piperine is a potent inhibitor of drug metabolism’, J. Pharmacol. Exp. Ther, 232(1), pp. 258–262. [PubMed] [Google Scholar]
- Aziz MH et al. (2005) ‘Chemoprevention of skin cancer by grape constituent resveratrol: Relevance to human disease?’, FASEB J, 19(9), pp. 1193–1195. doi: 10.1096/fj.04-3582fje. [DOI] [PubMed] [Google Scholar]
- Baur JA and Sinclair DA (2006) ‘Therapeutic potential of resveratrol: The in vivo evidence’, Nat. Rev. Drug Discov, pp. 493–506. doi: 10.1038/nrd2060. [DOI] [PubMed] [Google Scholar]
- Bhat KPL and Pezzuto JM (2002) ‘Cancer chemopreventive activity of resveratrol’, in Ann. N. Y. Acad. Sci, pp. 210–229. doi: 10.1111/j.1749-6632.2002.tb02918.x. [DOI] [PubMed] [Google Scholar]
- Bhullar KS and Udenigwe CC (2016) ‘Clinical evidence of resveratrol bioactivity in cardiovascular disease’, Curr. Opin. Food Sci doi: 10.1016/j.cofs.2016.03.005. [DOI] [Google Scholar]
- Burkon A and Somoza V (2008) ‘Quantification of free and protein-bound trans-resveratrol metabolites and identification of trans-resveratrol-C/O-conjugated diglucuronides - Two novel resveratrol metabolites in human plasma’, Mol. Nutr. Food Res, 52(5), pp. 549–557. doi: 10.1002/mnfr.200700290. [DOI] [PubMed] [Google Scholar]
- Chen CJ et al. (2009) ‘Resveratrol protects cardiomyocytes from hypoxia-induced apoptosis through the SIRT1-FoxO1 pathway’, Biochem. Biophys. Res. Commun doi: 10.1016/j.bbrc.2008.11.110. [DOI] [PubMed] [Google Scholar]
- Crescente M et al. (2009) ‘Interactions of gallic acid, resveratrol, quercetin and aspirin at the platelet cyclooxygenase-1 level: Functional and modelling studies’, Thromb. Haemost doi: 10.1160/TH09-01-0057. [DOI] [PubMed] [Google Scholar]
- Feng M et al. (2016) ‘Resveratrol treatment inhibits proliferation of and induces apoptosis in human colon cancer cells’, Med. Sci. Monit doi: 10.12659/MSM.897905. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heebøll S et al. (2016) ‘Placebo-controlled, randomised clinical trial: High-dose resveratrol treatment for non-alcoholic fatty liver disease’, Scand. J. Gastroenterol doi: 10.3109/00365521.2015.1107620. [DOI] [PubMed] [Google Scholar]
- Johnson JJ et al. (2011) ‘Enhancing the bioavailability of resveratrol by combining it with piperine’, Mol. Nutr. Food Res, 55(8), pp. 1169–1176. doi: 10.1002/mnfr.201100117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson JJ, Bailey HH and Mukhtar H (2010) ‘Green tea polyphenols for prostate cancer chemoprevention: A translational perspective’, Phytomedicine, 17(1), pp. 3–13. doi: 10.1016/j.phymed.2009.09.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Junsaeng D et al. (2019) ‘Comparative pharmacokinetics of oxyresveratrol alone and in combination with piperine as a bioenhancer in rats’, BMC Complement. Altern. Med doi: 10.1186/s12906-019-2653-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kang L et al. (2010) ‘Resveratrol modulates adipokine expression and improves insulin sensitivity in adipocytes: Relative to inhibition of inflammatory responses’, Biochimie. doi: 10.1016/j.biochi.2010.02.024. [DOI] [PubMed] [Google Scholar]
- Kinoshita Y et al. (2006) ‘Expression of prostate-specific membrane antigen in normal and malignant human tissues’, World J. Surg, 30(4), pp. 628–636. doi: 10.1007/s00268-005-0544-5. [DOI] [PubMed] [Google Scholar]
- Lambert JD et al. (2004) ‘Piperine Enhances the Bioavailability of the Tea Polyphenol (−)-Epigallocatechin-3-gallate in Mice’, J. Nutr, 134(8), pp. 1948–1952. doi: 10.1093/jn/134.8.1948. [DOI] [PubMed] [Google Scholar]
- De Leo A et al. (2011) ‘Resveratrol inhibits proliferation and survival of Epstein Barr virus-infected Burkitt’s lymphoma cells depending on viral latency program’, Mol. Cancer Res doi: 10.1158/1541-7786.MCR-11-0145. [DOI] [PubMed] [Google Scholar]
- Mukherjee S, Dudley JI and Das DK (2010) ‘Dose-dependency of resveratrol in providing health benefits’, Dose-Response, pp. 478–500. doi: 10.2203/dose-response.09-015.Mukherjee. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ndiaye M, Kumar R and Ahmad N (2011) ‘Resveratrol in cancer management: Where are we and where we go from here?’, Ann. N. Y. Acad. Sci, 1215(1), pp. 144–149. doi: 10.1111/j.1749-6632.2010.05851.x. [DOI] [PubMed] [Google Scholar]
- Osmond GW et al. (2012) ‘Enhancing melanoma treatment with resveratrol’, J. Surg. Res doi: 10.1016/j.jss.2010.07.033. [DOI] [PubMed] [Google Scholar]
- Palsamy P and Subramanian S (2010) ‘Ameliorative potential of resveratrol on proinflammatory cytokines, hyperglycemia mediated oxidative stress, and pancreatic β-cell dysfunction in streptozotocin-nicotinamide-induced diabetic rats’, J. Cell. Physiol doi: 10.1002/jcp.22138. [DOI] [PubMed] [Google Scholar]
- Patel KR et al. (2011) ‘Clinical trials of resveratrol’, Ann. N. Y. Acad. Sci, 1215(1), pp. 161–169. doi: 10.1111/j.1749-6632.2010.05853.x. [DOI] [PubMed] [Google Scholar]
- Reen RK et al. (1993) ‘Impairment of UDP-glucose dehydrogenase and glucuronidation activities in liver and small intestine of rat and guinea pig in vitro by piperine’, Biochem. Pharmacol, 46(2), pp. 229–238. doi: 10.1016/0006-2952(93)90408-O. [DOI] [PubMed] [Google Scholar]
- Shoba G et al. (1998) ‘Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers’, Planta Med, 64(4), pp. 353–356. doi: 10.1055/s-2006-957450. [DOI] [PubMed] [Google Scholar]
- Srinivasan K (2007) ‘Black pepper and its pungent principle-piperine: A review of diverse physiological effects’, Crit. Rev. Food Sci. Nutr, pp. 735–748. doi: 10.1080/10408390601062054. [DOI] [PubMed] [Google Scholar]
- Subramanian L et al. (2010) ‘Resveratrol: Challenges in translation to the clinic - A critical discussion’, Clin. Cancer Res, pp. 5942–5948. doi: 10.1158/1078-0432.CCR-10-1486. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thazhath SS et al. (2016) ‘Administration of resveratrol for 5 wk has no effect on glucagon-like peptide 1 secretion, gastric emptying, or glycemic control in type 2 diabetes: A randomized controlled trial’, Am. J. Clin. Nutr doi: 10.3945/ajcn.115.117440. [DOI] [PubMed] [Google Scholar]
- Tomayko EJ et al. (2014) ‘Resveratrol Supplementation Reduces Aortic Atherosclerosis and Calcification and Attenuates Loss of Aerobic Capacity in a Mouse Model of Uremia’, J. Med. Food doi: 10.1089/jmf.2012.0219. [DOI] [PubMed] [Google Scholar]
- Turner RS et al. (2015) ‘A randomized, double-blind, placebo-controlled trial of resveratrol for Alzheimer disease’, Neurology. doi: 10.1212/WNL.0000000000002035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vaz-da-Silva M et al. (2008) ‘Effect of food on the pharmacokinetic profile of trans-resveratrol’, Int. J. Clin. Pharmacol. Ther, 46(11), pp. 564–570. doi: 10.5414/CPP46564. [DOI] [PubMed] [Google Scholar]
- de Vries K, Strydom M and Steenkamp V (2018) ‘Bioavailability of resveratrol: Possibilities for enhancement’, J. Herb. Med doi: 10.1016/j.hermed.2017.09.002. [DOI] [Google Scholar]
- Wang D, Xu Y and Liu W (2008) ‘Tissue distribution and excretion of resveratrol in rat after oral administration of Polygonum cuspidatum extract (PCE)’, Phytomedicine. doi: 10.1016/j.phymed.2008.02.009. [DOI] [PubMed] [Google Scholar]
- Wenzel E and Somoza V (2005) ‘Metabolism and bioavailability of trans-resveratrol’, Mol. Nutr. Food Res doi: 10.1002/mnfr.200500010. [DOI] [PubMed] [Google Scholar]
- Wightman EL et al. (2014) ‘Effects of resveratrol alone or in combination with piperine on cerebral blood flow parameters and cognitive performance in human subjects: A randomised, double-blind, placebo-controlled, cross-over investigation’, Br. J. Nutr doi: 10.1017/S0007114514000737. [DOI] [PubMed] [Google Scholar]
- Yiu EM et al. (2015) ‘An open-label trial in Friedreich ataxia suggests clinical benefit with high-dose resveratrol, without effect on frataxin levels’, J. Neurol doi: 10.1007/s00415-015-7719-2. [DOI] [PubMed] [Google Scholar]
- Zhang F et al. (2013) ‘Resveratrol protects cortical neurons against microglia-mediated neuroinflammation’, Phyther. Res doi: 10.1002/ptr.4734. [DOI] [PubMed] [Google Scholar]
- Zhu W et al. (2012) ‘Trans -resveratrol alters mammary promoter hypermethylation in women at increased risk for breast cancer’, Nutr. Cancer doi: 10.1080/01635581.2012.654926. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zortea K et al. (2016) ‘Resveratrol Supplementation in Schizophrenia Patients: A Randomized Clinical Trial Evaluating Serum Glucose and Cardiovascular Risk Factors’, Nutrients, 8(2), p. 73. doi: 10.3390/nu8020073. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.