2018 Volume 41 Issue 2 Pages 281-284
A beverage made of red wine vinegar and grape juice (Yamanashi-no-megumi™) was developed as a supplemental fluid containing polyphenols, which has been clinically shown to enhance the colonic transit. In this study, we assessed the mechanism of its prokinetic action by analyzing the effects on both the colonic phosphodiesterase activity of rats (n=4) and the isolated colonic strip preparation of guinea pigs (n=4). The 7% (v/v) solution of the beverage significantly decreased the phosphodiesterase activity by 9% (n=4). The beverage in concentrations of 0.7, 2.1 and 7% (v/v) relaxed the colonic strips pre-contracted by 1 µmol/L of carbachol in a concentration-related manner with 50, 58 and 79%, each response of which was diminished to 11, 19 and 46%, respectively in the presence of 100 µmol/L of L-nitro-arginine methyl ester. These results obtained by biochemical, functional and pharmacological analyses suggest that the beverage could relax the colon through both cAMP-associated and nitric oxide-dependent pathways, which may partly explain clinically observed prokinetic effect of the beverage.
More than 200000 people have taken the beverage made of red wine vinegar and grape juice (Yamanashi-no-megumi™) after its launch onto the commercial market in 1999. The excellent cardiovascular effects, including the anti-hypertensive action observed during non-clinical studies,1–3) have been confirmed in the responses from the consumers and in the results from a limited number of patients with mild hypertension (n=15).4) We have also noticed that the beverage enhanced the colonic transit in patients with constipation,4) which may be another potential efficacy of this beverage. However, there has been limited information on how the beverage may modify the function of the gastrointestinal motility.5)
In the gastrointestinal tract motor function, cAMP-dependent pathways have been reported to play a pivotal role in the physiological as well as pathophysiological regulations.6) For example, phosphodiesterase inhibitors like 3-isobutyl-1-methylxanthine (IBMX) increase the cellular cAMP level, thereby relaxing the gastrointestinal smooth muscles7) and inhibiting their pacemaker activity of slow waves.8) We demonstrated that the extent of the inhibition of phosphodiesterase activity in the colonic tissue paralleled that of smooth muscle relaxation, which can be used as an excellent surrogate marker for predicting the efficacy of drugs in patients with constipation.9)
In order to clarify the mechanism of prokinetic action of the beverage, we initially analyzed its effects on the activity of phosphodiesterase hydrolyzing cAMP in the membrane preparations obtained from colon of rats. We used IBMX as a reference compound for biochemical assay.9) Next, we examined the relaxant effects of the beverage on carbachol-induced contraction of the isolated colonic strip preparation of guinea pigs. Furthermore, L-nitro-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, was used to confirm whether NO-dependent pathway may be involved in the beverage-induced relaxation of colonic strip, since the beverage was shown to activate the NO synthase activity in the vessels.2)
All experiments were approved by the Committee for Research at Yamanashi Research Center of Clinical Pharmacology (No. 2008-04) and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Yamanashi Research Center of Clinical Pharmacology.
Drugs, Test Substances and EnzymesCommercially produced and widely available, red wine vinegar beverage (Yamanashi-no-megumi™; which was called Budo-no-megumi™ before) was generously gifted from Asaya Foods Co., Ltd. (Yamanashi, Japan). The beverage consists of 25% (v/v) of red wine vinegar containing 4.5% acetic acid, 5% (w/v) of originally manufactured 100% grape juice, and appropriate amount of honey, oligosaccharides, vitamin C, citrate, glucose and calcium lactate. The beverage has been shown to contain >1.5 mg/mL of polyphenols as gallic acid units.1) All other enzymes, substrates and chemicals were obtained from Sigma-Aldrich Co. LLC. (St. Louis, MO, U.S.A.).
Experiment 1: Effects of the Beverage on Colonic Phosphodiesterase ActivityProduction of Membrane PreparationThe female Sprague-Dawley rats (Charles River, Yokohama, Japan) weighing 200–300 g (n=4) were anesthetized with thiopental sodium (40 mg/kg, intraperitoneally (i.p.)). Immediately after euthanization, portion of colon was stripped. After removing the fatty tissue from this segment, it was placed in ice-cold SET buffer (0.25 mol/L sucrose; 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA); and 5 mmol/L Tris-acetate, pH 7.4) and homogenized. The homogenates were filtered (Nitex filter; Tetko, Los Angeles, CA, U.S.A.) and centrifuged at 10000×g for 5 min at 4°C. The pellets were resuspended in SET buffer and the mixtures were centrifuged under the same setting. After this procedure was repeated twice more, the pellets were finally suspended in SET buffer. Protein concentration was determined using a protein assay reagent (Pierce, Rockford, IL, U.S.A.). The membrane suspensions were diluted with SET buffer to a concentration of 3–5 mg protein/mL and they were stored at –80°C until their phosphodiesterase activities were measured.9)
Assessment of Colonic Phosphodiesterase ActivityThe assay consists of two parts; hydrolysis of externally added cAMP by phosphodiesterase in the membrane preparation, and measurement of residual cAMP concentration.9) Fifty microliters of reaction mix (100 mmol/L Tris-acetate, pH 7.4; 10 mmol/L MgCl2; 0.4 mg/mL bovine serum albumin; and 20 µmol/L cAMP) was added to each microcentrifuge tube in duplicate with or without either of IBMX (2, 20 and 200 µmol/L) or the beverage (1.4 and 14%). Next, the membrane suspension in a volume of 50 µL was added to each tube on ice. The enzyme reaction was initiated by placing the tubes in a water bath maintained at 37°C. After 20 min, the reaction was terminated by heating at 95°C for 5 min. The mixture was vortexed 3 times and centrifuged at 10000×g for 5 min. A volume of 2 µL of the supernatant was transferred to a 10×75 mm assay tube (Iwaki Lab Ware, Tokyo, Japan) in triplicate. For cAMP standard, 2 µL of a known amount of cAMP solutions were added to the tubes. The cAMP concentrations were assayed with enzymatic fluorometric technique.9)
Experiment 2: Effect of the Beverage on Isolated Colonic ContractionPreparation of Colonic StripsThe male Hartley guinea pigs (Japan SLC, Inc., Hamamatsu, Japan) weighing 300–450 g (n=4) were anesthetized with thiopental sodium (40 mg/kg, i.p.). Immediately after euthanization, the colon was isolated and cut out in length of 10–15 mm, which was suspended in a 10 mL of an organ bath filled with oxygenated (95% O2 and 5% CO2) Tyrode’s solution (136.8 mmol/L NaCl, 2.7 mmol/L KCl, 1.8 mmol/L CaCl2, 1.1 mmol/L MgCl2, 0.4 mmol/L NaH2PO4, 11.9 mmol/L NaHCO3, 5.6 mmol/L glucose) at 31°C. One end of colonic strip was attached to a stainless steel tissue holder and the other to an isotonic transducer (TD-112S; Nihon Kohden, Tokyo, Japan) in the Magnus system (MYOBATH-4. World Precision Instruments, Inc., Sarasota, FL, U.S.A.). One gram of basal tension was applied to each strip that was equilibrated for >30 min.9)
Assessment of Relaxant EffectThe colonic strips were divided into the following 3 groups; beverage-treated group without L-NAME (n=4), beverage-treated group with 100 µmol/L of L-NAME (n=4) and vehicle-treated group (n=4). The colonic strips were contracted with 1 µmol/L of carbachol. After confirming the reproducibility of the responses of the colonic strips, they were contracted with 1 µmol/L of carbachol again. After the contraction reached a steady state level, which was usually observed about within 10 min after the administration of carbachol, the following treatment was performed. In both beverage-treated groups, 7 times concentrated beverages in a volume of 10, 20 and 70 µL were sequentially added into an organ bath filled with 10 mL of Tyrode’s solution at 31°C to obtain final concentrations of 0.7, 2.1 and 7% (v/v), respectively, in a cumulative manner every 3–5 min. Concentrations of the beverage were selected based on our previous study.2) In vehicle-treated group, distilled water in a volume of 10, 20 and 70 µL was sequentially administered in the same manner. In preliminary experiments, the pH of Tyrode’s solution was not changed by addition of either concentration of the beverage.
Data Analysis and StatisticsThe data are presented as mean±standard error of the mean (S.E.M.) Relaxation was expressed as percent reduction from the carbachol-induced contraction. Statistical comparisons of mean values among the groups were assessed by one-way factorial ANOVA followed by Fisher’s protected least significant difference (PLSD) for multiple comparison test. A p-value of <0.05 was considered to be significant.
The effects of the beverage on phosphodiesterase activity of the membrane preparations are summarized in Fig. 1 (n=4). The basal phosphodiesterase activity was 195±10 (pmol/min/mg protein). The 7% solution of the beverage significantly suppressed the phosphodiesterase activity by 9%, whereas no significant change was detected by the 0.7% solution. Similarly, IBMX inhibited the phosphodiesterase activity in a concentration-related manner.
Note that the extent of inhibition by 7% of the beverage was close to that by 10 µmol/L of IBMX. Data are presented as mean±S.E.M. (n=4). ** p<0.01 between the basal activity (Basal) and those of pharmacologically treated membrane preparations.
Application of L-NAME to the organ bath did not alter the basal length of the colonic tissue. Typical trace showing the effect of 0.7% of the beverage on carbachol-induced contraction of the colonic strip in the absence of L-NAME is depicted in Fig. 2A, and the results of the beverage-treated group without L-NAME (n=4), beverage-treated group with 100 µmol/L of L-NAME (n=4) and vehicle-treated group (n=4) are summarized in Fig. 2B. The extent of the carbachol-evoked changes in the length of the colonic tissue before the beverage or vehicle treatment was 10±3 mm in the beverage-treated group without L-NAME, 10±1 mm in the beverage-treated group with L-NAME and 13±3 mm in the vehicle-treated group, among which no significant difference was detected. The beverage significantly relaxed the colonic strip in a concentration-related manner with and without L-NAME. Significant difference in the extent of relaxation was detected at 0.7, 2.1 and 7% between the beverage-treated group without L-NAME and vehicle-treated group; only at 7% between beverage-treated group with L-NAME and vehicle-treated group; and at 0.7 and 2.1% between the beverage-treated groups with and without L-NAME.
(A) Typical tracing showing the effect of 0.7% of the beverage on 1 µmol/L of carbachol-induced contraction of the colon strips. (B) Summary of the effects of the beverage with (triangles) and without (circles) 100 µmol/L of L-nitro-arginine methyl ester (L-NAME) on 1 µmol/L of carbachol-induced contraction of the colonic strips in comparison with that of distilled water (squares) (n=4 for each treatment). Note that 7% of the beverage relaxed the strip by 79% in the absence of L-NAME. Data are presented as mean±S.E.M. * p<0.05, ** p<0.01.
Although drugs that can selectively stimulate gut motor function have significant potential clinical usefulness, only a limited number of agents are available for clinical use.10) Thus, red wine vinegar beverage, which has been shown to enhance the colonic transit, may become a promising candidate for treating patients with constipation. Since information on how the beverage exerted prokinetic action in patients is lacking, we assessed its effects on phosphodiesterase activity of the colonic membrane preparation and smooth muscle tone of the colonic strip preparation.
We observed that 0.7, 2.1 and 7% solutions of the beverage decreased the smooth muscle tone in a concentration-related manner with 50, 58 and 79%, each response of which was diminished to 11, 19 and 46%, respectively by 100 µmol/L of L-NAME, suggesting that the beverage relaxed the colon through both NO-dependent and independent pathways. We also found that 7% solution of the beverage decreased phosphodiesterase activity, extent of which was similar to that of 10 µmol/L of IBMX as shown in Fig. 1. In a previous study using the isolated colonic tissue,9) we have confirmed that 10 µmol/L of IBMX effectively relaxed the colonic strip pre-contracted with carbachol by 23%, which was >3 times smaller than that was observed by 7% solution of the beverage in this study. These biochemical, functional and pharmacological analyses suggest that the beverage could relax the colon at least through cAMP associated and NO-dependent pathways.
Of high interest would be the correlation between the concentrations of the beverage which exerted biochemical and pharmacological effects and those in plasma attained after oral administration of the recommended dose of the beverage (3 mL/kg) in humans. Previous studies2,3) have suggested that 0.25 to 2% of plasma concentrations can be attained after oral administration of 3 mL/kg when the bioavailability of the beverage is 3 to 25%. Based on this information along with the prokinetic responses confirmed in patients,1) one can speculate that absorbed components of the beverage into the systemic circulation together with those directly reaching the intestinal tract may provide enough tissue concentration to activate the colonic motility in vivo.
In conclusion, the beverage can inhibit colonic phosphodiesterase activity, and relax the colon through both cAMP associated and NO-dependent pathways, which may be partly associated with its clinically observed prokinetic effect. Further study is required to estimate which components in the beverage are essential to the current results, and how the beverage influences water and electrolytes transport across the gut as well as the pacemaker activity. Also, long-term effects of the beverage need to be confirmed in patients to fully understand the causal link between the present in vitro observations and its clinical utility.
This study was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant number JP16K08559. The authors thank Ms. Misako Nakatani and Mrs. Yuri Ichikawa for their technical assistance.
A.S. was a member of developer team of Yamanashi-no-megumi™. The other authors indicated no potential conflict of interest.
