Enhancement or Suppression of ACE Inhibitory Activity by a Mixture of Tea and Foods for Specified Health Uses (FOSHU) That Are Marketed as “Support for Normal Blood Pressure”

The ACE inhibitory activities of mixtures of FOSHUs (Healthya, Goma-Mugicha, Lapis Support and Ameal) were examined in order to identify any antihypertensive interactions. Among combinations of Healthya with other samples that contain active peptides, only that with Ameal was found to have no inhibitory activity. Enhanced activity was observed in 2 other mixtures. The activity of a mixture of tea polyphenols and the whey component extracted from an Ameal solution was significantly decreased, thus demonstrating that whey protein lowered the ACE inhibitory activity of Healthya. Although oral administration of tea polyphenols alone significantly decreased SBP in SHR at 2 and 4 hr, combined administration with Ameal failed to decrease SBP at the same time points. In conclusion, the simultaneous intake of tea and FOSHUs that contain active peptides might affect daily self-antihypertensive management via enhancement or suppression of ACE inhibitory activity.

With rising health consciousness among consumers, particular attention has recently focused on green tea's action to counter obesity and hypertension, which are diagnostic criteria for metabolic syndrome [10]. Tea beverages that contain high levels of tea polyphenols and that are labeled "For individuals worried about body fat" are being sold in Japan as foods for specified health uses (FOSHUs).
Long-term consumption of tea has been reported to result in antihypertensive activity in spontaneously hypertensive rats (SHRs) [7,8] while another study reported antihypertensive activity with a single administration of tea [9]. The antihypertensive activity of tea polyphenols is thought to occur via the inhibition of angiotensin-converting enzyme (ACE) activity [11]. Moreover, other FOSHUs, many of which have active components consisting of peptides that inhibit ACE activity, are marketed to target "individuals with high blood pressure" [12][13][14].
Indeed, tea beverages and FOSHUs can be readily obtained, so there may be instances in which they are consumed together. However, for individuals with high blood pressure, little is known about the antihypertensive interaction between tea and FOSHUs, which are both thought to have the same mechanisms of action.
Thus, the aim of the current study was to examine the antihypertensive interaction when a tea beverage and a FOSHU "For individuals with high blood pressure" (Goma-Mugicha, Lapis Support, and Ameal) were consumed together.

Materials. The green tea beverage used was Healthya
Green Tea (Kao Corporation, Tokyo). The 3 FOSHU products were Ameal (Calpis Co., Tokyo), which is claimed to "help maintain a normal blood pressure level" and contains Val-Pro-Pro as its active ingredient [12], Lapis Support (Tokiwa Yakuhin Co., Ltd., Yamaguchi, Japan), which has Val-Tys as its active ingredient [13], and Goma-Mugicha (Suntory Foods Ltd., Tokyo), which has Leu-Val-Tys as its active ingredient [14]. Whey protein concentrate (WPC: Protein, Easy Sports Co., Ltd., Tokyo) was also used. Polyphenon CG (green tea extract, tea polyphenol content of at least 30%) was purchased from Mitsui Norin Co., Ltd. (Tokyo). Rabbit lung ACE was purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were obtained from Wako Pure Chemicals Co. (Tokyo).

Preparation of Ameal.
The whey fraction was used to determine the ACE inhibitory activity of Ameal. The whey fraction was obtained as follows [15]. The pH of Ameal was adjusted to 3.4 by adding 50% lactic acid. The Ameal was centrifuged at 6,000 × g for 10 min, 10 N NaOH was added to the supernatant to raise the pH to 8.3, and then the supernatant was centrifuged at 6,000 × g for 10 min. The whey fraction of Ameal was used as a sample.

Assay of ACE Inhibitory
Activity. ACE inhibitory activity was assayed according to the method of Suzuki et al. [16] with some modifications. Hippuryl-L-histidyl-L-leucine (Hip-His-Leu) was dissolved in 400 mM phosphate buffer (pH 8.5) containing 300 mM NaCl. Next, 0.1 mL of 4.7 mM Hip-His-Leu solution was mixed with 0.05 mL of sample solution (Healthya, Goma-Mugicha, Lapis Support, Ameal, and their mixtures) and then preincubated for 5 min at 37 • C. The reaction was initiated by the addition of 0.1 mL of ACE dissolved in distilled water (25 mU/mL), and the mixture was incubated for 30 min at 37 • C. After the reaction had been stopped by adding 0.3 N NaOH (0.75 mL), 0.05 mL of 2% o-phthaldialdehyde in methanol was added, the mixture was left at room temperature for 10 min, and then 0.1 mL of 0.1 N HCl was added. After incubation for 30 min at room temperature, the amount of liberated His-Leu was determined by measuring the fluorescent intensity of its adduct with o-phthaldialdehyde (excitation at 340 nm and emission at 455 nm). The extent of inhibitory activity was calculated as follows: where A is the fluorescent intensity in the presence of ACE and ACE inhibitory component, B is the fluorescent intensity without ACE inhibitory component, and C is the fluorescent intensity without ACE. Inhibition was expressed as the concentration of component that inhibits 50% of ACE activity (IC 50 ). One unit of ACE inhibitory activity was expressed as the potency showing 50% ACE inhibition under these conditions and was calculated for one bottle, daily intake, or maximum dosage.   Figure 1 was calculated as IC 50 based on the amount of functional components included in each food product for specified health uses. inhibitory activity of a mixture of Healthya and Ameal was not enhanced, the elemental composition of each FOSHU was investigated. The whey fraction used in this experiment was in fact extracted from an Ameal solution (Goma-Mugicha and Lapis Support do not contain any ingredients that originate from milk). Healthya that was prepared to determine the IC 50 was mixed with 3 μg/mL WPC, and the mixture's rate of ACE inhibitory activity was measured. The concentration of β-lactoglobulin, the main component of whey protein [18], was 1.28 μg/mL in 3 μg/mL WPC and 18.6 μg/mL in the whey fraction of Ameal (concentration of IC 50 , 445 ng/mL Val-Pro-Pro). WPC (3 μg/mL, as whey) and Healthya (as tea polyphenol) were used for measurement. Examination of the ACE inhibitory activity of a mixture of whey component and tea polyphenols revealed a significant decrease in the rate of ACE inhibitory activity of Healthya (56.9 ± 3.2%) as a result of the addition of 3 μg/mL WPC (34.9 ± 9.8%: P < 0.05) (Figure 2), thus demonstrating that whey protein lowered the rate of ACE inhibitory activity of Healthya.

Effects of Tea Polyphenols and Ameal on Antihypertensive Activity in SHRs.
To evaluate the antihypertensive activity of the interaction of tea polyphenols and Ameal, the SBP of SHRs was measured after a single oral administration. The baseline SBP before administration was 198.2 ± 3.0 mmHg. Tea polyphenols (polyphenon CG, 2 g/kg body weight) significantly lowered SBP 2 and 4 hr after administration (155.4 ± 8.4 mmHg and 163.8 ± 7.6 mmHg, resp.) in comparison to distilled water (195.3 ± 6.7 mmHg and 178.8 ± 5.8 mmHg, resp.) (P < 0.05). However, oral administration of polyphenon CG and Ameal failed to decrease SBP (178.9 ± 4.7 mmHg and 169.2 ± 8.3 mmHg, resp.) at the same times ( Figure 3). Therefore, combined administration of Polyphenon CG with Ameal appears to attenuate antihypertensive activity by interacting with the whey protein in the Ameal solution. Though possible mechanisms of the interaction of tea and milk protein have been discussed in previous reports [19][20][21], the actual mechanism is still not clear In this study, markedly larger amounts of tea catechins than humans would normally be consumed in typical foods that were administered to SHRs, and this study examined the interaction of these catechins. While many health foods contain much smaller amounts of substances than were used in the current experiments, numerous products are being sold in formulations with active components in higher concentrations than that would normally be consumed. In addition, the amount of health foods consumed is substantial when individuals have a strong predilection toward selfmedication. Many such individuals will presumably consume numerous types of health foods at the same time. Accordingly, large quantities of tea catechins were used in the current study, and the consumption of such amounts is indeed a possibility.
Moreover, this study focused on blood pressure, which was found to be affected soon after administration [9], but interaction between tea catechins and whey protein attenuated the activity of the two, much like other types of activity (e.g., antiviral, antioxidative, antimutagenic and anticarcinogenic, and antiobesity activity) are attenuated. Based on results of the current study, consuming multiple health foods at the same time enhanced the activity of individual components in some instances. That said, the results also suggested that such consumption led to interactions, diminishing the effectiveness of some of the components of these foods.

Conclusion
The findings of the present study suggest for the first time that interaction between tea and milk protein may attenuate antihypertensive activity and that the simultaneous intake of tea and FOSHUs that contain active peptides might affect daily self-antihypertensive management via the enhancement or suppression of ACE inhibitory activity.