Antihypertensive effect and relaxant activity of post-fermentation tea extract on
vascular smooth muscles.
Akio Nakamura1, Ritsuko Kawaharada2, Shinji Yoshiyama1, Kayo Takazawa2, Hiiro Shimizu2, Haruna
Masuda1, Natsuko Inoue3, Takahiko Inoue3
1: Dept. Mol. Phrmacol. & Oncol, Gunma Univ., Grad., Sch. Med. Japan, Japan
2: Dept. Health & Nutri., Takasaki Univ. of Health and Welf. Japan
3: Black tea Inst. Japan
Correspondent email: [email protected]
Summary
We investigated the relaxant activity of various commercial tea extracts by measuring the isometric
contraction of vascular smooth muscles in rats. The post-fermentation tea extract showed significant relaxant
activity on vascular smooth muscles. By measuring vascular smooth muscle tension, the relaxant activity of
n-Hexane, ethyl acetate, and butanol fractions from crude tea extracts were evaluated. An ethyl acetate
fraction showed the relaxation of vascular smooth muscles in a dose-dependent manner and significantly
reduced blood pressure in spontaneously hypertensive rats. These results suggest that ethyl acetate fraction
from post-fermentation tea extracts could potentially be used as a natural drug or functional food to prevent
hypertension.
Introduction
The contractile force of smooth muscle is derived from the interaction between actin and myosin. Smooth
muscle myosin light chain can be phosphorylated by myosin light chain kinase at Ser19 and Thr18, and it
also plays an important role in activating actomyosin-linked contractility in smooth muscle cells.
Vasodilators and vasoconstrictors are known as extracellular regulators of contraction and relaxation of
smooth muscle and are usually found in natural sources (2). We searched for natural vasodilators from
various tea extracts (green, oolong, black, and post-fermentation
tea extracts) that particularly act on vascular smooth muscles.
Pu-erh tea, a typical post-fermentation tea that is a popular
beverage in southwestern China and South Asian countries, is
produced by Aspergillus fermentations of Camellia sinensis.
Goishi and Awa-ban tea are produced in Japan via a two-step
fermentation process under aerobic and anaerobic conditions (3).
In this study, we obtained tea extracts and examined the effect
on rat femoral arteries by measuring the isometric contraction.
Finally, the vasodilatory effect of an ethyl acetate fraction
(EtOAc) from post-fermentation tea extracts (Pu-erh, Goishi, and
Awa-ban tea) was determined.
Materials and methods
Tea was extracted by an autoclave at 120°C for 30 min. The hot
water extract was subjected to sequential liquid–liquid extraction
with a solvent series of increasing polarity of n-hexane, EtOAc,
and butanol. Partitioning was performed 4 times in glass
separatory funnels by mixing 100 ml of the solvent during the
aqueous phase and shaking the mixture for 60 min; after standing,
the residue was removed during the organic phase (Figure. 1).
Rats were anesthetized with intravenous pentobarbital sodium.
The femoral artery was isolated and the endothelial layer was
removed. For tension measurement, the femoral artery was
dissected into small strips (width, 200 µm; length, 5 mm). The
strips were set between a hook and an isometric force transducer
(Figure 2). The femoral artery strip was stabilized in normal extracellular solution (NES). Phenylephrine (50
1
µM) stimulation caused a phasic contraction of the strip in the Ca2+-free NES. Various tea extracts were
added to the strip in the Ca2+-free NES in a bubble chamber. Six-week-old 15 male spontaneously
hypertensive rats (SHR) were raised in an air-conditioned room (25°C) for 1 week; SHRs were then
randomly divided into 3 groups, and green tea with GABA, Pu-erh, or Goishi tea was orally administered.
Arterial blood pressure (BP) was determined by a tail-cuff system. Rats were lightly supported in a mesh
holder made of cloth and maintained at 37°C. The tail systolic blood pressure (SBP) was measured using an
indirect blood pressure meter (BP-98-A, Softron, Japan).
Results and discussion
We examined the relaxant activity of the extract by adding various amounts of the extract to NES. With
Pu-erh and Goishi tea, muscle tension decreased by cumulative addition of EtOAc (from ×4 to ×14; Figure.
3). However, tension increased in response to green tea than to Pu-erh and Goishi tea. This increase in
tension may be influenced by potassium or caffeine in green tea. Because this strip did not contain
endothelial cells, EtOAc fraction in post-fermentation tea
extracts directly caused relaxation of vascular smooth
muscles, thereby serving as an endothelial-independent
vasodilator.
The antihypertensive effect of the tea extract was
evaluated by measuring SBP changes after a single, oral
administration. The SBP-lowering effect of the tea was
transient and reverted to the baseline level after 24 h. As
shown in Figure 4, the blood pressure-lowering effect of
EtOAc fraction of Goishi tea continued for over 24 h, while
that of SHRs returned to initial levels 28 h after
administration. Pu-erh and green tea decreased SBP 4 h or
8 h after administration, and blood pressure returned to the
initial levels 24 h after administration. Post-fermentation tea extracts showed blood pressure-lowering
activity when orally administrated to SHRs.
Thus, post-fermentation tea such as Pu-erh and Goishi
showed a strong relaxation effect on vascular smooth
muscles and demonstrated antihypertensive effects. Thus,
the novel finding of this report is that post-fermentation tea
extracts may contain natural vasodilators. There
vasodilators directly relax vascular smooth muscle;
however, this effect is not the same as that associated with
angiotensin I-converting enzyme inhibitor and angiotensin
receptor blockers. However, the chemical composition of
relaxant has not yet been elucidated in detail. Therefore, we
would like to isolate the compound for vasodilators and
determine the chemical structure.
Acknowledgements
This work is supported by Japan Science and Technology Agency (JST), A-step feasibility study program
(#AS231Z02014G).
References
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in smooth muscle. Annu. Rev. Pharmacol. Toxicol. 25:593-620.
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Hum. Hypertens. 14:617-630
(3): Gong, Z, et al. (1993) Compositional change of pu-erh tea during processing. Biosci. Biotech. Biochem.
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