Objective: Long-term intake of Miso attenuates hypertension in Dahl salt-sensitive (Dahl S) rats through an increased urinary sodium excretion. We examined whether a bolus injection into the peritoneal cavity (i.p.) or a continuous subcutaneous infusion of a Miso extract attenuates hypertension in Dahl S rats. Materials and Methods: We investigated the effects of a bolus, i.p. injection of 50 mg Miso extract in 0.5 mL on hypertension in Dahl S rats, and examined whether a long-term subcutaneous infusion of the Miso extract (50 mg Miso/day), using an osmotic mini-pump working for 14 days, attenuates hypertension in Dahl S rats. Results: A bolus, i.p. injection of 50 mg Miso extract decreased SBP in 2 hrs. The reduction was significant at 4 hrs, and SBP returned to the baseline at 24 hrs. The SBP reduction at 4 hrs after the injection was increasingly greater each day during the 4 days. The SBP reduction by the Miso extract was dose-dependent and the antihy-pertensive activity occurs in a <5 kDa fraction of the extract. The subcutaneous infusion of 50 mg Miso extract/day for 14 days significantly attenuated hypertension in Dahl S rats. The SBP reduction was associated with significant decreases in the heart and kidney weights. Urinary protein excretion significantly decreased in the Miso group. The SBP reduction was not associated with increases in either urinary sodium excretion or fractional excretion of sodium. Conclusions: SBP reduction by very low-dose of the Miso extract may be mediated partly by mechanisms other than renal sodium handling.
Miso is a traditional Japanese food that was originally described in Taiho Ritsurei in 701 AD. Miso is manufactured using soybeans along with malted rice and salt; a significant amount of salt is needed for complete fermentation. One serving of Miso soup generally contains 1 - 1.5 g salt which supports the long-standing belief that traditional Miso soup is to blame for the high incidence of salt-sensitive hypertension and subsequent cerebral stroke in Japan.
Interestingly, however, we and investigators from other laboratory have reported in the previous study that long-term intake of Miso soup or Miso attenuates development of salt-induced hypertension in Dahl salt-
sensitive (Dahl S) rats when compared with rats given the same amount of saline solutions as the salt contents in Miso soup [
In our preliminary study using metabolic cages, sodium absorption in the intestine was not influenced by the long-term intake of Miso in normotensive Wistar rats (unpublished data) [
In the present study, we proposed the hypothesis that Miso extract attenuates hypertension through vasodilator effects on vascular walls via the circulation. To test this hypothesis, we injected a Miso extract into the abdominal cavity or subcutaneous tissue and we then examined the blood pressure in Dahl rats with salt-induced hypertension.
Protocol of the study
We purchased 4-week-old male Dahl salt-sensitive (Dahl S/Iwai) rats from Sankyo Laboratories, Inc., Tokyo, Japan. The Dahl S rats were fed a high-salt (4%NaCl) diet to develop salt-induced hypertension. Dahl S rats aged 7 - 8 weeks old were used for assessing the antihypertensive activity of a bolus intra-peritoneal (i.p.) injections of Miso extract.
Manufacture of Miso
Miso is manufactured from soybeans with malted rice and salt. A significant amount of salt is needed for complete fermentation. One serving of Miso soup contains 1 - 2 g salt. A representative Japanese meal is composed of rice, the main dish, several side-dishes, e.g., vegetables, etc., and Miso soup. Therefore, when Miso soup is served 3 times a day, 3 g of salt or more is consumed in addition to the usual salt intake from other foods.
Preparation of Miso extract
Miso utilized in the present study was offered by the Central Miso Research Institute (Tokyo, Japan). One thousand kilogram Miso is regularly made of 293 kg soybeans, 144 kg rice (malted), 130 kg NaCl, and water (Kome Miso full-boiled and colored light-yellow). Miso was fermented for 3 months [
Assay of antihypertensive activity
We injected 0.5 mL of the extract from 50 or 25 mg Miso into the peritoneal cavity of hypertensive Dahl S rats aged 7 - 8 weeks and administered 0.5mL of 1.3% or 0.65% saline solutions corresponding to the concentrations of salt in the Miso extract to the control hypertensive rats. The systolic blood pressure (SBP) was determined using the tail-cuff method of Freedman and Freed with a slight modification in the detection system (Natsume Seisakujo Model KN-210-1; Tokyo, Japan) [
Sixteen 4-week-old Dahl S rats were fed a high-salt diet (4% NaCl, w/w) for 4 weeks and six 4-week-old Dahl S rats were fed a low-salt diet (0.3% NaCl, w/w) diet for 4 weeks (low-salt DS). The Dahl S rats fed the high-salt diet (4% NaCl, w/w) were divided into 2 groups at the age of 6 weeks; 1) 8 Dahl S rats fed the high-salt with sham-operation (DS-high), and 2) 8 Dahl S fed the high-salt with a subcutaneous continuous infusion of the Miso extract (50 mg/day) using an osmotic mini-pump (ALZET model 2ML2; 5.0 μL/h working for 14 days, Cupertino, CA) (DS-miso) [
At the end of the study, each rat was placed in a metabolic cage for 3 days consecutively, and 24-h urine samples were collected on the last day for testing. Urine samples were stored at −80˚C until assayed. A single investigator measured the blood pressure of all animals in a quiet warm room from 13:00 to 16:00. The blood samples and organs of interest were obtained from rats under pentobarbital anesthesia (75 mg/kg body weight).
We used the osmotic mini-pump ALZET model 2ML2 for subcutaneous administration of the Miso extract [
Plasma and urinary concentrations of creatinine were measured using an autoanalyzer (Model Hitachi 736; Hitachi Co., Ltd., Tokyo, Japan). Concentrations of Na were determined by an Na+-sensitive electrode and Na METER TS-999i determination system (Tokyo Chemical Laboratories, Co, Ltd., Tokyo) [
All statistical analyses were performed using STATISTICA software (StatSoft, Tulsa, OK). Values were expressed as means ± SD. Differences were assessed by one-way factorial analysis of variance (ANOVA) followed by the post-hoc least significant difference (LSD) test. p-values less than 0.05 were considered statistically significant.
We followed the guidelines for experimental animal handling, and our study was approved by the Animal Care Committee of the Kyoritsu Women’s University (No. 13003, 2013 and No. 14001, 2014). The experiment was conducted in accordance with the guidelines of the National Institutes of Health.
A bolus i.p. injection of 50 mg Miso extract in 0.5 mL decreased SBP of Dahl S rats after 2 hrs. The reduction was statistically significant at 4 hrs and the SBP was equal to the baseline after 24 hrs (
The bolus injection once a day was continued for 4days. The SBP values were determined at 4 hrs after the injection on day 2 and day 4 (
The changes in SBP after administration of different doses of the Miso extract were shown in
Dahl S rats in a dose-dependent manner.
The crude Miso extract was passed through the membrane sieves (cutoff point <5 kDa) and we obtained <5 kDa fraction of the Miso extract. At 4 hrs after the i.p. injection, we measured and compared the antihypertensive activity between the crude (50 mg Miso) and fractionated extract (<5 kDa). There was no difference in the antihypertensive activity between the 2 samples, suggesting that the antihypertensive factor(s) was expected to be present in the <5 kDa fraction (
We examined the long-term effects of the low-dose (50 mg/day) Miso extract on SBP in Dahl S rats with salt-induced hypertension using a continuous, osmotic mini-pump working for 14 days. As shown in
Systolic blood pressure increased in a time-dependent manner in the DS-high group when compared with that in the DS-low group (
The heart weight of the DS-high group increased as compared with that of the DS-low group (
Next, to understand the role of the kidneys in sodium handling with respect to the reduction in blood pressure by the subcutaneous infusion of the Miso extract, we examined the parameters indicating renal sodium metabolism (
the other hand, free water clearance was significantly decreased in the DS-miso group compared with that in the DS-high group.
In addition, it is noted that Na/K in urine was significantly lower in Dahl S rats fed a low-salt diet than in Dahl S rats fed a high-salt diet, suggesting that Na/K exchange was enhanced in the low-salt Dahl S rats through up-regulated renin-angiotensin system on a low-salt diet. In DS-miso, however, the Na/K balance was not changed when compared with DS-high.
Fermented Miso is an integral part of Japanese cuisine. Miso usually contains 10% - 13% salt (w/w) and 10 - 30 g Miso/day is served as Miso soup. This means that the intake of salt from Miso averages 1 - 3.9 g/day, because of which, in Japan, Miso is believed to be responsible for incidence of hypertension. To our knowledge, however,
there are no studies showing that the intake of Miso soup induces hypertension in humans. Our preliminary, cross-sectional survey on subjects at an annual medical checkup found that the more frequent intake of Miso soup consuming 3.0 - 4.5 g salt is not associated with an elevation of blood pressure, compared with less than once a day intake of Miso soup consuming less than 1.5 g/day, suggesting that Miso contains some nutrients that attenuates blood pressure elevation due to higher salt intake (unpublished data).
To test the possibility that Miso contributes to hypertension, we examined the influence of long-term oral intake of Miso soup in salt-induced hypertension of Dahl S rats. We found that long-term intake of Miso prevents salt-induced hypertension as well as attenuates heart and kidney damage [
Organ | Unit | DS-high | DS-miso | DS-low |
---|---|---|---|---|
Heart weight | g/100g BW | 0.450 ± 0.025 | 0.392 ± 0.022* | 0.420 ± 0.106 |
Aortic weight | mg/mm2 | 0.327 ± 0.078 | 0.293 ± 0.036 | 0.260 ± 0.025* |
Kidney weight | g/100 BW | 0.592 ± 0.056 | 0.550 ± 0.013* | 0.468 ± 0.015** |
*p < 0.05, **p < 0.001 vs DS-high.
Variables | DS-high | DS-miso | DS-low |
---|---|---|---|
UV | 14.47 ± 1.46 | 9.69 ± 2.53** | 2.64 ± 0.40** |
UNaV | 4.45 ± 0.41 | 3.81 ± 0.99** | 0.09 ± 0.01** |
UKV | 1.10 ± 0.14 | 1.04 ± 0.32 | 0.90 ± 0.20 |
UNa/Uk | 4.0 ± 0.3 | 3.7 ± 0.6 | 0.1 ± 0.0** |
Ccr | 1.74 ± 0.18 | 1.46 ± 0.20* | 1.00 ± 0.22** |
CNa | 30.55 ± 3.18 | 27.65 ± 7.01 | 0.74 ± 0.10** |
FENa | 1.76 ± 0.24 | 1.89 ± 0.45 | 0.07 ± 0.01** |
POsm | 314 ± 4 | 310 ± 2* | 307 ± 4** |
UOsm | 15.3 ± 1.3 | 15.7 ± 3.4 | 6.3 ± 0.9** |
COsm | 48.9 ± 4.4 | 50.6 ± 11.0 | 20.7 ± 3.2** |
−34.4 ± 4.2 | −40.9 ± 8.7* | −18.1 ± 3.3** |
UV, urine volume (mL/100g BW/day); UNaV, urinary excretion of sodium (mEq/100g BW/day); UKV, urinary excretion of potassium (mEq/100g BW/day): UNa/Uk, ratio of urinary sodium/potassium; Ccr, creatinine clearance (L/100g BW/day); CNa, clearance of spdum (mL/100g BW/day); FENa, fractional excretion of sodium (%); POsm, plasma osmolality (mOsm/KgH2O); UOsm, urinary osmolality (Osm/KgH2O); COsm, clearance of osmolality (mOsm/KgH2O/100g BW/day); CH2O, free water clearance (mL/100g BW/day). p values indicate statistically significant between DS-high and DS-miso. *p < 0.05, **p < 0.001 vs DS-high.
the oral intake of 5 - 10 g Miso/day was associated with enhanced natriuresis/diuresis, likely mediated by the urinary dopaminergic system [
Thus, the present study examined whether a Miso extract decreases blood pressure via the circulation. We demonstrated that a bolus i.p. injection of Miso extract decreases the SBP in salt-induced hypertension in Dahl S rats fed a high-salt diet. The finding suggests that the Miso extract contains a substance(s) that decreases SBP in Dahl S rats via the circulation. Furthermore, the antihypertensive substance(s) is heat-stable and less than 5 kDa in molecular weight. These data suggested that there are several substances to decrease blood pressure in Miso, and we provided evidence for the occurrence of antihypertensive substance(s) that are different from the activity shown in the previous studies.
We administered 0.5 mL of the extract prepared using 50 mg Miso (10% Miso solution). The amount of Miso given via the i.p. route is much lower than that given via the oral route as reported previously [
Such a reduction in blood pressure was not accompanied by increases in natriuresis and diuresis that was observed for administration via the oral route. Additionally, neither osmotic nor free water clearance changed, suggesting that the Miso extract does not affect urinary excretion of solutes. These data indicate that the mechanism of antihypertension via the circulation is different from the mechanism via the oral route. The mechanism via the subcutaneous route is not clear; however, some reports indicate that peptides from soybeans may suppress the renin-angiotensin system, thereby decreasing the blood pressure [
We did not infuse the Miso extract directly into vascular vessels mainly because the influence by the operation was minimized. We believe that the direct determination of blood pressure was the next step in studying pharmacological properties of the antihypertensive activity along with purification and identification.
The Dietary Approaches to Stop Hypertension (DASH) diet has been reported to decrease the blood pressure compared with an American style diet [
In conclusion, the present study shows that treatment with very low doses of Miso extract via the i.p. or subcutaneous routes decreases blood pressure in rats with salt-induced hypertension. This blood pressure reduction is not mediated by increases in sodium excretion from the kidneys. The blood pressure reduction via the subcutaneous route is associated with the attenuation of cardiovascular stress following hypertension. Miso contains several substances beneficial for health [
We acknowledge Kyoko Nakano and Yoshihide Obata, Central Miso Research Institute, Tokyo, Japan for the gift of Miso. This study is supported in part by Grant-in-Aids from Center for Interdisciplinary Studies of Science and Culture, Kyoritsu Women’s University & Junior College (2012) and a grant from Central Miso Research Institute (2012).
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.