Accepted Manuscript

Antihypertensive effects of orally administered eggplant (Solanum melonge-

na) rich in acetylcholine on spontaneously hypertensive rats

Shohei Yamaguchi, Kento Matsumoto, Masahiro Koyama, Su Tian, Masanori

Watanabe, Akihiko Takahashi, Koji Miyatake, Kozo Nakamura

PII: S0308-8146(18)31782-5
DOI: https://doi.org/10.1016/j.foodchem.2018.10.017
Reference: FOCH 23678

To appear in: Food Chemistry

Received Date: 6 April 2018

Revised Date: 24 September 2018
Accepted Date: 3 October 2018

Please cite this article as: Yamaguchi, S., Matsumoto, K., Koyama, M., Tian, S., Watanabe, M., Takahashi, A.,
Miyatake, K., Nakamura, K., Antihypertensive effects of orally administered eggplant (Solanum melongena) rich
in acetylcholine on spontaneously hypertensive rats, Food Chemistry (2018), doi: https://doi.org/10.1016/
j.foodchem.2018.10.017

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Antihypertensive effects of orally administered eggplant (Solanum melongena) rich in

acetylcholine on spontaneously hypertensive rats

Shohei Yamaguchia, Kento Matsumotoa, Masahiro Koyamab, Su Tianc, Masanori Watanabed,

Akihiko Takahashie, Koji Miyatakef and Kozo Nakamuraa,g,*

a
Department of Agriculture, Graduate School of Science and Technology, Shinshu University, 8304,

Minamiminowa, Nagano 399-4598, Japan

b
Wellnas. Co., Ltd., 508-2, Fii building, 3-15-1, Tokida, Ueda, Nagano 386-8567, Japan

c
Department of Nutrition and Food Hygiene, School of Public Health, Hebei Medical University,

Hebei 050017, China

d
Department of Food, Life and Environmental Science, Faculty of Agriculture, Yamagata University,

1-23 Wakaba-machi, Tsuruoka, Yamagata 997-8555, Japan

e
Kochi Agricultural Research Center, 1100 Hataeda, Nankoku, Kochi 783-0023, Japan

f
Institute of Vegetable and Floriculture Science, NARO, 360 Kusawa, Ano-cho, Tsu, Mie 514-2392,

Japan

g
Institute of Agriculture, Academic Assembly, Shinshu University, 8304, Minamiminowa, Nagano

399-4598, Japan

1
*Corresponding author

2
ABSTRACT

Our previous results (Nakamura et al., 2013 and 2016) indicated that acetylcholine (ACh) in orally

administered foods exerts antihypertensive effects. Eggplants (Solanum melongena) contain

abundant ACh (Horiuchi et al., 2003), and their food functionality was discovered, using

spontaneously hypertensive rats, by measuring blood pressure after oral administration of a

suspension of lyophilized eggplant powder. We found that lyophilized eggplant powder induced

significantly lowered acute and chronic blood pressure levels at very low doses of 0.0650 mg/kg

body weight (b.w.) and 0.821 mg/(kg b.w.•day), respectively. Chronic administration suppressed

adrenaline and noradrenaline excretion in the urine, and aorta assays showed that eggplant acted on

the M3 muscarinic ACh receptor (M3 mAChR). ACh was conclusively shown to function as the

main component of eggplant contributing to antihypertensive activity by suppressing sympathetic

nervous activity via M3 mAChR. This report reveals a new food functionality of eggplant and its

potential as a novel antihypertensive food.

Keywords: Eggplant, Acetylcholine, Blood pressure, Spontaneously hypertensive rats,

Catecholamine

3
1. Introduction

Eggplant (Solanum melongena), a popular vegetable that is consumed globally on a daily basis, is

rich in dietary fibre and minerals, low in calories and protein, and contains vitamins such as B6, C,

and folate (USDA ARS, 2015). The National Institute of Diabetes and Digestive and Kidney

Diseases recommends reducing caloric intake and body weight to prevent type-2 diabetes, and the

American Diabetes Association showed that eggplant is a high-fibre and non-starchy vegetable

(NIDDK 2016, ADA 2011 and 2015). Recently, nasunin (Casati et al., 2016), γ-aminobutyric acid

(GABA) (Horie et al., 2013), and chlorogenic acid (CA) (Singh et al., 2009) were reported as

functional components of eggplant. Nasunin, which is a purple pigment in eggplant, contained in the

peel, has antioxidant activity (Noda et al., 2000). CA is the main polyphenol in eggplant and has

been reported to have antidiabetic, antihyperlipidemic (Wan et al., 2013), and hepatoprotective

effects (Shi et al., 2009). GABA is known to have relaxation effects in vivo (Abdou et al., 2006) as

well as antihypertensive effects (Hayakawa, Kimura & Kamata, 2002). However, to our knowledge,

only a few food functionalities of eggplant in vivo have been reported, including cardioprotection

(Das et al., 2011) and antioxidant activity (Sudheesh et al., 1999) in rats, and cholesterol-lowering

effects in hypercholesterolemic subjects (Guimarães et al., 2000).

Acetylcholine (ACh) is a well-known animal neurotransmitter, and Horiuchi et al. reported that

ACh was present in eggplant (Horiuchi et al., 2003). Other reports showed that food materials such

4
as milk (Whittaker, V. P., 1958), bamboo shoots, Bacillus subtilis, and Shiitake mushrooms

(Kawashima, 2007) also contain ACh. Previously, we reported that fermented buckwheat sprout

(FBS) was capable of lactic fermentation and exerted both a dose-dependent antihypertensive effect

at low doses (10 and 100 µg/kg of body weight [b.w.]) in spontaneously hypertensive rats (SHRs)

and a vasorelaxation effect, as determined in aorta assays (Nakamura et al, 2013). Several

angiotensin I-conversion enzyme (ACE)-inhibitory peptides were isolated from FBS (Koyama et al.,

2013) and we recently reported the isolation of choline esters, ACh, and lactoylcholine as

vasodilation compounds, which indicated that choline esters may be responsible for the blood

pressure-lowering effect of FBS (Nakamura et al., 2016). The report demonstrated the first isolation

of natural lactoylcholine and showed the potential of choline esters as functional food ingredients.

These data led to the hypothesis that eggplant containing ACh reduced blood pressure in SHRs.

To test this hypothesis, the blood pressure-lowering effects of oral eggplant intake in SHRs were

investigated in this study. The acute and chronic effects of eggplant on blood pressure in SHRs were

evaluated by conducting single and 28-day oral-administration tests. Previously, it was reported that

the activity of tyrosine hydroxylase (a rate-determining enzyme of catecholamine biosynthesis) was

high in the adrenal medulla of SHRs compared with that in normotensive Wistar Kyoto rats, and the

higher catecholamine levels were considered a cause of essential hypertension (Kumai et al., 1994).

Therefore, to investigate the hypotensive mechanism of eggplant, chronic adrenalin (AD),

5
noradrenalin (NAD), and dopamine (DA) levels were determined by liquid chromatography–tandem

mass spectrometry (LC–MS/MS) and aorta assays to estimate the site of action of ACh. The ACh,

GABA, and CA contents in eggplants were quantified by LC–MS/MS analysis to estimate the main

functional food compound in eggplant.

2. Materials and methods

2.1. Chemicals

Ultra-pure water with a specific resistance of 18.2 MΩ/cm was produced in an Arium 611

ultrapure water system (Sartorius Co., Germany) and used in our experiments. Methanol

(HPLC-grade), formic acid, 5 N hydrochloric acid, acetic acid, sodium chloride (NaCl), and

2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) were purchased from Nacalai

Tesque, Inc. (Kyoto, Japan). Acetonitrile (HPLC grade), ACh chloride (Cica-Reagent, special grade),

CA, magnesium sulfate (MgSO4), potassium chloride (KCl), potassium dihydrogen phosphate

(KH2PO4), D-(+)-glucose, calcium chloride dihydrate (CaCl2•2H2O), sodium bicarbonate (NaHCO3),

citric acid monohydrate, sodium acetate, phenylephrine (PE), and papaverine were purchased from

Kanto Chemical Co., Inc. (Tokyo, Japan). The reagent 1,1-dimethyl-4-diphenylacetoxypiperidinium

iodide (4-DAMP) was purchased from Enzo Life Sciences, Inc. (Farmingdale, USA). GABA,

DL-AD, hydrogen tartrate L-NAD monohydrate, DA hydrochloride, and sodium 1-octane sulfonate

6
(IPC-ALKS-8) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Isoflurane

was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

2.2. Animals and ethics statement

Male 14-week-old SHR/NCrlCrlj rats (Charles River Laboratories Japan, Inc., Kanagawa, Japan)

were used in vasorelaxant assays of thoracic aorta rings and single oral-administration tests, and

8-week-old rats were used in chronic oral-administration tests. SHRs were housed individually under

a 12-h light–dark cycle at 23 ± 4°C, with 50 ± 20% humidity. Male 8-week-old SHRs were housed

in individual KN-646 metabolic cages (Natsume Seisakusho Co., Ltd., Tokyo, Japan). These rats had

free access to laboratory feed (MF, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water. All

experiments and surgical procedures were performed with the approval of the Animal Care

Committee of the Faculty of Shinshu University (approval number: 290061).

2.3. Sample preparation

A commercially available Japanese eggplant landrace and a cultivar with an oval shape and dark

purple skin were purchased in June and November, respectively. The ‘Mizunasu’ landrace was

cultivated in the Osaka prefecture in Japan, and the ‘Tosataka’ cultivar was cultivated in the Kochi

prefecture. ‘Mizunasu’ was used in the aorta assay and single oral-administration test, and ‘Tosataka’

7
was used in the chronic oral-administration test. After washing the fresh eggplant fruit, a calyx was

cut off and sliced into pieces with a 1–2 cm width, stored at –80ºC for 2 days, and a freeze-dried

product was prepared using a freeze dryer (FDU-2000, Tokyo Science Instrument Co., Ltd.). The

dried yields of ‘Mizunasu’ and ‘Tosataka’ were 6.08% (155.4 g fresh weight [FW], 9.45 g dry weight

[DW]) and 6.47% (84.12 g FW, 5.44 g DW), respectively. To prepare lyophilized eggplant powders,

the lyophilized matter was powdered using a mill mixer (MNN-2001, Tokyo Unicom) for 1.5 min

(31,000 rpm).

2.4. Quantitation of ACh, GABA, and CA in eggplants

ACh, GABA, and CA levels in the eggplants were analyzed by LC–MS/MS, using an Acquity

UPLC system and a Quattro Micro API mass spectrometer (Waters Co., Milford, MA) under the

following conditions. Two hundred microliters of 50 mM hydrochloric acid were added to 10 mg of

lyophilized powder and vortexed for 3 min. The supernatant was recovered after centrifugation

(8070  g, 3 min). This extraction process was repeated twice, the collected supernatants were mixed

together, and the volume was adjusted to 1 ml with mobile phase. The sample was diluted 400-fold

with mobile phase, filtered with a 0.45-μm pore size syringe filter, and subsequently analyzed.

LC-based separation was performed using a YMC Triart-PFP (4.6 mm × 250 mm) column at 40°C,

and 50% (V/V) methanol containing 0.010% formic acid was used as the mobile phase, with a flow

8
rate of 0.50 ml/min and an injection volume of 50 µl. The mass spectrometer was operated in

positive mode, and electrospray ionization (ESI) was used as the ionization source. Other

experimental parameters were as follows: a 3.5 kV capillary voltage, a desolvation gas (N2) flow rate

of 650 L/h, a cone gas (N2) flow rate of 50 L/h, a source temperature of 120°C, and a desolvation

temperature of 350°C. Detection was performed in multiple-reaction mode (MRM). The optimized

MRM transition, cone voltage, and collision voltage of each compound were 146.2>87.0, 20 V, and

10 V (ACh); 104.01>86.94, 20 V, and 10 V (GABA); and 355.15>163.06, 15 V, and 10 V (CA),

respectively. The identities of compounds in each peak were confirmed by comparing their retention

times and observed m/z ratio with those of standards. Quantitation was conducted by comparison

with a calibration curve obtained by analyzing standards. The standards were prepared by diluting

them in the mobile phase, after which they were stored at –80°C until use. Three samples from each

eggplant cultivar were analyzed by LC–MS/MS.

2.5. Measuring the vasorelaxant effect of eggplant in SHR thoracic aorta rings

SHRs were sacrificed by exsanguination via an abdominal aorta under anesthesia with isoflurane

(3.0% isoflurane initially, followed by 1.5% isoflurane to maintain anesthesia during exsanguination).

A thoracic aorta was isolated, the surface was immediately cleaned in Krebs–Henseleit solution (119

mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2•2H2O, 1.1 mM KH2PO4, 1.2 mM MgSO4, 11 mM glucose,

9
and 25 mM NaHCO3; pH 7.4) to remove fat and connective tissue, and cut into 2–3 mm rings. The

rings were mounted in an organ bath (5.0 ml) filled with Krebs–Henseleit solution bubbled with 95%

O2 and 5% CO2 at 37°C, and equilibrated at a resting tension of 1.5 g for 60 min, while exchanging

the solution every 15 min. The change in tension was measured using a UFER UM-203 isometric

transducer (Iwashiya Kishimoto Medical Instruments Co., Ltd., Kyoto, Japan) with a UFER UC-5A

magnus system (Iwashiya Kishimoto Medical Instruments Co., Ltd.). The data were recorded and

analyzed with a PowerLab data-acquisition device and LabChart®7 software (ADInstruments Pty.

Ltd., New South Wales, Australia). After the rings were contracted in the presence of 0.30 µM PE,

active integrity of the vascular endothelium was confirmed by vasodilation with 0.10 mM ACh,

where >90% expansion was considered passing. The rings were stabilized at the maximum

contraction by re-exposure to PE, after which ACh solution and eggplant powder suspension in

Krebs–Henseleit solution were cumulatively added to final ACh concentrations ranging from 1.00 

10-3 to 1.00  10-0.5 µM (i.e., 0.337 to 114 µg of eggplant powder) in the organ bath. In

receptor-inhibition assays, the selective M3 muscarinic acetylcholine receptor (M3 mAChR)

antagonist 4-DAMP was used at 10 µM. Each experiment was performed six times. The change of

tension from maximum contraction was recorded as the % vasorelaxation. From these results, a 50%

relaxant concentration (EC50) was calculated from the relaxation curve.

10
2.6. Measurement of the acute antihypertensive effect of eggplant

Male 14-week-old SHRs (n = 6) were fasted for 15 h and orally administered 0.0650 mg of

eggplant powder/kg b.w. (ACh equivalent of 1.00  10-9 mol/kg b.w.) suspended in pure water (1.00

ml), using a feeding needle. GABA and CA were administered at dosages of 0.456 µg/kg b.w. and

0.853 µg/kg b.w., respectively. A control group (n = 6) was administered pure water only. Before and

3, 6, 9, and 24 h after oral administration, systolic blood pressure (SBP) and diastolic blood pressure

(DBP) were measured by the tail-cuff method using Softron BP-98 (Softron Co., Tokyo, Japan) after

the SHRs were warmed in a thermostat at 38°C. Blood pressure changes were calculated from blood

pressure measurements at the beginning of the experiment and at each subsequent time point.

2.7. Measurement of the chronic antihypertensive effect of eggplant

2.7.1. Chronological changes in the blood pressure of SHRs

SHRs were assigned to two groups with almost the same average systolic blood pressure (control:

186.7 ± 2.6 mm Hg, eggplant: 188.9 ± 1.7 mm Hg) and body weight (control: 260.2 ± 3.3 g,

eggplant: 258.1 ± 6.0 g) at 1 week after delivery. Then after a 1-week acclimation (Takenaka, 2014),

repeated oral eggplant powder administration was conducted when the rats were 10–14 weeks old,

and urine sampling was initiated one day before eggplant powder administration began. A water

suspension of eggplant powder was administered to SHRs in the test group (equivalent in ACh 1.00

11
 10-8 mol/[kg b.w.•day]; n = 6) and pure water was administered to the control group (n = 6) using a

feeding needle. Administration in each group continued for 28 days. The ingested amount of

eggplant powder was 0.821 mg/kg b.w. per day. Daily food and water consumption, body weights,

and 24-h urine volumes were recorded during the experimental period. Before and 7, 14, 21, and 28

days after the start of administration, SBP and DBP were measured using the method described in

Section 2.6.

2.7.2. Quantitation of AD, NAD, and DP levels in 24-h urine samples

Quantitative analysis of the AD, NAD, and DP levels in urine sampled on the day before the oral

administration test was performed, and on days 6, 13, 20, and 27 (one day before the blood pressure

measurements) was conducted using a high-performance liquid chromatography-electrochemical

detector (HPLC-ECD). A Prominence HPLC system (Shimadzu Co., Kyoto, Japan) equipped with an

ED723 detector (GL Sciences Inc., Tokyo, Japan) was used for the experiments. Six hundred

microliters of each urine sample were thawed and centrifuged (1000  g, 3 min). Next, 200 µl of the

supernatant was mixed with 400 μl of 1.5 mM HEPES-NaOH buffer (binding solvent, pH 8.5) and

added to a Monospin PBA column (GL Sciences, Inc.), which was pre-treated with an aqueous 1%

acetic acid solution (elution solvent) and 100 mM HEPES–NaOH buffer (wash solvent, pH 8.5).

After centrifugation (5,000  g, 2 min), the eluent was recovered, added back to the column, and

12
centrifuged under the same conditions. The column was rinsed with 200 μl of the wash solvent, and

the adsorbed catecholamines were eluted with 200 μl of the elution solvent. The eluents were used

for quantitative analysis. A mixture of acetate–citrate buffer (citric acid monohydrate, 1.64 g/l;

sodium acetate, 4.20 g/l; and IPC-ALKS-8, 1.0 g/l) and acetonitrile (100/16 v/v) was used as a

mobile phase. Separation was conducted using an Inertsil ODS-4 column (4.6 × 250 mm, 5 µm; GL

Science, Inc.) at 35°C, with an injection volume of 20 µl and a flow rate of 0.80 ml/min. Diamond

and Ag/AgCl were used as the working and reference electrodes for detection, respectively, at a

voltage of 800 mV. Quantitation was conducted by interpolation of a calibration curve obtained by

analyzing standards. The standards were prepared by dissolving and diluting each catecholamine

standard in the elution solvent, and were stored at –80°C until use.

2.8. Statistical analysis

All results are expressed as the mean ± standard error (S.E.). Standard differences were considered

significant at p < 0.05, as determined by Student’s t-test.

3. Results

3.1. Detection and quantitation of ACh, GABA, and CA levels in eggplants

A representative MS chromatogram showing the simultaneous quantitation of ACh, GABA, and

13
CA by LC–MS/MS analysis is shown in supplementary data (Fig. S1), and the contents detected in

‘Mizunasu’ landrace and ‘Tosataka’ cultivar are summarized in Table 1. The LC conditions used

resulted in complete separation of the compounds in order of CA, GABA, and ACh, and the

calibration curve equations were y = 15.5 + 82.7 (R2 = 0.999, range: 0 to 500 ng/ml), y = 113.5 +

620.1 (R2 = 0.995, range: 0 to 250 ng/ml), and y = 1274.8 + 2914.3 (R2 = 0.998, range: 0 to 100

ng/ml), respectively. Other validation parameters, such as the recovery, relative standard deviation

(R.S.D.), and limit of quantitation (LOQ), are shown as supplementary data (Table S1). CA was the

most abundant compound detected, and GABA was the second most abundant. ACh was measured at

the same level in both eggplant samples used.

Table 1. Contents of ACh, GABA, and CA in eggplants (n = 3)

Mizunasu Tosataka

(mg/100 g FW) (mg/100 g FW)

ACh 13.7 ± 0.9 11.5 ± 0.1

GABA 42.6 ± 0.2 37.8 ± 0.09

CA 79.8 ± 1.4 120.6 ± 1.3

3.2. Vasorelaxation effect of eggplant

14
 The compound 4-DAMP is a known selective antagonist of the M3 mAChR and was used to

identify the active site of M3 mAChR with thoracic aorta rings from SHRs (Nakamura et al, 2013).

Fig. 1 shows a concentration–vasorelaxation relationship observed after adding ACh and eggplant

powder. An ACh concentration ranging from 1.00  10-3 to 1.00  10-0.5 µM (derived from the

eggplant powder) showed similar concentration-dependent vasorelaxation compared to an ACh

standard, and the EC50 values of the ACh standard and ACh from eggplant powder were 0.0369 ±

0.005 and 0.0372 ± 0.008 µM, respectively, and no significant difference was found between the

groups (p = 0.980; determined by Student’s t-test). Fig. 1 also shows that vasorelaxation, both with

eggplant powder and ACh, significantly decreased in the presence of 4-DAMP. These results

indicated that the bioavailability of ACh in eggplant was equivalent to that of the standard and

activated the M3 mAChR on blood vessels to exert vasoactivity.

3.3. Acute antihypertensive effect

Fig. 2 shows changes in the SBP and DBP before and after oral eggplant powder suspension

administration. The SBP in the eggplant group significantly decreased (p < 0.05) at 3 and 9 h by 4.81

and 10.0 mm Hg, respectively, after administration compared with the control group. The DBP also

decreased at 3 and 9 h post-administration, although the differences were not significant. The blood

pressure of both groups returned to the initial values after 24 h.

15
3.4. Chronic antihypertensive effect

The general condition of SHRs was good throughout the experimental period. As shown in Fig. 3,

daily water consumption, body weights, and urine volumes were not different between either group,

although food consumption in the eggplant group was significantly higher on day 27, compared with

the control group. Fig. 4 shows serial changes in blood pressures. The SBP of the eggplant group

was significantly lower than that of the control group on days 7, 14, and 28 (by 10.1, 12.3, and 16.1

mm Hg, respectively). The DBP also decreased (by 10.4 mm Hg) on day 28. Quantitative analysis of

the catecholamine levels in urine is shown in Fig. 5. AD and NAD excretion in the eggplant group

were significantly lower on days 7 and 21, compared with the control group. In addition, urine AD,

NAD, and DA levels were significantly lower on day 27, compared with the control group.

4. Discussion

In this study, we demonstrated that eggplant powder could lower acute and chronic blood pressure

levels in SHRs by oral administration. The main compound in the eggplant powder responsible for

the antihypertensive effect was concluded to be ACh, as discussed below.

Green coffee beans and tomatoes (Solanum lycopersicum) have been reported to lower blood

pressure in vivo (Suzuki et a0000, 2001, Yoshimura et al., 2010). Green coffee beans were used as

16
green coffee extract or diet containing it, GABA-rich tomato was used as 0.5% methyl cellulose

solution or diet containing dried powder. CA and GABA were found to mediate the blood

pressure-suppressive effects of single and chronic oral SHR administration, respectively. A diet

containing 1% (w/w) green coffee extract (28% CA content) and a diet containing 8% tomato

powder (0.18% GABA content) were used for the chronic administrations. The effective doses of

coffee bean extract and CA were estimated to be 177 mg/kg b.w. and 0.501 mmol/kg b.w.,

respectively, in SHRs of the same age that were administered the same average food intake (20 g/rat

per day) (DMCRA, 2010). In addition, the effective doses of the dried tomato powder and GABA

were calculated to be 10.8 mg/kg b.w. and 0.105 mmol/kg b.w., respectively. The eggplant samples

used in this study contained GABA and CA, and the ingested amounts in the chronic-administration

test were 0.0465 and 0.0432 µmol of GABA and CA per kg b.w., respectively. These studies showed

that the blood pressure-lowering effect of these compounds is dose-dependent; thus, the levels of

GABA and CA in the eggplant samples were probably too low to exert blood pressure-suppressing

effects in this study. Thus, compounds other than GABA and CA were potentially responsible for the

antihypertensive effect, although the contents of GABA and CA were more abundant than that of

ACh.

The SHR aorta assays revealed that an ACh standard and ACh from eggplant powder showed

nearly the same effective concentration in terms of vasodilation, suggesting that ACh in eggplant

17
was responsible for this effect. The influence of potential contaminants was estimated to be low

because the concentration of the powder was only 0.466 µg/ml; therefore, the bioactivity of ACh in

eggplant powder was equal to that of the standard. The vasodilating activity of the eggplant powder

was likely mediated through the M3 mAChR on the blood vessels, considering that the effect was

significantly inhibited in the presence of 4-DAMP, an M3 mAChR antagonist. The M3 mAChR also

exhibits effects on the digestive system (Wess, 2004), and orally administered ACh from eggplants

could act on the receptor in the digestive system as well.

Excretion of AD and NAD into the urine decreased following eggplant administration in SHRs

and was linked with blood pressure suppression, suggesting that the antihypertensive mechanism of

eggplant involves suppressing the secretion of hypertensive catechol amines. These compounds are

secreted from sympathetic synaptic terminals, transferred into the blood, and excreted in the urine.

Therefore, the decreased urinary NAD and AD levels indicated that suppression of sympathetic

nerve activity occurred in SHRs. Orally administered bethanechol, a cholinergic agent, acts on the

M3 mAChR and stimulates the parasympathetic nerve to promote gastric secretion, gastrointestinal

peristaltic motion, and urination (Eisai Co., Ltd., 2013). Increased parasympathetic nerve activity

usually suppresses sympathetic nervous activity by reciprocal inhibition. Based on the results

obtained in this study, the ACh was estimated to be the main compound of eggplants responsible for

antihypertensive activity, which may have occurred by suppressing sympathetic nervous activity.

18
 Peptides derived from sardines, which can function as orally available ACE inhibitors, were

previously investigated for antihypertensive effects in SHRs. For example, the oral administration of

an ACE-inhibitory fraction from sardines showed a chronic hypotensive effect at a dose of 10 mg/kg

b.w. (Seki., 1999). In this study, the chronic effective dose of the eggplant powder in SHRs was only

0.821 mg/kg b.w. per day. The chronic effective dose for an adult person weighing 60 kg was

estimated to be 13.1 mg/day of the eggplant powder, based on Kleiber's law (Kleiber., 1961). Hence,

eggplant can potentially serve as an important antihypertensive food. The "Family Income and

Expenditure Survey" and "Retail Price Survey" (Ministry of Internal Affairs and Communications,

Statistics Bureau, Japan, 2015) provide estimates showing that individuals consume an average of

11.1 eggplants (90 g/fruit) a year, equivalent to 2.7 g of fresh eggplant per day per person, where the

amount of eggplant powder was calculated to be 164–175 mg based on the yields of eggplant

powder found in this study. Thus, individuals may already be benefiting from eggplant consumption

in the hypertensive condition.

ACh is easily decomposed by ACh esterase in vivo; thus, orally administrated ACh has not been

investigated. In addition, studies on ACh in vegetables have been conducted only in terms of the

ACh content or bio-physiology in the plant body (Kawashima, 2010). We demonstrated the

antihypertensive effects of eggplants rich in ACh and propose that ACh lowered hypertension by

suppressing sympathetic nerve activity through the M3 mAChR, although the relevance of the novel

19
function of ACh needs to be investigated in more detail in terms of the mechanism and effective

dosage. Though people consume ACh in daily dishes, it is difficult to estimate the quantities of ACh

expected for an antihypertensive effect, based on previous research (Kawashima, 2010). The present

research suggests that eggplant powders could provide antihypertensive effects at small amount in

SHRs. In the future, the effect of daily ingestion of eggplant powder on blood pressure and the

effective dosage should be investigated in a clinical trial. Globally, an enormous amount of eggplant

(51 million tons in 2016, FAO) is produced, as it is a popular vegetable; thus, its safety has been

empirically demonstrated. We hope that this report will be beneficial by publicizing the health

effects of eggplant and promoting further studies and applications of eggplant as an antihypertensive

food.

5. Conclusion

Our results validated the hypothesis that the oral administration of eggplants can induce

hypotensive effects in spontaneously hypertensive rats. These rats have genetic factors causing

hypertension with ageing, and are used as a model of human essential hypertension, indicating that

eggplant can be expected to exert hypotensive effects on people with similar genetic factors.

Hypertension is a major symptom of lifestyle-related diseases, and our results also suggest the

potential of eggplant as a functional food to prevent hypertension and its complications in daily life.

20
Acknowledgements

We would like to thank Editage (www.editage.com) for English language editing and

publication support.

Declarations of interest: none

Role of the funding source

This work was supported by the Special Scheme Project on Vitalizing Management

Entities of Agriculture, Forestry and Fisheries from the NARO Biooriented Technology Research

Advancement Institution (Project Number 16932813). The funding source had no role in the study

design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the

decision to submit the article for publication.

21
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Figure legends

Fig. 1. Concentration–vasorelaxation curves of thoracic aorta rings from SHRs. (a) ○: treatment

with ACh (n = 6), □: co-treatment with an ACh standard and 4-DAMP (n = 6). (b) ●: treatment

with eggplant powder suspension (n = 6), ■: co-treatment with eggplant powder suspension and

4-DAMP (n = 6). Each data point and error bar represent the mean ± S.E. *p < 0.05, **p < 0.01,

versus treatment with ACh (a) or eggplant powder (b), as evaluated by Student’s t-test. 4-DAMP,

1,1-dimethyl-4-diphenylacetoxypiperidinium iodide

Fig. 2. Changes in the SBP (a) and DBP (b) of SHRs after a single oral administration of eggplant

suspension. SHRs were administered pure water as a control. ●: treatment with eggplant powder

suspension (n = 6), 〇: control treatment (n = 6). Each data point and error bar represent the mean ±

S.E. *p < 0.05, versus the control group, as evaluated by Student’s t-test. SBP, systolic blood

pressure; DBP, diastolic blood pressure

Fig. 3. Daily food consumption (a), water consumption (b), body weight (c), and 24-h urine volume

(d) of SHRs during chronic administration of eggplant powder suspension. SHRs were administered

pure water as a control. ●: treatment with eggplant powder suspension (n = 6), 〇: control

treatment (n = 6). Each data point and error bar represent the mean ± S.E. **p < 0.01, versus the

27
control group, as evaluated by Student’s t-test.

Fig. 4. Changes in SBP (a) and DBP (b) of SHRs during chronic administration of eggplant powder

suspension. The control group was administered pure water. ●:treatment with eggplant powder

suspension (n = 6), 〇: control treatment (n = 6). Each data point and error bar represent the mean ±

S.E. *p < 0.05, **p < 0.01, versus the control group, as evaluated by Student’s t-test. SBP, systolic

blood pressure; DBP, diastolic blood pressure

Fig. 5. Excretion of adrenaline (AD) (a), noradrenaline (NAD) (b), and dopamine (DA) (c) in 24-h

urine samples of SHRs during chronic administration of eggplant suspension. The control group was

administered pure water. ●:treatment with eggplant suspension (n = 6), 〇: control treatment (n =

6). Each data point and error bar represent the mean ± S.E. *p < 0.05, **p < 0.01, ***p < 0.001,

versus the control group, as evaluated by Student’s t-test.

28
Highlights

 Oral eggplant administration induced acute and long term antihypertension in

SHRs.

 Eggplant was found to be rich in ACh.

 ACh activated the M3mAChR, causing vasorelaxation comparable to a standard

reagent.

 Eggplant consumption lowered AD, NAD, and DA levels in the urine.

 ACh may be the major antihypertensive component in eggplants.

29
(a) 350 (b) 5000
300 4500

NAD excretion (ng)

4000
AD excretion (ng)

250 3500
200 3000
2500
150
2000 *
** ***
100 1500 eggplant
* eggplant 1000
50 control
control
500
0 0
0 7 14 21 28 0 7 14 21 28
Day Day
(c) 12000

10000
DA excretion (ng)

8000

6000
**
4000
eggplant
2000 control
0
0 7 14 21 28
Day
(a) 40 (b) 40
Change in SBP (mm Hg)

Change in DBP (mm Hg)

30 30

20 20

10 * ** 10
* *

0 eggplant 0 eggplant
control control
-10 -10
0 7 14 21 28 0 7 14 21 28
Day Day
(a) 24 (b) 36
Food consumption (g)

Water consumption (g)

22 34

32
20
**
30
18
28
16 eggplant eggplant
control 26 control
14 24
3 7 10 14 17 21 24 28 3 7 10 14 17 21 24 28
Day Day

(c) 360 (d) 22

20

Urine volume (mL)

340 18
Body weight (g)

16
320
14
300 12
10
280 eggplant eggplant
control 8 control
260 6
3 7 14 21 28 0 7 14 21 28
Day Day
(a) 10 (b) 10

Change in DBP (mm Hg)

Change in SBP (mm Hg)

0 0

-10 -10
*

-20 -20
eggplant eggplant
*
control control
-30 -30
0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24
Time after administration (h) Time after administration (h)
 (a) (b)
* * ** **
0 ** ** 0

20 20
Vasorelaxant (%)

Vasorelaxant (%)
40 40

60 60 eggplant powder
ACh
co-treatment with
80 co-treatment with 80 eggplant powder and 4-DAMP
ACh and 4-DAMP

100 100
0 10-3 10-2 10-1 10-0.5 0 10-3 10-2 10-1 10-0.5
ACh concentration in organ bath (µmol/L) ACh concentration in organ bath (µmol/L)