Journal of Ethnopharmacology 72 (2000) 53 – 59

www.elsevier.com/locate/jethpharm

Effects of aqueous extracts of Apocynum 6enetum leaves on

spontaneously hypertensive, renal hypertensive and
NaCl-fed-hypertensive rats
Dong-Wook Kim, Takako Yokozawa, Masao Hattori *, Shigetoshi Kadota,
Tsuneo Namba
Institute of Natural Medicine, Toyama Medical and Pharmaceutical Uni6ersity, 2630 Sugitani, Toyama 930 -0194, Japan

Received 30 April 1999; received in revised form 1 February 2000; accepted 22 February 2000

Abstract

Effects of aqueous extracts of Apocynum 6enetum leaves (Luobuma extracts) on the blood pressure were evaluated
in hypertensive animal models, such as spontaneously hypertensive rats (SHR), renal hypertensive rats and
NaCl-induced hypertensive rats. In SHR, administration of Luobuma (heat-processed and unprocessed leaves)
extracts at a dose of 70 mg/rat per day significantly decreased the systolic blood pressure value, but their decreasing
effects were weaker than that of captopril. The urine volume, and the urinary Na+, K+ and protein excretions were
not significantly different between Luobuma-treated and untreated groups. In 3/4 nephrectomized rats, the Luobuma
extracts significantly decreased the systolic blood pressure value, accompanied by significant increases of the urine
volume and the urinary Na+ and K+ excretions. Furthermore, they decreased the blood urea nitrogen (BUN) level.
In NaCl-induced hypertensive rats, the Luobuma extract decreased the systolic blood pressure value. However, it did
not change the urinary excretions of Na+, K+ and protein. The BUN level was lower than that of control rats, but
the serum total cholesterol (TC) level did not changed. From these findings, the Luobuma extracts have an
anti-hypertensive effect, possibly due to amelioration of the kidney functions in the three experimental animal models.
© 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Apocynum 6enetum L.; Spontaneously hypertensive rat; Renal hypertensive rat; Sodium-induced hypertensive rat

1. Introduction used in traditional medicine. According to Zhu

Apocynum 6enetum L. (Luobuma in Chinese;
Rafuma in Japanese) growing wild in the central
and northwestern areas of China has long been
* Corresponding author. Fax: +81-76-4345060.
E-mail address: saibo421@ms.toyama-mpu.ac.jp (M. Hat-
tori).
and Jiu-Huang-Ben-Cao (Zhu, Ming dynasty: a
Chinese herbal book named ‘‘Herbs that Save
Famine’’), Luobuma was described to have
medicinal effects, such as clearing heat, nourish-
ing heart, tranquilizing spirit, and eliminating
edema through diuresis in terms of traditional
Chinese medicine.

0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 1 9 7 - 5
54 D.-W. Kim et al. / Journal of Ethnopharmacology 72 (2000) 53–59
As regards pharmacological effects of Luobuma
leaves, diuretic (Qing et al., 1988), cholesterol-
lowering (Kim et al., 1998a), anti-LDL oxidation
(Kim et al., 1998b) and anti-lipid peroxidation
(Yokozawa et al., 1997) effects have been re-
ported hitherto.
In the present paper, we describe anti-hyperten-
sive effects of aqueous extracts of Luobuma
leaves, together with changes in biological
parameters, such as urinary and serum elec-
trolytes, blood urea nitrogen (BUN) and total
serum cholesterol levels, following the administra-
tion of Luobuma extracts to spontaneously hyper-
tensive rats (SHR), renal hypertensive rats and
NaCl-induced hypertensive rats.
2. Materials and methods
2.1. Plant materials and a herbal prescription
Luobuma A was A. 6enetum leaves collected in
China. Luobuma B was twice roasted leaves of
Luobuma A at 150– 200°C. This treatment leads
to better taste and aroma of Luobuma tea prod-
ucts, and the product comes onto the Japanese
market as Luobuma tea. Luobuma A (546 g) and
Luobuma B (1.3 kg) were extracted with water
under reflux at 70°C to give water extracts in
yields of 115 (21.1%) and 231 g (17.8%), respec-
tively. Choto-san is the most popular herbal pre-
scription used for hypertensive patients in
traditional Chinese medicine (Sekine et al., 1986;
Ishii et al., 1987), which consists of eleven crude
drugs: Gypsum Fibrosum (5 g), Aurantii Nobilis
Pericarpium (3 g), Ophiopogonis Tuber (3 g),
Pinelliae Tuber (3 g), Hoelen (3 g), Uncariae
Ramulus et Uncus (3 g), Ginseng Radix (2 g),
Ledebouriellae Radix (2 g), Chrysantheni Flos (2
g), Glycyrrhizae Radix (1 g) and Zingiberis Rhi-
zoma (1 g). In this experiment, a Choto-san ex-
tract was prepared by extracting the above crude
drug mixture (28 g) with boiling water (1000 ml)
for 60 min, filtrating the solution with gauze
and evaporating the solvent under reduced pres-
sure.
2.2. Effect of Luobuma extracts on spontaneously
hypertensi6e rats (SHR)
Male SHR/Izm rats with a body weight of
200–210 g were placed in metabolic cages, kept at
259 2°C and 6095% relative humidity in a room
with a 12 h light-dark cycle, fed a commercial
pellet chow (Type CE-2, CLEA Japan, Tokyo,
Japan). Rats were divided into four groups of
eight rats each (control and Luobuma-A-, Lu-
obuma-B- and captopril-treated groups). Animal
experiments were carried out over a period of 40
consecutive days starting at 8 weeks of age. The
blood pressure of rats was measured on days 0,
10, 20, 30 and 40.
2.3. Effect of Luobuma extracts on renal
hypertensi6e rats
The experiment was performed in three groups
(control, Luobuma A and Luobuma B) of male
Wistar strain rats, 7 weeks old. All rats were
anesthetized with sodium pentobarbital (50 mg/
kg, i.p.), and two poles of the left kidney were cut
following tight stricture with a silk suture. After
10 day, an artery of the right kidney was ligated
with a silk suture. All rats were fed rat chow
(Type CE-2). On 10 days later, all rats were
placed in metabolic cages for collecting urine dur-
ing 2 days, and then fed a commercial diet and
treated with Luobuma extracts for 100 days. The
blood pressure of rats was measured at an interval
of 20 days through the entire experimental period.
2.4. Effect of Luobuma extracts on NaCl-
induced hypertensi6e rats
Male Wistar strain rats with 200–210 g were
placed in metabolic cages. During the adaption
period of 7 days, the rats were fed a commercial
chow (type CE-2). The rats were then divided into
four groups of eight rats each (control, Luobuma-
B-, Choto-san- and captopril-treated groups).
They were administered powdered chow (MM3,
Sankyo Labo Service, Tokyo, Japan) containing
5% NaCl, 1% cholesterol and 0.5% cholic acid.
This experiment was carried out over a period of
60 days.
 D.-W. Kim et al. / Journal of Ethnopharmacology 72 (2000) 53–59
Throughout the experimental period, no statis-
tically significant difference in body weight was
observed between test sample-treated and un-
treated rats. The food intake of each rat was
essentially proportional to its change in weight.
Animals were periodically placed in metabolic
cages, and then the 24 h urine was collected for
determination of Na+, K+ and protein excretions
and urine volume.
During the experimental period, aqueous solu-
tions of Luobuma extract, Choto-san extract and
captopril were given orally to rats every day as
drinking water. The dose of Luobuma extract or
Choto-san extract was adjusted to 70 mg/rat per
day by regulating its concentration in relation to
water consumption. The dose of captopril was
adjusted to 30 mg/rat per day. Control rats were
given an equivalent amount of water.
2.5. Measurement of blood pressure, and urine
and serum constituents
Systolic, mean or diastolic blood pressure in
each conscious rat was determined by a tail pulse-
up method and recorded with an automatic
sphygmotonograph (Muromachi Kikai, Tokyo,
Japan). Blood pressure values were expressed as
the means of four consecutive determinations.
Analysis of Na+ and K+ was performed with an
electrolyte measurement apparatus (AHS/Japan
Corporation, Tokyo, Japan) based on an ion elec-
Fig. 1. Effect of Luobuma extracts on systolic blood pressure in SHR (A), 3/4 nephrectomized- (B) and NaCl-fed (C) rats ,
control;
, Luobuma A;
, Luobuma B; , Choto-san; , captopril; statistical significance: aPB 0.05, bPB 0.01, cPB 0.001
versus control rats.
55
trode method. The urinary protein concentration
was measured by precipitation with 3% sulfosali-
cylic acid. The BUN level was determined with
commercial kits (BUN kainos, Kainos, Tokyo,
Japan) according to a modified urease-indole-phe-
nol method. The serum total cholesterol level was
enzymatically determined with commercial kits
(Chol I E, Wako Pure Chemical Industries, Os-
aka, Japan).
2.6. Statistics
The significance of difference between control
and sample-treated groups was evaluated byDun-
nett’s t-test. The values were expressed as mean 9
SE.
3. Results
3.1. Effects of Luobuma extracts on blood
pressure
Fig. 1A shows that the effects of oral adminis-
tration of Luobuma extracts on the systolic blood
pressure in SHR for 40 days. At the starting point
of experiment, the systolic blood pressure in all
groups was approximately 180–190 mmHg in
SHR, but the value in a control group (water-ad-
ministered group) was increased up to 224 mmHg
on day 40. However, in both of Luobuma-A- and
56 D.-W. Kim et al. / Journal of Ethnopharmacology 72 (2000) 53–59
B (twice roasted leaves)-treated groups, the sys-
tolic blood pressure, as well as the mean and
diastolic blood pressures (data not shown), was
decreased significantly, compared to that of the
control; on day 40, the systolic blood pressure
in the Luobuma-treated groups was decreased
by ca. 10 mmHg, to the control value. The de-
creasing effects of the Luobuma extracts were
less than that of captopril (an inhibitor of an-
giotensin-converting enzyme). Similarly, the
mean and diastolic blood pressures were also
decreased by administration of Luobuma ex-
tracts and captopril (data not shown).
Fig. 1B shows the effects of Luobuma ex-
tracts on the systolic blood pressure in 3/4
nephrectomized rats. After nephrectomization,
the systolic blood pressure was gradually in-
creased to ca. 145 mmHg, the value being ap-
preciably higher than that of normal rats (8
weeks old) (ca. 120 mmHg). Furthermore, the
blood pressure of a control group (3/4 nephrec-
tomized) was increased up to ca. 155 mmHg
during 100 days, indicating to be in the mild
hypertensive state. However, in the Luobuma-
treated groups, the systolic blood pressure was
significantly decreased. This effect was marked
as the administration period was extended over
40 days, when the hypertension of the control
group became more evident. On day 100, the
blood pressure in the Luobuma-treated groups
was close to that of normal rats. There was no
appreciable difference in systolic blood pressure
between groups treated with Luobuma A and B.
Fig. 1C shows that the effects of the Lu-
obuma extract (twice roasted leaves) on the sys-
tolic blood pressure in NaCl-induced
hypertensive rats. The systolic blood pressure in
rats administered with 5% NaCl containing 1%
cholesterol, began to increase on day 30, and its
value reached to ca. 145 mmHg (mild hyperten-
sion value) on day 60. However, the systolic
blood pressure in the Luobuma B-treated group
was significantly decreased from day 10, when
compared to that of the control group, and its
decreasing effect continued during the whole pe-
riod of experiment.
A group treated with Choto-san, a herbal pre-
scription used for the treatment of hypertension
in traditional Chinese medicine, resulted in a
significant decrease of the systolic blood pres-
sure. The effect of Luobuma was comparable in
potency to that of a Choto-san decoction. Ad-
ministration of captopril led to an abrupt de-
crease in systolic blood pressure on day 10 (ca.
102 mmHg), and the decreasing effect, com-
pared to the control value, was continued dur-
ing the whole period of experiment. The similar
findings were observed for the mean and dias-
tolic blood pressures (data not shown).
3.2. Effects of Luobuma extracts on urinary
parameters
In the SHR, the urinary parameters were not
significantly different between the control and
the Luobuma-treated group. However, the
parameters, such as urine volume, and Na+ and
K+excretions, were significantly higher in the
captopril-treated group on days 20 and 40, com-
pared to the control values, except for the
protein excretion value (data not shown).
In the 3/4 nephrectomized rats, the urine vol-
ume of the Luobuma-treated groups was slightly
increased from day 60, compared with that of
the control group (Table 1). Then, the volume
was significantly increased in the Luobuma-A-
treated group on day 80 and 100 (PB 0.01 and
PB 0.05, respectively), and in the Luobuma B-
treated group on day 100 (PB 0.01). The uri-
nary Na+ and K+ excretions were increase in
both Luobuma-treated groups. Especially, in the
Luobuma-B-treated group, the Na+ excretion
was significantly increased on day 60 and 100
(PB 0.05), and the K+ excretion was increased
in the almost entire experimental period. The
urinary protein excretion was also significantly
decreased on day 40 and 60 in the Luobuma-B-
treated group.
On the other hand, in the NaCl-fed rats, the
urine volume was increased approximately 2.1–
2.6 times on day 20–60, compared to that on
day 0. However, no significant change in urinary
parameters were observed in rats given the Lu-
obuma B extract (data not shown).
 D.-W. Kim et al. / Journal of Ethnopharmacology 72 (2000) 53–59 57

Table 1
Effect of Luobuma extracts on urine volume and urine constituents in 3/4 nephrectomized rats

Day Group Urine volume (ml/24 h) Na (mM/24 h) K (mM/24 h) Protein (mg/24 h)

0
Control 32.292.3 1.74 9 0.06 3.50 9 0.10 18.3 92.3
Luobuma A 35.89 2.7 1.72 9 0.12 3.87 9 0.31 15.5 9 2.3
Luobuma B 36.39 5.2 1.67 9 0.16 3.42 9 0.34 16.4 95.9
20
Control 29.59 2.5 1.41 9 0.13 3.03 9 0.19 22.3 9 2.9
Luobuma A 28.29 1.7 1.57 9 0.11 3.80 9 0.15c 24.4 9 3.9
Luobuma B 29.2 9 2.7 1.38 90.12 3.66 90.19c 17.6 9 5.4
40
Control 26.09 1.5 1.34 9 0.10 3.23 9 0.19 26.0 9 2.9
Luobuma A 26.3 9 3.1 1.30 90.18 3.01 9 0.40 28.6 9 5.5
Luobuma B 30.29 3.1 1.41 90.17 3.33 9 0.21 17.5 9 5.9a
60
Control 29.89 1.7 1.66 9 0.08 3.35 9 0.09 38.8 9 4.2
Luobuma A 33.2 9 2.7 1.53 90.12 3.52 9 0.24 42.5 9 6.1
Luobuma B 34.49 4.5 1.78 90.16a 3.86 9 0.19b 24.0 9 8.1b
80
Control 31.59 1.7 2.08 9 0.12 3.49 9 0.15 43.5 9 4.1
Luobuma A 39.59 4.3b 2.48 9 0.11b 4.11 9 0.09c 48.8 9 10.6
Luobuma B 35.1 9 3.9 2.16 9 0.21 4.05 90.27b 29.2 9 11.5
100
Control 34.69 2.1 2.06 9 0.08 3.76 9 0.19 57.1 9 7.9
Luobuma A 41.79 4.9a 2.15 9 0.11 3.55 90.31 60.1 914.5
Luobuma B 46.0 93.7b 2.43 9 0.30a 4.83 9 0.35c 41.2 9 12.9

a
Statistical significance PB0.05 versus control rats.
b
Statistical significance PB0.01 versus control rats.
c
Statistical significance PB0.001 versus control rats.

3.3. Effect of Luobuma extracts on serum 4. Discussion

constituents
Table 2 shows the effects of Luobuma extracts
on serum constituents in SHR, 3/4 nephrec-
tomized rats and NaCl-fed rats. The Na+ and K+
concentrations in the Luobuma and other samples
treated groups were not significantly changed,
compared with those of the control group. How-
ever, the BUN levels in the 3/4 nephrectomized
rats given Luobuma A and B extracts were signifi-
cantly decreased (PB 0.05 and P B 0.01, respec-
tively). The BUN level of the Luobuma- B-treated
group in NaCl-fed rats was significantly lower
than that of the control group. The TC levels
were not significantly changed.
The therapeutic agents for hypertension, such as
diuretic, a- and b-blockers, Ca2 + -channel blockers,
vasodilators and angiotensin-converting enzyme
inhibitors, have strong depressive effects on blood
pressure, but show some adverse effects, related to
lipid metabolism, proteinuria etc. For this reason,
many studies have been conducted to find more
suitable antihypertensives from natural sources.
The present study showed that chronic oral
administration of the Luobuma extract decreased
the blood pressure in SHR, 3/4 nephrectomized-
and NaCl-induced hypertensive rats, although the
decreasing effects were not so strong as that of
captopril.
58 D.-W. Kim et al. / Journal of Ethnopharmacology 72 (2000) 53–59

Table 2
Effects of Luobuma extracts on serum parameters

Experiment number Group Na (mM/l) K (mM/l) Protein (g/dl) BUN (mg/dl) TC (mg/dl)

1
Control 144.49 0.3 8.32 90.13 6.25 9 0.06 – -
Luobuma A 144.8 9 1.0 8.879 0.29 6.29 9 0.10 – –
Luobuma B 145.29 0.8 8.33 9 0.24 6.32 9 0.15 – –
Captopril 144.09 0.6 8.16 90.13 6.16 9 0.08 – –
2
Control 149.49 0.9 7.80 90.58 5.70 9 0.14 40.3 9 3.6 –
Luobuma A 150.0 9 1.0 7.259 0.17 5.46 9 0.10 35.3 9 2.1a –
Luobuma B 149.89 0.6 7.129 0.17 5.52 90.21 32.5 91.6b –
3
Control 145.59 0.9 7.46 90.35 – 21.4 9 0.71 139.4 9 3.7
Luobuma B 146.09 0.5 7.06 90.07 – 19.4 9 0.5a 133.0 96.2
Chotosan 146.3 9 0.6 7.34 90.21 – 21.6 9 0.7 130.2 910.1
Captopril 146.69 0.7 7.01 9 0.15 – 20.8 90.9 126.8 911.8

a
Statistical significance: PB0.05 versus control rats.
b
Statistical significance: PB0.01 versus control rats.

The Luobuma-treated groups (Luobuma A and
B) in the SHR were not significantly changed in
urinary Na+ and K+ excretions, compared to the
control group. Furthermore, the Luobuma-
treated groups in 3/4 nephrectomized rats showed
statistically significant differences in urine volume
and urinary Na+ and K+ excretions. Our finding
was in agreement with that of Qing et al. (1988),
who reported that the Luobuma leaves in nor-
motensive animals showed diuretic effects, in-
creasing the urine volume and urinary Na+ and
K+ excretions. Nishibe et al. (1986) also reported
that the Luobuma extract and its phenolic con-
stituents decreased the blood pressure by single
injection (10–50 mg/kg, i.v.) to SHR.
On the other hand, the BUN levels of the
Luobuma-treated groups in 3/4 nephrectomized-
and NaCl-induced hypertensive rats were lower
than that of the control group. This finding on the
BUN level, as well as on the Na+ and K+ excre-
tions, suggested the increase of glomerular filtra-
tion, which may be explained by the increase of
bradykinin or decrease of angiotensin II produc-
tion (Terragno et al., 1975; Hutchinson et al.,
1980; Koike et al., 1980). Since urea is a final
product in the protein metabolism, being pro-
duced in the liver, circulated in the blood, and
excreted to urine through the kidney, the BUN
level is important for the evaluation of the kidney
function.
Based on these findings, it is suggested that the
Luobuma extract has anti-hypertensive effects in
hypertensive model animals, due to amelioratinon
of the kidney functions, such as the increased
excretions of urine volume and urine electrolytes
and the decreased BUN levels. However, the anti-
hypertensive mechanism of the Luobuma extract
should be clarified further in connection with the
renin-angiotensin-aldosterone and kallikrein-kinin
systems.
References
Hutchinson, J.S., Mendelssohn, F.A.O., Doyle, A.E., 1980.
Blood pressure responses of conscious normotensive and
spontaneously hypertensive rats to intracerebral and pe-
ripheral administration of captopril. Hypertension 2, 546 –
550.
Ishii, K., Kano, T., Ando, J., 1987. Pharmacological effect of
Diao-Teng-San, a blended traditional; Chinese herb
medicine, in spontaneously hypertensive rats (SHR) and
normo-tensive Wistar – Kyoto (WKY) rats. J. Trad. Med.
4, 107 – 115.
Kim, D.W., Yokozawa, T., Hattori, M., et al., 1998a. Effects
of aqueous extracts of Apocynum 6enetum leaves on hyper-
cholesterlaemic rats. Phytother. Res. 12, 46 – 48.
 D.-W. Kim et al. / Journal of Ethnopharmacology 72 (2000) 53–59
Kim, D.W., Yokozawa, T., Hattori, M., et al., 1998b. Lu-
obuma leaf inhibits oxidation of low-density lipoprotein in
cholesterol-fed rats. J. Trad. Med. 15, 40–44.
Koike, H., Ito, K., Miyamoto, M., Nishino, H., 1980. Effects
of long- term blockade of angiotensin converting enzyme
with captopril (SQ 14 225) on hemodynamics and circulat-
ing blood volume in SHR. Hypertension 2, 299–303.
Nishibe, S., Sakushima, A., Yasui, S., et al., 1986. Phenolic
compounds of Apocynum 6enetum leaves and pharmaco-
logical effect. J. Trad. Med. 3, 244–245.
Qing, Z.N., Song, L.H., Gu, Z.L., et al., 1988. An experimen-
tal observation on the diuretic effect of an extract of
Luobuma (Apocynum 6enetum) leaves. Bull. Chin. Mater.
Med. 13, 44 – 46.
59
Sekine, I., Shichijo, K., Nishimori, I., et al., 1986. Effect of
Oren-gedoku-to and Tyoto-san on hypertensive lesions of
stroke-prone spontaneously hypertensive rats (SHRSP). J.
Trad. Med. 3, 71 – 76.
Terragno, D.A., Crowshaw, K., Terragno, N.A., McGriff,
J.C., 1975. Prostaglandin synthesis by bovine mesenteric
arteries and veins. Circ. Res. Suppl 36, 76 – 80.
Yokozawa, T., Dong, E., Kashiwagi, H., et al., 1997.
In vitro and in vivo studies on anti-lipid peroxidation
effect of extract from Luobuma leaves. Nat. Med. 51,
325 – 330.
Zhu L., Jiu-Huang-Ben-Cao, Ming dynasty, a Chinese
herbal book named ‘‘Herbs that Save Famine’’ China,
p.4.