Effect of Shosaikoto, Daisaikoto and Sannoshashinto (traditional Japanese and Chinese medicines) on experimental hyperlipidemia in rats

255

Journal of Ethnopharmacology, 26 (1989) 255-269 Elsevier Scientific Publishers Ireland Ltd.

EFFECT OF SHOSAIKOTO, DAISAIKOTO AND SANNOSHASHINTO (TRADITIONAL JAPANESE AND MEDICINES) ON EXPERIMENTAL HYPERLIPIDEMIA

MASAOMI

UMEDA,

SAKAE

AMAGAYA

and YUKIO

Department of Pharmacognosy, Faculty of Pharmaceutical Z-1, Tanube-dori, Mizuho-ku, Nagoya 467 (Japan1 (Accepted

CHINESE IN RATS

OGIHARA Sciences,

Nagoya City University,

January 31, 1989)

Summary Effect of Sannoshashinto, Shosaikoto and Diasaikoto, Japanese and Chinese traditional medicinal mixtures (kampohozail, on cholesterol-induced hypercholesterolemia, aging-induced hyperlipidemia and cholesterol turnover were studied in rats. Sannoshashinto, Shosaikoto and Daisaikoto reduced the hypercholesterolemia induced by a high cholesterol diet and Sannoshashinto and Daisaikoto improved the atherogenetic index. Liver total cholesterol as increased by a high cholesterol diet was reduced by all three kampohozai. Furthermore, the increases of serum and liver triglyceride were also inhibited. In an experiment using untreated aging rats, both serum total cholesterol and serum triglyceride levels were increased. The aging-induced increases of serum total cholesterol were inhibited by Sannoshashinto and Daisaikoto and the increases of serum triglyceride were reduced by all three kampohozai. These drugs showed no effect on cholesterol biosynthesis in liver. Sannoshashinto, however, appeared to accelerate the disappearance of cholesterol from blood, while Daisaikoto inhibited the cholesterol absorption from the intestine.

Introduction

Hypercholesterolemia is one of the risk factors for atherosclerosis (Gore et al., 1960; Hirst et al., 1960; Marek et a!., 1962; Consensus Conference, 1985; Gotto et al., 19861 and two different types of hypercholesterolemia are known, an exogenous hypercholesterolemia and an endogenous hypercholesterolemia. These are brought about by an excessive intake of cholesterol and an excessive production of cholesterol in liver or a lowering catabolism of cholesterol, respectively. Furthermore, these phenomena are accelerated by aging. Endogenous hypercholesterolemia is associated with a genetic defect 037%8741/89/$05.60 0 1989 Elsevier Published and Printed in Ireland

Scientific Publisher Ireland Ltd.

256

relating to the lack of low-density lipoprotein (LDL) receptors which catabolise LDL in organs. Experimentally in animals, hypercholesterolemia can be produced by the administration of a high cholesterol diet (HCD). On the other hand, aging produces many diseases, and many appear related to hyperlipidemia and/or atherosclerosis (Eisenberg et al., 1969; McNamara et al., 1971; Berensen, 1977; Bierman and Ross, 1977; Sadoshima et al., 19801. In the present study, a natural onset hyperlipidemia obtained by allowing rats to age for 110 weeks and a HCD-induced hyperlipidemia were used to evaluate the actions of three kampohozai (Japanese and Chinese traditional medicines). These kampohozai have been clinically used to prevent atherosclerosis. Among these, Sanoshashinto is perhaps the most well known and has also been widely used to treat hypertension and internal fever. Compound Sannoshashinto consists of Coptis japonica rhizome, whose main ingredient is berberine, Scutelluriu baicalensis roots, whose main ingredients are baicalin and baicalein and Rheum tanguticum rhizome, whose main ingredients are sennosides and tannins. Berberin is reported to show anti-inflammatory (Fujimura et al., 19701, anti-atherogenetic (Kuwano, 19701 and central nervous system regulating effects (Yamahara, 19761. Baicalin and baicalein are known to possess anti-allergy (Koda et al., 1979 a,bl and an antiatherosclerotic (Aonuma et al., 1957) activity in addition to an ability to inhibit cyclooxygenase and lipoxygenase (Sekiya and Okuda, 19821. The tannins present may possess strong anti-oxidant and anti-inflammatory actions. Daisaikoto has been recently reported to reverse hypercholesterolemia (Teramoto et al., 19861 and atherosclerosis (Haranaka et al., 19861 in rats. This herbal mixture contains Rheumm tanguticum rhizome, Zingiber officinale rhizome, Paeonia lactiflora root and Bupleurum falcatum root. Zingiber officinale rhizome has been reported to inhibit cyclooxygenase activity (Kiuchi et al., 19821, and the paeonol and paeoniflorine found in Paeoniae luctiflora root are reported to show anti-inflammatory (Yamahara et al., 1982; Kubo et al., 1984; Sugishita et al., 19841, ant-allergy (Kubo et al., 1984) and antithrombosis (Kubo et al., 19821 actions. Saikosaponins contained in Bupleurum falcatum root are reported to show anti-inflammatory (Yamamoto et al., 1975a1, antihepatotoxic (Shibata et al., 19761, anti-ulcer (Shibata et al., 19731 and antihypercholesterolemic (Yamamoto et al., 197513; Yokoyama et al., 1981; Yamamoto et al., 1985) actions. Although the composition of Shosaikoto is similar to that of Daisaikoto, Shosaikoto lacks Rheum tanguticum rhizome, Citrus aurantium fruit and Paeonia luctiflora root and adds Panax ginseng Root and Glycyrrhiza glabra root. Panax ginseng root which contains ginsenosides is reported to show central nervous stimulating (Takagi, 19821, antistress (Takagi, 19821, antidiabetic (Takagi, 1982; Yokozawa et al., 19841, antihypertensive (Kishi et al., 19851 and antihypercholesterolemic (Takagi, 1981; Yamamoto et al., 1985) effects. Glycyrrhizin is known to possess anti-inflammatory (Takagi, 19811, anti-ulcer (Takagi, 19811 and ant-atherosclerotic capacity (Shibata, 19621. The reports cited above support the anti-atherosclerotic actions of these

257

kampohozai to some extent. In this paper, the effects of Sannoshashinto, Daisaikoto and Shosaikoto on hyperlipidemia, an important factor of atherosclerosis, and on cholesterol metabolism are studied. Materials and methods Animals

Male Wistar rats weighing 160-220 g were obtained from Nippon BioSupplies Center (Tokyo, Japan). Male Fischer Fad4 rats, 24 weeks old, were obtained from Charles River (Atsugi, Japan). They were kept in an air-conditioned room and given commercial diet and water ad libitum. Preparation

of kampohozai

Each raw herbal drug in Sannoshashinto, Shosaikoto and Daisaikoto was authenticated and provided by Tsumara Co. Ltd. (Tokyo, Japan). Sannoshashinto, Shosaikoto and Daisaikoto are the lyophilized extracts of the mixtures of 3, 8 and 7 raw herbal drugs, respectively, as shown in Table 1. Each raw herbal mixture was extracted with 700 ml of boiling water for 1 h and the decoction was lyophilized to give a powdered extract. The yields were 3.6 g (Sannoshashintol, 7.2 g (Shosaikotol and 7.2 g (Daisaikotol of kampohozai in the present study was as the powdered extract dissolved in 2 ml of water per rat and all doses were expressed as the weight of powdered extract. Reagents

dl-a-Tocopherolnicotinate was supplied from Tsumura Co., Ltd. (Tokyo, Japan). Clofibrate was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Standard diet (CR-21 and high-cholesterol chow containing 1% cholesterol, 0.2% cholic acid and 2.5% olive oil were purchased from Nihon Clea Ltd. (Tokyo, Japan). [1-14C]Acetic acid (55 mCi/mmol) and [4-14C]cholesterol (53.2 mCi/mmoll were purchased from New England Nuclear (Boston, MA, U.S.A.). Preparation

of cholesterol-induced

hypercholesterolemiu

in rats

Wistar rats were used for the cholesterol-induced hypercholesterolemia. The cholesterol non-treated group was divided into 5 subgroups with 10 rats each. The control group was fed a normal diet for 10 days. The Sannoshashinto group was fed a normal diet and orally administered Sannoshashinto at a dose of 0.25 g/kg once a day. Shosaikoto and Daisaikoto groups were fed normal diets and orally administered Shosaikoto and Diasaikoto, respectively, at a dose of 0.5 g/kg once a day. The clofibrate group was fed a normal diet and orally administered clofibrate by dissolving this compound in 2

258

TABLE

1

CRUDE

DRUG

Plant

name

glabra L.

ginseng

C.A. Meyer

Bupleurum

falcatum

Scutellaria

baicalensis

L. Georgi

officinale Roscoe

Zingiber Roscoe Paeonia

lactiflora Pallas

Pine&a

ternata Breitenbach

Zizyphus Rheum

OF THE

KAMPOHOZAI

Family (Part used)

Glycyrrhiza Panax

COMPOSITION

vulgaris

Lam.

tanguticum

Maxim.

Citrus aurantium

L.

Coptis japona’ca

Makino

Legminosae (Root) Araliaceae (Root) Umbelliferae (Root) Labiatae (Root) Zingiberaceae (Root) Paeoniaceae (Root) Araceae (Tuber) Rhamnaceae (Fruit) Polygonaceae (Rhizomal Rutaceae (Fruit) Ranunculaceae (Root)

Composition

(gl

Sannoshashinto

Shosaikoto

-

2.0

-

3.0

_

-

7.0

6.0

1.0

3.0

3.0

-

1.0

1.0

-

-

3.0

-

5.0

4.0

-

3.0

3.0

1.0

_

2.0

-

_

2.0

1.0

-

Daisaikoto

ml of distilled water once a day and giving it at a dose of 0.1 g/kg. The cholesterol-treated group was also divided into five subgroups. Each group was fed a high cholesterol diet (HCD) and orally administered a kampohozai or clofibrate by mixing in 0.2 ml of distilled water at the doses mentioned above. Preparation

of aging rats

Male Fischer F,,, rats were maintained on a normal diet in an air-conditioned room till 72 weeks of age. At that time, the rats were divided into five groups. The control group was bred and maintained on a normal diet. The other four groups were fed normal diets containing Sannoshashinto extracts at a dose of 0.05 g/kg per day, Shosaikoto extracts at a dose of 0.12 glkg per day, Daisaikoto extracts at a dose of 0.12 g/kg per day, and dl-atocopherolnicotinate at a dose of 0.02 g/kg per day as a standard, respectively. All groups were fed the diets mentioned above till 110 weeks of age.

Determination

of serum

and hepatic

lipids

Blood was collected by heart puncture under ether anesthesia and the serum was obtained by centrifugation at 3000 x g for 10 min. Serum total cholesterol (serum TC) and serum triglyceride (serum TGl were determined enzymatically (Iatrolipo TC, New Cleantech TG: Iatron Laboratories Inc., Tokyo, Japan). High density lipoprotein (HDL) was separated using a precipitant (HDL-CZ precipitant, Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan). Hepatic lipids were extracted with chloroform/methanol (2: 1, v/v) after the removal of liver at the end of feeding and homogenization by a Potter homogenizer with a Teflon pestle. Liver total cholesterol (liver TC) and liver triglyceride (liver TG) were determined according to the previously described enzymatic methods. Cholesterol

biosynthesis

According to the method of Demattets (19681, male Wistar rats were killed at 01: 00 h and the liver was quickly removed under ether anesthesia, washed with cold saline solution and blotted on filter paper. An amount of liver weighing about 1 g was placed in a screw-capped tube. After adding 2 ml of 15% ethanolic KOH, the mixture was saponified at 80°C for 2 h until totally dissolved. The non-saponifiable lipids were then extracted three times each with 6 ml of petroleum ether. The combined petroleum ether layer was evaporated in vacua. The residue was dissolved in 1 ml of acetone/ethanol (1: 1, v/v) to which 1 ml of 0.5% of digitonin solution had been added and then allowed to stand overnight. The digitonin-precipitable sterols (DPS) were isolated by centrifugation for 10 min at 1200 x g, washing the resultant precipitate first with 2 ml of acetone/ether (1: 2, v/v) and then with 2 ml of ether. The washed DPS was dissolved in 1 ml of methanol, transferred quantitatively to a scintillation vial and counted after adding toluene. Sannoshashinto at a dose of 0.25 g/kg, Shosaikoto at a dose of 0.5 g/kg and Daisaikoto at a dose of 0.5 glkg were administered once a day for 16 days before killing the rats. [1-14C]Acetic acid (20 &i/O.5 ml isotonic NaCl solution/rat) was intraperitoneally injected 50 min prior to death. Determination

of blood concentration

of [P4C]cholesterol

The intravenous dose of [4-14C]cholesterol was prepared by dissolving in 95% ethanol. Colloidal cholesterol was obtained by adding 0.5 ml of 0.9% NaCl. Following the method of Yagasaki et al. (19841, the resulting mixture was injected intravenously within 1 h of its preparation; each rat was injected 0.5 ml. The oral dose of [4-14C]2 &i of [4-14C]cholesterol per cholesterol was prepared as follows: 2 &i, was dissolved with 6 mg of

260

carrier cholesterol in 156 mg of triolein, the oil phase of which was then suspended by sonication in water containing 7.5 mg of sodium cholate and the final volume was adjusted with water to 2 ml. Each rate therefore received 6 mg of cholesterol plus 2 &i of [4J4C]cholesterol per 2 ml. Kampohozai at doses identical to those in the experiment on cholesterol biosynthesis were administered to rats for 8 days before the cholesterol treatment. Blood was collected 24 h after the labeled cholesterol treatment. The ratio of oral cholesterol absorption at 24 h after isotope administration was calculated as isotope ratio (Zilversmit and Barry Hughes, 1974) as follows: % of the oral dose in an aliquot of blood % of the intravenous

dose in the same aliquot of blood

x 100

Statistics The significance of differences between the control treated groups was tested by means of Student’s t-test.

and kampohozai-

Results

Hypercholesterolemiu

induced

by high cholesteorl

diet (HCDI

Table 2 shows the changes of body weight and liver weight when hypercholesterolemia was induced in rats by feeding a HCD. The doses of

TABLE

2

EFFECT OF KAMPOHOZAI ON HYPERCHOLESTEROLEMIC RATS’ Diet

Drug

Dosage

BODY

N

(g/kg per day) N

N N N N HCD HCD HCD HCD HCD

N SAN SHO DA1 CL0 C SAN SHO DA1 CL0

_ 0.25 0.50 0.50 0.10 0.25 0.50 0.50 0.10

10 10 10 10 10 10 10 10 10 10

AND

LIVER

WEIGHT

IN

NORMAL

OR

Body weight

Relative liver weight

(g)

(g/100 g gody weight)

285 231 288 274 246 248 223 240 223 238

f 7 & i’* f 8 f 7 f lo* f 5 + 4** -c 10 2 11 5 7

4.38 4.07 4.41 4.33 4.08 5.23 4.86 4.93 4.96 5.76

f -+ + 2 + + f 2 -c +

0.02 0.01 0.01 0.01 0.01 0.07 0.15** 0.11** 0.35 0.14**

‘N, Normal; C, control; HCD, high-cholesterol diet; SAN, Sannoshashinto; SHO, Shosaikoto; Daisaikoto; CLO, clofibrate. Each tabular value indicates the mean -C S.E.M. of 10 rats. Significance: *P < 0.05 vs. N/N; **P < 0.05 vs. HCDIC.

DAI,

261

kampohozai were equivalent to five times the doses for humans per day. The normally expected gain of body weight was inhibited by cholesterol feeding (N/N vs. HCD/Cl. In the normal diet groups, Sannoshashinto and clofibrate treatments reduced the gain of body weight, although the other kampohozai showed no effects. In the HCD-treated group, Sannoshashinto further decreased the body weight gain while the other kampohozai showed no significant effect on the HCD-induced decrease of body weight. The liver weight of the HCD-treated controls increased relative to that in the normal controls. In the normal diet groups, all kampohozai and clofibrate showed no effects on liver weight, but in the high-cholesterol groups, all kampohozai showed a tendency to reduce the liver weight, although clofifbrate increased it. Figure 1 shows the serum and liver lipid levels in rats receiving the normal diet. Clofibrate decreased serum TC levels, although the kampohozai showed no effect. However, Sannoshashinto and Daisaikoto, in addition to clofibrate, decreased serum TG levels. Liver TC levels were reduced by Shosaikoto, although the other kampohozai and clofibrate showed no effects on either TC or TG levels. Cholesterol feeding increased TC and TG levels in both serum and liver as shown in Fig. 2. All kampohozai and clofibrate inhibited the cholesterol-induced increase of TC and TG levels in both serum and liver in a significant manner. The action of the three kampohozai and clofibrate appeared equivalent. Table 3 summarizes the data on serum high density lipoprotein (HDLl-cholesterol and atherogenetic index (AI). Cholesterol serum 0 C SAN

liver

TC(mg/dl) 50

100

c

(A)

f

-

P

SHO

Ir

CL0 serum

liver) 2

Es-

(B)

--’

ll++ 0

TC(mg/g 1

*

F

DA1

0

TG(mg/dl) 50

’ 100

0

liver

TG(mg/g 5

liver) 10

C

ii

SAN

b

CD)

SHO DA1 CL0

Fig. 1. Effect of kampohozai on serum and liver total cholesterol (TC) and triglyceride (TG) levels in rats fed a normal diet (Nl. C, control; SAN, Sannoshashinto-treated; SHO, Shosaikoto-treated; DAI, Daisaikoto-treated; CLO, clofibrate-treated. (A) serum TC level, (Bl liver TC level, (Cl serum TG level, (Dl liver TG level. Each column indicates the mean 2 S.E.M. of 10 rats. Significance: *P < 0.05 and **P < 0.01 vs. control.

262

serum

TC(mg/dl)

0

liver

100

200

0

TC(mg/g

liver)

10

20

C

(D)

SAN SHO DA1 CL0

TG(mg/dl )

serum

Fig. 2. Effect levels in rats treated: SHO, koto-treated;

of kampohozai on serum and liver total cholesterol (TC) and triglyceride (TG) fed a cholestrol diet (HCD). C, HCD control; SAN, HCD- and SannoshashintoShosaikoto-treated; SHO, HCD- and Shosaikoto-treated; DAI, HCD- and DaisaiCLO, HCD- and clofibrate-treated. (A) serum TC level, (B) liver TC level, (Cl

serum TG level, (D) liver TG level. Each column indicates nificance: *P < 0.05 and **P < 0.01 vs. HCD control.

TABLE

the mean

2 S.E.M.

of 10 rats.

Sig-

3

EFFECT OF KAMPOHOZAI HYPERCHOLESETEROLEMIC

ON HDL-CHOLESTEROL RATS GIVEN A HIGH

AND ATHEROGENETIC CHOLESTEROL DIETa

INDEX

Diet

Drug

Dosage (g/kg per day)

HDL-cholesterol (mgldl serum)

Atherogenetic

N HCD HCD HCD HCD HCD

N C SAN SHO DA1 CL0

_ 0.25 0.50 0.50 0.10

46 31 33 33 36 31

0.21 4.09 2.58 3.03 2.00 2.76

r r r 2 r r

2* 2 2 3 5 2

+ ” ? f + -r-

IN

index

0.03* 0.45 0.55* 0.52 0.46* 0.21*

“Atherogenetic indesx = (Total cholesterol - HDL-cholesterol)/HDL-cholesterol. N, normal: C, control: HCD, high cholesterol diet; SAN, Sannoshashinto; SHO, Shosaikoto; Daisaqikoto; CLO, clofibrate. Each tabular value indicates the mean + S.E.M. of 10 rats. Significance: *P < 0.05 vs. HCD/C.

DAL

263

feeding decreased the HDL-cholesterol levels, but all kampohozai showed no The AI value was remarkably normalizing effect on this phenomenon. increased by the HCD treatment and all the kampohozai and clofibrate tended to normalize this effect, although the effect of Shosaikoto was not statistically significant. Hyperlipidemia

in aging rats

Aging increased the body, aorta and liver weights as shown in Table 4. In this experiment, the doses of kampohozai were equivalent to the human doses per day. Treatment with all of the kampohozai and with dl-a-tocopherolnicotinate inhibited the normal body weight gain seen in the control group. Body weights in llO-week-old rats treated with Shosaikoto and dl-a-tocopherolnicotinate were lowered to the level of 50-week-old rats not treated with drugs. The increase of aorta weight in llO-week-old rats was inhibited by Sannoshashinto to the level o 50-week-old rats and Daisaikoto also tended to show a decrease. All kampohozai and dl-a-tocopherolnicotinate showed no effect on liver weight. Figure 3 summarizes the results of serum lipid levels in aging rats. Aging increased the serum TC levels, and the increased serum TC levels in llO-week-old rats were higher than those found in 6- or 50-weekold rats. Serum TG levels also increased with aging, and the serum TG levels in 50- and llO-week-old rats were drastically higher than those in g-weekold rats. At 110 weeks, Sannoshashinto and Daisaikoto inhibited the aginginduced increased of serum TC, and all of kampohozai and dl-atocopherolnicotinate inhibited the aging-induced increase of serum TG. Liver lipid levels were not significantly changed by the kampohozai and dl-tocopherolnicotinate treatment, although liver TC increased slightly in IlO-weekold rats. TABLE EFFECT

4 OF KAMPOHOZAI

Group (age)

N

ON BODY, AORTA Body weight (g)

c (110 W) SAN (110 W)

8 7

SHO (110 W) DA1 (110 W) TOC (110 W) c (50 W) C (6 W)

10 8 10 10 7

399 f 377 f 357 373 348 353 150

6 6*

f 8++ f 4** 2 ll** f 4** 2 7**

AND LIVE WEIGHT

IN AGED RATS”

Aorta weight (mglg body wt.)

Liver weight (g/100 g body wt)

0.311 f 0.237 f

0.019 0.022*

3.43 * 0.07 3.78 f 0.16

0.338 0.276 0.307 0.235 0.289

0.022 0.027 0.021 0.015** 0.057

3.53 3.61 3.40 3.04 4.23

2 f f rt f

f f 2 f f

0.16 0.05 0.10 0.08** O.lO**

‘C, control; SAN, Sannoshashinto (0.05 g/kg per day); SHO, Shosaikoto (0.12 g/kg per day); DAI, Diasaikoto (0.12 g/kg per day); TOC: tocopherolnicotinate (0.02 g/kg per day). Each tabular value indicates the mean f S.E.M. of 7-10 rats. Significance: *P < 0.05, **p < 0.01 vs. c (110 W) group.

264

serum

dose(g/Kg/day)

iT(mg/dl)

0

100

0

serum TG(mg/dl) 50

C

100 (6)

SAN SHO DA1 TOC C( 5OW) C(W

Fig. 3. Effect of kampohozai on serum lipid total cholesterol (TC) and triglyceride (TGl levels in aging rats. C (llOW1, IlO-week-old control group; SAN, IlO-week-old Sannoshashintotreated group; SHO, llO-week-old Shosaikoto-treated group; DAI, IlO-week-old Daisaikototreated group; TOC, IlO-week-old dl-o-tocopherolnicotinate-treated group; C (5OW1, 50.weekold control group; C (6W1, 6-week-old control group. (Al serum TC level, (Bl serum TG level. Each column indicates the mean f S.E.M. of 10 rats. Significance: *P < 0.05 vs. C (llOW1.

Turnover

of cholesterol

To establish the antihyperlipidemic mechanism of action of kampohozai, the effects of Sannoshashinto, Shosaikoto and Daisaikoto on the biosynthesis and metabolism of cholesterol were studied. The doses of the kampohozai used were equivalent to five times the human dose per day. The action of kmapohozai on liver cholesterol biosynthesis from [lJ4C]acetic acid is summarized in Figure 4. The rate of cholesterol biosynthesis was not significantly changed by kampohozai treatment. Figure 5 shows the disappearance of labeled cholesterol orally or intravenously given after the administration of kampohozai. Sannoshashinto accelerated the excretion of intravenously injected cholesterol and orally administered cholesterol to the same degree.

Hepatic 0

sterol

synthesis 1 I

rate(cpm/5Omin/g 2

liver) 3 I

x103

C SAN

.

SHO DA1

Fig. 4. Effect of kampohozai on biosynthesis of cholesterol in liver. C, control: SAN, Sannoshashinto-treated (0.25 g/kg); SHO, SHosaikoto-treated (0.5 g/kg); DAI, Daisaikoto-treated (0.5 g/kg). Each column indicates the mean f S.E.M. of 10 rats.

265

[4-“C]Cholesterol(p.o.) 0

(xlO”dpm/O. 4

[4-“C]Cholesterol(i.v.)

5ml blood)

(x104dpm/0. 8

0

5ml blood) 1.5

SHO

tr

DA1

H

Fig. 5. Effect of kampohozai on serum-labeled cholesterol level after the oral or intravenous treatment. C, control; SAN, Sannoshashinto-treated (0.25 g/kg); SHO, Shosaikoto-treated (0.5 g/kg); DAI, Daisaikoto-treated (0.5 g/kg). Each column indicates the mean f S.E.M. of 10 rats. Significance: *P f 0.05 vs. control.

This indicates that Sannoshashinto accelerates the excretion of cholesterol from blood. On the other hand, Daisaikoto decreased orally administered cholesterol only. Figure 6 shows the ratio of oral cholesterol absorption (isotope ratio). Daisaikoto decreased the ratio of oral cholesterol absorption. These data indicate that Daisaikoto may inhibit the absorption of cholesterol from the intestine. Discussion How hypercholesterolemia is produced after feeding HCD has not well resolved, but most of the cholesterol is recovered as a fraction of It is well known that the catabolism of LDL is performed by specific receptors, and that about 60% of LDL is processed in liver (Kovanen,

isotope

c

ratio

been LDL. LDL19811.

(X)

OW

SAN SHO

DA1

Fig. 6. Effect of kampohozai on oral cholesterol absorption (isotope ratio). C, control, SAN, Sannoshashinto-treated (0.25 g/kg); SHO, Shosaikoto-treated (0.5 g/kg); DAI, Daisalkototreated (0.5 g/kg). Each column idicates the mean f S.E.M. of 10 rats. Significance: *P < 0.05 vs. control.

266

It is assumed that hypercholesterolemia is due to the slow catabolism of LDL in liver owing to the feeding of HCD. This model is well suited for the screening of drugs to reduce hypercholesterolemia. It was observed that serum and liver TC levels were remarkably increased when rats were fed HCD for 10 days. Furthermore, the abundant intake of cholesterol increased both serum and liver TG contents. In HCD-treated animals, Sannoshashinto, Daisaikoto and Shosaikoto showed a marked inhibitory effect on the increased serum and liver TC and TG levels. AI value, a ratio of HDL-cholesterol against serum TC and an index of atherosclerosis, was reduced by treatment with Sannoshashinto and Daisaikoto as well as clofibrate. Shosaikoto has been reported to reduce serum TC in a HCD-treated model (Yamamot0 et al., 19851 and Daisaikoto has also been reported to inhibit cholesterol-induced increases of serum TC (Ohminami et al., 19851. Furthermore, Daisaikoto has been reported to inhibit apoprotein B synthesis in a HCD model (Teramoto, 19851. Apoprotein B is a main apoprotein of very low density lipoprotein WLDL) and is considered to be the regulator of VLDL secretion. In an another hyperlipidemia model which seems to occur naturally as a product of aging, serum TG levels increased drastically in addition to an increase of serum TC. Sannoshashinto and Daisaikoto inhibited the aginginduced increase of serum TC, although Shosaikoto showed no inhibition. However, all the kampohozai and dl-a-tocopherolnicotinate inhibited the aging-induced increase of seurm TG. In an experiment of cholesterol metabolism, Sannoshashinto appeared to accelerate cholesterol removal from blood, while Daisaikoto appeared to inhibit cholesterol absorption from intestine. This action of Daisaikoto is supported by the report of Ohminami et al. (19851. In regard to Shosaikoto, cholesterol removal from blood and cholesterol absorption from the intestine were not clearly changed, but Shosaikoto showed a tendency to inhibit the cholesterol from the intestine. Our data suggest that Sannoshashinto and Daisaikoto would be more useful clinically in the treatment of hypercholesterolemia. Shosaikoto, however, did not inhibit the increase of serum TC in the HCD-treated model and inhibited the aging-induced increase of serum TG. These data indicate that Shosaikoto may also be effective on hyperlipidemia. The inhibitory action of Shosaikoto on aging-induced increase of serum TG is attractive and further detailed studies are needed to make clear the action mechanisms. In our previous paper (Umeda et al., 19881, Shosaikoto strongly inhibited the development of atherosclerosis in rabbits fed a 0.75% cholesterol-diet for 6 months, while Daisaikoto inhibited it slightly. Shosaikoto significantly reduced the hardening of aorta, the increase of aortic collagen content and the tearing of elastic lamina in media. Shosaikoto further decreased the accumulation of calcium deposits and necrotic substances on the surface of thoracic aorta. The fact that the hyperlipidemic action of Daisaikoto is more effective than that of Shosaikoto suggests that Shosaikoto modifies atheros-

267

clerosis by other actions such as the regulation of the blood coagulationfibrinolysis system or the prevention of aortic endothelial or smooth mustle cells from the injury induced by hyperlipidemia. Body weight was not influenced by treatment with Shosaikoto, but Sannoshashinto inhibited the increase of body weight in both the normal and the HCD-treated groups and Daisaikoto also tended to decrease it in the HCD-treated group. There was no decreasing effect in the aging-induced model. The different results for body weight in both experiments may be due to the lower doses of the kampohozai used in the aging model. Sannoshashinto is famous for its puragative action and the main active compounds have been reported to be the sennosides (Fujimura, 1985) found in Rheum tunguticum rhizome. Daisaikoto also contains Rheum tanguticum rhizome. This purgative action of sennosides may account for the decrease of body weight and the decreased absorption of cholesterol from the intestine and the lowered serum TC levels. Duplication of this work using equivalent amounts of the pure sennosides appears indicated. However, the different mechanisms of actions of Sannoshashinto and Daisaikoto suggest that other ingredients may play a functional role in their antihyperlipidemic actions. References Aonuma, S., Mimura, T. and Tarutani, M. (1957) Effects of coptis, scutellaria, rhubarb, and bupleurum on the serum cholesterol and phospholipids of rabbits. Yukugaku Zasshi 77, 1033 - 1307. Berensen, E.L. (19771 Risk factors in children - The early natural history of atherosclerosis. A theTos&ToSis 4, 489 - 495. Bierman, E.L. and Ross, R. (19771 Aging and atherosclerosis. Atherosclerosis Review 2, 79-111. Consensus Conference (19851 Lowering blood cholesterol to prevent heart disease. Journal of the American Medical Association 253, 2080 - 2086. Demattets (19681 Stimulation of liver cholesterol synthesis by actinomycin D. Biochemical JOUTnal 109. 775-785. Eisenberg, S., Stein, Y. and Stein, 0. (19691 Phospholipases in arterial tissue. IV. The role of phosphatide acyl hydrolase, lysophosphatide acyl hydrolase, and sphingomyelin choline phosphohydrolase in the regulation of phospholipid composition in the normal humane aorta with age. Journal of Clinical Investigation 48, 2320 - 2329. Fujimura, H., Sawada, T. and Goto, M. (19701 Screening test of anti-inflammatory action on the constituents of crude drugs and plants. Yakugaku Zasshi 90, 782-783. Fujimura. H. (19831 Purgative action of Daio (rhubarb) and its constituents. Journal of Z’raditional &no-Japanese Medicine 4, 44 - 48. Gore, I., Rofertson, W., Hirst, A.E., Hadley, G.C. and Koseki, T. (19601 Geographic differences in the severity of aortic and coronary atherosclerosis. The United States, Jamaica, West India, South India and Japan. American Journal of Pathology 36, 559-574. Gotto, Jr. A.M. and Jones, P.H. (19861 Rationale and criteria for lowering serum cholesterol. Atherosclerosis Reviews 14, l-4. Haranaka, R., Hasegawa, R., Kosoto, H., Owada, S., Hirama, N., Hanawa, J., Iimura, F. and Nakagawa, S. (19861 Anti-atherosclerotic effect of traditional Chinese medicines (Ba-Wei-DiHuang-Wan, Chai-Hu-Jia-Long-Gu-Mu-Li-Tang, Da-Chai-Hu-tang, Huang-Lian-Jie-Du-Tang) in experimental 51-57.

animals.

Journal

of Medical

and Pharmaceutical

Society

for

Wakan-Yaku

3,

268

Hirst, A.E., Gore, I., Hadley, G.G. and Gault, E.W. (19601 Gross estimation of atherosclerosis in aorta, coronary and cerebral arteries. A comparative study in Los Angeles and South India. AMA Archives of pathology 69, 578- 584. Kishi, K., Watanabe, T.X. and Sokabe, H. (19851 Effects of steamed ginseng radix on blood pressure in hypertensive rats. Journal of Medical and Pharmaceutical Society for Wakan-Yaku 2, 7-13. Kiuchi, FR., Shibata, M. and Sankawa, (19821 Inhibitors of prostaglandin biosynthesis from ginger. Chemical and Pharmaceutical Bulletin 30, 754 - 757. Koda, A., Nagai, H. and Wada, H. (1970a) Pharmacological actions of baicalin and baicalein. I: Active anaphylaxis. Folia Pharmacology of Japonica 66, 194-213. Koda, A., Nagai, H. and Wada, H. (1970bl Pharmacological actions of baicalin and baicalein. II: Passive anaphylaxis. Folia Pharmacology of Japonica 66, 237 - 247. Kovanen, P.T. (19811 Regulately role for hepatic low density lipoprotein receptors in wivo in the dog. Proceedings of the National Academy Sciences KJSAI 78, 1194-1198. Kubo, M., Matsuda, H., Izumi, S., Tani, T., Arich, S., Yoshikawa, M. and Kitagawa, I. (19821 Studies on moutan cortex (VI). Inhibitory effects on the intravascular coagulation (Part 11. Shoyakugaku Zasshi 36, 70 - 77. Kubo, M., Matsuda, H., Tani, T., Arichi, S. and Kitagawa, I. (1984) Inhibitory effects on histamine release from rat peritoneal mast cells in vitro. Shoyakugaku Zasshi 38, 276-278. Kuwano, S. (19701 Pharmacological effects of coptis rhizome and the constituents. Shoyakugaku Zasshi 24, l-5. Marek, Z., Jaegerman, K. and Ciba, T. (19621 Atherosclerosis and level of serum cholesterol in postmortem investigation. American Heart Journal 63, 768 - 774. McNamara, J.J., Malot, M.A., Stremole, J.F. and Cutting, R.T. (19711 Coronary artery disease in combat casualties in Vietnam. Journal of American Medical Association 216, 1185- 1187. Ohinami, K., Matsuoka, E. and Okuda, H. (19851 Effect of Daisaikoto on hyperlipidemia. Yakuri to Chiryo 13, 5097 - 5099. Sadoshima, S., Kurozumi, T. and Tanaka, K. (19801 Cerebral and aortic atherosclerosis in Hisayama. Japanese Atherosclerosis 36, 117 - 126. Sekiya, K. and Okuda, H. 119821 Selective inhibition of platelet lipoxygenase by baicalein. Biochemical and Biophysical Research Communications 105, 1090 - 1095. Shibata, M., Yoshida, R., Motohashi, S. and Fukushima, M. (19731 Pharmacological studies on Bupleurum falcatum L. IV: Some pharmacological effects of crude saikosides, saikogenin A and syrupy residue. Yakugaku Zasshi 93, 1660- 1667. Shibata, M., Isomura, Inoue, T. and Nagai, M. (1976) Some pharmacological studies on the crude drugs possessing anti-inflammatory properties - On the root of Bupleurum and the leaves of Fig. Shoyakugaku Zasshi 30,62-66. Shibata, N. (19621 Effect of glycyrrhizin on the experimental atherosclerosis in rabbit, especially on the antihypercholesteremic action. Medical Journal of Osaka University 12, 297-313. Sugishita, E., Amagaya, S. and Ohigara, Y. (19841 Studies on the combination of glycyrrhizae radix in Shakuyakukanzo-to. Journal of Pharmacobio-Dynamics 7, 427 - 435. Takagi, K. (19811 The pharmacological action of glycyrrhizae radix. Journal of Traditional SinoJapanese Medicine, 12,3437. Takagi, K. (19821 Pharmacology of ginseng. Journal of Traditional &no-Japanese Medicine 3, 47 -54. Teramoto, T. (1985) Metabolism of apoprotein B. Z’aisha 22, 611-620. Teramato, T., Kato, T. and Kinoshita, M. (1986) Jikkenteki koshiketsu rat ni okeru Daisaikoto no koshiketsu sayo. The Clinical Report 20, 105- 109. Umeda, M., Amagaya, S. and Ogihara, Y. (19881 Effects of Shosaikoto and Daisaikoto on experimental atherosclerosis in rabbits. Journal of Medical and Pharmaceutical Society for WakanYaku 5, 154 - 162. Yakasaki, K., Okada, K., Takagi, K. and Irikura, T. (19841 Effect of 4-(4’.chlorobenzyloxy) benzyl nicotinate (KCD-232) on cholesterol metabolism in rats fed an amino acid imbalance diet. Agricultural and Biological Chemistry, 48, 1417-1423.

269

Yamahara, J. (1976) Behavioral pharmacology of herherine-type alkaloids. I: Central depressant effect of tetrahydro berberine (THBl and related compounds. Folia Pharmacology of Juponica 72,909 - 927. Yamahara, J., Yamada, T., Kimura, H., Sawada, T. and Fujimura, H. (19821 Biologicaly active principles of crude drugs. II: Anti-allergic principles in “Shoseiryu-to”. Anti-inflammatory properties of paeoniflorine and its derivatives. Journal of Phannacobio-Dynamics 5, 921-929. Yamamoto. M., Kumagai, A. and Yamamura. Y. (1975al Structure and actions of saikosaponins isolated from Bupleurum f&&urn L. I: Anti-inflammatory action of saikosaponins. Arzneimittel Furschung 25, 1021- 1023. Yamamoto, M., Kumagai. A. and Yamamura, Y. (1975bl Structure and action of saikosaponins isolated from Bupleurum fulcatum L. II: Metabolic actions of saikosaponins, especially a plasma cholesterol-lowering action. Arzneimittel Forschung 25, 1240- 1243. Yamamoto, M., Uemura, T., Nakama. T., Uemiya, Y., Tanaka, T. and Minamoto, S. (19851 Effects of saponins from ginseng and bupleurum, extracts from Xiai-Chai-Hu-Tang and Gui-Zhi-FuLing-Wan, on blood lipoproteins, apoproteins and prostanoids on hepatic lipids in experimental hyperlipidemia. Journal of Medical and Pharmaceutical Society for Wakan-Yaku 2, 377385. Yokoyama, H., Hiai, S. and Oura, H. (19811 Chemical structures and corticosterone secretioninducing activities of saikosaponins. Chemical and Pharmaceutical Bulletin 29, 500- 504. Yokozawa. T., Kobayashi, T., Oura, H. and Kawashima, Y. (19841 Improving effects of ginsenoside-Rb, in streptozotocin diabetic rats with hyperglycemia and hyperlipemia. Journal of Medical and Pharmacological Society for Wakan- Yaku 1, 22 - 28. Zilversmit, D.B. and Barry Hughes, L. (19741 Validation of a dual-isotope plasma ratio method for measurement of cholesterol absorption in rats. Journal of Lipid Research 15, 465- 473.