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Hypoglycemic Effect of 13-Membered Ring Thiocyclitol,

a Novel -Glucosidase Inhibitor from Kothala-himbutu (Salacia reticulata)

Hiromi OE and Sei OZAKIy

Research Institute, Fuji-Sangyo Co., Ltd., Kagawa 763-0071, Japan

Received February 25, 2008; Accepted April 2, 2008; Online Publication, July 7, 2008

[doi:10.1271/bbb.80118]

A novel 13-membered ring thiocyclitol, isolated from

an aqueous extract of Kothala-himbutu (Salacia retic-

ulata), inhibited -glucosidase in vitro. The inhibitory

activity was investigated by maltose- and sucrose-

loading on Wistar rats. This study found significantlowering of postprandial glucose levels, and the potency

of 13-membered ring thiocyclitol was confirmed in vivo.

Key words: Kothala-himbutu; thiocyclitol; -glucosi-

dase inhibitor; postprandial glucose level

Kothala-himbutu (Salacia reticulata WIGHT; family,

Hippocrateaceae) has received much attention as an

effective herb for the treatment of diabetes. It has long

been a very popular medicinal plant in Sri Lanka and

the southern region of India.1,2) Many studies have

confirmed that the anti-diabetic effect of this plant is to

be especially attributed to -glucosidase inhibitoryactivity.3,4) Salacinol and kotalanol (shown in Fig. 1),

with an internal salt of spiro-like configuration, have

been identified as potent -glucosidase inhibitors in

Salacia spp. by Yoshikawa et al.4) On the other hand,

our investigation recently confirmed the existence of a

novel inhibitor, which was identified as 13-membered

ring thiocyclitol (13-MRT, shown in Fig. 1).5) To

determine the effects of 13-MRT, assessment of -

glucosidase inhibition, referring to the method of Toda

et al.,6) was carried out. A crude enzyme was prepared

from rat intestinal acetone powder (Sigma Aldrich

Japan, Tokyo). Maltase and sucrase inhibitory activitiesrespectively were measured using maltose and sucrose

as a substrate. It was confirmed that 13-MRT showed

more potent -glucosidase inhibitory activities (IC50:

maltase, 0.23mM; sucrase, 0.19mM) than those of

salacinol (IC50: maltase, 9.6mM; sucrase, 2.5 mM).7) This

evidence suggested that 13-MRT might be a contributive

constituent in the anti-hyperglycemic effect of Kothala-

himbutu. In this study, we investigated the effect of

13-MRT on postprandial glucose levels in maltose- and

sucrose-loaded rats.

Twenty-four male Wistar rats (5 weeks old) were

purchased from Japan SLC. All animals were housed inan air-conditioned room under controlled temperature

(24 2 C) and a relative humidity of50 10% with a

12-h light/dark cycle. They were fed on a standard diet

(CE-2, Clea Japan, Tokyo) and tap water ad libitum

throughout the study. After an acclimatization period of

1 week, they were randomly divided into four groups,

each group consisting of six animals. This procedure

was carried out on each test sample, including an

aqueous extract of Kothala-himbutu (KOT), 13-MRT,

salacinol, and voglibose. KOT was prepared by follow-

ing method: stems of Kothala-himbutu were extracted

Salacinol

Kotalanol

13-MRT

SHO

HO OH

OHOH

OSO3

SHO

HO OH

OHOH

OSO3 OH

OH

OH

S

OH

OH

OH

OHHO

HO

HO OH

O

Fig. 1. The Structures of Salacinol,4) Kotalanol,4) and 13-MRT.

y To whom correspondence should be addressed. Tel: +81-877-25-3221; Fax: +81-877-25-0567; E-mail: s ozaki@fuji-sangyo.co.jp

Abbreviations: KOT, an aqueous extract of Kothala-himbutu; 13-MRT, 13-membered ring thiocyclitol

Biosci. Biotechnol. Biochem., 72 (7), 19621964, 2008

Note

http://dx.doi.org/10.1271/bbb.80118http://dx.doi.org/10.1271/bbb.80118

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40

80

120

160

200

0 60 120

Time (min)

Blood

glucose(mg/dl)

Control25 mg/kg50 mg/kg75 mg/kg

**

**

A

40

80

120

160

200

0 60 120

Time (min)

B

loodglucose(mg/dl)

Control0.10 mg/kg0.15 mg/kg0.20 mg/kg

***

B

40

80

120

160

200

0 60 120

Time (min)

Bloodglucose(mg/dl)

Control1.25 mg/kg

2.50 mg/kg5.00 mg/kg

*

*

C

40

80

120

160

200

0 60 120

Time (min)

Bloodglucose(mg/dl)

Control0.30 mg/kg0.60 mg/kg

0.90 mg/kg

**

**

a

D

Fig. 2. Effects of KOT, Voglibose, Salacinol, and 13-MRT on Blood

Glucose Levels in Maltose-Loaded Rats.

After a 16-h fast, each rat was orally administered maltose

solution (2.5 g/kg) and the test samples: A, KOT at doses of 25, 50,

and 75mg/kg; B, Voglibose at 0.10, 0.15, and 0.20mg/kg; C,

Salacinol at 1.25, 2.50, and 5.00 mg/kg; D, 13-MRT at 0.30, 0.60,

and 0.90 mg/kg. The control group was given only maltose solution,

and the test samples were suspended in maltose solution. The blood

glucose level was measured 0, 60, and 120 min after an oral

administration. Each value represents the mean SD (n 6). P

values are calculated by Dunnetts test. Significant difference inglucose level vs. that in the control group: ap < 0:1, p < 0:05,p < 0:01.

60

90

120

150

180

0 60 120

Time (min)

Blood

glucose(mg/dl)

Control10 mg/kg20 mg/kg40 mg/kg

**

*

A

60

90

120

150

180

0 60 120Time (min)

Blo

odglucose(mg/dl)

Control0.05mg/kg0.10mg/kg0.15mg/kg

****

****

**

B

60

90

120

150

180

0 60 120

Time (min)

B

loodglucose(mg/dl)

Control0.30 mg/kg0.60 mg/kg1.20 mg/kg

*

*

C

60

90

120

150

180

0 60 120

Time (min)

Bloodglucose(mg/dl)

Control0.15 mg/kg0.30 mg/kg0.45 mg/kg

**

*

D

Fig. 3. Effects of KOT, Voglibose, Salacinol, and 13-MRT on Blood

Glucose Levels in Sucrose-Loaded Rats.

After a 16-h fast, each rat was orally administered sucrose

solution (2.5 g/kg) and the test samples: A, KOT at doses of 10, 20,

and 40mg/kg; B, Voglibose at 0.05, 0.10, and 0.15mg/kg; C,

Salacinol at 0.30, 0.60, and 1.20 mg/kg; D, 13-MRT at 0.15, 0.30,

and 0.45 mg/kg. The control group was given only sucrose solution,

and the test samples were suspended in sucrose solution. The blood

glucose level was measured 0, 60, and 120min after oral admin-

istration. Each value represents the mean SD (n 6). P values arecalculated by Dunnetts test. Significant difference in glucose level

vs. that in the control group: p < 0:05, p < 0:01.

Effect of a Novel -Glucosidase Inhibitor from Kothala-himbutu 1963

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with water at 90 C for 2 h. 13-MRT and salacinol were

isolated from KOT by HPLC with a YMC-Pack Poly-

amine-II (250 20mm I.D., YMC, Kyoto, Japan) and a

Daisopak SP-120-5-ODS-BP column (250 20mm

I.D., Daiso, Osaka, Japan). Still, KOT (100 g) contained

13-MRT (241.8mg) and salacinol (252.5 mg).5) Vogli-

bose was used as a positive control. This study wascarried out in accordance with Guideline for Animal

Experimentation no. 6, established by the Prime

Ministers Office of Japan in 1980.

Maltose- and sucrose-loading tests8) were carried out

to examine maltase and sucrase inhibitions of 13-MRT.

All animals were fasted for 16 h, followed by oral

administration of water solution of maltose and sucrose

(2.5 g/kg), and the test materials suspended in sugar

solutions were administered simultaneously. The control

group was administered with only substrate solution.

Blood samples were collected from the tail veins of the

animals at 0 (prior to administration), 60, and 120 minafter oral administration. Serum samples were obtained

by centrifugation to measure glucose levels, which were

measured with a commercially available kit (Glucose

CII-test Wako, Wako Pure Chemical Industries, Osaka,

Japan). All data were expressed as means SD. Multi-

ple comparisons were carried out by Dunnetts test. p

Values of less than 0.05 were considered significant.

The maltase inhibitory effects of the inhibitors are

shown in Fig. 2. The postprandial glucose levels of

KOT (50 and 75 mg/kg, Fig. 2A), voglibose (0.15 and

0.20 mg/kg, Fig. 2B), and salacinol groups (2.50 and

5.00 mg/kg, Fig. 2C) were significantly lower than

those of the control group at 60 min after oral admin-istration. These results agree with the reported data,4,8)

and support the validity of this study. As shown in

Fig. 2D, the blood glucose level of 13-MRT group

(0.30 mg/kg) was not significant as compared with that

of the control group. In contrast, the 13-MRT groups

(0.60 and 0.90 mg/kg) were significantly suppressed in

comparison with the control group. Next, the sucrase

inhibition of 13-MRT was also investigated, by a similar

method (Fig. 3). As shown in Fig. 3D, the 13-MRT

groups (0.30 and 0.45 mg/kg) highly suppressed hyper-

glycemia as compared with the control group. This

evidence confirmed that both maltase and sucraseinhibition of 13-MRT was more potent than that of

salacinol in vivo, but was several times weaker than

that of voglibose. In addition, we observed that this

compound significantly suppressed the elevation of

postprandial glucose levels after starch-loading (1.5 g/

kg), but its effect failed as to dose-response. Further-

more, we confirmed that 13-MRT had no effect on

hyperglycemia in glucose-loaded (2.5 g/kg) rats (H., Oe

and S., Ozaki, unpublished results). The results revealed

that the hypoglycemic effect of 13-MRT was achieved

through -glucosidase inhibitory activities in the small

intestine. As regards structure-activity relationship, it

is still unclear. Further studies on 13-MRT, such as

elucidation of its absolute structure and investigation ofthe active site of the inhibitor, are required. These are

currently in progress. On the other hand, -glucosidase

inhibitors might induce the onset of symptoms such as

abdominal distention, diarrhea, and soft feces,9) while

ingestion of 13-MRT did not affect this under this

condition.

In conclusion, this study found that 13-MRT retarded

the absorption of carbohydrates by -glucosidase

inhibitory activities in the small intestine, not be due

to -amylase inhibition.

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p. 43 (1994).

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and their active constituents. Phytomedicine, 2, 137189

(1995).

3) Karunanayake, E. H., Welihinda, J., Sirimanne, S. R., and

Sinnadorai, G., Oral hypoglycaemic activity of some

medicinal plants of Sri Lanka. J. Ethonopharmacol., 11,

223231 (1984).

4) Matsuda, H., Yoshikawa, M., Morikawa, T., Tanabe, G.,

and Muraoka, O., Antidiabetogenic constituents from

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1964 H. OE and S. OZAKI