Hypoglycemic Effects

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The Hypoglycemic Effects of Camellia assamica var. kucha Extract

Guo XIE,1;2 Rong-rong HE,1 Xia FENG,1 Tun YAN,3 Fang LAN,4

Min-zhi WU,2 and Hiroshi KURIHARA1;y

1Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou 510632, China2Zhongshan Institute, University of Electronic Science and Technology of China, Zhongshan 528402, China3School of Life Science and Biopharmaceutics, Shengyang Pharmaceutical University, Shengyang 110016, China4College of Traditional Chinese Materia Medica, Shengyang Pharmaceutical University, Shengyang 110016, China

Received August 19, 2009; Accepted October 23, 2009; Online Publication, February 7, 2010

[doi:10.1271/bbb.90618]

Kucha (Camellia assamica var. kucha) is an endemic

tea that grows in cloudy and foggy highlands in southwest

China. Its hypoglycemic effects were studied here. The

postprandial blood glucose levels in mice after sucrose

and soluble starch loading were significantly reducedby Kucha administration. The glycosidase inhibitory

activities were effective both in vitro and in vivo. It

is concluded that the hypoglycemic effects of Kucha

might be due to inhibition of disaccharidases activities.

Key words: Camellia assamicavar.kucha; carbohydrate

tolerance; disaccharidase

Camellia assamica var. kucha (Kucha) is a wild

species ofCamellia sinensis L. in Jinping, China.1) The

leaves of Kucha are consumed as a healthy beverage,

and the local people have drunk it for a long time.

Recently, epidemiological research revealed that therewere significantly lower incidences of hyperglycemia

and diabetes mellitus patients in these areas.2) However,

experimental research on the hypoglycemic action of

Kucha has not yet been reported. In this study, we

examined the effects of Kucha on blood glucose levelsand the inhibitory activities of related glycosidases.

Kucha leaves were obtained from plants growing in the

wild in the Jinping highlands of Yunnan Province, south-

west China, in April 15, 2007. Dry leaves were treated 3

times with 30 parts of hot water for 45 min at 80 C. After

filtration and evaporation of water, the residue was

powdered under freezing-decompression conditions. The

recovery ratio of Kucha was 21.3%, and the extractresidue was immediately dissolved in water before the

experiments. For quality control of Kucha, the chemical

pattern of Kuchaextract was obtained by RT-HPLC analy-

sis under the conditions used by Yanget al.3) (Fig. 1)

Seven-week-old male ICR mice were purchased from

Guangdong Medical Laboratory Animals Center

(Guangzhou, China). The animals were housed ingroups inside plastic cages and kept in a dedicated

pathogen-free animal room at23 1 C at a humidity of

70% under a 12 h light-dark cycle, lights on from 6:00 to

18:00. They were provided standard laboratory chow

(GB 14924-2001, Guangdong Medical Laboratory Ani-

mals Center, China) and water. The animals were

acclimated for 1 week before the experiments. The care

and treatment of animals conformed to the Guide for

Care and Use of the Laboratory Animals published by

the U.S. NIH (publication no. 8523, revised 1996). The

carbohydrate tolerance and glycosidase inhibitory activ-ities of the small intestine mucous membrane weremeasured after oral administration of water, 6.25 mg/kg

of acarbose, 125 mg/kg of Kucha, and 500 mg/kg of

Kucha to the mice, which had been starved overnight for

18 h (15:009:00). Data are presented as mean S.E.M.

Two-way analysis of variance (ANOVA) was applied toanalyze the differences in biochemical parameters

among the experimental groups, followed by Dennetts

test for pair-wise multiple comparisons. Differences

were considered statistically significant at p < 0:05.

The effects of Kucha on postprandial glucose levels

are shown in Fig. 2. There was no statistically signifi-

cant difference in initial basal blood glucose levelsbetween the groups studied. The peak time for blood

glucose concentration was 15 min for glucose and

sucrose tolerance, but 30 min for starch tolerance. As

shown in Fig. 2A, no statistically significant difference

was observed in the blood glucose concentrations of thedifferent groups after glucose loading. However, the

blood glucose concentrations of 6.25 mg/kg of acarbose,

125 mg/kg of Kucha, and 500 mg/kg of Kucha groupwere significantly lower than the control group at 15 and

30 min after sucrose loading (Fig. 2B). Moreover,

compared with the control group, administration of

500 mg/kg Kucha significantly reduced blood glucose

concentrations at 30 and 90 min after starch loading(p < 0:05) (Fig. 2C). Studies have suggested that

EGCG, other catechins, and theaflavins help prevent

hyperglycemia,4) and that blood glucose levels in the

rats are reduced by black tea,5,6) green tea,7,8) and

EGCG.810) As shown in Fig. 1, many tea polyphenols

and methyl xanthine alkaloids, which might be the

active components of Kucha, were separated. Theseresults indicate that the Kucha decoction caused sig-

nificant decreases in the hyperglycemic peaks during the

sucrose and starch tolerance tests, like the drug

acarbose, but not on the glucose tolerance test. Hence,

glycosidase inhibitory activities bothin vitro and in vivo

were evaluated for hypoglycemic effects.

y To whom correspondence should be addressed. Tel/Fax: +86-20-85221559; E-mail: Hiroshi [email protected]: Caff, caffeine; Tb, theobromine; Tp, theophylline; Tc, theacrine; GA, gallic acid; C, ()-catechin; EC, ()-epicatechin;

GC, ()-gallocatechin; ECG, ()-epicatechin gallate; GCG, ()-gallocatechin gallate; EGCG, ()-epigallocatechin gallate; CG, ()-catechingallate; EGC, ()-epigallocatechin; PNPG, p-nitrophenyl--D-glucopyranoside

Biosci. Biotechnol. Biochem., 74 (2), 405407, 2010

Note
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The intestinal mucosa glycosidase inhibitory activities

of-glucosidase, sucrace, -amylase were measured bythe method of Matsumoto et al.11) As shown in Fig. 3,

compared to the control group, both 125 and 500 mg/kg

Kucha significantly inhibited small intestine mucous

membrane glycosidase activities in a dose-dependent

manner (p < 0:01). The inhibitory activities of acarbose

showed similar potency. On the other hand, the

inhibitory activities on glycosidases in vitro were

assayed by the chromogenic method.12) The IC50valuesfor acarbose on -glucosidase, sucrase, and -amylase

were 1.13, 0.36, and 2.53 mg/ml respectively. The IC50values for Kucha were 1.95, 0.21, and 2.57 mg/ml,

which indicates that Kucha had glycosidase inhibitory

effects. The inhibitory activities of Kucha on glycosi-

dasesin vitroand in vivowere similar to acarbose, while

the dosage of Kucha was much higher than acarbose

in vivo. This might be due to the complexity and

diversity of Kuchas components and activities.

As is well known, -glucosidase plays a major role in

degradation of starch and oligosaccharides to mono-

saccharides before absorbance. It is proposed that

suppression of the activities of such digestive enzymesdelays the degradation of starch and oligosaccharides.

Furthermore, the inhibitor of -amylase delayed carbo-

hydrate digestion and prolonged the overall carbohy-

drate digestion time, causing a reduction in the rate

of glucose absorption and consequently blunting of

the postprandial plasma glucose level. A report ofMatsumoto indicated that -amylase activity can be

depressed by tea polyphenols,13) and the mechanism is

probably inhibition of the hydrolysis of -amylase and

reduction of the glucose transporter of intestinal epi-

thelial cells by the ECG and EGCG of tea polyphe-

nols.14) In addition, sucrase might also have been

strongly inhibited by the tea polyphenols in Kucha.

Since sucrase is a characterized small-intestinal disa-caccharidase, it played important roles in carbohydrate

digestion by forming a complex enzyme (SI complex)

on the brush border membranes. It has been reported that

the activity of sucrase, as well as other disacacchar-

idases, is abnormally high in diabetic animals and

human diabetes mellitus patients.15)

In conclusion, our aim was to study the hypoglycemicactivity of Kucha and to provide an approach to the

evaluation and validation of the properties of this plant

in diabetes prevention. To the best of our knowledge,

this is the first study to investigate and analyze the

hypoglycemic activity of Kucha. The mechanism of the

hypoglycemic effect of Kucha might be related toinhibition of glycosidases activities. The hypoglycemic

effects were the results of delayed hydrolysis of

polysaccharides and glucose absorption in the intestinal

mucosa.

A

B

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 17.5 18.8 20.0 21.3 23.0

-500

0

500

1,000

1,500

2,000

2,500 06-9-2 #30 7 UV_VIS_3

mAU

min

WVL:231 nm

GA

Tb

GC

TpCaff

EGC

Tc

C EC

EGCG

GCG

ECG

CG

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 17.5 18.8 20.0 21.3 23.0

-100

0

125

250

375

500

625

750

875

1,000 06-9-2 #33 kucha UV_VIS_3

mAU

min

WVL:231 nm

GCGA

EGC

Tc

CaffEC

EGCG

GCG ECG

Fig. 1. Chemical Fingerprint of Kucha Analyzed by HPLC.

A, Thirteen standard compounds, and B, Kucha extract. The analysis was performed with a Cosmosil 5PE-MS column (4:6mm 150mm) at

40

C. Compounds were eluted (solvent A, 5:95:0.05 v/v/v acetonitrile:H2O:85% H3PO4; solvent B, 50:50:0.05 v/v/v acetonitrile:H2O:85%H3PO4) at a flow rate of 1 ml/min using a gradient program (10% solvent B content at 0 min, 10% at 5 min, 30% at 8 min, 30% at 10 min, 80% at

15 min, and 80% at 23 min), and detected at a wavelength of 231nm.

406 G. XIE et al.


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Acknowledgments

We are very grateful to the teachers and students of

Yaoshan Primary School in Jinping County for a

generous supply of Kucha leaves, and to Dr. J.K. Xu

for technical support in HPLC measurements.

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A

50

150

250

350

0 15 30 45 60 75 90

Time (min)

Bloodglucose(mg/dl)

Control (water)

Acarbose 6.25 mg/kg

Kucha 125 mg/kg

Kucha 500 mg/kg

B

50

150

250

350

0 15 30 45 60 75 90

Time (min)

Bloodglucose(mg/dl)

Control (water)

Acarbose 6.25 mg/kg

Kucha 125 mg/kg

Kucha 500 mg/kg

bb

a

b

a

C

50

150

250

350

0 15 30 45 60 75 90

Time (min)

Bloodglucose(mg/dl)

Control (water)

Acarbose 6.25 mg/kg

Kucha 125 mg/kg

Kucha 500 mg/kg

a a

b a

Fig. 2. Effects of Kucha on Carbohydrate Tolerance Tests.

A, Glucose tolerance test; B, sucrose tolerance test; C, starch

tolerance test. Oral administration of 125mg/kg of Kucha, or

500 mg/kg of Kucha, 6.25mg/kg of acarbose, or water was

performed on mice that had been deprived of food for 18 h.

Solutions of 20% glucose, 40% sucrose, and 60% soluble starch

were given 30 min after drug administration at 10 ml/kg of body

weight respectively. Blood glucose levels were analyzed chrono-

logically, and the results were represented as mean S.E.M.of theblood glucose levels obtained from seven animals each time. The

significance of difference from the normal control group was

represented as ap < 0:05, bp < 0:01.

0

25

50

75

100

-Glucosidase Sucrase -Amylase

I

nhibitoryactivity(%)

Control

Acarbose 6.25 mg/kg

Kucha 125 mg/kg

Kucha 500 mg/kgc

b

c

c

b

c

cc

c

Fig. 3. Effects of Kucha on Glycosidase of the Small Intestine

Mucous Membrane.

The activities of the small intestine mucous membrane were

measured 30 min after administration of 125mg/kg of Kucha, or

500 mg/kg of Kucha, 6.25 mg/kg of acarbose, or water. The results

were represented as the mean S.E.M. from the various experi-

ments. Each experiment was performed with seven mice in each

group, and significance of difference was compared with the control

group at ap < 0:05, bp < 0:01, and cp < 0:001.

The Hypoglycemic Effects of Kucha 407

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Hypoglycemic Effects