全 文 :262 Journal of Chinese Pharmaceutical Sciences http://www.jcps.ac.cn
Antihyperglycemic, insulinsensitizing and antioxidant activities of
the active fractions from Anoectochilus chapaensis
Jinyan Cai 1* , Lin Zhao 2 , En Zhu 1 , Chengxi Wang 1
1. School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China
2. School of Life Science and Biopharmaceutical, Guangdong Pharmaceutical University, Guangzhou 510006, China
Abstract: Anoectochilus chapaensis Gagnep. (Orchidaceae), an indigenous and valuable Chinese folk medicine, has been widely
used in China to treat diabetes. However, few reports are available about its constituents and activity. The present experiment was
conducted to investigate the active fractions from A. chapaensis in diabetic rat model induced by highfat diet plus streptozotocin.
The total EtOH extract from the whole herbs of A. chapaensis, half of which was partitioned in sequence with petrol ether (PE), ethyl
acetate (EtOAc), nBuOH and H2O, thus yielding four fractions, all of them were orally administered with an identical dose
amount to 4 g/kg dried crude herbs once a day for consecutive two weeks to further investigate the antihyperglycemic activity.
The EtOAc fraction caused a significant fall in the nonfasting blood glucose level of diabetic rats from (402.66±82.26) to
(226.26±62.10) mg/dl, which may be attributed to ameliorating insulin resistance, modulating the activity of enzymatic antioxidants,
reducing the content of NO, etc. Much more intact β cells in the islets of Langerhans in EtOAc fractiontreated groups than the
negative control were observed, which greatly supported the morphological and functional elucidation. The OGTT evidenced
that EtOAc fraction could promote the endurance capacity of acute glucose increase in diabetic rats. The EtOAc fraction of
A. chapaensis contains some hypoglycemic and antioxidant principles with the potential to be developed further for the treatment
of diabetes specifically associated with an insulin resistance state.
Keywords: Anoectochilus chapaensis; Antihyperglycemic; ISI; Active fraction
CLC number: R962 Document code: A Article ID: 1003–1057(2014)4–262–06
Received: 20131218; Revised: 20140222; Accepted: 20140227.
Foundation items: National Natural Science Foundation of China
(Grant No. 81001628) and Guangdong Natural Science Foundation
(Grant No. S2013010014771);
* Corresponding author. Tel.: +8615920107845; Fax: +862039352129;
Email: caijy928@163.com
http://dx.doi.org/10.5246/jcps.2014.04.037
1. Introduction
Diabetes mellitus is a group of metabolic disorders
characterized by hyperglycemia [1] . The number of
diabetic patients has been increasing rapidly [2] ; however,
the control of diabetes and its complications remains
a challenge. There is an increasing demand by patients
to use the natural products with antidiabetic activity,
due to obvious side effects associated with the use of
insulin and oral hypoglycemic agents.
In China, a country with one of the highest rates of
diabetes in the world, Traditional Chinese Medicine
(TCM) is popular and used for the treatment of many
ailments. Many patients in China choose a combination
of western medicine and TCM to treat diabetes [3] .
The genera Anoectochilus (Orchidaceae) are perennial
herbs which comprise more than 40 species and are
widespread in the tropical regions. Of those species,
A. chapaensis, an indigenous and valuable Chinese folk
medicine, has been used as a popular herbal drug in China
and other Asian countries. It is also called “king medicine”
along with A. roxburghii because of its diverse pharma
cological effects [4] . The whole dried plants have been
widely used in China to treat diabetes, nephritis and
venomous snake bite, etc. Because of a low budding
and growth rate in natural surroundings, predatory mass
collection, and damages to the ecological environment,
the natural resources of A. chapaensis are becoming
exhausted as well as A. roxburghii. In recent years much
research has been performed on A. roxburghii [5–8] , however,
few reports have been available about A. chapaensis’s
constituents and antidiabetic activity.
In this study, highfat diet plus STZ induced hyper
glycemia model on rats were employed to evaluate the
antihyperglycemic activity of different fractions from
A. chapaensis.
2. Materials and methods
2.1. Plant materials
The herbs of A. chapaensis were collected in the
263 Cai, J.Y. et al. / J. Chin. Pharm. Sci. 2014, 23 (4), 262–267
September of 2010 from Yunnan Province, southwest
China. The plant was authenticated by Hongyan Ma,
School of Traditional Chinese Medicine, Guangdong
Pharmaceutical University. A voucher specimen was
deposited in the herbarium of School of Pharmacy,
Guangdong Pharmaceutical University.
2.2. Apparatus
Blood glucose levels were measured by Onetouch
Blood Glucose Monitoring System. Inverted microscope
(NIKON ECLIPSE TS100), KDC160HR high speed
low temperature freezing centrifuge, microplate reader
(BIORAD Model 680), superclean worktable, OLYMPUS
AU400 automatic biochemistry analyzer, and UV2201
visibleultraviolet spectrophotometer were employed.
2.3. Reagents
Streptozotocin (STZ) was purchased from Sigma
Aldrich Inc. (USA). Superoxidase dismutase (SOD), total
antioxidant capacity, and content of malonyldialdehyde
(MDA), NO were assayed using kits from Nanjing
Jiancheng Biological Reagents Company. Insulin in
plasma was determined with Rat Insulin Elisa Kit from
Blue Gene Company.
2.4. Sample preparation
Dry powdered herbs (1.6 kg) of A. chapaensis was
refluxed with 95% (v/v) ethanol for 2 h, and each
filtrate was concentrated to dryness in vacuo to render
the total EtOH extract, then half of which was suspended
in distilled water and partitioned in sequence with
petrol ether (PE), ethyl acetate (EtOAc), nBuOH and
H2O, thus yielding four fractions. The respective yields
of the PE fraction, EtOAc fraction, nBuOH fraction
and H2O fraction were 14.02%, 10.90%, 27.48%, and
48.19%, respectively.
2.5. Experimental animals and treatment protocol
Male SpragueDawley rats (specificpathogen free grade,
weight 80–110 g) were obtained from the Laboratory
Animal Breeding and Research Center of Guangdong
Province, and fed with a highfat diet (formula: yolk
2.5%, sugar 20%, lard 10% and basal feed 67.5%) and
tap water for four weeks. Animals were housed in an air
conditioned room at (23±2) °C with natural light.
Diabetes mellitus was artificially induced to overnight
fasted rats by intraperitoneal injection of STZ (30 mg/kg
body weight dissolved in citrate buffer, 0.1 M, pH 4.5).
After 10 d, the rats (showing stabilized diabetes) with
nonfasting blood glucose level above 199.8 mg/dl, were
selected for the study. Diabetic rats were randomly
divided into seven groups (each group contains eight
rats): A. EtOH extract, B. PE fraction, C. EtOAc fraction,
D. nBuOH fraction, E. H2O fraction, F. metformin,
G. vehicle. And each group was treated with either
vehicle, metformin (60 mg/kg in water), total extract or
fractions all at the same dose amount to 4 g/kg dried
crude herbs in vehicle in a similar volume for two weeks,
respectively, the treatment of which was defined according
to a doseresponse experiment previously.
The fractions, metformin or vehicle were orally
administered once a day and lasted for 14 d. Body
weight, urine glucose level, food consumption and
faeces were observed and recorded daily, and blood
glucose level was monitored with the trace blood
samples from the tail vein every week. After two weeks,
the OGTT test was also performed to evaluate the
endurance performance besides measuring glucose
concentration. At the end of the experiment, blood was
collected in heparin treated tubes from abdominal aorta
for assessment after callisection, and pancreas was
treated with formaldehyde for histological examination.
Blood was centrifuged at 2500 r/min for 10 min. Insulin
level was evaluated with Enzyme Linked Immunosorbent
Serologic Assay (ELISA). Spectrophotometric test kits were
used to evaluate relative biomarkers in plasma including
total antioxidant activity, activity of total SOD, content
of MDA, and content of NO, an important active factor.
2.6. Glucose tolerance test
The hypoglycemic effects of all administrations in
diabetic rats were assessed by the improvement of
glucose tolerance. Glucose tolerance test was performed
on diabetic controlled and extracts treated rats after 14 d.
Hence, glucose tolerance was investigated on basis of
all groups (each group has eight rats): A. EtOH extract,
B. PE fraction, C. EtOAc fraction, D. nBuOH fraction,
E. H2O fraction, F. metformin, G. vehicle. All the groups
were administered by glucose (2.5 g/kg) after overnight
fasting. Blood samples were collected just prior to
glucose administration (0 h) and 0.5, 1, 1.5, 2 h after
the glucose loading.
264 Cai, J.Y. et al. / J. Chin. Pharm. Sci. 2014, 23 (4), 262–267
2.7. Data analysis
Data were shown as the mean±standard deviation (SD).
Insulin sensitivity index (ISI) was calculated by this
formula: ISI=1/(fasting blood glucose × fasting plasma
insulin). Statistical analysis was performed by oneway
analysis of variation (ANOVA) followed by Dunnett
ttest for multiple comparisons. A difference with a
P value of <0.05 was considered statistically different.
3. Results
3.1. Blood glucose and body weight
A significant and steady increase in blood glucose
levels were observed after 10 d (the induction of diabetes).
The effects of different fractions and metformin on
nonfasting blood glucose and body weight were shown
in Table 1. After 14 d treatment, nonfasting blood glucose
was significantly lower for metformintreated diabetic rats
in the range from (296.10±31.32) to (127.80±4.32) mg/dl.
Daily administration of the EtOAc fraction also caused
a significant fall in the nonfasting blood glucose levels of
diabetic rats from (402.66±82.26) to (226.26±62.1) mg/dl,
furthermore, in which the body weight was much
higher than vehicle group. Then minor variation can be
observed in other groups including EtOH extract (from
(363.24±68.4) to (354.78±121.32) mg/dl), PE fraction
(from (327.60±63.0) to (318.06±104.22) mg/dl), nBuOH
fraction ((348.48±51.48) to (297.90±70.02) mg/dl), H2O
fraction ((427.86±64.44) to (376.20±88.02) mg/dl).
3.2. Plasma insulin and insulin sensitivity index
Chronic administration of EtOH extract, EtOAc fraction
and nBuOH fraction resulted in a promising improvement
of insulin resistance in diabetic rats induced by highfat
food plus STZ, even better than metformin (Table 2),
the reference drug used in this study.
3.3. Serum SOD, total antioxidant activity and content
of MDA
A significant increase in SOD activity was observed
after the treatment with PE fraction, EtOAc fraction,
nBuOH fraction and metformin. Total antioxidant
activity was significantly increased after the treatment
with PE fraction, EtOAc fraction, H2O fraction and
metformin (Table 3). Whereas MDA values in plasma
were lower in the EtOH extract, PE fraction, and nBuOH
fractiontreated groups than the STZ+vehicle group.
3.4. Content of NO in plasma
As shown in Figure 1, much lower content of NO
were observed in EtOH extract, PE fraction, EtOAc
fraction, nBuOH fraction and the metformin treated
rats than those of vehicle group.
Groups
0 d 14 d
Blood glucose (mg/dl) Body weight (g) Blood glucose (mg/dl) Body weight (g)
Vehicle 373.14±87.66 248.00±29.00 309.06±66.24 222.00±36.00
Metformin 296.10±31.32 240.00±18.40 127.80±4.32 b 240.50±18.60 b
EtOH extract 363.24±68.40 241.63±20.25 354.78±121.32 223.71±33.21
PE fraction 327.60±63.00 253.00±18.57 318.06±104.22 247.00±20.57 b
EtOAc fraction 402.66±82.26 251.67±5.71 226.26±62.10 b 259.83±13.62 b
nBuOH fraction 348.48±51.48 251.38±13.17 297.90±70.02 244.25±23.72 b
H2O fraction 427.86±64.44 285.00±10.29 376.20±88.02 250.17±13.84
Groups Fasting blood glucose (mg/dl) Plasma insulin (ng/mL) Insulin sensitivity index (ISI) (×10 –3 )
Vehicle 144.00±12.06 2.233±0.456 2.83±0.21
Metformin 133.20±5.40 1.637±0.737 4.61±0.15 b
EtOH extract 135.54±11.52 0.791±0.521 9.33±0.06 b
PE fraction 158.40±19.26 2.212±0.809 2.84±0.10
EtOAc fraction 92.70±15.30 1.925±0.290 5.61±0.03 b
nBuOH fraction 124.20±20.34 1.141±0.479 7.06±0.08 b
H2O fraction 122.40±10.62 2.400±0.281 3.39±0.04
Table 1. Effect of the fractions on body weight and blood glucose
b P<0.05 vs. vehicle group.
b P<0.05 vs. vehicle group.
Table 2. Effect of the fractions on fasting blood glucose, plasma insulin and insulin sensitivity index
265 Cai, J.Y. et al. / J. Chin. Pharm. Sci. 2014, 23 (4), 262–267
3.5. Histological examination
Figure 2 showed the typical views of the pancreas by
HE staining. Much more intact β cells in pancreatic
islet issue in PE fraction, EtOAc fraction, nBuOH
fraction and the metformin treated rats than those in the
negative control group. These results showed that this
plant could be useful in reversing the injury of β cells
in pancreatic islet and the protective effect may be
closely related with its improving function.
3.6. Oral glucose tolerance test
Endurance capacity, especially the value and time of
peak blood glucose, was evaluated by blood glucosetime
curve. As shown in Figure 3, a significant improvement
(P<0.05) in the endurance performance can be observed
in all fractionstreated groups, especially in the EtOAc
fraction, PE fraction and EtOH extractadministered
groups.
Groups SOD activity (U/mL) Total antioxidant capacity (U/mL) Content of MDA (nmol/mL)
Vehicle 232.33±21.48 0.987±0.178 5.780±1.420
Metformin 355.00±113.22 b 1.603±0.292 b 5.208±0.880
EtOH extract 96.12±5.904 0.678±0.128 4.453±0.440 b
PE fraction 257.00±22.82 b 1.141±0.316 b 3.125±0.210 b
EtOAc fraction 493.44±23.88 b 1.603±0.267 b 5.781±0.000
nBuOH fraction 401.26±137.98 b 0.216±0.032 3.724±0.254 b
H2O fraction 233.97±8.35 1.529±0.281 b 6.281±1.373
Table 3. Effect of the fractions on the total antioxidant capacity, contents of SOD and MDA
b P<0.05 vs. vehicle group.
Figure 1. Effect of all fractions on contents of NO. A: Vehicle;
B: EtOH extract; C: PE fraction; D: EtOAc fraction; E: nBuOH
fraction; F: H2O fraction; G: Metformin.
Figure 3. Blood glucosetime curves in OGTT of all groups. A: EtOH
extract; B: PE fraction; C: EtOAc fraction; D: nBuOH fraction;
E: H2O fraction; F: Metformin; G: Vehicle.
100
80
60
40
20
0
C
on
te
nt
o
f N
O
(
μm
ol
/L
)
A B C D E F G
Groups
35
30
25
20
15
10
5
0
B
lo
od
g
lu
co
se
(
m
m
ol
/L
)
0.0 0.5 1.0 1.5 2.0 2.5
t (h)
A B C D
E F G
Figure 2. Photomicrographs of histopathological changes of the pancreatic islet issue in diabetic rats. A: EtOH extract; B: PE fraction; C: EtOAc
fraction; D: nBuOH fraction; E: H2O fraction; F: Metformin; G: Vehicle.
A B C D
E F G
266 Cai, J.Y. et al. / J. Chin. Pharm. Sci. 2014, 23 (4), 262–267
4. Discussion
EtOAc fractiontreatment revealed the most promising
hypoglycemic effect among all the administrations,
which significantly improved the activity of insulin,
prevented weight loss in diabetic rats, and ameliorated
βcells damage caused by oxidative stress and NO.
The bioactivity screening of different fractions may
give an effective guide of the isolation of the active
compounds.
It is found that the metabolic syndrome compared
with impaired fasting glucose status correctly allocates
the risk of newonset diabetes in a higher proportion of
people (62% vs 38%) [9] . Insulin resistance is a metabolic
disorder, the prevalence of which is increasing alarmingly
in populations worldwide. It occurs when the body
tissues becoming increasingly resistant to insulin,
leading to a marked decrease in glucose metabolism,
which is associated with type 2 diabetes [10] . Indeed, the
effects of EtOH extract, EtOAc fraction and nBuOH
fraction on insulin resistance generally compared
favorably with those of metformin. A. chapaensis
may contain one or more principles which have the
potential to be developed further for the treatment
of diabetes specifically associated with a insulin
resistance state.
Hyperglycemia complications in diabetes mellitus not
only generate elevated level of reactive oxygen species
(ROS) but also alter antioxidative machinery through
glycation of the scavenging enzymes. Oxidative stress
has been considered to be a common pathogenic factor
of diabetic complications including nephropathy [11] .
The increase in SOD activity in diabetic animals is
possibly due to increased dismutation of superoxide anion
to molecular oxygen and hydrogen peroxide as an adaptive
response to increased oxidative stress. The increase
in SOD activity was proposed to protect catalase and
GPx against inactivation by superoxide anions which
are known to inactivate catalase and GPx [12] . The
significant increase in the SOD and total antioxidant
capacity as well as the decreased content of MDA in
the diabetic rats suggests that peroxidative injury and
imbalance of the cellular defence system be involved
in the development of diabetic disorders. The results
demonstrated that the EtOAc fraction significantly
reduced the blood glucose level, meanwhile the SOD
activity and total antioxidant activity was significantly
increased. These findings in this paper also suggest
that PE fraction, EtOAc fraction, nBuOH fraction from
A. chapaensis had antioxidant effects and could protect
tissues from lipid peroxidation. It would be essential to
further study its antioxidant activities.
Nitric oxide (NO) could be considered as one of the
key proinflammatory mediators for β cell dysfunction
and destruction via affecting mitochondrial functions
and later endoplasmic reticulum stress [13,14] . EtOH
extract, PE fraction, EtOAc fraction, nBuOH fraction
obviously reduced the content of NO.
With late type2 diabetes, chronic cytokine attack,
glucolipotoxicity, and other extracellular stresses lead
to a massive loss of β cells [15,16] . This paper also
demonstrated the extract or fractions from A. chapaensis,
especially the EtOAc fraction possessed a reversing
or protective effect against β cells’ damage, which
contributed to the antihyperglycemic principle underlying
a protective effect at the cellular and molecular level.
The result is in agree with our previous report [17] . The
histological examination (as shown in Fig. 2) showed
the EtOH extract, PE fraction, EtOAc fraction, nBuOH
fraction had a protective and ameliorative effect on
β cells’ structure and function against oxidative stress and
NO factor. Furthermore, it showed a marked improve
ment in the glucose tolerance of the diabetic rats.
5. Conclusions
In view of the protective property in the antihyper
glycemic and antioxidant activity and its relatively
nontoxic nature, this plant and its components would
be promising candidates for the development as anti
diabetic agents in human. This pharmacological profile
provides a scientific support for the claimed ethnomedical
use of A. chapaensis. Further studies are warranted and
undertaken to confirm our results and to insight into
the accuracy mechanisms of action.
In conclusion, A. chapaensis herb extract exhibits
a potent blocking up effect on the progression of
highfat diet associated insulin resistance in rats. Our
findings are encouraging, and suggest that, with further
research and development, A. chapaensis herb extract
may prove to be a useful treatment for diabetes and
related symptoms.
Acknowledgements
The authors thank National Natural Science Foundation
of China (Grant No.81001628) and Guangdong Natural
Science Foundation (Grant No.S2013010014771) for
financial support.
267 Cai, J.Y. et al. / J. Chin. Pharm. Sci. 2014, 23 (4), 262–267
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滇越金线兰活性部位降血糖、增敏胰岛素和抗氧化活性研究
蔡金艳 1* , 赵林 2 , 朱恩 1 , 王成蹊 2
1. 广东药学院 药科学院, 广东 广州 510006
2. 广东药学院 生命科学与生物制药学院,广东 广州 510006
摘要: 采用高糖高脂饮食联合低剂量STZ诱导造成糖尿病大鼠模型, 实验组大鼠分别灌胃给予滇越金线兰总膏和溶剂
萃取法所得各极性部位, 测定给药后大鼠血糖、胰岛素敏感性、抗氧化活性及NO水平等相关指标。胰腺切片, 进行病理
学检查。与模型组相比, 乙酸乙酯部位给药组的随机血糖可见明显降低, 从给药前402.66至226.26 mg/dl (P<0.05), 而且
该组大鼠的体重值明显高于模型对照组, 同时能提高胰岛素敏感性, 改善高糖负荷以后的糖耐量; 胰腺细胞形态较模型组
有明显改善; 同时SOD活性增加, NO含量均显著降低。表明滇越金线兰的乙酸乙酯部位是其降血糖的主要活性部位,
其作用机制可能与改善胰岛素抵抗、增强机体抗氧化活性和降低血浆NO等机制有关。
关键词: 滇越金线兰; 降血糖;胰岛素敏感指数;活性部位