Original Article

Fatemeh Yousefi (MSc)

1

Soleiman Mahjoub (PhD)
2, 3
Mahdi Pouramir (PhD)
*3, 4

Fatemeh Khadir (MSc)
1








1- Babol University of Medical
Sciences, Babol, Iran.
2- Infertility and Reprodactive
Health Research Center, Babol
University of Medical Sciences,
Babol, Iran.
3- Department of Biochemistry,
Babol University of Medical
Sciences, Babol, Iran.
4- Cellular and Mollecular Biology
Research Center, Babol University
of Medical Sciences, Babol, Iran.



* Correspondence:
Mahdi Pouramir, Cellular and
Mollecular Biology Research
Center, Babol University of
Medical Sciences, Babol, Iran.




E-mail: [email protected]
Tel: 0098 111 3236591
Fax: 0098 111 2224152








Received: 22 April 2013
Revised: 18 June 2013
Accepted: 1 July 2013

Hypoglycemic activity of Pyrus biossieriana Buhse leaf
extract and arbutin: Inhibitory effects on alpha amylase
and alpha glucosidase


Abstract
Background: The mechanism of hypoglycemic and hypolipidemic activities of Pyrus
biossieriana Buhse leaf extract (PbBLE) and its phytochemical component arbutin, have
not been well determined. The present study was performed to understand the
hypoglycemic activity mechanisms of pbBLE and arbutin more clearly.
Methods: In vitro enzymatic carbohydrate digestion with PbBLE and arbutin was assessed
using -amylase and -glucosidase powders. The enzyme solutions were premixed with
PbBLE and arbutin at different concentrations (0.1, 1, 10 and 100 mg/ml). Substrate
solutions and colorimetric reagents were added to the reaction. The release of glucose was
determined by spectrophotometric method. Acarbose was used as the positive control.
Results: The extract (10, 100 mg/ ml) completely inhibit - amylase and - glucosidase
activities. The extract produced higher reduction of -amylase and -glucosidase activity
than arbutin. Inhibition at various concentrations (0.1, 1, 10, 100 mg/ml) were significantly
different (p<0.05).
Conclusion: Our results exhibited that both the extract and arbutin were able to suppress
the enzymes strongly.
Keywords: Hypoglycemic; Pyrus biossieriana Buhse extract; Arbutin; -Amylase/ -
glucosidase inhibitors

Caspian J I ntern Med 2013; 4(4): 763-767

D
iabetes mellitus (DM) is a lifelong disease and there is not yet a cure, but
symptoms and problems can be prevented (1). DM is a chronic disease, resulting from
insulin deficiency or decreased responsiveness of the organs to insulin or both (2) and has
a significant impact on health, quality of life and life expectancy of patient, as well as on
the healthcare system (3). Two types of DM are currently known, Type 1, insulin-
dependent diabetes mellitus (IDDM), and type 2, non-insulin-dependent diabetes mellitus,
(NIDDM) (3). The patients with type 1 DM are absolutely dependent upon insulin for life
but the management of type 2 often needs combined regiments, including diet, medicine
and exercise (4). The progressive nature of the disease necessitates continuous
reassessment of glycemic control in people with diabetes and appropriate adjustment of
therapeutic regiment. Due to the life-long treatment, prohibitive cost and unavailability of
treatment in rural areas, the disease is a great burden to the patients (1). Herbal medicines
have received significant attention because of their effectiveness, availability, fewer side
effects and relatively low cost (5). Carbohydrates are the major constituents of the human
diet (6). Pancreatic -amylase and intestinal -glucosidase digest carbohydrates and
facilitate absorption of monosaccharide such as glucose and fructose. One of the
therapeutic approaches for reducing blood glucose in patients with diabetes is to prevent
the absorption of carbohydrates after food uptake (7). In this regard, -amylase and -
glucosidase inhibitors play a critical role in reducing the postprandial increase of blood
glucose level after a mixed carbohydrate diet (8-10).

Caspian J Intern Med 2013; 4(4): 763-767
764 Yousefi F, et al.
In addition, the commercially available drugs for DM
(such as acarbose) are limited by gastrointestinal disturbance
effects (11). Therefore, there is an urgent need for searching
therapies that may have less severe or no side effects.
In this study, we have selected Pyrus biossieriana Buhse
leaf extract (PbBLE) and its phytochemical components,
arbutin, to evaluate their -amylase and -glucosidase
inhibitory properties. Pyrus biossieriana Buhse (locally
known as wild pear) is a species of pear that is native to Iran.
The leaves are used for treatment of inflammation of the
bladder, bacteriuria, high blood pressure, urinary stones and
also used as diuretic (12). The dried leaves of this tree
contain a considerable amount of arbutin that is abundant in
a number of different plant species including Ericaceae and
Rosaceae (Pyrus biossieriana Buhse) (13). Arbutin is a
glycosilated hydroquinone that inhibits melanin formation by
blocking tyrosinase, it also has been shown to have
antioxidant (14), antihyperglycaemic and antihyperlipidemic
properties (15).
In this study, in vitro models were used to understand the
mechanism of action of PbBLE and its active component on
carbohydrate metabolism.


Methods
Materials: -amylase from porcine pancreas, -
glucosidase type1 from bakers yeast, acarbose, arbutin, p-
nitrophenyl -D-glucopyranoside and 3, 5 dinitrosalicylic
acid, potato starch were purchased from Sigma-Aldrich
(USA).
Plant material and preparation: The leaves of Pyrus
biossieriana Buhse were obtained from their natural habitat
in the north of Iran; Babol. The plant material was identified
and authenticated by the Mazandaran Department of
Agricultural Sciences and Natural Resources. The
authenticated dried leaves of Pyrus biossieriana Buhse were
extracted with aqueous methanol (63%) for 36 h room
temperature. The extract was evaporated to dryness under
reduced pressure with a rotator evaporator. The dried residue
was dissolved in water and used for in vitro experiments (13,
15).
Assay for -amylase inhibitory activity: The -amylase
inhibitory assay was performed by the methods of Tingting
Wu (16) and Monica R. Louizzo (17) with a little
modification. The -amylase solution was prepared by
mixing 1 mg of enzyme in 1 ml phosphate buffer (0.002 M
sodium phosphate buffer, pH 6.9 with 0.006M sodium
chloride). The colorimetric reagent was prepared by mixing
sodium potassium tartrate solution (12.0 g sodium potassium
tartrate tetrahydrate in 8.0 ml of 2M NaOH) and 90 mM of 3,
5 dinitrosalicylic acid solution (0.2 g of 3, 5 dinitrosalicylic
acid in 10 ml of distilled water) 1:1 (v/v). The methanolic
extract of Pyrus biossieriana Buhse and arbutin at different
concentrations (0.1, 1, 10 and 100 mg/ml) were premixed
with -amylase solution and then were pre incubated in 25
o
C
for 10 minutes. The reaction was initiated by adding 1%
starch solution dissolved in sodium phosphate buffer. The
mixtures were incubated at 25
o
C for another 10 minutes.
After incubation, the reaction was stopped with 200l of
colorimetric reagent. The test tubes were maintained in a
boiling water bath for 5 minutes and cooled to room
temperature. The reaction mixture was then diluted by
adding 4 ml distilled water and absorbance was measured at
540nm.
The absorbance of the sample blanks (buffer instead of
enzyme solution) and the control (buffer in place of samples)
were recorded as well. Acarbose was used as the positive
control. The inhibitory activity of the extract and arbutin was
calculated by the following equation:
Inhibition activity (%) =




Assay for -glucosidase inhibitory activity: The -
glucosidase inhibitory assay was performed according to the
procedures reported by Suresh V. Nampoothiri (6) with
modifications. The methanolic extracts at various
concentrations (200l) and -glucosidase (20l, 10 U/ml) in
0.005M phosphate buffer (pH 6.8) were mixed and incubated
for 5 minutes at 37
o
C. Para-nitrophenyl--D-
glucopyranoside (1mM, 200l) was added to initiate the
reaction and the mixture was further incubated at 37
o
C for 20
minutes. The reaction was terminated by the addition of
500l of 1M Na
2
CO
3
and diluted by adding 500l of
distilled water. The absorbance was recorded at 405nm.
Acarbose was used as the positive control.
Inhibition activity (%) =




Caspian J Intern Med 2013; 4(4): 763-767
Hypoglycemic activity of Pyrus biossieriana Buhse leaf extract and arbutin 765

Statistical analysis: Each experiment was performed in
triplicate. The results are presented as mean standard
deviation (SD). The statistical analysis was performed using
one-way analysis of variance (ANOVA) with subsequent
post hoc comparisons by LSD (SPSS 16.0). The criterion for
statistical significance is expressed as p < 0.05.


Results
Both the extract and arbutin exhibited inhibitory activity
in a dose-dependent manner (figures 1, 2). An almost
complete inhibition of the -amylase and -glucosidase
activities were obtained at 10 and 100mg/ml dosages of the
extract, respectively. Arbutin, at the highest concentration
was used (100mg/ml), inhibited only 75% of -glucosidase
activity and 81% of -amylase activity.












Figure 1. -glucosidase inhibitory activities of the extract and
arbutin. Inhibition by various concentrations are significantly
different (p<0.05)












Figure 2. -amylase inhibitory activities of the extract
and arbutin. Inhibition by various concentrations are
significantly different (p<0.05)
Discussion
Diabetes mellitus is an endocrine disease that is
developing along with an increase in both obesity and ageing
in the general population (2). The treatment aim of diabetic
patients is to maintain near normal levels of glycemic
control, in both the fasting and postprandial states. In
particular, -amylase and -glucosidase participate in
glucose digestion and are considered as key enzymes that
can control postprandial hyperglycemia. In this present
study, we have investigated the inhibitory effects of PbBLE
and arbutin on -amylase and -glucosidase activities.
Several biological activities of PbBLE and arbutin have been
described by some colleagues. According to the previous
studies, PbBLE has an antioxidant activity in serum, liver,
kidney and pancreas in normal rats (18). In addition, PbBLE
can reduce the glucose diffusion and absorption through
dialysis bag in laboratory models (19). In other studies, the
antibacterial, antifungal, antilarvae, antioxidant,
anticholinestrase (13) and antihyperglycemic activities of
PbBLE and arbutin have been performed (15). Arbutin also
has protective effects against tert-butyl hydroperoxid
induced toxicity in hepatic cell lines (20). In this in vitro
experiment, we focused to study the possible mechanism of
PbBLE and arbutin as -amylase and -glucosidase
inhibitors.
The results of this in vitro experiment showed that
PbBLE were able to inhibit both the enzymes significantly.
Arbutin also showed strong inhibition of -amylase and -
glucosidase, though less potent than the extract, that it can be
attributed to other compounds found in the extract such as
flavonoids, phenols and glycosides (21). Some of these
inhibitors have been used in clinical trials, for example, in a
randomized design, Salacia oblonga extract was fed to a
number of healthy subjects and results show a great
reduction in plasma glucose and insulin. Also, breath
hydrogen excretion was 60% greater (P<0.001) compared
with control (22). At the same time, other studies have
reported the high efficacy and safety of -glucosidase
inhibitors added to metformin compared with metformin
alone in patient with type 2 diabetes (23).
Daily consumption of Reliv Glucaffect was found to
statistically significantly be lower blood fasting glucose from
145.3 mg/dL to 101.1 mg/dL and body weight from an
average of 88.5 kg (BMI 26.8 kg/m2) to 81.3 kg (BMI 24.5
kg/m2) as compared to the control group (24). Our research
is the first study of Pyrus biossieriana Buhse and its active
0
10
20
30
40
50
60
70
80
90
100
-1 0 1 2
i
n
h
i
b
i
t
i
o
n

%
log concentration
Arbutin
Extract
0
10
20
30
40
50
60
70
80
90
100
-1 0 1 2
i
n
h
i
b
i
t
i
o
n

%
log concentration
Arbutin
Extract

Caspian J Intern Med 2013; 4(4): 763-767
766 Yousefi F, et al.
component, arbutin, in relation to their -amylase and -
glucosidase inhibitory activities. Based on the results
presented here, we can claim that besides any other possible
mechanism of action, PbBLE and arbutin have inhibitory
effects on -amylase and -glucosidase. With these results,
we can further support the traditional use of the plants based
on their inhibitory activities on glucose absorption in the
intestine.
The weakness of this study was related to the old
fashioned equipment used in colorimetric assay. This study
confirmed the potential anti-diabetic activity of the PbBLE
and arbutin, focusing on the inhibitory effects on -amylase
and -glucosidase. In our in vitro model, the results give
scientific support to the use of Pyrus biossieriana Buhse leaf
extract and arbutin for the treatment of diabetes, however,
further investigation is required to validate its use prior to
clinical implementation as therapeutic agent.


Acknowledgments
We gratefully thank the Cellular and Molecular Biology
Research center of Babol University of Medical Sciences for
supporting this study.

Funding: This research was financially supported by the
Babol University of Medical Sciences. (Grant No: 9133019)
Conflict of Interest: There was no conflict of interest.


References
1. Deutschlander MS, van de Venter M, Roux S, Louw J,
Lall N. Hypoglycaemic activity of four plant extracts
traditionally used in South Africa for diabetes. J
Ethnopharmacol 2009; 124: 619-24.
2. Bhandari MR, Jong-Anurakkun N, Hong G, Kawabata
GH. alpha-Glucosidase and alpha-amylase inhibitory
activities of Nepalese medicinal herb Pakhanbhed
(Bergenia ciliata, Haw.). Food Chem 2008; 106: 247-
52.
3. Kwon YI, Apostolidis E, Shetty K. In vitro studies of
eggplant (Solanum melongena) phenolics as inhibitors of
key enzymes relevant for type 2 diabetes and
hypertension. Bioresource Technol 2008; 99: 2981-8.
4. Jong-Anurakkun N, Bhandari MR, Kawabata J. alpha-
Glucosidase inhibitors from Devil tree Alstonia scholaris.
Food Chem 2007; 103: 1319-23.
5. Nampoothiri SV, Prathapan A, Cherian OL, et al. In vitro
antioxidant and inhibitory potential of Terminalia
bellerica and Emblica officinalis fruits against LDL
oxidation and key enzymes linked to type 2 diabetes.
Food Chem Toxicol 2011; 49: 125-31.
6. Ieyama T, Gunawan-Puteri M, Kawabata J. alpha-
Glucosidase inhibitors from the bulb of Eleutherine
americana. Food Chem 2011; 128: 308-11.
7. Kim YM, Wang MH, Rhee HI. A novel alpha-
glucosidase inhibitor from pine bark. Carbohydr Res
2004; 339: 715-7.
8. Zhang L, Hogan S, Li J, et al. Grape skin extract inhibits
mammalian intestinal alpha-glucosidase activity and
suppresses postprandial glycemic response in
streptozocin-treated mice. Food Chem 2011; 126: 466-
71.
9. Ortiz-Andrade RR, Garcia-Jimenez S, Castillo-Espana P,
et al. alpha-glucosidase inhibitory activity of the
methanolic extract from Tournefortia hartwegiana: An
anti-hyperglycemic agent. J Ethnopharmacol 2007; 109:
48-53.
10. Saijyo J, Suzuki Y, Okuno K, et al. a-Glucosidase
Inhibitor from Bergenia ligulata. J Oleo Sci 2008; 57:
431-5.
11. Ooi K, Muhammad TS, Tan ML, Sulaiman SF.
Cytotoxic, apoptotic and anti-alpha-glucosidase activities
of 3,4-di-O-caffeoyl quinic acid, an antioxidant isolated
from the polyphenolic-rich extract of Elephantopus
mollis Kunth. J Ethnopharmacol 2011; 135: 685-95.
12. Zargari A. Medicinal plants. 6th ed. Tehran: Tehran
University Publications 1996; pp: 231-45. [In Persion]
13. Azadbakht M, Marston A, Hostettmann K, et al.
Biological activity of leaf extract and phenolglycoside
arbutin of Pyrus Boissieriana Buhse. J Medicinal Plants
2004; 3: 9-14.
14. Gerich JE. Matching treatment to pathophysiology in
Type 2 Diabetes. ClinTherapeutics 2001; 23: 64659.
15. Shahaboddin ME, Pouramir M, Moghadamnia AA, et al.
Pyrus biossieriana Buhse leaf extract: An antioxidant,
antihyperglycaemic and antihyperlipidemic agent Food
Chem 2011; 15: 1730-3.
16. Wu T, Zhou X, Deng Y, et al. In vitro studies of Gynura
divaricata (L.) DC extracts as inhibitors of key enzymes
relevant for type 2 diabetes and hypertension. J
Ethnopharmacol 2011; 22: 305-8.

Caspian J Intern Med 2013; 4(4): 763-767
Hypoglycemic activity of Pyrus biossieriana Buhse leaf extract and arbutin 767

17. Loizzo MR, Saab AM, Tundis R, et al. In vitro inhibitory
activities of plants used in Lebanon traditional medicine
against angiotensin converting enzyme (ACE) and
digestive enzymes related to diabetes. J Ethnopharmacol
2008; 2: 109-16.
18. Gholizadeh N, Khaunbabapoor Z, Habibnejad F, Lakzaei
M, Pouramir M. Effects of Pyrus Boissieriana Buhse
Leaves Extract on Antihyperglycemic, Antioxidant and
Antilipidproxidative in Rats. J Babol Univ Med Sci
2009; 11: 12-18.
19. Asgharpour F, Pouramir M, Moghadamnia A.A, et al.
Evaluation of viscosity of traditional medicinal
antihyperglycemic plant extracts and relationship with
glucose diffusion in vitro. Medicinal plants 2011; 18: 38-
42.
20. Seyfizadeh N, Mahjoub S, Zabihi E, et al. Cytoprotective
Effects of Arbutin Against Tert-Butyl
Hydroperoxid Induced Toxicity in Hep-G2 Cell Line. Wold
Appl Sci J 2012; 19: 163-7.
21. Andrade-Cetto A, Becerra-Jimenez J, Cardenas-Vazquez
R. Alfa-glucosidase-inhibiting activity of some Mexican
plants used in the treatment of type 2 diabetes. J
Ethnopharmacol 2008; 28: 27-32.
22. Collene A, Hertzler S, Williams J, Wolf BW. Effects of a
nutritional supplement containing Salacia oblonga extract
and insulinogenic amino acids on postprandial glycemia,
insulinemia, and breath hydrogen responses in healthy
adults. Nutrition 2005; 21: 848-54.
23. Liu S, Tu YK, Chien MN, Chien KL. Effect of
antidiabetic agents added to metformin on glycaemic
control, hypoglycaemia and weight change in patients
with type 2 diabetes: a network meta-analysis. Diabetes
Obes Metab 2012; 14: 810-20.
24. Belcaro G, Cesarone M, Silvia E, et al. Daily
consumption of Reliv Glucaffect for 8 weeks
significantly lowered blood glucose and body weight in
50 subjects. Phytother Res 2009; 23: 1673-7.