Alpha-glucosidase inhibitory effects of propolis extracts in

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Inhibitory properties of aqueous ethanol extracts of
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propolis on alpha-glucosidase
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Hongcheng Zhang , Guangxin Wang , Trust Beta , Jie Dong1,3*
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(1 Bee Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100093,
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China; 2 Department of Food Science, University of Manitoba, Winnipeg, R3T 2N2,
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Canada; 3 National Research Center of Bee Product Processing, Ministry of Agriculture,
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Beijing, 100093, China)
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*Corresponding author: Tel.: +86 10 62594264; Fax: +86 10 62594264
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Email: [email protected] (J. Dong)
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Address: Bee Research Institute,
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Chinese Academy of Agricultural Sciences,
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Xiangshan, Beijing, 100093, China
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Abstract
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The objective of the present study was to evaluate the inhibitory properties of various
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extracts of propolis on alpha-glucosidase from baker’s yeast and mammalian intestine.
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Inhibitory activities of aqueous ethanol extracts of propolis were determined by using
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4-nitrophenyl-D-glucopyranoside, sucrose and maltose as substrates, and acarbose as
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a positive reference. All extracts were significantly effective in inhibiting
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α-glucosidase from baker’s yeast and rat intestinal sucrase in comparison with
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acarbose (p<0.05). The 75% ethanol extracts of propolis (75% EEP) showed the
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highest inhibitory effect on α-glucosidase and sucrase and was a noncompetitive
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inhibition mode. 50% EEP, 95% EEP and 100% EEP exhibited a mixed inhibition
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mode; while water extracts of propolis (WEP) and 25% EEP demonstrated a
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competitive inhibition mode. Furthermore, WEP presented the highest inhibitory
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activity against maltase. These results suggest that aqueous ethanol extracts of
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propolis may be used as nutracueticals for the regulation of postprandial
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hyperglycemia.
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Key Word: Propolis; Alpha-glucosidase; Inhibitory properties; Postprandial
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hyperglycemia
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1 Introduction
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Diabetes mellitus is the most common endocrine disorder disease caused by
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inherited or acquired deficiency in insulin excretion and by decreased responsiveness
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of the organs to secreted insulin. According to the recent reports, the number of
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diabetic patients in the world would amount to 366 million in 2030 and death related
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to diabetes mellitus account for about 9% global mortality, particularly type 2 diabetes
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mellitus (Adolfo, Jaime, & René, 2008). In China, more than 92 million adults have
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diabetes, and 95 percent of them are type 2 diabetes mellitus, according to the survey
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of current epidemiology (Dai & Wang, 2011). Because of more-affluent lifestyles, the
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number of people with diabetes is predicted to increase in the future. Patients with
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type 2 diabetes mellitus usually tend to have long-term complications, such as
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retinopathy, cataract, atherosclerosis, neuropathy, nephropathy and impaired wound
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healing (Ahmed, 2005). Diabetes mellitus is characterized by abnormally high plasma
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glucose. Hyperglycemia has played a central role in pathogenesis of complications
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related to diabetes mellitus. In treatment of type 2 diabetes, suppression of
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postprandial hyperglycemia can decrease the risk of those complications. Hence,
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control of postprandial hyperglycemia should be a primary goal in the prevention and
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management of type 2 diabetes.
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Alpha-glucosidases are a series of enzymes, including sucrase and maltase,
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located in the brush-border surface of intestinal cell, which catalyze the final step in
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the digestive process of carbohydrates to release absorbable monosaccharides
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resulting in increased blood glucose levels (Shai et al., 2010). If α-glucosidases are
3
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inhibited, the liberation of D-glucose from dietary complex carbohydrates can be
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retarded. Thus, α-glucosidase inhibitors have become candidates of hot pursuits to
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delay the digestion and absorption of carbohydrates and restrain postprandial
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hyperglycemic excursions. Alpha-glucosidase inhibitors as one of therapeutic
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approaches for diabetes mellitus have been known since the early 1990s (Ye, Shen, &
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Xie, 2002). At present, some α-glucosidase inhibitors, such as acarbose, miglitol and
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voglibose, have been approved for clinical use in the management of type 2 diabetes,
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as well as the treatment of obesity. However, some synthetic α-glucosidase inhibitors
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have side-effects, such as flatulence, diarrhea and abdominal cramping, all of which
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are associated with incomplete carbohydrate absorption (Ghadyale, Takalikar,
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Haldavnekar, & Arvindekar, 2012). As a result, many researchers have focused on
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novel α-glucosidase inhibitors from natural materials, which are used to develop
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functional foods or lead compounds for antidiabetic treatment including Syagrus
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romanzoffiana, Adhatoda vasica Nees, Syzygium cumini (Linn.) seed kernel (Matsui,
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et al, 2001; Murai, et al, 2002; Shinde, et al, 2008). Some medicinal plant species
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have more potent α-glucosidase inhibitory activities than powerful synthetic
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α-glucosidase inhibitors such as acarbose (Andrade, Becerra, & Cárdenas, 2008).
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Propolis is a colored and aromatic colloidal substance collected by honeybees
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through adding their saliva secreted to the resinous plant exudate, which is used to
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build honeycomb and to fight against the invasion of pathogenic microorganism. The
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chemical composition of propolis varies and depends mainly upon the local flora in
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the region of collection. At present, more than 300 components have been identified
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including flavonoids, phenolic acids, alcohols and their esters, ketones, and inorganic
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compounds (Banskota, Tezuka, & Kadota, 2001). Propolis has been applied in popular
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folk medicine since 3000 BC due to possessing a broad spectrum of biological
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activities such as antioxidant, antimicrobial, anti-inflammatory, antiviral, anticancer
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and antihepatotoxic properties (Bankova, 2005). Recent studies show that ethanol and
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water extracts of propolis can control the glycemia and modulate glucose and lipid
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metabolism in STZ-induced diabetic rats (Hu, et al.，2005; Yan, et al. 2008). Ethanolic
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extracts of Brazilian green propolis were also shown to possess therapeutic potential
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in STZ-induced diabetic rats (Búfalo et al., 2009). In China, propolis has been
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approved for use in functional foods carrying a health claim of controlling glycemia in
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1999 by the Ministry of Health (State Administration of Traditional Chinese Medicine,
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1999). Moreover, propolis has been accepted as adjuvant therapy drugs for diabetes
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mellitus in Chinese Pharmacopoeia in 2005 (State Pharmacopoeia Commission, 2005).
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However, there are few reports whether controlling glycemia of propolis is related to
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its inhibitory activities against α-glucosidase.
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In the present study, we assessed inhibitory effects of aqueous ethanol extracts of
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propolis on alpha-glucosidase and the kinetics of enzyme inhibition by using the
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Michaelis-Menten model.
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2 Material and Methods
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2.1 Chemicals and reagents
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Alpha-glucosidases (EC 3.2.1.20) from baker’s yeast, rat intestinal acetone powder,
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p-Nitrophenyl-α-D-glucopyranoside (PNP-glucoside), Sucrose, Maltose,
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Piperazine-1,4-bisethanesulfonic acid (PIPES), Folin-Ciocalteu, and Glucose-Trinder
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100 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dimethyl
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sulphoxide (DMSO, cell culture grade) was purchased from Applichem Co.
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(Darmstadt, Germany). The crude propolis was procured from Bee Research Institute,
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Chinese Academy of Agricultural Science (Beijing, China). All reagents were of
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analytical grade.
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2.2 Preparation of aqueous ethanol extracts of propolis
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Proplis extracts were prepared as described by Huang et al. (2009) with slight
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modification. Crude propolis (10 gram) was respectively extracted with 200ml
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aqueous ethanol solvent at concentrations ranging from 0%, 25%, 50%, 75%, 95% to
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100% (in water, v/v) by using ultrasonic extract for 4 hours (ultrasonic extractor,
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DCTZ-1000, Beijing Hongxianglong Biotechnol Co. Ltd., Beijing, China). The
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suspensions were centrifuged at 12,000×g for 30 min to obtain supernatants which
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were concentrated at 45℃ in a rotary evaporator (Rotavapor, R-215, Buchi Co., Ltd,
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Switzerland) and then freeze-dried. Extracts were stored in ziplock bag at 4℃ in
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darkness. The extracts were dissolved by DMSO until application.
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2.3 Determination of total phenolic contents
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Total polyphenol contents in propolis extracts were determined according to the
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Folin-Ciocalteu colorimetric method with slight modifications (Kumazawa, et al.,
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2002). A standard curve was built with gallic acid solution. Aliquots ranging from 0 to
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1.2 ml of standard solution (100 µg/ml) were pipetted into 10 ml volumetric flasks
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containing 6 ml distilled water. 0.5 ml of 2 mol/L Folin-Ciocalteu solution and 1.5 ml
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of 10% Na2CO3 (w/v) were added and the volume made up to 10 ml with distilled
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water. Following mixing, the solution was measured at 765nm after 10min by using
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UV-2500/Ultraviolet visible spectrophotometer with UVProbe software (Shimadzu
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Co., Ltd., Tokyo, Japan). The blank was also prepared without addition of the
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standard aliquots. For determination of the total phenolic contents in propolis extracts,
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20μl of aqueous solution at a concentration of 1 mg/ml was used. Total phenolic
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contents were expressed as milligrams gallic acid equivalents per gram extract (mg
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GAE / g).
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2.4 Determination of total flavonoid contents
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Contents of flavonoid in propolis extracts were determined according to the
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method of Kaijv, Sheng, and Chao (2006), with minor modifications. A standard curve
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was built with chrysin solution. Aliquots ranging from 0 to 12 ml of standard solution
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(100 µg/ml) were pipetted into 25 ml volumetric flasks containing 1.0 ml aliquots of
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5% NaNO2 (w/v) solution. After 6 min, 1.0 ml 10% AL(NO3)3 (w/v) solution was
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added and thoroughly mixed. 10ml 4.3% NaOH (w/v) solution were added after 6min
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and the volume was made up with ethanol. The blank was prepared by using ethanol
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instead of standard solution or sample solutions. After 15 min, the absorbance was
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measured at 510 nm by using UV-2500/Ultraviolet visible spectrophotometer with
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UVProbe software (Shimadzu Co., Ltd., Tokyo, Japan). For determination of the total
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flavonoid contents in propolis extracts, 0.5 ml of aqueous solution at a concentration
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of 1mg/ml were used. Total flavonoid contents were expressed as milligramms
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chrysin equivalents of per gram extract (CE).
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2.5 Inhibitory activity assay for baker’s yeast alpha-glucosidase
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Alpha-glucosidase inhibitory effect of propolis extracts was assayed according to
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the procedure described previously by Kim et al. (2005) with slight modifications.
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The enzyme reaction was performed using PNP-glycoside as a substrate in 0.1M
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PIPES buffer (pH 6.8). 1ml PNP-glycoside (1.0 mM) was premixed with 0.1 ml
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propolis extracts at various concentrations (2μg/ml-200μg/ml) in 2.05ml PIPES buffer
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and the reaction system was preheated at 37oC for 30 min. Then, 0.5 ml enzyme
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solution (0.11 U/ml) was added into mixture and absorbances were immediately
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measured at 30 second intervals for 15 min at 415nm using UV-2500/Ultraviolet
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visible spectrophotometer with UVProbe software (Shimadzu Co., Ltd., Tokyo, Japan).
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The inhibition activity was calculated by the equation: Inhibition (%) = [1-(A
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sample/A control)] ×100% (Kim et al. 2005). Acarbose was also assayed as a positive
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reference. Concentrations of extracts resulting in 50% inhibition of enzyme activity
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(IC50 values) were determined graphically. Different propolis extracts were compared
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on the basis of their IC50 values estimated from the dose response curves.
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2.6 Inhibitory activity assay for rat intestinal sucrase
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Rat intestinal sucrase inhibitory effects of propolis extracts was assayed
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according to a procedure described previously (Nishioka et al., 1998) with slight
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modifications. Briefly, 100 mg rat intestinal acetone powder was homogenized in 15
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ml 0.9 % NaCl solution (w/v). After centrifugation at 12,000g for 40 min, the
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supernatant was stored at 4oC as enzyme solution. 0.8 ml 56mmol/L Sucrose and 0.75
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ml 0.1 M PIPES buffer (pH 7.0) were premixed with 0.1ml propolis extracts of
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various concentrations (2μg/ml-200μg/ml). After pre-incubating for 5 min at 37 oC,
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0.7 ml of the enzyme solution was added and the reaction was carried out at 37oC for
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30 min. The reaction was terminated by adding 0.75 ml of 2 mol/L Tris-HCl buffer
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(pH 6.9). For the blank, 0.1 ml DMSO was used in place of propolis extract. The
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concentration of glucose released from the reaction mixtures was determined
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colorimetrically using Glucose-Trinder 100 (Hwang et al., 2000) and the absorbance
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measured at 505 nm after incubation at 37oC for 20 min. The inhibition activity was
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calculated by the equation: Inhibition (%) = [1-(A sample/A control)] ×100%.
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Acarbose was also assayed as a positive reference.
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2.7 Inhibitory activity assay for rat intestinal maltase
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Rat intestinal maltase inhibitory effect of propolis extracts was assayed
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according to a procedure described previously (Toda, Kawabata and Kasai, 2000). The
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assay for rat intestinal maltase inhibitory activity was carried out in a similar manner
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as the rat intestinal sucrase inhibitory activity, except that 10mM maltose in 0.1M
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PIPES buffer (pH 7.0) was used as a substrate. Acarbose was also assayed as a
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positive reference.
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2.8 Inhibition kinetics on alpha-glucosidase activity
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Inhibition model of different extracts of propolis on baker yeast’s α-glucosidase
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was tested according to procedure described previously (Shai et al., 2010) with minor
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modifications. Alpha-glucosidase activity was measured with increasing
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concentrations of PNP-glucoside (0.4-6.0 mM) in the absence or presence of propolis
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extracts at various concentrations (0.1-2.0 mg/ml). The reaction was initiated by
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addition of enzyme and the reaction measured at 415 nm at 30 seconds interval for 15
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min. The enzyme assay data containing various concentration of PNP-glucoside and
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propolis extracts were used to construct the Lineweaver-Burk plots to determinate
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inhibition model, Vmax and Km values.
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2.9 Inhibition constant analysis
The inhibition constant of propolis on alpha-glucosidase was calculated
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by
Ki 
[I ]
α  1 , where [I] represents the concentration of inhibitor, and α is confirmed
v
V max/α [ S ]
Km  [ S ] . Vmax and Km values were obtained from the above assay, and [S]
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by
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represents the concentration of substrate.
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2.10 Statistical analysis
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Experimental results were expressed as mean ± standard deviation (SD) of
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triplicate measurements. The data were subjected to one way analysis of variance
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(ANOVA). Significance differences at p＜0.05 among treatment means were obtained
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using Duncan’s multiple range test using SAS version 9.1 (SAS Institute Inc., Cary,
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NC, USA).
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3 Results and Discussion
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3.1 Total phenolic and flavonoid contents of aqueous ethanol extracts of propolis
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Phenolics are the predominant bioactive materials in propolis which have been
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reported to have multiple biological effects, including antidiabetes. Therefore,
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measurement of total phenolic contents (TPC) and total flavonoid contents (TFC) was
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inevitable. Total phenolic and flavonoid contents in various aqueous ethanol extracts
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of propolis are presented in Table 1. TPC ranged from 273.94 to 386.49 mgGAE/g
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extracts increasing in the following order: 25% EEP > WEP > 50% EEP > 75% EEP >
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95% EEP > 100% EEP. TPC was not significantly different among WEP, 25% EEP,
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and 50% EEP. TFC ranged from 352.32 to 697.36 mg CE/g extracts increasing in the
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following order: 75% EEP > 100% EEP > 95% EEP > 50% EEP > 25% EEP > WEP.
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TFC was not significantly different among 100% EEP, 95% EEP, and 75% EEP.
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The total phenolic and flavonoid contents of propolis extracts varied with
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different concentrations of hydrous ethanol. A similar report shows that ethanol/water
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concentrations correlates with the amount and composition of phenolic compounds
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and flavonoids of propolis extracts (Cottica et al. 2011). Moreover, propolis from
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various areas of China was found to contain a wide variety of bioactive compounds,
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mainly phenolic acids and flavonoids (Zhou et al., 2008). In the current study, while
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ethanol concentrations in hydrous ethanol were less than 50% as extraction solvent,
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the TPC of these extracts were significantly higher than those containing higher
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ethanol concentrations (p<0.05). These propolis extracts may mainly contain more
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hydrophilic phenolic compounds, cinnamic acid derivatives (Nakajima et al, 2007).
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On the other hand, when ethanol concentrations were higher than 50%, TFC of the
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extracts were significantly higher compared to those with lower ethanol
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concentrations (p<0.05). These propolis extracts mainly contain a significant increase
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in the ratio of more hydrophobic flavonoid compounds, such as apigenin, kaempferol
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and chrysin (Zhou et al., 2008).
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3.2 Inhibition of aqueous ethanol extracts of propolis against alpha-glucosidase
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The α-glucosidase inhibitory activity of various propolis extracts is shown in table
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2. The propolis extracts had ever been found to be reversibly bound to α-glucosidase
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in our previous report. The effectiveness of enzymatic inhibition of the various
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extracts was determined by calculating IC50. The lower of the value showed the higher
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of the enzymatic inhibition. IC50 values of propolis extracts ranged from 7.24 to 20.01
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μg/ml against baker’s yeast α-glucosidase and from 32.34 to 53.12 μg/ml against rat
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intestinal sucrase. These values were significantly (p<0.05) lower than 177.5 μg/ml
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and 538.3μg/ml of acarbose, a positive reference, respectively. Thus, all propolis
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extracts possessed much stronger inhibitory effects on α-glucosidase from baker’s
246
yeast and sucrase compared to acarbose. The 75% EEP showed the highest inhibitory
247
effect on α-glucosidase from baker’s yeast and sucrase, and IC50 values accounted for
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only 4% and 6% of that of acarbose, respectively. For rat intestinal maltase, WEP had
249
the highest inhibitory activity among all extracts with IC50 value of 32.67 μg/ml that
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was significantly higher than that of acarbose of 22.5μg/ml (p<0.05). All propolis
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extracts showed weak inhibitory effects on maltase in comparison to acarbose.
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Many plant extracts from food and Chinese traditional medicine have been
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reported to have anti-diabetic activity (Ye, Shen, Xie 2002). These anti-diabetic
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phytochemicals are probably comprising phenolic compounds, such as flavonoids and
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phenolic acids (Tadera, Minami, Takamatsu, & Matsuoka, 2006). Propolis extracts
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contain phenolic compounds which are classified into two major categories, phenolic
257
acids and flavonoids. As shown in table 1, TPC and TFC of various ethanol extracts of
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propolis were different. Similarly, the inhibitory effects of various propolis extracts on
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alpha-glucosidases were also different (Table 2). The 75% EEP possessed the highest
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flavonoid contents and the strongest inhibitory effect on α-glucosidases from baker’s
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yeast and rat intestinal sucrase among all extracts. Wang, et al. (2010) also reported
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that some flavonoids have the higher inhibitory effect on rat sucrase than that of rat
263
maltase. Moreover, the study revealed WEP and 25% EEP had higher TPC and
264
stronger inhibitory effects on rat intestinal maltase among all extracts. Some phenolics
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such as chebulanin, chebulagic acid and chebulinic acid were reported to have the
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same potential against rat intestinal maltase (Kumar et al., 2011). Kamiyama (2010)
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similarly found that some catechin derivatives had better inhibitory activity against rat
268
intestinal maltase than rat intestinal sucrase. Therefore, it seems to assume that
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extracts with higher TFC could have better inhibition against rat intestinal sucrase;
270
similarly extracts with higher TPC could possess better inhibition against rat intestinal
271
maltase.
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3.3 Inhibitory kinetics of aqueous ethanol extracts of propolis against baker’s
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yeast alpha-glucosidase
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Yeast α-glucosidase is readily available in a pure form and has been widely used
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for anti-diabetic nutraceutical and medicinal investigations as a model for screening
276
potential inhibitors and studying inhibitory mechanism (Ye, Shen, & Xie, 2002). To
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find the inhibition mechanism of aqueous ethanol extracts of propolis, inhibitory
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kinetics against yeast α-glucosidase was analyzed by Lineweaver-Burk plots using
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data derived from enzyme assay containing different concentrations of PNP-glucoside
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in the absence or presence of different concentration of inhibitor.
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As can be seen in Table 3, various aqueous ethanol extracts of propolis had
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different inhibition modes. Double-reciprocal plots of enzyme kinetics demonstrated
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that the inhibition of WEP and 25% EEP was competitive inhibition mode (Fig.1A
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and B), and the Ki values were 10.43μg/ml and 9.85µg/ml (Table 3). Plots also
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indicated that the type of 50% EEP, 95% EEP and 100% EEP was mixed inhibition
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mode (Fig.1C, E and F), and the Ki values were 9.92 μg/ml, 9.65 μg/ml and 10.89
287
μg/ml, respectively (Table 3). Moreover, inhibitory mode of 75% EEP was
288
noncompetitive inhibition (Fig.1D), and the Ki value was 15.18 μg/ml, a value
289
significantly higher than those of other extracts (p<0.05).
290
Phenolic compounds are able to inhibit the activities of carbohydrate-hydrolysing
291
enzymes due to their ability to bind with proteins (Shobana, Sreerama, & Malleshi,
292
2009). As can be seen, different aqueous ethanol extracts of propolis were revealed to
293
have different inhibition modes against α-glucosidase. Probably that is because
294
phenolic compounds in different EEP have different bound modes with α-glucosidase.
295
The inhibition of WEP and 25% EEP was a competitive mode characterized by the
296
fact that the substrate and inhibitor compete for the same binding site in the enzyme,
297
the so-called active site or substrate-binding site. It indicated that phenolic compounds
298
in WEP and 25% EEP can bind to the active site of α-glucosidase. The inhibition of
299
75%EEP was a noncompetitive mode in that inhibitor is not binding to the active site
300
on α-glucosidase. Inhibition mode was a mixed inhibition for 50%EEP, 95%EEP and
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100%EEP. For the noncompetitive and mixed inhibition mode, the binding of
302
inhibitor can influence the binding of substrate by changing the conformation of the
303
enzyme. Different inhibition modes shown by various EEP were likely due to the
304
different bioactive compounds in the extracts. 75% EEP was found to contain the
14
305
highest TFC among EEP and exhibit non-competitive inhibition. TFC of 95%EEP and
306
100% EEP was lower but not significantly different than 75% EEP and showed mixed
307
inhibition. A similar trend was observed that many flavonoids exhibited mixed or
308
noncompetitive type of inhibition (Tadera, Minami, Takamatsu, & Matsuoka, 2006;
309
Xu, 2010). WEP and 25%EEP contained the higher TPC and exhibited a competitive
310
inhibition mode. The ethanol extract of G. montanum rich in phenolic composition
311
also showed competitive inhibition against yeast α-glucosidase (Ramkumar, 2010). It
312
seems to assume that inhibition of aqueous ethanol extracts of propolis with the higher
313
TPC is likely a competitive mode while those with higher TFC tend to have a
314
noncompetitive or mixed mode.
315
All aqueous ethanol extracts of propolis showed stronger inhibition against the
316
yeast α-glucosidase compared to acarbose, especially for those extracts using the
317
higher concentration of ethanol as the solvent. It is probably that the ability to bind to
318
wide regions of enzyme other than the active site enables these propolis extracts as
319
noncompetitive or mixed inhibitors a broader specificity of inhibition, compared with
320
acarbose, a competitive inhibitor. Another advantage of aqueous ethanol extracts of
321
propolis over acarbose is that these propolis extracts, noncompetitive or mixed
322
inhibitors, may not be affected by the higher concentration of the substrate in contrast
323
to acarbose which is a competitive inhibitor. It is reported that, with higher
324
carbohydrate food intake, higher concentrations of acarbose as a competitive inhibitor
325
would be needed to show the same effect. For mixed inhibition, the inhibitor would be
326
still effective at lower concentrations (Ghadyale et al., 2012).
15
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328
4. Conclusion
In the present study, all aqueous ethanol extracts of propolis show stronger
329
inhibitory effects on yeast α-glucosidase and rat intestinal sucrose than that of
330
acarbose as a positive reference. Our findings seem to prove that the controlling
331
glycemia of propolis may be related to the inhibitory activities against α-glucosidase.
332
Thus, aqueous ethanol extracts of propolis may be used as nutracueticals for the
333
regulation of postprandial hyperglycemia. For further study, the fractions from
334
propolis having inhibitory activity against α-glucosidase are being purified and their
335
chemical structures are being characterized.
336
Acknowledgements
337
This research was supported by a grant project of 12th Five Years’ National
338
Science and Technology Support Programme (2011BAD33B04) from the Ministry of
339
Science & Technology of P. R. China.
340
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440
Table 1 Total phenolic and flavonoid contents of various ethanol extracts of propolis
Aqueous ethanol extracts of
propolis (EEP)
Water extracts of propolis (WEP)
25% ethanol extracts of propolis
(25% EEP)
50% ethanol extracts of propolis
(50% EEP)
75% ethanol extracts of propolis
(75% EEP)
95% ethanol extracts of propolis
(95% EEP)
100% ethanol extracts of propolis
(100% EEP)
Total phenolic contents (mg
Total flavonoid contents (mg
GAE/g extracts)
CE/g extracts)
ab
352.32±0.67a
369.31±26.87
386.49±17.36a
504.18±32.10b
355.71±31.35ab
620.80±4.08c
341.05±14.89b
697.36±6.15d
312.65±24.13cb
637.84±8.13cd
273.94±1.76d
673.24±5.96d
441
Note: Water extracts of propolis was expressed as WEP. Extracts of propolis using 25 %, 50%, 75%,
442
95% and 100% (in water, v/v) aqueous ethanol solvents were expressed as 25% EEP, 50% EEP, 75%
443
EEP, 95% EEP and 100% EEP, respectively. TPC was expressed as milligram of gallic acid equivalent
444
per gram of propolis extracts (mg GAE/g extracts). TFC was expressed as milligram of rutin equivalent
445
per gram of propolis extracts (mg RE/g extracts). Dates are mean ±standard deviation (n=3).Values in
446
the same column followed by the same lower case letter are not significantly different by Duncan ’s
447
multiple range test (p<0.05).
448
21
449
450
Table 2 Inhibition of propolis extracts against yeast and rat intestinal
alpha-glucosidase
IC50(μg/ml)
Aqueous ethanol extracts
of propolis (EEP)
Baker’s yeast
alpha-glucosidase
Rat intestinal
sucrase
Rat intestinal
maltase
WEP
16.0±1.38bc
37.91±0.52ab
32.67±0.24e
25% EEP
11.32±2.23ab
40.25±0.97ab
44.80±0.25d
50% EEP
12.90±1.14b
53.12±0.95b
100.60±2.95b
75% EEP
7.24±1.16a
32.34±0.61a
71.82±2.95c
95% EEP
16.26±0.43bc
32.91±0.54a
102.76±1.47b
c
ab
131.12±1.83a
100% EEP
20.01±1.54
Acarbose
177.47±6.28d
38.06±0.56
538.30±26.69c
22.46±0.86f
451
Note: Water extracts of propolis was expressed as WEP. Extracts of propolis using 25 %, 50%, 75%,
452
95% and 100% (in water, v/v) aqueous ethanol solvents were expressed as 25% EEP, 50% EEP, 75%
453
EEP, 95% EEP and 100% EEP, respectively. Dates are mean ±standard deviation (n=3).Values in the
454
same column followed by the same lower case letter are not significantly different by Duncan’s
455
multiple range test (p<0.05). Inhibition of propolis extracts against yeast and rat intestinal
456
alpha-glucosidase were both expressed as IC50 (concentration of total phenolics able to scavenger 50%
457
of alpha-glucosidase activity)
458
459
460
461
462
22
463
464
Table 3 Inhibitory kinetics and Ki values of various propolis extracts against baker’s
yeast alpha-glucosidase
Aqueous ethanol extracts of
propolis (EEP)
WEP
25% EEP
50% EEP
75% EEP
95% EEP
100% EEP
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
Kinetic mode
Ki (μg/ml)
competitive inhibition
competitive inhibition
mixed inhibition
noncompetitive inhibition
mixed inhibition
mixed inhibition
10.43±1.41b
9.85±2.48b
9.92±0.43b
15.18±0.58a
9.65±1.54b
10.89±2.84b
Note: Water extracts of propolis was expressed as WEP. Extracts of propolis using 25 %, 50%, 75%,
95% and 100% (in water, v/v) aqueous ethanol solvents were expressed as 25% EEP, 50% EEP, 75%
EEP, 95% EEP and 100% EEP, respectively. The inhibition constant of propolis on alpha-glucosidase
was expressed Ki. Dates are mean ± standard deviation (n=3).Values in the same column followed by
the same lower case letter are not significantly different by Duncan’s multiple range test (p<0.05).
23
499
(A)
Control
1/V(mM/min)-1
100
0.3mg/ml
80
1.0mg/ml
60
1.5mg/ml
40
20
0
-6
-3
0
3
-20
9
12
15
-40
500
501
6
1/[S](mM)-1
(B)
180
Control
0.5mg/ml
150
1/V(mM/min)-1
1.0mg/ml
120
1.5mg/ml
90
2.0mg/ml
60
30
0
-6
-4
-2
0
2
4
6
-30
1/[S ](mM)
502
503
8
10
12
14
-1
-60
(C)
Control
180
0.5mg/ml
150
1.0mg/ml
1.5mg/ml
1/V(mM/min)-1
120
2.0mg/ml
90
60
30
0
-9
-6
-3
0
3
-30
504
505
6
-1
1/[S ](mM)
9
12
15
-60
(D)
Control
90
0.1mg/ml
80
0.25mg/ml
70
0.5mg/ml
1/V(mM/min)-1
1.0mg/ml
60
50
40
30
20
10
0
506
-10
-5
0
1/[S](mM)-1
5
10
15
24
507
(E)
140
Control
0.5mg/ml
120
1.0mg/ml
1.5mg/ml
80
2.0mg/ml
1/V(mM/min)-1
100
60
40
20
0
-6
-4
-2
-20
2
4
6
8
10
12
14
1/[S](mM)-1
-40
508
509
0
(F)
1/V(mM/min)
-1
Control
160
0.5mg/ml
140
1.0mg/ml
120
1.5mg/ml
2.0mg/ml
100
80
60
40
20
-6
-4
-2
0
-20 0
-40
510
511
512
513
514
515
2
4
6
8
10
12
14
1/[S](mM)-1
-60
Fig. 1 Lineweaver-Burk plots of inhibition kinetics of yeast alpha-glucosidase inhibitory effects by
WEP (A), 25% EEP (B), 50% EEP (C), 75% EEP (D), 95% EEP (E) and 100% EEP (F). Water extracts
of propolis was expressed as WEP. Extracts of propolis using 25 %, 50%, 75%, 95% and 100% (in
water, v/v) aqueous ethanol solvents were expressed as 25% EEP, 50% EEP, 75% EEP, 95% EEP and
100% EEP, respectively.
25