Plant Foods Hum Nutr

DOI 10.1007/s11130-016-0581-2

ORIGINAL PAPER

Evaluation of Total Flavonoids, Myricetin, and Quercetin

from Hovenia dulcis Thunb. As Inhibitors
of α-Amylase and α-Glucosidase
Yonghong Meng 1 & Anping Su 1 & Shuang Yuan 1 & Huaguo Zhao 2 & Siyuan Tan 1 &
Chingyuan Hu 3 & Hong Deng 1 & Yurong Guo 1

# Springer Science+Business Media New York 2016

Abstract This study was designed to investigate the inhibi-
tion effect and mechanism of total flavonoids, myricetin and
quercetin extracted from Hovenia dulcis Thunb. on α-amylase
and α-glucosidase in order to explore the potential use of
Hovenia flavonoids in alleviating postprandial hyperglyce-
mia. The results demonstrate that total flavonoids, myricetin,
and quercetin were effective inhibitors of α-amylase with IC50
values of 32.8, 662 and 770 μg ml−1, respectively. And all
three were effective inhibitors of α-glucosidase with IC50
values of 8, 3 and 32 μg ml−1, respectively. Enzyme kinetics
tests and Lineweaver-Burk results showed the inhibition ef-
fects of total flavonoids, myricetin and quercrtin on α-amylase
were all reversible and competitive, and the effects on α-
glucosidase were all reversible but non-competitive. This
study revealed that Hovenia flavonoids, especially myricetin,
are effective and promising functional foods in alleviating
type 2 diabetes mellitus.
Electronic supplementary material The online version of this article
(doi:10.1007/s11130-016-0581-2) contains supplementary material,
which is available to authorized users.
* Yonghong Meng
mengyonghong@snnu.edu.cn
1
Shaanxi Engineering Lab for Food Green Processing and Security
Control, College of Food Engineering and Nutritional Science,
Shaanxi Normal University, 620 West Chang’an Avenue, Chang’an,
Xi’an 710119, People’s Republic of China
2
Xi’an Healthful Biotechnology Co., Ltd., HangTuo Road, Chang’an,
Xi’an 710100, People’s Republic of China
3
Department of Human Nutrition, Food and Animal Sciences, College
of Tropical Agriculture and Human Resources, University of Hawaii
at Manoa, 1955 East West Road, AgSci 216, Honolulu, HI 96822,
USA
Keywords Flavonoids . T2DM . α-amylase .
α-glucosidase . Hovenia dulcis Thunb
Abbreviations
ESI-MS The electrospray ionization mass
spectrometry
H. dulcis Thunb. Hovenia dulcis Thunb.
NMR Nuclear magnetic resonance
T2DM Type 2 diabetes mellitus
TFs Total flavonoids
Introduction
Type 2 diabetes mellitus (T2DM) is a serious chronic meta-
bolic disease which represents more than 90 % of all diabetes
patients [1]. Diet plays an important role in T2DM develop-
ment [2]. Regulating and retarding the activity of the major
digestive enzymes, mainly pancreatic α-amylase and α-glu-
cosidase, has been reported to reduce the absorption rate of
carbohydrates in the gastrointestinal tract, control or even pre-
vent T2DM and alleviate postprandial hyperglycemia [3].
Pancreatic α-amylase and α-glucosidase are responsible for
the hydrolysis of carbohydrates into disaccharides and mono-
saccharides, which are absorbed into the bloodstream from the
small intestine [4]. Thus, inhibition of these two enzymes
maybe an effective tool in alleviating hyperglycemia, and con-
sequently beneficial for T2DM patients.
Many studies have demonstrated that plant flavonoids have
the function of inhibiting enzymes to reduce blood sugar. For
example, Kim et al. [5] investigated the potential of sorghum
in the development of effective anti-diabetic agents. Lin et al.
[6] revealed the flavonoids extracted from guava fruit signif-
icantly reduced the glucose and blood urea levels in the

diabetic mice model. And Dalar et al. [7] found that
E. bornmuelleri leaf extracts can modulate metabolism of
sugars through inhibition of the relevant digestive enzymes.
In this study, we were interested in finding more potent α-
amylase and α-glucosidase inhibitors from Hovenia dulcis
Thunb. (H. dulcis Thunb.), a natural plant, which belongs to a
small genus of Rhamnaceae family, being widely distributed in
southern and southwestern China [8]. The ancient Chinese med-
icine records showed that the H. dulcis Thunb. (known as the
longevity fruit in China) has many beneficial properties, such as
antifebrile, detoxifying alcoholic intoxication, slaking thirst and
helping to produce saliva, strengthening the stomach, and pro-
moting digestion [9]. The main chemical constituents of
H. dulcis Thunb. are flavonoids, including kaempferol, apigenin,
myricetin, quercetin, dihydromyricetin and anthraquinones em-
odin. etc. [8, 10]. We found the total flavonoids (TFs), myricetin
and quercetin among Hovenia chemical ingredients are quality
inhibitors of α-amylase and α-glucosidase, and their inhibition
ratio and mechanisms were elucidated by examining their enzy-
matic dynamics. Our results provide new knowledge for devel-
oping Hovenia flavonoids as a high value-added functional food.
Materials and Methods
Materials and Reagents
Mature H. dulcis Thunb. seeds were harvested from Xunyang
County of Shaanxi province of China. D101 macroporous
resin (5.0 i.d. ×100 cm) was purchased from Nankai
Hecheng S&T (Tianjin, China). The pancreatic α-amylase,
α-glucosidase, rutin and 4-nitrophenylα-D-glucopyranoside
(pNPG) were purchased from Sigma-Aldrich (St. Louis,
MO, USA). The acetic acid and acetonitrile were HPLC grade
and purchased from Tianjin Hedong Red Cliff Chemical
Reagent Factory (Tianjin, China). All other reagents were of
analytical grade.
Extraction, Quantification and Isolation of Main Active
Compositions from TFs
Dried mature seeds of H. dulcis Thunb. were milled into fine
powder and screened through a 60-mesh sieve to ensure their
homogeneity. Powdered samples (10 g) were reflux extracted
with 100 ml of 75 % ethanol for three times (1 h each time).
After using rotary evaporator to remove ethanol from the ex-
tract, the residual extract fraction was frozen to dryness [11].
TFs was assayed using the method of Xie et al. [12]. The
concentration of TFs in extract was expressed as mg of rutin
equivalents per gram dry weight of extract. The calibration
curve was determined using the following equation:
Y = 0.031 X + 0.0347, R2 = 0.9992. Where X is absorbance
value of sample, Y is sample concentration.
Plant Foods Hum Nutr
Then the extracts were concentrated under vacuum to a
liquid density range of 1.02–1.05 g ml−1, and then filtered
for subsequent studies. D101 macroporous resin was applied
to separate the extracts with resin sample loading amount of
125 mg g−1. The impurities were first removed using distilled
water and followed by 5–10 % (v/v) ethanol. The eluent was
collected using 65 % (v/v) ethanol, concentrated by vacuum,
and then subjected to a silica gel chromatography column,
eluted by petroleum ether (PE): ethyl acetate (EA) system.
Myricetin fraction was collected using PE:EA = 5:2 (v/v),
quercetin fraction using PE:EA = 3:1 (v/v). The ultimate elu-
ate was concentrated and dried once again. The dried sample
was repeatedly recrystallized with 100 % methanol to obtain
highly purified myricetin and quercetin. These purified pow-
ders were used in the subsequent experiments for the determi-
nation of chemical and enzymology analyses.
Determination of Structures of Main Active Compositions
of TFs
The electrospray ionization mass spectrometry (ESI-MS)
spectra were measured on a Bruker Apex III FT ion cyclotron
resonance mass spectrometer in the positive ionization mode
at a potential of 4.0 kV (on the discharge needle). The 1H
nuclear magnetic resonance (NMR) spectra were recorded
on a Bruker AMX 400 spectrometer from Bruker Biospin
(Rheinstetten, Germany) operating at a proton frequency of
400.13 MHz. The resulting spectra were manually phased and
the baselines were corrected manually.
Chemical Analysis of Myricetin and Quercetin
Thermo U3000 HPLC system and C18 column
(4.6 × 250 mm, Venusil, USA) were used for the analysis.
The flow rate of mobile phase consisted of 0.1 % acetic acid
in pure water and pure acetonitrile at the flow rate of
1 ml min−1. Eluent program was 20 % pure acetonitrile for
3 min, to 38 % for 24 min, to 90 % for 1 min, and then held at
90 % for 5 min, to 20 % for 6 min. The injection volume was
10 μL, and UV absorbance was measured at 368 nm [13].
Inhibition Activity and Mechanism Study
One unit of α-amylase is the amount of enzyme that catalyzes
and reduces 1 μmol sugar per minute at pH 6.9 and 25 °C. The
rate of α-amylase reaction was assayed using the method of
Oboh et al. [14] with slight modification. Briefly, 0.5 ml of
0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl),
0.5 ml of 0.54 U ml−1 α-amylase solution, and 0.5 ml of 1 %
starch solution in 0.02 M sodium phosphate buffer were
mixed and incubated at 25 °C for 3 min; enzyme reaction
stopped using 1 ml of dinitrosalicylic acid color reagent,
cooled to room temperature, and change in absorbance at
Plant Foods Hum Nutr
Fig. 1 HPLC chromatogram of
TFs and standard myricetin and
quercetin. The structures of
standard myricetin and quercetin
are shown above their peaks
540 nm per minute was measured using a UV
spectrophotometer.
One unit of α-glucosidase is the amount of enzyme that
catalyzes the hydrolysis of 1 mmol substrate (pNPG)
per minute at pH 6.8 and 37 °C. The rate of α-
glucosidase reaction was assayed according to Xi et al.
[15] with slight modification. The 2 ml reaction system
consisted of 35 U ml−1 α-glucosidase, 2.02 × 10−4 mol L−1
substrate pNPG, and 0.1 mol L−1 sodium phosphate buffer
(pH 6.8). After incubated at 37 °C for 10 min, the change in
absorbance at 405 nm per minute was measured using a UV
spectrophotometer.
When inhibition mode on α-amylase was tested, the final
concentrations of 32 μg ml−1 TFs, 100 μg ml−1 myricetin and
200 μg ml−1 quercetin solution were separately pre-incubated
with 1 % starch solution and different concentrations of each
enzyme at 25 °C for 20 min. When inhibition mode on
α-glucosidase was tested, the final concentrations of
0.4 μg ml−1 TFs, 0.5 μg ml−1 myricetin and
5 μg ml−1 quercetin solution were separately preincubat-
ed with 5 mmol L−1 pNPG different concentrations of
each enzyme at 37 °C for 20 min. There is no inhibitor in
control system. Then the enzyme reaction was started as
Table 1 Inhibitory effect of flavonoids from H. dulcis Thunb. on α-amylase and α-glucosidase
α-amylase
Regression equation
TFs y = 1.1*10−3 × 3–0.061 × 2 + 1.51× + 26.34 32.8
Myricetin y = 56.008 × 4–249.51 × 3 + 345.27 × 2–113.42× + 35.42 662
Quercetin y = 178.78 × 3–276.45 × 2 + 154.35× + 13.28 770
Bx^ represents the different inhibitor concentration, By^ represents the inhibition rate
previously described. Nonlinear fitting was employed to cal-
culate the IC50 values.
In order to study the inhibitory effects of Hovenia flavonoids
on α-amylase kinetics, we used various concentrations (0.6,
0.8, 1, 1.2, 1.4, and 1.6 %) of starch solution, various inhibitors
(8 μg ml−1 TFs, 50 μg ml−1 myricetin and 50 μg ml−1 quercetin
solution) in the presence of 0.54 U/ml α-amylase. And for α-
glucosidase, various concentrations (0.75, 1.0, 1.25, 1.5, 1.75,
and 2 μg ml−1) of substrate, and various inhibitors (0.4 μg ml−1
TFs, 1 μg ml−1 myricetin and 10 μg ml−1 quercetin solution) in
the presence of 35 U/ml α-glucosidase were used. Then the
Lineweaver-Burk plots were used to determine the inhibition
mode by calculating the inhibition constant through nonlinear
regression. IC50 value is the concentration of a test substance
required to achieve half maximal inhibition rate of a given
reaction. The inhibition rate is calculated using the following
formula:
.
The inhibition rateð%Þ ¼ ðA−AI Þ A*100%
A = absorbance of reaction system without inhibitor.
AI = absorbance of reaction system with addition of
inhibitor.
α-glucosidase
IC50 Regression equation IC50
(μg ml−1) (μg ml−1)
y = −2*10−5 × 4–0.0096 × 3–0.36 × 2 + 4.71× + 32.86 8
y = −2*107 × 3 + 134599 × 2 + 2919.7× + 40.09 3
y = 2*106 × 4-251237 × 3 + 6163.4 × 2 + 504.7× + 33.76 32
 Plant Foods Hum Nutr

Fig. 2 Kinetics curves of different inhibitors (TFs, myricetin, and quercetin) on α- amylase and α-glucosidase (a α-amylase; b α-glucosidase)

Competitive inhibition formula: Statistical Analysis

 
1 Km ½I  1 1 All values were expressed as mean ± standard deviation, and
¼ 1þ þ ð1Þ
V Vmax Ki ½S  Vmax each value was the mean of separate experiments in triplicate.

Non-competitive inhibition formula:

   
1 Km ½I  1 1 ½I  Results and Discussion
¼ 1þ þ 1þ ð2Þ
V Vmax Ki ½S  Vmax Ki
Isolation of Active Flavonoid Ingredients
Where V is the initial reaction rate; Vmax is the maximum and its Structural Analyses
reaction rate; Km is the Michaelis-Menten constant; Ki is the
inhibition constant. [S] is the concentration of substrate; [I] is From the MS and NMR data, two main pure compounds were
the inhibitor concentration. identified as myricetin and quercetin with the molecular

Fig. 3 Lineweaver-Burk plot of different inhibitors on α-amylase (a TFs; b myricetin; c quercetin) and on α-glucosidase (d TFs; e myricetin; f
quercetin)
Plant Foods Hum Nutr

Table 2 Related parameters on

inhibition kinetics of flavonoids Double reciprocal R2 Km Vmax (OD Ki Inhibition
from H. dulcis Thunb. on α- equation (mg ml−1) min−1) (μg ml−1) type
amylase
TFs y = 48.57× + 2.07 0.99 24.28 0.5 12 competitive
Myricetin y = 49.58× + 1.99 0.99 24.79 0.5 70 competitive
Quercetin y = 46.48× + 1.99 0.99 23.24 0.5 90 competitive
Control y = 29.63× + 2.01 0.99 14.68 0.5 / competitive

Bx^ represents the reciprocal of substrate concentration, By^ represents the reciprocal of rate of catalysis reaction
rate, B/^ represents no inhibition constant (Ki) value

weight of 317.02 and 301.03, respectively (MS and NMR
spectrum were shown in Supplementary Fig.1-3). HPLC re-
sults (Fig.1) show that myricetin and quercetin are the major
components in H. dulcis Thunb., representing 63.2 and
12.1 % in TFs, respectively.
IC50 of the TFs, Myricetin and Quercetin for α-Amylase
and α-Glucosidase
Table 1 shows the inhibition rate regression equation and IC50
of three tested substrates on α-amylase and α-glucosidase. R2
for each regression equation was 0.99, indicating good fitting
degree. TFs was the most effective inhibitor of α-amylase
followed by myricetin, and quercetin with calculated IC50
values of 32.8, 662 and 770 μg ml −1 , respectively.
Myricetin, however, was the most effective inhibitor of α-
glucosidase followed by TFs, and quercetin with calculated
IC50 values of 3, 8 and 32 μg ml−1, respectively.
Acarbose is a well-known and typical anti-diabetic drug
because its IC50 for α-glucosidase is 17.8 μg ml−1 [4]. In
contrast, IC50 of myricetin (3 μg ml−1) for α-glucosidase
was six times lower than that for acarbose. And the IC50 of
TFs for α-glucosidase was 8 μg ml−1, its effectiveness was
also better than that of acarbose. Therefore, Hovenia flavo-
noids exerted a higher inhibition effect on α-glucosidase,
which means Hovenia flavonoids are potentially better alter-
natives for T2DM patients than acarbose. In addition, tradi-
tional oral hypoglycemic drugs for diabetes have undesirable
side effects, such as liver toxicity, kidney damage and adverse
gastrointestinal symptoms [5, 16]. It is worth noting that pre-
vious reports have demonstrated that mild α-amylase inhibi-
tory activity is useful to alleviate gastrointestinal malaise,
because excessive inhibition of α-amylase could lead to ab-
normal bacterial fermentation of undigested starch in the co-
lon [17]. Interestingly, it is significant to note that myricetin
had the most inhibition (IC50 = 3) for α-glucosidase but lower
inhibition (IC50 = 662) for α-amylase, which suggests that
myricetin most likely will produce less side effect. In addition,
other studies have shown that myricetin has various other
functions without toxicity, which may alleviate complications
of T2DM [11, 18]. Taken together, we have demonstrated that
myricetin, one of the promising components extracted from
H. dulcis Thunb. is a promising compound to alleviate post-
prandial hyperglycemia or to be used as health care products.
The Inhibition Mode and Inhibition Kinetics
of α-Amylase and α-Glucosidase
Effect of three tested compounds on initial reaction rates was
measured as described in the methods section. When there is a
reversible inhibitor in the reaction system, its rate curve goes
through the origin. From Fig. 2 we can see that all the rate
lines went through the origin, so the inhibition type of TFs,
myricetin and quercetin for α-amylase and α-glucosidase was
all reversible inhibition. Reversible inhibition mode doesn’t
have accumulation effect in vivo, thus it should be safer to
be used as a functional food.
Lineweaver-Burk plots were generated to clarify inhibition
mode. As shown in Fig. 3a, b, c, all inhibition kinetics curves
crossed y axis, so the inhibition type of TFs, myricetin and
quercetin for α-amylase was competitive. From the
Michaelis-Menten equation in Table 2, the value of the verti-
cal axis intercept (1/Vmax) remained unchanged with all three
compounds, indicating that the TFs, myricetin and quercetin

Table 3 Related parameters on

inhibition kinetics of flavonoids Double reciprocal R2 Km Vmax Ki Inhibition type
from H. dulcis Thunb. on α- equation (mg ml−1) (OD min−1) (μg ml−1)
glucosidase
TFs y = 57.32× + 40.98 0.99 1.4 0.02 0.22 Non-competitive
Myricetin y = 81.20× + 58.73 0.99 1.4 0.01 0.32 Non-competitive
Quercetin y = 57.07× + 40.37 0.99 1.4 0.02 50 Non-competitive
Control y = 21.16× + 14.21 0.99 1.4 0.07 / Non-competitive

Bx^ represents the reciprocal of substrate concentration, By^ represents the reciprocal of rate of catalysis reaction
rate, B/^ represents no inhibition constant (Ki) value

were competitive inhibitor for α-amylase. And the value of
the inhibition constant (Ki) was 12, 70 and 90 μg ml−1, re-
spectively. In Fig. 3d, e, f, all inhibition kinetics curves
crossed x axis, so the inhibition type of TFs, myricetin and
quercetin for α-glucosidase was non-competitive. From the
Michaelis-Menten equation in Table 3, the value of the trans-
verse axis intercept (Km) remained unchanged with all three
compounds, indicating that TFs, myricetin and quercetin were
non-competitive inhibitor for α-glucosidase. And the value of
the inhibition constant (Ki) is 0.22, 0.32 and 50 μg ml−1, re-
spectively. Because there are combined actions between in-
hibitor and enzyme as well as between inhibitor and enzyme-
substrate complex in the non-competitive enzyme reaction,
the inhibition effect of non-competitive mode was stronger
than competitive mode. This explains why Hovenia flavo-
noids have a lower IC50 for α-glucosidase.
In conclusion, Hovenia flavonoids offer a prospective al-
ternative for the management of postprandial hyperglycemia.
Myricetin exerted promising inhibition effect on two major
hydrolytic enzymes, α-amylase and α-glucosidase, and it ex-
hibits similar reversible inhibition mechanism to that of
acarbose. Furthermore, myricetin is more effective than
arcobase due to non-competitive inhibition on α-glucosidase.
This study indicates that H. dulcis Thunb. may be used as a
functional food with the purpose of T2DM alleviation as well
as to be utilized by the nutraceutical industry as a source of
myricetin.
Acknowledgments This research was supported by the overall plan-
ning of Shaanxi science and technology (Project No. 2015KTCQ02-07),
Doctoral Scientific Fund Project of the Ministry of Education of China
(20130202120007), National Key Technology R&D Program
(2015BAD16B02) and an earmarked fund for the China Agriculture
Research System (CARS-28).
Compliance with Ethical Standards
Conflict of Interest All authors declare that they have no conflict of
interest.
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