Journal of Ethnopharmacology 186 (2016) 189–195

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Screening for potential α-glucosidase and α-amylase inhibitory

constituents from selected Vietnamese plants used to treat type
2 diabetes
Binh T.D. Trinh, Dan Staerk, Anna K. Jäger n
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100
Copenhagen, Denmark

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: The 18 plant species investigated in this study have been used as herbal
Received 11 November 2015 antidiabetic remedies in Vietnamese traditional medicines. This study aimed to evaluate their ability to
Received in revised form inhibit α-glucosidase and α-amylase, two key enzymes involved in serum glucose regulation.
29 March 2016
Materials and methods: Chloroform, ethanol and water extracts of 18 plants were screened for α-glu-
Accepted 30 March 2016
cosidase and α-amylase inhibitory activity. Analytical-scale HPLC was subsequently used to investigate
Available online 31 March 2016
the most active extracts, where samples with low level of tannins were identified and fractionated into
Keywords: 96-well microplates, followed by α-glucosidase and α-amylase assessment of each well. High-resolution
Type 2 diabetes α-glucosidase and α-amylase inhibition profiles constructed from these assays allowed identification of
α-amylase HPLC peaks correlated with α-glucosidase and α-amylase inhibitory activity. The active constituents
α-glucosidase were subsequently isolated using preparative-scale HPLC and their structure was elucidated by HR-
High-resolution inhibition profile
ESIMS and NMR.
Vietnamese plants
Phyllanthus
Results: Ethanol extracts of Nepenthes mirabilis, Phyllanthus urinaria, and Kandelia candel significantly
inhibited α-glucosidase with IC50 values of 32.7 76.3, 39.77 9.7, and 35.47 13.9 μg/mL, respectively.
Water extracts of N. mirabilis, Phyllanthus amarus, P. urinaria, Lagerstroemia speciosa, Syzygium cumini,
Rhizophora mucronata, and K. candel showed IC50 values of 3.3 70.8, 34.9 7 1.5, 14.6 74.6, 5.4 70.5,
20.9 71.8, 3.37 0.6, and 4.07 0.8 μg/mL, respectively. In the α-amylase inhibition assay, ethanol extracts
of K. candel and Ficus racemosa showed IC50 of 7.67 0.9 and 46.7 7 23.6 μg/mL, respectively. Showing low
tannin constituents as seen from HPLC profiles, P. amarus and P. urinaria water extracts and F. racemosa
ethanol extract were subjected to microfractionation. Only high-resolution α-glucosidase inhibition
profiles of P. amarus and P. urinaria water extracts showed several active compounds, which were iso-
lated and identified as corilagin (1), repandusinic acid A (2), and mallotinin (3). IC50 of these compounds
were 1.707 0.03, 6.10 7 0.10, and 3.76 70.15 μM, respectively. Kinetics analysis revealed that 1 displayed
a mixed type mode of inhibition with Ki and Ki′ values of 2.37 70.90 and 2.61 7 0.61 μM, respectively,
whereas 2 and 3 competitively inhibited α-glucosidase with Ki values of 4.01 70.47 and 0.65 7 0.11 μM,
respectively.
Conclusion: Corilagin (1), repandusinic acid A (2), and mallotinin (3) were potent α-glucosidase in-
hibitors contributing significantly to the inhibitory effect observed for the water extracts of P. amarus and
P. urinaria.
& 2016 Published by Elsevier Ireland Ltd.

1. Introduction
Diabetes mellitus, a chronic metabolic disease, has become a
worldwide health problem. In 2010, 285 million people between
Abbreviations: HPLC, High-performance liquid chromatography; NMR, Nuclear
Magnetic Resonance; HR-ESIMS, High-resolution electrospray ionization mass
spectrometry
n
Corresponding author.
E-mail address: anna.jager@sund.ku.dk (A.K. Jäger).
the age of 20 and 79 were affected, and this number is predicted to
increase to 439 million people by 2030 (Shaw et al., 2010). Treat-
ment and strategies for prevention of diabetes amounted to 376
billion USD in 2010, and is expected to rise to 490 billion USD in
the next two decades. (Zhang et al., 2010).
Accounting for roughly 90–95% of all diabetes cases worldwide,
type 2 diabetes is characterized by high and/or fluctuating blood
glucose due to insulin resistance. (Alberti et al., 2004; Interna-
tional Diabetes Federation, 2013). This is associated with severe
complications such as high blood pressure, blindness, kidney

http://dx.doi.org/10.1016/j.jep.2016.03.060
0378-8741/& 2016 Published by Elsevier Ireland Ltd.
190 B.T.D. Trinh et al. / Journal of Ethnopharmacology 186 (2016) 189–195

failure, lower limb amputation, heart disease, and stroke (Fowler, Table 1
2008). One way of maintaining lower and more stable blood glu- Plants tested for α-glucosidase and α-amylase inhibitory activity
cose is by inhibiting the carbohydrate hydrolyzing enzymes α-
Scientific name Family Part used Voucher #
glucosidase and α-amylase in the digestive system (Toeller, 1994).
Secreted from saliva and pancreas, α-amylase catalyzes the clea- Nepenthes mirabilis (Lour.) Druce Nepenthaceae Whole VN-01
vage of α-1,4 glycosidic bonds to convert polysaccharides into plant
smaller oligosaccharides such as maltose, maltotriose, and a Ludwigia octovalvis (Jacq.) P.H. Onagraceae Aerial part VN-02
Raven
number of α-1,4 and α-1,6-oligoglucans. These fragments are Phyllanthus amarus Schumach. & Phyllanthaceae Whole VN-03
subsequently involved in further degradation by α-glucosidase Thonn. plant
located in the brush border of the small intestine. This enzyme is Phyllanthus urinaria L. Phyllanthaceae Whole VN-04
responsible in the hydrolysis of terminal non-reducing 1,4 linked plant
α-glucose residues leading to the release of absorbable mono- Phyllanthus reticulatus Poir. Phyllanthaceae Stem and
leaf
VN-05

saccharides to enter the blood stream (Alagesan et al., 2012a, Scoparia dulcis L. Scrophulariaceae Whole VN-06
2012b; Chiba, 1997; de Sales et al., 2012). Therefore, inhibition of plant
these enzymes can delay digestion of carbohydrates causing a Mirabilis jalapa L. Nyctaginaceae Whole VN-07
reduction in the rate of glucose absorption and consequently a plant
Cassia fistula L. Leguminosae Leaf VN-08
suppression of postprandial hyperglycemia (Kumar et al., 2011). Lagerstroemia speciosa (L.) Pers. Lythraceae Leaf VN-09
In recent years, several studies have been focused on α-glu- Syzygium cumini (L.) Skeels Myrtaceae Fruit VN-10
cosidase and α-amylase inhibitors (AGIs and AAIs) from medicinal Euphorbia hirta L. Euphorbiaceae Whole VN-11
plants (Kasabri et al., 2011; Mojica et al., 2015; Nampoothiri et al., plant
Rhizophora mucronata Lam. Rhizophoraceae Bark VN-12
2011; Thilagam et al., 2013; Wang et al., 2010). Although bioassay-
Kandelia candel (L.) Druce Rhizophoraceae Bark VN-14
guided fraction has been successfully applied for identification of Cardiospermum halicacabum L. Sapindaceae Whole VN-14
AGIs and AAIs, this approach is time-consuming, and the repeated plant
preparative-scale separations may cause minor constituents with Pandanus odoratissimus L.f. Pandanaceae Fruit VN-15
interesting bioactivity to be missed (Pieters and Vlietinck, 2005). Morinda citrifolia L. Rubiaceae Fruit VN-16
Ficus racemosa L. Moraceae Fruit VN-17
In this study, we therefore used high-resolution inhibition profil- Pithecellobium dulce (Roxb.) Leguminosae Stem and VN-18
ing, in which eluate from analytical-scale high-performance liquid Benth. leaf
chromatography is microfractioned into microplates followed by
bioassaying of the material in all wells (Giera et al., 2009). High-
resolution inhibition profiles constructed from these assays can 2.2. Plant material
allow fast pinpointing of individual chromatographic peaks re-
sponsible for the bioactivity, which is an essential step for tar- A list of 18 species of medicinal plants was obtained based on
geting subsequent isolation and structure elucidation. High-re- literature about plants that have been used widely in Vietnamese
solution inhibition profiling has already proven a promising and folk medicine to treat diabetes (Pham, 2007; Vo, 1997). Samples
efficient method for identification of the bioactive constituents were collected from the Southern part of Vietnam in June 2014,
from natural sources such as microorganisms (Wubshet et al., and identified by Son V. Dang, Curator of the VNM Herbarium,
2013a), food (Schmidt et al., 2014; Wiese et al., 2013; Wubshet Institute of Tropical Biology, Vietnam. Voucher specimens are
et al., 2013b), and medicinal plants (Kongstad et al., 2015; Liu et al., stored at the General Herbarium of Vascular Plants, National His-
2015; Tahtah et al., 2015). tory Museum, University of Copenhagen. All the plant species, the
Vietnam is a tropical country with more than 10,000 plant parts used and voucher numbers are presented in Table 1.
species, many of which have been traditionally used to treat dia-
betes (Pham, 2007; Vo, 1997), yet little research has been carried 2.3. Plant extraction
out to evaluate the their ability to control hyperglycemia. There-
fore, the aim of this study was to screen crude extracts of 18 The chloroform and ethanol extracts were prepared by soni-
Vietnamese medicinal plants for bioactive components against α- cating 1 g of the dried and coarsely powdered plant material in
glucosidase and α-amylase in order to develop functional foods or 10 mL solvent each for 30 min and macerating the material over-
identify lead compounds for use against type 2 diabetes. night. After filtration through No. 4 filter paper (Whatman, USA),
the filtrate was evaporated to dryness under reduced pressure at
35 °C by a Savant SPD121P speed vacuum concentrator (Thermo
2. Materials and methods Scientific, Waltham, MA) coupled with an OFP400 vacuum pump
and a RVT400 refrigerated vapor trap. The aqueous extracts were
2.1. Chemicals prepared by boiling 1 g of the dried and coarsely powdered plant
material with 10 mL water in a flask fitted with reflux condenser
Dimethyl sulphoxide, methanol-d4 (99.8% of deuterium), so- for 30 min. The mixture was then filtered and concentrated as
dium phosphate monobasic dihydrate, sodium phosphate dibasic, described above.
sodium azide, sodium chloride, acarbose, p-nitrophenyl α-d-glu-
copyranoside (PNPG), α-glucosidase type I (EC 3.2.20, from Sac- 2.4. In vitro α-glucosidase inhibition assay
charomyces cerevisiae, lyophilized powder), NaCl, 2-chloro-4-ni-
trophenyl-α-d-maltotrioside (CNP-G3) and α-amylase type VI-B α-Glucosidase inhibition was assessed using the method de-
(EC 3.2.1.1, from porcine pancreas, lyophilized powder) were pur- scribed by Schmidt et al. (2012). In brief, 90 μL of 0.1 M phosphate
chased from Sigma-Aldrich (St. Louis, MO). Calcium acetate and buffer (pH 7.5, 0.02% NaN3), 10 μL test sample dissolved in DMSO,
formic acid was from Merck (Darmstadt, Germany). HPLC-grade and 80 μL of enzyme solution (well concentration 0.05 U/mL)
acetonitrile and chloroform were obtained from VWR Interna- were added to each well. The mixture was incubated at 28 °C for
tional (Fontenay-sous-Bois, France). Water was purified by 10 min before adding PNPG to a final volume of 200 μL (final well
0.22 mm membrane filtration and deionization (Milli-Q Plus sys- concentration 1.0 mM). The blank was carried out in a similar
tem from Millipore, Billerica, MA, USA). manner, with the test sample replaced by solvent. The hydrolysis
 B.T.D. Trinh et al. / Journal of Ethnopharmacology 186 (2016) 189–195
rate of PNPG to release p-nitrophenolate was monitored at 405 nm
every 30 s for 35 min. Incubation and absorbance measurements
were performed with a Multiscan FC microplate photometer with
built-in incubator (Thermo Scientific, Waltham, MA), controlled by
SkanIt ver. 2.5.1 software. The α-glucosidase inhibitory activity
was expressed as percentage inhibition and was calculated using
the following formula:
% inhibition ¼(Slope blank – Slope sample)/ Slope blank  100.
Acarbose was used as positive control and all measurements
were performed in triplicate.
2.5. In vitro α-amylase inhibition assay
The α-amylase inhibition assay was conducted according to the
method described by Okutan et al. (2014). In brief, 80 μL of 0.1 M
phosphate buffer (pH 6.0, 0.02% NaN3), 20 μL test sample dissolved
in DMSO, and 80 μL of enzyme solution (well concentration
0.05 U/mL) were added to each well. After incubation at 37 °C for
10 min, the reaction was started by adding CNP-G3 to a final vo-
lume of 200 μL (final well concentration 1.0 mM). The blank was
carried out in a similar manner, with the test sample replaced by
solvent. Absorbance was measured at 405 nm every third minute
for 30 min on the same instrument as described above. The α-
amylase inhibitory activity was expressed as percentage inhibition
and was calculated using the following formula:
% inhibition ¼(Slope blank–Slope sample)/ Slope blank  100.
Acarbose was used as positive control and all measurements
were performed in triplicate.
2.6. High-resolution α-glucosidase and α-amylase biochromatogram
Extracts were dissolved in methanol to a concentration of
30 mg/mL and filtered using a Nylon Target Syringe Filter (0.45 mm
pore size). Analytical-scale HPLC separation was performed with
an Agilent 1200 series instrument (Santa Clara, CA, USA) consisting
of a quaternary pump, a degasser, a thermostatted column com-
partment, a photodiode-array detector, a high-performance auto
sampler, and a fraction collector, all controlled by Agilent Chem-
Station ver. B.03.02 software and equipped with a reversed-phase
Luna C18(2) column (Phenomenex, 150  4.6 mm, 3 mm, 100 Å)
maintained at 40 °C. The flow was maintained at 0.5 mL/min using
a binary gradient mixture of acetonitrile: water (5:95, v/v, solvent
A) and acetonitrile: water (95:5, v/v, solvent B); both acidified with
0.1% formic acid. For a single injection of 10 μL, the elute from 5 to
30 min was fractionated into 2  88 wells of two 96-well micro-
plates, leading to a resolution of 7.0 points per min. The following
HPLC gradients were used: gradient I for separation of P. urinaria:
0 min, 0% B; 1 min, 5%B; 16 min, 15% B; 26 min, 40% B; 31 min,
100% B; 36 min, 100% B; 37 min, 0% B; and 5 min of equilibration;
gradient II for separation of P. amarus: 0 min, 0% B; 1 min, 5% B;
10 min, 10% B; 20 min, 20% B; 30 min, 40% B; 31 min, 100% B;
36 min, 100% B; 37 min, 0% B; and 5 min of equilibration; gradient
III for separation of F. racemosa: 0 min, 0% B; 30 min, 100% B;
35 min, 100% B; 36 min, 0% B; and 5 min of equilibration. The
microplates were subsequently evaporated to dryness using a Sa-
vant SPD121P speed vacuum concentrator. The α-glucosidase and
α-amylase inhibition activities for each well were determined as
described above, but replacing DMSO of test samples by DMSO for
dissolution of fractionated material. For each microplate, a positive
control (acarbose) as well as a negative control (blank) was in-
cluded in triplicate. High-resolution inhibition profiles were plot-
ted by representing α-glucosidase and α-amylase inhibitory ac-
tivity against chromatographic retention time.
191
2.7. Isolation and structure determination of active compounds
Separation of P. urinaria and P. amarus extracts was performed
by preparative-scale HPLC with an Agilent 1100 series instrument
(Santa Clara, CA, USA) consisting of a quaternary pump, a degasser,
a thermostated column compartment, a photodiode-array de-
tector, a high-performance auto sampler, and a fraction collector,
all controlled by Agilent ChemStation ver. B.01.01 software and
equipped with a reversed-phase Luna C18(2) column (Phenom-
enex, 250  21.2 mm, 5 mm, 100 Å). The column was operated at
room temperature and the flow rate was maintained at 20 mL/min
using the same solvent gradient profile as described above. 900 μL
of a 50 mg/mL solution of crude extracts was injected consecutive
times and peaks were collected based on UV absorption
thresholds.
NMR experiments were recorded with a Bruker Avance 600
instrument (1H, 600.13 MHz) at 300 K, using methanol-d4 as
solvent. 1H and 13C chemical shifts were referenced to the residual
solvent signal at δ 3.31 and δ 49.00, respectively. NMR data pro-
cessing was performed using Bruker Topspin version 3.2 software.
Mass spectra were acquired in negative ion mode, using Bruker
micrOTOFQ II mass spectrometer equipped with an electrospray
ionization (ESI) interface (Bruker Daltonik, Bremen, Germany).
2.8. Kinetics of α-glucosidase inhibition
Enzyme kinetics of isolated α-glucosidase inhibitiors was
measured in triplicate using the standard assay conditions de-
scribed above. Reactions were carried out with increasing con-
centrations of PNPG (0.125, 0.25, 0.5, 1.0, 2.0, and 4.0 mM) as
substrate in the absence and presence of test samples (3 con-
centrations). The mode of inhibition was determined graphically
from the Lineweaver-Burk plots. Kinetic parameters were calcu-
lated by fitting the data to the Michaelis-Menten equation using
non-linear regression analysis (GraphPad Prism 6.0, GraphPad
Software Inc.).
3. Results and discussion
3.1. In vitro α-glucosidase and α-amylase inhibition assay of crude
extracts
A total of 54 extracts from the 18 plant species in Table 1 were
assessed for inhibitory activity against α-amylase and α-glucosi-
dase. Crude extracts were dissolved in DMSO to obtain a stock
concentration of 10 mg/mL and serial ten-fold dilutions were
performed 6 times for IC50 determinations. Crude extracts can be
considered as having a significant inhibitory effect if the IC50 value
is below 50 μg/mL. Based on this criteria, ethanol and water ex-
tracts of N. mirabilis, P. urinaria, K. candel and water extracts of P.
amarus, L. speciosa, S. cumini, and R. mucronata significantly in-
hibited α-glucosidase (IC50 of acarbose ¼581.1 719.4 μg/mL,
Schmidt et al., 2012). In the α-amylase inhibition assay, only
ethanol extracts of K. candel and F. racemosa fulfilled the criteria of
an IC50 value below 50 μg/mL (IC50 of
acarbose ¼0.11 7 0.02 μg/mL). None of the chloroform extracts
inhibited α-amylase and α-glucosidase to an extent that allowed
determination of IC50 values. The IC50 values of active extracts
were calculated from the dose-response curves (Supplementary
data, Fig. S1–S2) and summarized in Table 2.
Previously, α-glucosidase inhibitory activity has been reported
for 80% ethanol extract of P. urinaria (IC50 ¼28.4 μg/mL, Mediani
et al., 2015), the ash of P.amarus (IC50 ¼ 982 mg/mL, Wongnawa
et al., 2014), ethanol extract of R. mucronata
(IC50 ¼0.08 71.82 μg/mL, Lawag et al., 2012), and water extract of
192 B.T.D. Trinh et al. / Journal of Ethnopharmacology 186 (2016) 189–195

Table 2
IC50 for the active plant extracts

Scientific name α-Glucosidase assay IC50 (μg/mL) α-Amylase assay IC50 (μg/mL)

EtOH extracts H2O extracts EtOH extracts H2O extracts

Nepenthes mirabilis (Lour.) Druce 32.7 76.3 3.3 7 0.8 73.6 7 10.2 296.7 7 121.0
Ludwigia octovalvis (Jacq.) P.H. Raven 213.7 7 35.1 n.d n.d n.d
Phyllanthus amarus Schumach. & Thonn. 926.5 739.0 34.9 7 1.5 n.d n.d
Phyllanthus urinaria L. 39.7 79.7 14.6 7 4.6 998.9 7 14.0 n.d
Lagerstroemia speciosa (L.) Pers. 1114.9 7 26.5 5.4 7 0.5 n.d n.d
Syzygium cumini (L.) Skeels n.d 20.9 7 1.8 n.d n.d
Euphorbia hirta L. 331.5 771.6 n.d n.d n.d
Rhizophora mucronata Lam. 126.6 718.2 3.3 7 0.6 185.5 7 83.7 573.6 7 90.1
Kandelia candel (L.) Druce 35.47 13.9 4.0 7 0.8 7.6 7 0.9 206.07 75.0
Cardiospermum halicacabum L. 956.4 724.4 n.d n.d n.d
Ficus racemosa L. 278.6 7 49.1 n.d 46.7 7 23.6 742.7 795.0
Pithecellobium dulce (Roxb.) Benth. 1203.2 7187.3 n.d n.d n.d

n.d: Not detected.

IC50 of all CHCl3 extracts were not detected.
Values are expressed as mean7 standard deviation (n¼ 3).

Fig. 3. Dose-response curves of corilagin (1, A), repandusinic acid A (2, B) and
mallotinin (3, C). Each point represents the average of triplicate measurements.

S. cumini (IC50 ¼800 717 μg/mL, Alagesan et al., 2012a, 2012b).

Fig. 1. HPLC trace at 254 nm (black) and high-resolution inhibition profiles (red) of
water extracts of Phyllanthus urinaria (a) and Phyllanthus amarus (b). (For inter-
pretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
From another work, three new hydrolyzable tannin including ja-
mutannins A and B, isooenothein C, together with eight other
known phenolic compounds were isolated from butanol extract of
S. cumini and evaluated along with α-glucosidase inhibitor

Fig. 2. Structure of corilagin (1), repandusinic acid A (2) and mallotinin (3).
 B.T.D. Trinh et al. / Journal of Ethnopharmacology 186 (2016) 189–195
Fig. 4. Lineweaver–Burk plots of inhibition kinetics of α-glucosidase inhibitory effects by corilagin (1, A), repandusinic acid A (2, B) and mallotinin (3, C). Each point
represents the average of triplicate measurements.
Table 3
IC50, Ki and mode of inhibition for corilagin (1), repandusinic acid A (2) and mal-
lotinin (3) on α-glucosidase enzyme.
Compound IC50 (μM) Ki (μM) Ki′ (μM) Mode of
inhibition
Corilagin (1) 1.707 0.03 2.377 0.90 2.617 0.61 Mixed
Repandusinic acid A 6.107 0.10 4.017 0.47 n.a Competitive
(2)
Mallotinin (3) 3.767 0.15 0.65 7 0.11 n.a Competitive
n.a: not available.
Values are expressed as mean7 standard deviation (n¼ 3).
acarbose. Results showed that isooenothein C was the most active
with IC50 of 8.2 70.8 μM. This value was significant when com-
pared to that of acarbose (IC50 ¼208.6 73.4 μM, Omar et al., 2012).
α-Amylase activity has been reported for 50% aqueous methanolic
extract of P. urinaria with 98.0 75.8% inhibitory activity at the
concentration of 5 mg/mL (Gunawan-Puteri et al., 2011). The
hexane and ethanol extract of P. amarus exhibited appreciable α-
amylase inhibitory activity (IC50 ¼ 48.92 73.43 μg/mL and
36.05 74.01 μg/mL, respectively) when compared to acarbose
(IC50 ¼83.337 0.34 μg/mL, Tamil et al., 2010). Besides, P. amarus
hexane extract was reported in another work to demonstrate very
strong inhibition to α-amylase (IC50 ¼32 μg/mL). From this extract,
oleanolic acid and ursolic acid (2:1) mixture was revealed a potent
α-amylase inhibitor with IC50 ¼ 2.01 μg/mL (4.41 μM, Ali et al.,
2006). α-Amylase or α-glucosidase activity has also been shown in
extracts of other Phyllanthus species, namely P. acidus, P. emblica
and P. niruri (Hossain et al., 2008; Sulaiman and Ooi, 2014; Med-
iani et al., 2015). α-Amylase activity has been also reported for
water extract of L. speciosa (IC50 ¼ 530 μg/mL, Suzuki et al., 2001),
though the reported IC50 values were too high to be considered
active. P. urinaria, P. amarus, L. speciosa, S. cumini and F. racemosa
have shown ability to lower blood glucose in vivo (Higashino et al.,
1992; Verma et al., 2014; Evi et al., 2011; Raphael et al., 2002;
Shetti et al., 2012; Mbagwu et al., 2011; Adeolu et al., 2013; Sau-
mya et al., 2011; Shareef et al., 2013; Barun et al., 2009; Kumar
et al., 2008; Singh and Gupta, 2007; Helmstädter, 2008; Zulfiker,
2011).
3.2. High resolution α-glucosidase and α-amylase inhibition profiles
A total of 11 extracts with IC50 below 50 μg/mL were subjected
to analytical-scale HPLC. The chromatograms are shown in Sup-
plementary data, Fig. S3, and shows that most extracts contain
large amounts of tannins based on the large broad peak from 7 to
22 min. Tannins are polyphenolic compounds widely distributed
in many species of plants, and are generally considered as non-
specific inhibitors, which have low priority for drug discovery.
Thus, based on the HPLC chromatograms, only water extracts of P.
193
amarus and P. urinaria and ethanol extract of F. racemosa had low
levels of tannins, and were therefore subjected to micro-
fractionation followed by bioassaying of the content in each well.
The high-resolution α-glucosidase inhibition profiles of P. urinaria
and P. amarus water extracts showed several discrete peaks cor-
related with peaks in the HPLC chromatogram - and thereby al-
lowed pinpointing of potential α-glucosidase inhibitors (Fig. 1).
The high-resolution α-amylase inhibition profile of F. racemosa
(Supplementary data, Fig. S4) showed no discrete peaks and
therefore no further investigation of this extract was performed.
3.3. Isolation and structure determination of active compounds
Peaks 1a-3a, 1b and 2b in Fig. 1 were selected for isolation and
structure elucidation due to their pronounced α-glucosidase in-
hibition (more than 80% inhibition) and their strong absorption in
the HPLC chromatogram at 254 nm. From 7.0 g dried material of P.
urinaria, 1.3 g water extract was obtained, subsequently subjected
to a preparative scale HPLC with the solvent gradient profile de-
scribed above, to obtain the material eluted as peak 1a (28.0 mg),
2a (12.2 mg) and 3a (4.5 mg). Similarly, the material eluted as peak
1b (22.0 mg) and 2b (10.7 mg) were isolated from extract of 7.0 g
dried material of P. amarus.
On the basis of HRMS as well as 1D and 2D NMR spectra, the
material eluted as peaks 1a-3a were identified as corilagin (1),
C27H22O18, HR-ESIMS(-) m/z 633.0942 [M-H]-, 1H NMR (600 MHz,
methanol-d4) ppm: δ 7.05 (2H, s, H-G2,6), 6.69 (1H, s, H-B3), 6.66
(1H, s, H-A3), 6.36 (1H, d, J ¼2.0 Hz, H-1), 4.96 (1H, dd, JE 10.8 Hz,
H-6B), 4.80 (1H, br s, H-3), 4.52 (1H, br dd, J ¼10.8, 8.0 Hz, H-5),
4.46 (1H, br s, H-4), 4.16 (1H, dd, J ¼10.8, 8.0 Hz, H-6A), 3.98 (1H,
br dd, J ¼3.5, 2.0 Hz, H-2), 13C NMR (150 MHz, methanol-d4) ppm:
δ 170.1 (C-A7), 168.5 (C-B7), 166.7 (C-G7), 146.4 (C-G3,5), 146.1, 145.2
(C-A6, B6), 145.6 (C-A4), 145.3 (C-B4), 140.4 (C-G4), 138.2 (C-B5),
137.7 (C-A5), 125.5, 125.4 (C-A2, B2), 120.6 (C-G1), 117.2 (C-B1), 116.7
(C-A1), 111.0 (C-G2,6), 110.2 (C-B3), 108.3 (C-A3), 95.0 (C-1), 76.2 (C-
5), 71.7 (C-3), 69.5 (C-2), 65.0 (C-6), 62.5 (C-4) (Sudjaroen et al.,
2012); repandusinic acid A (2), C41H30O28, HR-ESIMS(-) m/z
969.1259 [M-H]-, 1H NMR (600 MHz, methanol-d4) ppm: δ 7.10
(1 H, s, H-5′), 7.07 (1H, s, H-3″ ), 7.06 (2H, s, H-G2,6), 6.72 (1H, s,
H-A3), 6.71 (1H, s, H-B3), 6.21 (1H, d, J ¼2.8 Hz, H-1), 5.59 (1H, d,
J¼ 3.3 Hz, H-4), 5.43 (1H, d, J ¼2.2 Hz, H-3′), 5.35 (1H, d, J ¼2.2 Hz,
H-2′), 4.87 (1H, dd, J ¼11.3, 8.5 Hz, H-6B), 4.82 (1H, br s, H-3), 4.53
(1H, dd, J E8.5 Hz, H-5), 4.23 (1H, dd, J ¼11.3, 8.5 Hz, H-6A), 4.06
(1H, m, H-2), 13C NMR (150 MHz, methanol-d4) ppm: δ 172.2 (C-
1′), 170.0 (C-A7), 169.2 (C-6′), 168.2 (C-B7), 167.2 (C-7″ ), 166.6 (C-
G7), 166.0 (C-7′), 146.6, 140.5 (C-4″ , C-5″ ), 146.4 (C-G3,5), 146.1 (C-
A4), 145.6 (C-B4), 145.3, 145.1 (C-A6, B6), 144.2 (C-6″ ), 140.6 (C-G4),
140.1 (C-4′), 138.3 (C-B5), 137.7 (C-A5), 133.3 (C-5′), 125.4 (C-A2),
1251. (C-B2), 120.6 (C-G1), 117.7 (C-2″ ), 117.3 (C-B1), 116.9 (C-1″ ),
116.4 (C-A1), 110.9 (C-G2,6), 110.2 (C-B3), 109.3 (C-3″ ), 108.8 (C-A3),
95.2 (C-1), 79.7 (C-2′), 74.0 (C-5), 71.2 (C-3), 69.7 (C-2), 65.3 (C-4),
194 B.T.D. Trinh et al. / Journal of Ethnopharmacology 186 (2016) 189–195
65.0 (C-6), 36.1 (C-3′) (Saijo et al., 1989); and mallotinin (3)
C41H30O28, HR-ESIMS(-) m/z 969.1176 [M-H]-, 1H NMR (600 MHz,
methanol-d4) ppm: δ 7.23 (1H, s, H-3″ ), 7.08 (2H, s, H-G2,6), 6.95
(1H, s, H-B3), 6.68 (1H, s, H-A3), 6.57 (1H, d, J¼ 4.7 Hz, H-1), 5.40
(1H, br d, J ¼4.7 Hz, H-2), 5.07 (1H, d, J ¼1.4 Hz, H-2′), 5.00 (1H, d,
J ¼1.4 Hz, H-3′), 4.90 (1H, m, H-3), 4.69 (1H, dd, J¼ 11.0, 9.8 Hz,
H-6B), 4.59 (1H, dd, J¼ 9.8, 7.7 Hz, H-5), 4.52 (1H, br d, J ¼3.1 Hz,
H-4), 4.24 (1H, dd, J ¼11.0, 7.7 Hz, H-6A), 3.25 and 3.20 (each 1H,
J ¼16.8 Hz, H-5′B and H-5′A), 13C NMR (150 MHz, methanol-d4)
ppm: δ 176.0 (C-7′), 173.8 (C-1′), 172.0 (C-6′), 169.7 (C-A7), 168.5 (C-
B7), 166.6 (C-G7), 165.7 (C-7″ ), 148.1 (C-6″ ), 147.8 (C-4″ ), 146.5 (C-
A4, G3,5), 146.0, 145.2 (C-A6, B6), 145.8 (C-B4), 140.5 (C-G4), 137.9 (C-
B5), 137.8 (C-A5), 136.4 (C-5″ ), 125.7, 125.3 (C-A2, B2), 123.3 (C-2″),
120.4 (C-G1), 116.9 (C-B1), 116.7 (C-A1), 116.4 (C-1″), 112.8 (C-3″),
110.7 (C-G2,6), 110.2 (C-B3), 108.6 (C-A3), 91.9 (C-1), 89.6 (C-4′), 83.8
(C-2′), 77.7 (C-5), 74.0 (C-3), 72.4 (C-2), 65.2 (C-6), 62.5 (C-4), 52.5
(C-3′), 39.1 (C-5′) (Saijo et al., 1989). The material eluted as peaks
1b and 2b were also identified as corilagin (1) and repandusinic
acid A (2), respectively. The structures of 1, 2 and 3 are shown in
Fig. 2.
3.4. Inhibitory activity of isolated compounds
The α-glucosidase inhibitory activity of isolated compounds
was assessed in vitro with concentrations in the range 0.01–
100 μg/mL. Dose-response curves are shown in Fig. 3, and from
these results IC50 values of 1.70 70.03 μM (corilagin, 1),
3.76 70.15 μM (mallotinin, 3) and 6.10 70.10 μM (repandusinic
acid A, 2) were obtained. These results show that all compounds
display a strong inhibition of α-glucosidase, with IC50 values sig-
nificantly lower than that of acarbose (581.1 719.4 μg/mL or
900 73 μM, Schmidt et al., 2012). Corilagin (1) has previously
been reported to possess α-glucosidase activity with an IC50 value
of 2.64 μM (Li et al., 2014), which is in agreement with our find-
ings. No previous investigations of the α-glucosidase inhibitory
activity of repandusinic acid A (2) and mallotinin (3) have been
reported.
3.5. Kinetics of α-glucosidase inhibition
In order to examine the mode of inhibition of α-glucosidase by
compounds 1–3, kinetic analyses were carried out. As shown in
Fig. 4, all the data lines on the Lineweaver-Burk plot of corilagin (1)
intersected in the second quadrant, reflecting a mixed type mode
of inhibition in which both EI and ESI complexes are formed. The
inhibitor constants, Ki and Ki′ (KEI and KESI), were determined to be
2.37 70.90 μM and 2.617 0.61 μM, respectively. From the kinetic
data, the lower value of Ki indicates the more favorable formation
of the EI complex than ESI complex. Corilagin has previously been
reported to be competitive inhibitor of α-glucosidase (Li et al.,
2014); however, the results of the present study showed corilagin
to act as a mixed type inhibitor. Our data also revealed that both
repandusinic acid A (2) and (3) were competitive inhibitors as the
Km values increased with increasing concentration of inhibitors
while Vmax remained unchanged (Fig. 4). During competitive in-
hibition, the substrate competes with the inhibitor for the access
to the active site of the enzyme. Thus, despite compounds 1, 2 and
3 are ellagitannins, compound 2, 3 and partly 1 show specific ac-
tivity against α-glucosidase, instead of the unspecific binding to
protein of other tannins. Competitive inhibition can be overcome
at sufficiently high substrate concentrations, with the Vmax re-
maining unaffected. In this type of inhibition, Ki is the most im-
portant measurement for a compound, as it is an indication of the
affinity for the enzyme inhibitor site. Ki values of 2 and 3 were
calculated to be 4.01 70.47 μM and 0.65 70.11 μM, respectively,
suggesting that 3 is more tightly bound to the enzyme, and hence
much more potent than 2. The Ki values and modes of inhibition
for compounds 1–3 are summarized in Table 3.
4. Conclusion
In conclusion, α-glucosidase and α-amylase inhibitory activity
of chloroform, ethanol and water extracts of 18 Vietnamese
medicinal plants were measured in vitro. Our study showed that 11
ethanol and water extracts significantly suppressed α-glucosidase
and α-amylase. For most extracts, tannins were responsible for the
activity. It was not possible to identify compounds with α-amylase
inhibitory activity. The study points to P. amarus and P. urinaria as
the most promising species for use in herbal medicine as α-glu-
cosidase inhibitors, and also shows that high-resolution inhibition
profiling is a useful method for identification of the main active
compounds in even very complex plant extracts. The Phyllanthus
species are widely distributed in Asian countries, where they are
used in traditional medicine for diabetes, jaundice and urinary
tract problems (Lai et al., 2008; Gunawan-Puteri et al., 2011; Jo-
seph and Raj, 2011; Wasnik, 2012; Sarin et al., 2014; Mediani et al.,
2015). This is the first report of the α-glucosidase and α-amylase
inhibitory effect of these 18 Vietnamese plants, and thus for some
of them giving a scientific support for their traditional uses in folk
medicine for the treatment of type 2 diabetes.
Acknowledgements
Author Binh T.D. Trinh is thankful to Vietnam Ministry of
Education and Training for a scholarship under Project 911 and
Son V. Dang for assistance in verifying all collected plants. HPLC
equipment used for obtaining high-resolution α-glucosidase and
α-amylase inhibition profiles was obtained via a grant from The
Carlsberg Foundation.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.jep.2016.03.060.
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