Bioassay guided isolation of -glucosidase inhibitory

compound,

in vivo

postprandial anti hyperglycemia

and docking study of the isolated compound from the

leaves of the methanolic extract of

Quercus serrata

Maibam B. Chanu

, Rajendra K. Labala

, Yunus Sheikh

, Jagat C. Borah

, Surajit K. Ghosh

Dinabandhu Sahoo

, Oinam J. Singh

, Anshul Shakya

and Biseshwori Thongam

Natural Products Chemistry and Pharmacology Programme, Institute of Bioresources and Sustainable

Development, Imphal-795001, Manipur, India

Distributed Information Sub-Centre, Institute of Bioresources & Sustainable Development, Imphal -795001,

Manipur, India

Natural Product Chemistry, Institute of Advanced Study in Science & Technology, Guwahati -781035,

Assam, India.

Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam 786004, India

Microbial Resources Programme, Institute of Bioresources and Sustainable Development, Imphal-795001,

Manipur, India

Department of Pharmacology, Jawaharlal Nehru Institute of Medical Sciences, Imphal 795001, Manipur, India

Plant Systematic Conservation Laboratory, Institute of Bioresources and Sustainable Development,

Imphal-795001, Manipur, India

ABSTRACT

Diabetes mellitus is rapidly emerging creating major health problem worldwide. Though synthetic drugs are available, due to their

association with side effect, there is always interest for search of herbal formulation. Quercus serrata is a plant used by traditional

healers of Manipur, India as a folk remedy to treat diabetes mellitus. The objective of this study is bioassay guided isolation of

-glucosidase inhibitory compound from the leaves of Quercus serrata and to check the postprandial antihyperglycaemic effect

of the isolated compound in STZ-induced diabetic albino mice. And to perform molecular docking studies to predict the bind-

647

Pharmaceutical

Communication

Biosci. Biotech. Res. Comm. 11(4): 647-657 (2018)

ARTICLE INFORMATION:

Corresponding Authors: b_thongam07@yahoo.com

Received 30

Sep, 2018

Accepted after revision 21

Dec, 2018

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC / Clarivate Analytics USA

Mono of Clarivate Analytics and Crossref Indexed

Journal Mono of CR

NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196

Online Contents Available at:

http//www.bbrc.in/

DOI: 10.21786/bbrc/11.4/16

Maibam B. Chanu et al.

648 BIOASSAY GUIDED ISOLATION OF -GLUCOSIDASE INHIBITORY COMPOUND BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

ing interaction of the isolated compounds with -glucosidase.The methanolic extract of Quercus serrata leaves was screened for

-glucosidase inhibitory activity and fractionated into n-butanol, ethyl acetate and water fraction to isolate the active compound.

Quanti cation of isolated compound was done by HPLC-PDA. Postprandial antihyperglycaemia effect was check in normal and

STZ (100mg/kg) + nicotinamide (120mg/kg) induced diabetic mice after sucrose load (2g/kg). Molecular docking study was done

using AutoDock 4.2.6. Rosmarinic acid was identi ed as the active compound present in Quercus serrata leaves responsible for

the inhibition of -glucosidase with IC50 0.23 ± 0.02 gmL-1 (0.636µmolmL-1). Postprandial blood sugar and Area Under Curve

were signi cantly (p<0.05) reduced by treatment with rosmarinic acid in normal and diabetic mice. Additionally, in silico dock-

ing study elaborated the possible binding between rosmarinic acid and -glucosidase. Above  nding con rmed the anti-diabetic

potential of traditionally used Quercus serrata leaves and the isolated compound, rosmarinic acid.

KEY WORDS: -GLUCOSIDASE, DIABETES MELLITUS, MOLECULAR DOCKING, POSTPRANDIAL HYPERGLYCEMIA, QUERCUS SERRATA,

ROSMARINIC ACID

INTRODUCTION

Diabetes mellitus and the spectrum of complications

associated with it impose uncertain healthcare chal-

lenges and economic burdens to the global population.

The catastrophic prevalence of diabetes mellitus, pre-

dominantly type 2 diabetes, has become a global health-

care problem affecting 425 million people worldwide,

(Zimmet et al., 2014, IDF Diabetes Atlas, 2017). In India,

it is reaching extreme epidemic level. A recent study

revealed that prolongation of diabetes leads to neuropa-

thy (24.6%), the most common complication, followed

by cardiovascular complications (23.6%), renal prob-

lems (21.1%), and retinopathy (16.6%) (Kaveeshwar and

Cornwall, 2014).

Diabetes is a life style disease and food habit plays

a major role in the management of diabetes. Con-

trol of postprandial hyperglycemia is also one of the

measures to control severity of the disease. Acarbose,

miglitol and voglibose are available clinically used

drug to control postprandial absorption of glucose by

inhibiting the enzyme -glucosidase and -amylase.

Although substantial quantum of efforts has been made

towards conquering the disease, it is still imperative to

put rigorous effort in research to mitigate the disease.

There is always search for new herbal formulation to

avoid the side effect associated with the synthetic drug,

(Polonsky, 2012, Nakatsu et al., 2017, Brito-Arias et al.,

2018).

Quercus serrata Murray is widely used by traditional

healers of Manipur, India as a folk remedy to treat dia-

betes mellitus (Sheikh et al 2015). No elaborate study

has been reported on the medicinal use of Quercus ser-

rata leaves and the chemical constituent present. So,

the present study is aim at bioassay guided isolation of

the active constituent responsible for the inhibition of

the enzyme -glucosidase, checking its antihypergly-

caemic activity and to do molecular docking study of

the isolated compound to predict the possible binding

interaction of the isolated compound with the enzyme,

-glucosidase.

MATERIALS AND METHODS

Leaves of Quercus serrata, Murray were collected from

Kangla Siphai, Manipur, India and authenticated by

Dr. Biseshwori Thongam, Scientist D, Pant Taxonomist,

IBSD, Manipur. Voucher specimen (No. IBSD/M-202)

was deposited in the IBSD herbarium. Analytical or

HPLC grade organic solvents (Merck Millipore, India)

were used for experiments. For column chromatog-

raphy, 100-200 mesh size silica gel (Merck) was used.

-Glucosidase (Maltase, EC 3.2.1.20), p-nitrophenyl--

D-glucopyranoside and streptozotocin were purchased

from Sisco Research Laboratory. HPLC (Shimadzu LC-

20AD) was performed using Photo diode array (PDA)

detector. NMR spectra were recorded on a Bruker Avance

500 MHz instrument with TMS as an internal standard.

Chemical shifts were expressed in  values. Agilent

6520 Accurate mass Q-TOF/LC-MS was used to deter-

mine molecular weight. Absorbance was measured by

Thermo Scienti c Multiskan spectrometer.

Extraction and isolation of active compound: Air-dried

leaves of Quercus serrata (3.5 kg) were extracted three

times with methanol (MeOH) (15 L). Then 150 mL water

was added to the concentrated methanolic extract (3.0

L) to make hydro-alcoholic solution. This solution was

washed with 3.0 L of petroleum ether to remove fatty

matter present in the leaves and then concentrated to

yield 400 g of dried extract. A suspension was prepared

from this using 500 mL of water and fractionated into

ethylacetate , n-butanol and water to yield 152 g of ethyl

acetate fraction, 103 g of butanol fractions and 135g

of water fraction respectively. The butanol fraction was

found to be most active and was subjected to silica gel

column chromatography with increasing polarity: petro-

leum ether-chloroform 1:1 (Fr 1, 1 L), chloroform (Fr 2,

1 L), and chloroform – methanol 9:1 (Fr 3, 2 L), chloro-

form – methanol 4:1 (Fr 4, 2 L), chloroform – methanol

1:1 (Fr 5, 2.5 L) and chloroform – methanol 3:7 (Fr 6,

2.5 L). The sub-fraction 6 (SFr 6) was found to be most

active in inhibition of -glucosidase. Further, SFr 6 was

Maibam B. Chanu et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIOASSAY GUIDED ISOLATION OF -GLUCOSIDASE INHIBITORY COMPOUND 649

subjected to semi- preparative HPLC to yield compound

1 (rosmarinic acid).

Analytical and semi-preparative HPLC: Shimadzu LC-

20AD with PDA detector (SPD-M20A)  tted with either

an analytical (CAPCELL PAK C18 MGII S5, 5µ C18 250

× 4.6 mm) or semi-preparative (CAPCELL PAK C18 MGII

S5, 5µ C18 250 × 10.00 mm) column was used for HPLC

analysis. A solution of 10 mg/mL of enriched fraction

(SFr 6) was prepared in HPLC grade water and metha-

nol (7:3) and  ltered through 0.45µm Millex-HN syringe

 lter. A 20µL or 200µL aliquot of  ltered sample solu-

tion was injected for analysis for analytical and semi

preparative separation respectively. The mobile phase

used consisted of 1% acetic acid in water (solvent A)

and methanol (solvent B) following linear gradient over

a total run time of 23min: initially a linear increase of

B up to 100% in 21 min and back to 100% A in 23

min. Flow rate was 1 mL/min and 3 mL/min for analyti-

cal and semi-preparative respectively. Individual peaks

eluting from the column were carefully collected and

the solvents were evaporated in a rotary vacuum evapo-

rator. Instrument control and data handling were per-

formed with the LC solution software on a PC.

Quantitative analysis by HPLC-PDA: Quantitative

analysis was done by analytical HPLC in the protocol

described above. Standard stock solution of 1mg/mL of

rosmarinic acid was prepared and then diluted to yield

the desired test concentrations. Quantitative estimation

of the major active compound present in the methanolic

extract, n-butanol fraction and enriched fraction (SFr6)

was done using calibration curve of the standard solu-

tion and plotted concentration versus area using excel

2007 (Sheikh et al., 2016).

-Glucosidase inhibitory assay: A 0.5U/mL stock solu-

tion of readily available -glucosidase (from Saccharo-

myces cerevisiae) was prepared in 0.1M phosphate buffer

(pH 6.8) and diluted with the same buffer to desired test

concentration. Stock solution of plant extract, fractions

and compounds were prepared in dimethyl sulfoxide

(DMSO) and diluted for assay to the required concen-

tration with same buffer maintaining DMSO concentra-

tion below 1% v/v. -glucosidase inhibitory activities

were determined spectrophotometrically based on an

earlier reported method by using p-nitrophenyl--D-

glucopyranoside as substrate (Laishram et al., 2014)

Acarbose was used as positive control and the unin-

hibited enzyme was taken as negative control (DMSO

control). The assay was performed in three independent

experiments.

Experimental animals: Swiss albino mice (25-30 g)

were used in all the experiment and were procured from

Regional Institute of Medical Sciences (RIMS), Manipur,

India. Ethical clearance was obtained from the Insti-

tutional Animals Ethical Committee (Approval No.–

RIMS.171/IAEC/2011) prior to the experiments. Animals

were acclimatized in laboratory condition for 7 days in

polypropylene cages lined with husk under standard

environmental conditions. Animals had free access to

water and were fed on pelleted diet.

Acute oral toxicity study :The acute toxicity study

was performed as per Organisation for Economic Co-

operation and Development (OECD) guideline no. 423

(Annexure-2c) adopted by the Committee for the pur-

pose of control and supervision of experiments on ani-

mals (CPCSEA), Government of India (Veeraraghavan,

2000). Mice were kept under observation for 14 days

with special attention for the  rst 24 h after dosing.

Induction of diabetes: Diabetes was induced in mice

by single intraperitoneal (i.p.) injection of nicotinamide

(120mg/kg i.p.) and then 100 mg/kg b.w. of streptozo-

tocin (freshly dissolved in 0.1M citrate buffer, pH 4).

Mice showing marked hyperglycemia (fasting blood glu-

cose ≥ 250 mg/dl) at 48 h after streptozotocin treatment

were considered as diabetic and selected for the study,

(Nakatsu et al., 2017)

Effect of rosmarinic acid on postprandial hyperglyce-

mia :Both normal and diabetic mice were fasted over-

night and divided into  ve groups (n=6). Group I served

as normal control and received vehicle, group II served

as positive control and received acarbose (10 mg/kg

b.w.). Group III, IV and V received rosmarinic acid (5,

10 and 20 mg/kg) suspended in gum acacia (2% w/v)

(Chandramohon et al., 2015). All groups received 2 g/kg

b.w. sucrose orally along with their respective test sam-

ples (Miura et al., 2004). Blood glucose levels were esti-

mated from the tail vein from each group before (0 min)

and at 30, 60 and 120 min after sucrose load. Blood glu-

cose was measured by using Accu-Check Active, Roche

Diagnostic Mannheim, Germany. AUC was calculated by

using the software, GraphPad Prism 5.

In silico structure prediction of -glucosidase: S. cer-

evisiae -glucosidase protein sequence (MAL12, Acces-

sion Number: P53341) was retrieved from UniProt (www.

uniprot.org). NCBI CDD

(Marchler-Bauer et al., 2015)

was

used to  nd out the conserved domain and other impor-

tant catalytic sites. Structural 3D model for the protein

was built using automated comparative protein mod-

eling server SWISS-MODEL (https://swissmodel.expasy.

org). Homology based search was performed by the

server in Protein Data Bank (PDB) and SWISS-MODEL

template library (SMTL) repositories for template struc-

tures (Biasini et al., 2014). Suitable template with similar

biological property and highest similarity was selected

for creating the 3D model. Further checking accuracy of

Maibam B. Chanu et al.

650 BIOASSAY GUIDED ISOLATION OF -GLUCOSIDASE INHIBITORY COMPOUND BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

the newly generated structure was done by using PRO-

CHECK (Laskowski et al.,1993) and ProSA-web (Sippl,

1993; Wiederstein and Sippl, 2007) for evaluation of

Ramachadran plot.

Docking study: 3D structure of rosmarinic acid

(PubChem CID: 5281792) was obtained from NCBI

PubChem database (https://pubchem.ncbi.nlm.nih.gov/).

Surface topology was calculated using CASTp server

(Dundas et al., 2016) for potential binding pockets in

the structural model of -glucosidase. Initially whole

protein was considered for docking. Polar hydrogen

atoms and Gasteiger charge were added to the protein

molecule. Grid size of 96 Å × 120 Å × 110 Å with a

spacing of 0.619 Å was set to cover the entire protein.

Lamarckian genetic algorithm search was considered to

generate 100 conformations and rest of the parameter

was set to default. Later, a cluster analysis was carried

out by considering the root mean square deviation of

conformations to identify major occupied binding sites

in the protein. On the basis of major CASTp pockets and

top AutoDock clusters information, a smaller grid box,

62 Å × 60 Å × 70 Å with a spacing of 0.464 Å was

determined for the second set of docking simulation. All

other parameters and procedureof the docking program

were kept as earlier.

The best docked conformation of rosmarinic acid

was selected based on lowest binding energy. Minimiza-

tion of the docked complex was then performed using

CHARMm force- eld with smart minimizer (2000 steps)

by considering Generalized Born with Molecular Volume

(GBMV) implicit solvent model. Binding free energy (∆G)

of the docked complex was calculated by using the same

GBMV model. Implicit Distance-Dependent Dielectric

model was used to calculate total and individual inter-

action energy (IE) possessed by the amino acid residues.

Molecular graphics, analysis and depiction were per-

formed using UCSF Chimera

(Pettersen et al., 2004) and

BIOVIA Discovery Studio Visualizer Version 4.5

(http://

www.3dsbiovia.com).

Statistical analysis: Results were expressed as Mean ±

S.D., where n=6. Differences among data were deter-

mined using one way ANOVA followed by Tukey’s Mul-

tiple Comparison test (Graph Pad Prism software, ver-

sion 7).

p<0.05 was considered statistically signi cant.

RESULTS AND DISCUSSION

Quantitative HPLC analysis: Rosmarinic acid (Fig. 1)

was found to be the major compound present in the

enriched fraction Sfr 6. Fig. 2 showed the HLPC chro-

matogram of rosmarinic acid in methanolic extract,

n-butanol fraction, enriched fraction (SFr 6) as well as

that of isolated rosmarinic acid. The calibration curve

(Fig. 3) of the standard rosmarinic acid was found to be

linear (r

=0.993) in the concentration range of 0.01 to

0.09 mg/ml. 54.28±4.14 mg, 239±1.21 mg and 426±8.36

mg of rosmarinic acid was present per 1gm each of

methanol extract, n-butanol fraction and enriched frac-

tion (SFr 6) respectively.

Rosmarinic acid NMR interpretation:Compound 1 (Fig.

1) gave a molecular ion peak in HRMS at m/z = 360.3212

[M+H]

corresponding to the molecular formula C

The

C NMR in CD

OD indicated the presence of signals

attributed to all 18 carbon atoms. One ester carbonyl

at  = 167.06 displayed HMBC correlation with H-7’ (

7.58), coupled to a doublet of J = 14.5 Hz at  6.34

indicative of a cinnamoyl ester. Another carbonyl at 

172.06 showed HMBC correlation with signal of H-7 (

3.13, H-7

and  3.05, H-7

), coupled to a methene signal

at  5.20, agreeing with the presence of a phenyl lactic

acid unit. Two ABX systems were noted in the aromatic

region of the

H NMR vouching for 3,4-dihydroxylation

pattern of both the rings. The linkage of the cinnamoyl

ester to the phenyl lactic acid moiety was inferred from

HMBC correlations displayed by the H-8 signal to both

the carbonyl peaks. Signals at  6.98 (1H, d, J = 2.0 Hz,

H-6’) and  7.06 (1H, dd, J = 8.5, 2.0 Hz, H-2’) show-

ing meta coupling between them and ortho coupling

between H-2’ and H-3’ with signal at  6.79 (1H, d, J =

8.5 Hz). Another ABX system with signal at  6.64 (1H,

d, J = 2.0 Hz, H-2),  6.70 (1H, dd, J = 9.0, 2.0 Hz, H-6)

and  6.69 (1H, d, J = 9.0 Hz, H-5) is observed. All sig-

nals of

H and

C, and the observed HMQC,

H-

H COSY

and HMBC correlations are in good agreement with lit-

erature reports on rosmarinic acid.

H NMR (500 MHz, CD

OD,  ppm): 7.58 (1H, d, J =

14.5 Hz, H-7

), 7.06 (1H, dd, J = 2.0, 8.5 Hz, H-2

), 6.98

(1H, d, J = 2.0 Hz, H-6

), 6.79 (1H, d, J = 8.5 Hz, H-3

6.70 (1H, dd, J = 2.0, 9.0 Hz, H-6), 6.69 (1H, d, J = 9.0

Hz, H-5), 6.64 (1H, d, J = 2.0 Hz, H-2), 6.34(1H, d, J =

14.5 Hz, H-8

), 5.20 (1H, m, H-8), 3.13 (1H, m, H-7

) and

3.05 (1H, m, H-7

).13C NMR (125 MHz, CD

OD,  ppm):

172.06 (C-9), 167.06 (C-9

), 148.34 (C-4

), 146.35 (C-7

145.41 (C-5

), 144.76 (C-4

), 143.89 (C-3), 127.84 (C-1),

126.25 (C-1

), 121.77 (C-2), 120.40 (C-6

), 116.18 (C-6),

FIGURE 1. Structure of Rosmarinic acid

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BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIOASSAY GUIDED ISOLATION OF -GLUCOSIDASE INHIBITORY COMPOUND 651

FIGURE 2. HPLC chromatogram of (a) methanol extract (b) n-butanol fraction (c) enriched frac-

tion SFr6 and (d) isolated rosmarinic acid, showing the peak of rosmarinic acid along with the

retention time.

115.10 (C-5), 114.89 (C-3

), 113.81 (C-8

), 112.99 (C-2

73.18 (C-8) and 36.51 (C-7). (Supp Fig. 1a, 1b)

Oral acute toxicity study: No fatality and ethological

changes were observed when administered a dose of

2000 mg/kg b.w. of the methanolic extract. No changes

in behavioral pattern were observed in the mice in oral

toxicity study. So, Quercus serrata leaves were non toxic

upto a dose of 2000mg/kg.

In vitro -glucosidase inhibitory effect: Blood glucose

levels are highly affected by the saccharides contained

in food which are converted into glucose by the actions

of digestive enzymes like -glucosidase. Carbohydrates

like sucrose are hydrolysed to monosaccharides (glu-

cose and fructose) by -glucosidase thereafter caused an

increased in blood glucose. The methanolic extract of

Quercus serrata leaves, ethyl acetate fraction, n-butanol

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652 BIOASSAY GUIDED ISOLATION OF -GLUCOSIDASE INHIBITORY COMPOUND BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 3. Calibration curve of standard

rosmarinic acid as obtained from HPLC

chromatogram. The graph is plotted, Area

against Concentration (mg/ml) from the

range 0.01 to 0.09mg/ml.

SUPP FIG. (1a) 1H NRM of rosmarinic acid (1b) 13C NMR of rosmarinic acid

fraction, enriched fraction (SFr 6) and rosmarinic acid

potentially inhibited -glucosidase, and the result is

shown as table 1. The above result signi es Quercus ser-

rata as a promising plant for postprandial management

of diabetes mellitus type 2. Moreover, rosmarinic acid

showed greater potential in inhibition of -glucosidase

in vitro when compared to that of acarbose.

Postprandial antihyperglycaemic effect of rosmarinic

acid: Rosmarinic acid is a polyphenol compound. It is

an ester of caffeic acid and 3,4-dihydroxyphenyllactic

acid (Petersen and Simmonds, 2003). The effect of ros-

marinic acid on postprandial blood glucose level after

sucrose load in overnight fasted normal and diabetic

mice were presented in the Fig 4A & B. In diabetic mice

postprandial blood glucose increased from 19.14±1.01 at

30 min to 20.81±0.62 mmol/L at 60 min and decreased

to 18.50±0.51 mmol/L at 120 min. Rosmarinic acid

at a dose of 20 mg/kg b.w signi cantly reduced the

postprandial blood glucose to 15.43±0.64, 15.01±0.48

and 14.19±0.74 mmol/L at 30 min, 60 min and 120

min which value was comparable to that of acarbose

15.45±0.23, 14.42±4.01 and 14.21±1.12 mmol/L at 30

min, 60 min and 120 min respectively. Rosmarinic acid

at all the doses caused signi cant fall in AUC (Table 2)

when compared to diabetic control (56.31±1.50 mmol.

min/L) indicating its effectiveness in lowering postpran-

dial glucose absorption. There are studies which reported

that medication which can  attens the peak blood glu-

cose level postprandial can reduce the AUC (Inoue et

al.1997). Postprandial blood glucose and AUC in normal

rats was in consistent with that of diabetic groups.

Docking: To understand the binding interaction between

rosmarinic acid and -glucosidase, molecular docking

was performed. Yeast and human glucosidase are similar

in their substrate speci city, pH optimum, and inhibitor

sensitivity. Thus, the yeast enzyme besides its affordabil-

ity serves as a good experimental model to learn more

about the structure, substrate speci city, and enzymatic

mechanism of human glucosidase (Brito-Arias et al.,

2018).

AutoDock 4.2.6 (Morris et al., 2009) incorporated in

MGL Tools version 1.5.6 (The Scripps Research Insti-

tute) was used to execute the docking simulations. In

this docking study -glucosidase was treated as rigid

molecule and rosmarinic acid as  exible. Due to non

availability of three-dimensional crystal structure of S.

cerevisiae -glucosidase, homology study is done. Based

on homology study , the protein structure of S. cerevi-

siae isomaltase (PDB ID: 3AJ7, Chain A at 1.3Å resolu-

tion) which got 72% similarity and 99% query coverage

when compare to S. cerevisiae -glucosidase, MAL12

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FIGURE 4. Effect of rosmarinic acid on postprandial blood glucose concentration

after sucrose load. Graph showed plasma blood glucose concentration (mmol/L)

on Y axis against time (min) on X axis. (a) on normal mice, (b) on diabetic mice.

Values are the Mean ± SD, n=6, *P < 0.05 statistically signi cant when comparing

to control.

FIGURE 5. Target-template alignment of MAL12 (-glucosidase protein sequence of S. cerevi-

siae) and 3AJ7 (Isomaltase protein sequence of S. cerevisiae (PDB ID: 3AJ7, Chain A)), showing

high sequence homology of important catalytic site residues.

was chosen for our study as the best template to build

the 3D structure for S. cerevisiae -glucosidase. (Fig 5)

Molecular docking study found that Domain 1 of

-glucosidase resembled -amylase catalytic domain

with a gap of 42 amino acids, named here as AamyGap42

(Fig. 6a). These AamyGap42 site residues are shaped

as -helices near the entry point amino acid residues

(HIS279, THR307, SER308, PRO309, PHE310, PHE311,

and ARG312)

of the active site pocket of the enzyme

(Yamamoto et al., 2010).

Maibam B. Chanu et al.

654 BIOASSAY GUIDED ISOLATION OF -GLUCOSIDASE INHIBITORY COMPOUND BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 6. (a) 3D homology model predicted by the SWISS-MODEL program presented

in rounded ribbon with designated three domains and AamyGap42 site. Catalytic

residues are represented in magenta spheres. (b) Rosmarinic acid and -glucosidase

docked complex in solid surfaces, representing AamyGap42 interacting residues in

blue color.

FIGURE 7. CASTp pockets and AutoDock conformation clusters at

-glucosidase. Major two CASTp identi ed pockets represented in spheres.

Top two AutoDock conformation clusters (stick representation) coinciding

with Pocket 1.

According to Benkert et al 2009, GMQE score which is

expressed between 0 and 1 is a score where higher value

indicate increased stability of the predicted structure.

GMQE score for our  nal 3D model was found to be 0.92

which imply good model accuracy. The evaluation of the

obtained models was done using the PROCHECK pro-

gram through Ramachandran plot. Ramachandran plot

revealed 88.8% residues were within the most favoured

regions, 10.6% of residues were in additional allowed

regions, 0.4% residues were in generously allowed

regions, and only 0.2% residues were found in the dis-

allowed regions. The percentage of residues in most

favorable regions showed the quality of protein mod-

els. The above result obtained indicated that the model

developed was quali ed to be used for molecular dock-

ing process.

Surface topology analysis, was done to know the

potential binding pockets in the structural model of

-glucosidase. Surface topology analysis on the protein

have identi ed two major pockets, named here as Pocket

Maibam B. Chanu et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIOASSAY GUIDED ISOLATION OF -GLUCOSIDASE INHIBITORY COMPOUND 655

FIGURE 8. Docking analysis: (a) 3D surface representation of rosmarinic acid and -glucosidase interactions,

representing hydrogen bonding sites (light green), Pi-interactions (pink), and van der Waals interactions (blue). (b)

Schematic representation of the interaction in detail with covalent and non-covalent bonds.

Table 1. Half maximum inhibitory concentration (IC

)

of the tested samples on -glucosidase

Samples IC

(μgmL

-1

)

MeOH extract 1.84 ± 0.11

Ethylacetate fraction 2.83 ± 0.07

n-butanol fraction 1.15 ± 0.06

Water fraction

No inhibition up to 500

gmL

-1

n-butanol Sub fraction (SFr 6) 0.66 ± 0.01

Rosmarinic acid

0.23 ± 0.02(0.636

µmolmL

-1

)

Acarbose 78.2 ± 0.17

Each value is the mean ± SD (n=3).

Table 2. AUC of postprandial glucose after sucrose

load in normal and diabetes mice

Group (mg/kg.bw)

AUC mmol.min/L

Normal rats Diabetic rats

Control 32.15±0.23 56.31±1.50

Acarbose(10) 19.95±0.41** 43.90±0.94*

Rosmarinic acid (5) 23.35±0.43* 48.82±0.57*

Rosmarinic acid (10) 22.71±0.25* 48.85±1.21*

Rosmarinic acid (25) 20.20±0.21** 44.80±1.33*

Each value is the mean ± SD (n=6). *p< 0.05 statistically signi cant

comparing to control.

performed,  rst by involving the whole protein, result-

ing in multiple conformations of the rosmarinic Acid.

Root mean square deviation cluster analysis of these

docked conformations showed two most populated clus-

ters, Cluster 1 and Cluster 2 (Fig. 7). Auxiliary observa-

tions have shown that all the pockets and clusters were

surrounding the AamyGap42 site with Cluster 1 directly

falling under it. The catalytic and active sites of the pro-

tein were falling under Pocket 2. CASTp pockets (Pocket

1 and Pocket 2) along with AutoDock major clusters

(Cluster 1 and Cluster 2) lead the target area in the pro-

tein to perform second set of docking experiment. This

targeted docking study showed that rosmarinic acid did

not directly bind at the active site present in Pocket 2 of

the protein, instead it attached to the AamyGap42 site of

Domain 1 with the lowest binding energy of -7.72 kcal/

mol (Fig. 6b).

Binding details showed that rosmarinic acid inter-

acted with 12 amino acids in the AamyGap42 site form-

ing total 6 hydrogen bonds, two each with amino acid

residue GLU402 and TYR404, and one each with LYS417

and LYS422. This interaction substantially increased the

binding af nity as these amino acid residues were also

found to be the major contributor of electrostatic inter-

action energy (IE). Moreover, the residues TYR413 and

LYS410 were involved in Pi-Pi T-shaped and Pi-alkyl

interactions respectively (Fig. 8a,8b). Pi-Pi T-shaped

interaction between TYR413 and  rst benzene ring of

the rosmarinic acid, Pi-Alkyl interaction with LYS410,

and van der Waals interaction with PRO400, ILE401,

GLU405, ASP406, VAL409, GLU414, strengthened the

stability of the enzyme and rosmarinic acid complex.

Binding free energy (∆G) between the receptor and ligand

was found to be -19.14 kcal/mol. The energy value sug-

1 and Pocket 2 with volume of 431.6 Å

and 384.7 Å

respectively (Fig. 7). Two sets of docking study were

Maibam B. Chanu et al.

656 BIOASSAY GUIDED ISOLATION OF -GLUCOSIDASE INHIBITORY COMPOUND BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

gested the possibility of inhibition of -glucosidase by

rosmarinic acid. The interaction energies (IE) between

the protein and ligand analysed for each amino acid

residues and overall residues showed electrostatic IE as

-44.70 kcal/mol and van der Waals IE as -31.22 kcal/mol

which showed good binding energy with the enzyme.

For the  rst time we have reported the anti-diabetic

effect of Quercus serrata leaves extract and the dominant

active constituent rosmarinic acid. Further, this study

reported the in silico binding analysis of rosmarinic acid

with the enzyme -glucosidase of S. cerevisiae.

Con ict of interest: The authors declare no competing

interest.

ACKNOWLEDGEMENTS

Authors are thankful to the Director, IBSD, Imphal and

Department of Biotechnology (DBT), Govt. of India and

the Director, Institute of Advanced Study in Science

and technology for providing necessary infrastructure

to carry out this work. Authors are also thankful to Dr.

Basudeb Achari and Dr. Bikash C Pal for their valuable

suggestions.

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