Naturally occurring Batatasins and their derivatives as α-glucosidase inhibitors

Article abstract of DOI:10.1039/c5ra15328j

Batatasins are endogenous plant hormones found in yams and other related plant species. These plants are widely consumed as botanical dietary supplements in many parts of the world. This study investigated the inhibitory effects and mechanisms of Batatasin I, III, IV, V against α-glucosidase regarding their antidiabetic activities. The results revealed that Batatasin I, III, IV inhibited α-glucosidase in a reversible and noncompetitive manner, with IC₅₀ values of 2.55, 1.89 and 2.52 mM respectively, while Batatasin V completely abolished its inhibitory activity even at the highest concentrations tested. Furthermore, a class of Batatasin-derived compounds with different substitution patterns was synthesized and subjected to the assay to clarify the structure-activity relationships, which suggested that the hydroxyl at the C-2′ position may play a significant role in improving the inhibitory activities. Their probable binding modes and the specificity of the binding sites were studied using molecule docking simulation. It was concluded that Batatasins, especially Batatasin III and IV, are promising α-glucosidase inhibitors, which therefore could be used as functional food to alleviate postprandial hyperglycemia and as potential candidates for the development of antidiabetic agents.

Full text of DOI:10.1039/c5ra15328j

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Page 1 of 19
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DOI: 10.1039/C5RA15328J
Naturally Occurring Batatasins and their Derivatives as
αꢀGlucosidase Inhibitors
Weiꢀping Hu a, Guoꢀdong Cao a, Jinꢀhua Zhu a, Jiaꢀzhong Lid, Xiuꢀhua Liu a,b,c*1
a
Institute of Environmental and Analytical Sciences, College of Chemistry and Chemical Engineering, Henan
University, Kaifeng, 475004, China
b
Key Laboratory of Natural Medicine and Immuneꢀengineering of Henan Province, Henan University, Kaifeng,
475004, China
c Institute of Chemistry and Biology of Henan University, Kaifeng, 475004, China
d
School of Pharmacy, Lanzhou University, Lanzhou,730000,China
1
Correspondence author: Tel: + 86037122199505;
Email address: ll514527@163.com , liuxiuhua@henu.edu.cn

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Abstract
Batatasins are endogenous plant hormones found in yams and other related plant species.
These plants are widely consumed as botanical dietary supplements in many parts of the world.
This study investigated the inhibitory effects and mechanisms of Batatasin I, III, IV, V against
αꢀglucosidase regarding their antidiabetic activities. The results revealed that Batatasin I, III, IV
inhibited αꢀglucosidase in a reversible and noncompetitive manner, with the IC50 value of 2.55,
1.89 and 2.52 mM respectively, while Batatasin V completely abolished its inhibitory activity
even at the highest concentrations tested. Furthermore, a class of BatatasinꢀDerived compounds
with different substitution patterns was synthesized and subjected to the assay to clarify the
structureꢀactivity relationships, which suggested that the hydroxyl at the Cꢀ2’ position may play a
significant role in improving the inhibitory activities. Their probable binding modes and the
specificity of the binding sites were studied using molecule docking simulation. It was concluded
that Batatasins, especially Batatasin III and IV, are promising αꢀglucosidase inhibitors, which
therefore could be used as functional food to alleviate postprandial hyperglycemia and as potential
candidates for the development of antidiabetic agents.
Keywords: Batatasins; αꢀglucosidase inhibitors; Chinese yam.

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Introduction
Typeꢀ2 diabetes, as a healthꢀthreatening concomitant disease all over the world, is a
chronic metabolic disease characterized by major imbalances in glucose metabolism and
abnormalities in fat.1,2 Therapies focusing on achieving normal glycemia are proven to be effective
in improving postprandial glycemic control and delaying the progression of longꢀterm diabetic
complications.3,4 One of the approaches currently used is the reversible inhibition of αꢀglucosidase
involved in the digestion of carbohydrates, which delays the absorption of glucose from the small
intestine and thus leads to a lowering effect on postprandial blood glucose and insulin levels.5,6
Beyond that, αꢀglucosidase is also involved in biosynthesis of glycoproteins, including promotion
of protein folding and stabilisation of cellꢀsurface glycoproteins.7,8 Hence, αꢀglucosidase inhibitors
have also been well appreciated as therapeutic agents for the treatment of such diseases as viral
diseases, cancer, and chronic heart failure.9ꢀ11
To date, only three antidiabetic agents, acarbose, miglitol, and voglibose belonging to this
category are clinically used to retard carbohydrate digestion to a moderate extent. However, they
are all structurally confined to glycosidic derivatives and required multiple steps from the
carbohydrates.12 Recent warnings about adverse effects of those drugs, such as flatulence, diarrhea
and hepatotoxicity in chronic therapy, highlight the urgent of alternative and safer treatments to
control postprandial glucose levels.13 In the course of looking for novel, easily accessible and
chemically stable αꢀglucosidase inhibitors, plantꢀbased foods and dietary bioactives are more
acceptable sources for the screening of practicable candidates due to their diversity and relative
safety. 14,15
Dioscorea opposita Thunb, commonly known as “yam”, is widely consumed as botanical

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dietary supplements in many areas of tropical America, Africa and Asia. This species, alone or in
combination with other food, has a long dietary therapy history for the diabetes and widely
cultivated in China. However, there is a limited understanding of its antidiabetic constituents and
corresponding mechanisms. Batatasins are endogenous plant hormones isolated from yams and
further related plant species like Dioscorea cayenesis Lam, D. dumetorum Pax, and D. composite
Hemsl,16ꢀ18 which have been recognised as an important factor in the dormancy of yams bulbils
related to gibberellic acid and abscisic acid metabolism. Numerous pharmacological studies had
indicated that Batatasins possessed variously beneficial health effects, such as antiꢀinflammatory,
antifungal, radical scavenging and immune system modulator.19ꢀ21 In the previous studies, we had
shown that Batatasin I led to a remarkable inhibitory effect against αꢀglucosidase.22 However, the
inhibitory effects of other Batatasins on αꢀglucosidase have not been assessed yet and their
pharmacological mechanisms remain to be clarified. In response to these issues, we synthesized
Batatasin I, III, IV, V and evaluated their inhibitory effects on αꢀglucosidase. LineweaverꢀBurk
plots were further employed in the kinetic enzymatic assays to determine the mechanism of
enzymatic inhibition. The probable binding modes and the specificity of the binding sites were
studied by using molecule docking simulation. To gain further insight into their structureꢀactivity
correlations, a class of Batatasin derivatives with hydroxyl and/or methoxyl substitutions was also
synthesized and subjected to the enzymatic assay, which provide the possible guidelines for future
rational design of potent BatatasinꢀDerived αꢀglucosidase inhibitors.
Materials and methods
Chemicals and materials

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All solvents used in this research were of analytical grade, and the chemicals used for
synthesizing the derivatives were of reagent grade and commercially available. The baker’s yeast
αꢀglucosidase (EC 3.2.1.20), pNPG (4ꢀnitrophenylꢀaꢀDꢀglucopyranoside), and acarbose were
purchased from SigmaꢀAldrich Chemical (St. Louis, MO, USA). All other chemicals, including
material and reagents, were purchased from Aladdin Chemical Ltd (Shanghai, China).
General experimental procedures
The NMR spectra were recorded at 400 MHz on a Bruker Avance 400 spectrometer with
TMS as an internal standard. J values were reported in Hertz. Highꢀresolution TOFꢀMS
experiments were carried with a Qstar Elite mass spectrometer (AB Sciex, America). The IR
spectra were measured with the AVATAR 360 infrared spectrometer (Nicolet, America).
Measurement of αꢀglucosidase activities were performed with the DNMꢀ9606 microplate reader
(Beijing, China).
Synthesis of Batatasins and their derivatives
Batatasins derivatives were prepared according to the method described for Batatasins with
modification.23 A generalized synthetic approach for the proposed compounds was illustrated in
Figure 1, which was efficiently synthesized via Wittig reaction and stilbene reduction procedure as
key steps. Briefly, functionallyꢀsubstituted benzyl alcohol 2dꢀ10d, which were synthesized
according to the procedures described in literatures,24ꢀ26 were reacted with phosphorus tribromide
respectively to obtain the Brꢀsubstituted intermediates 2cꢀ10c. These intermediates were then used
by adding stoichiometric triethyl phosphate to form the stilbene derivatives 2bꢀ10b, which
underwent the intramolecular ringꢀclosure via the Wittig olefination reaction at room temperature.
The intermediates 2bꢀ10b were submitted to the catalytic hydrogenolysis under a hydrogen

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atmosphere to give the target compounds 2ꢀ10. Compound 1 with a phenanthrene skeleton was
prepared by the following strategy, where the intermediate 1b (4ꢀbenzyloxyꢀ3,
3’,5ꢀtrimethoxystibene) was irradiated with UV light in the presence of catalytic iodine and then
adopted to the reduction procedure under the same condition as described above to obtain
Batatasin I.
Inhibitory activity against αꢀglucosidase
The αꢀglucosidase enzymatic assay was performed according to the methods described by
Chen and Wang27,28 with some minor modifications. This assay used pNPG as the substrate, which
was hydrolyzed by αꢀglucosidase to release pꢀnitrophenol, a color agent that can be monitored at
405 nm. Briefly, 20 ꢁL of a sample solution was mixed with 20 ꢁL of the enzyme solution (0.45
U/mL) in 0.1 M phosphate buffer (pH 6.8) and incubated at 37 °C for 5 min under shaking. After
that, 40 ꢁL of 2.5 mM pNPG solution in the above buffer was added to initiate the colorimetric
reaction. After incubation at 37 °C for 10 min, 80 ꢀL of 0.2 M sodium carbonate solution was
added to stop the reaction, the total volume was 160 ꢁL, and the released pꢀnitrophenol was
monitored at 405 nm by the microplate reader. Acarbose was used as a positive control. The
inhibitory activity was calculated by the following equation:
Inhibition (%) = ( Acontrol ꢀ Asample)/Acontrol × 100
where Acontrol is the pꢀnitrophenol resulting from the enzymatic hydrolysis without inhibitor, and
Asample is the pꢀnitrophenol resulting from the enzymatic hydrolysis in the presence of the inhibitor.
All of the analyses were performed in triplicate.
Kinetics of α-glucosidase inhibitors
In this study, the type of the inhibitive mode of the enzyme inhibitors were characterised by

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the kinetic method. Typically, different concentrations (0ꢀ25 ꢀM) of each inhibitor around the IC50
values were chosen. Under each concentration, αꢀglucosidase activity was assayed by varying the
concentration of PNP glycoside (0.25, 0.5, 0.75, 1.0 and 1.5 mM), respectively. The enzymatic
assay was performed according to the aboveꢀmentioned reaction conditions. The inhibitory
kinetics of the investigated compounds on αꢁglucosidase was analysed using the LineweaverꢀBurk
plots 1/v versus 1/[S].
Molecular modeling and docking
AutoDock Tools were employed to assign Gasteiger partial charges and merge nonꢀpolar
hydrogen atoms. The rigid protein structure of αꢀglucosidase (ID: 4J5T) was downloaded from the
Protein Data Bank as a receptor for the docking calculations. It was suggested that the Cꢀdomain
residues of Asp568 and Glu771 affect substrate orientation and metabolism that play critical roles in
substrate recognition and binding processes.29 Mutations of these residues often led to dramatic
changes in the kinetics of the catalytic reaction. The docking simulations were performed with the
Lamarckian genetic algorithm for at least 80 docking runs for each ligand. An extended model
was employed to estimate the energetics of the unbound states of the receptor and the ligands
when evaluating their binding free energies (ꢂG), which mainly involved two steps: (i) the
intramolecular energetics was estimated for the transition from unbound states to the conformation
of the ligand and protein in the bound state; (ii) evaluation of the intermolecular energetics of
combining the ligand and protein in their bound conformations.
Statistical analysis
All of the samples were run at least in triplicate. The data obtained in this study were dealt
with using GraphPad Prism 5 and Origin 8 software. Values were expressed as means ± standard

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deviations and the difference was considered significant at P ≤ 0.05.
Results and discussion
αꢀGlucosidase inhibitory activity and structureꢀactivity relationships
The enzymatic inhibitory activities of compounds 1ꢀ10 against αꢀglucosidase were assayed in
the experimental section. The IC50 values for all samples were calculated and listed in Figure 2.
Since the smaller IC50 value means the stronger inhibitory activity, Batatasin I, III, and IV
exhibited potent inhibitory activities against αꢀglucosidase, more than acarbose (IC50 = 3.52 mM).
The results showed that even 2.55 mM of Batatasin I, 1.89 mM of Batatasin III and 2.52 mM of
Batatasin IV had been enough to inhibit 50% of the αꢀglucosidase activity, which demonstrated
the potential of Batatasins, especially Batatasin III and IV, as a new type of natural αꢀglucosidase
inhibitors.
It is interesting to note that in the enzymatic activity assay, Batatasin V completely abolished
its inhibitory actions, which encouraged us to explore the structureꢀactivity relationships of
Batatasins and their derivatives. As an evident from the Figure 3, Compound 5 was the most
potent αꢀglucosidase inhibitor with an IC50 value of 0.95 mM, being about 4 times more potent
than acarbose. The remaining compounds, apart from compound 9, had significantly reduced the
enzymatic activities with IC50 in the range of 0.95ꢀ4.25 mM. As the main skeleton for all
compounds is same, the differences in their inhibitory potential would be due to the different
substitution patterns on bibenzyl. It is worthy to note that compound 3, 6, 7 and 8 bearing
hydroxyl at the Cꢀ2’ position exhibited considerably enhanced inhibitory activities, while
compounds 2 and 10, with the same substitution at the Cꢀ3’ position, led to a large decrease in
their inhibition effects suggesting that introduction of hydroxyl group at the Cꢀ2’ position plays a

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more important role in improving the inhibitory activities against αꢀglucosidase.
Moreover, Batatasin V did not show any inhibitory activity even at the highest concentrations
tested, illustrating that 3, 4, 5ꢀtrimethoxy substitutions on the A ring might be a crucial factor for
their ineffective inhibition effects, which was confirmed further by the similar phenomenon
observed in compound 9. Contrarily, when substituted with a 3, 5ꢀdimethoxy moiety, compound 5
drastically increased its inhibitory activity, suggesting the moderately deactivating groups at the
Cꢀ4 position could enhance the affinity between inhibitors and enzyme. These results indicated
that the relatively small structural changes in the skeleton of bibenzyl resulted in the significant
differences in their abilities to inhibit αꢀglucosidase.
The inhibition kinetics of αꢀglucosidase
In order to clarify the inhibitory characters of these compounds, the mechanisms of these
inhibitors binding to the αꢀglucosidase were further studied by analyzing their inhibitory kinetics.
The inhibition of αꢀglucosidase by compound 3 (Batatasin IV) and compound 5 (the most
effective specie) were illustrated in Figure 4 representatively. It showed that plots of the initial
velocity versus enzyme concentrations in the absence and presence of compounds gave a family of
straight lines, all of which passed through the origin. Increasing the inhibitor concentrations
resulted in lowering of the slope of the line, which suggested these compounds were reversible
inhibitors.30
Furthermore, the inhibition types of the representative αꢀglucosidase inhibitors were explored
by LineweaveꢀBurk plots, a linear doubleꢀreciprocal plot of the substrate concentration and
reaction velocity.31 As shown in Figure 5, the doubleꢀreciprocal plots for all of the inhibitors and
the control differed in slopes, and the intercepts on the xꢀaxis remained nearly constant, indicating

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that the type of inhibition belonged to the noncompetitive mode. Additionally, similar trends were
observed in the remaining compounds. The inhibition mode of Batatasins and their derivatives
belonging to the noncompetitive type was different from that of acarbose, which behaved as a
typical competitive inhibitor.32 It is suggested that competitive inhibition is usually reversible and
an excessive amount of substrate would displace the inhibitor, while noncompetitive inhibition
like that of Batatasins will not be influenced by the substrate concentrations. These above findings
supported the possibility that Batatasins and their derivatives bind to the noncompetitive domain
of yeast’s αꢀglucosidase rather than to its active site.
Thermodynamics of interaction between ligands and αꢀglucosidase
To provide further insight into the binding model of inhibitors toward αꢀglucosidase as well
as into their impacts on the activity of the enzyme at the noncompetitive domain, a molecular
docking study was performed using the program AutoDock4. Docking of Batatasins and their
derivatives to αꢀglucosidase resulted in a position for the ligand coinciding with that of the
resolved crystal structure (4J5T; 2.04 Å) and confirmed the validity of the docking procedure. As
shown in Figure 6, all these inhibitors were situated appropriately for enzymatic interaction with
Asp568 and Glu771
.
Interestingly, significant Hꢀbonding interactions of ligands with the amide backbone were
observed in these binding sites. For example, the Cꢀ5 and Cꢀ2’ hydroxyl groups of Batatasin IV
form Hꢀbonds with the key residues Asp568 and Glu771, while the Hꢀbonds of Cꢀ3’ hydroxyl of
Batatasin III was not observed, which verified the aforementioned inference that hydroxyl group
at the Cꢀ2’ position had more important impact than that of Cꢀ3’ position substitution. Similar
hydrogen bonds had also been found between the remaining compounds and the amide backbone

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residues, especially with Asp568, suggesting that Hꢀbonding interaction played a key role in the
binding modes of Batatasins and their derivatives. Beyond that, binding free energies in these
binding modes were calculated ranging from ꢀ4.7 to ꢀ6.6 kJ mol−1 with compound 5 being the
highest binding affinity, which was in full accordance with the kinetic results. Taken together, the
above results indicated that the strong binding interaction of Batatasins and their derivatives to the
residues Asp568 and Glu771 of αꢀglucosidase would probably perturb the protein structure, which
may lead to the significant inhibition activities toward αꢀglucosidase.
Conclusion
In summary, our results revealed that Batatasins, especially Batatasin III and IV are
promising αꢀglucosidase inhibitors, which could be used as potential candidates for the
development of antidiabetic agents. Kinetic enzymatic analysis indicated that they inhibit
αꢀglucosidase in a noncompetitive and reversible manner. In order to clarify the structureꢀactivity
relationships of Batatasins, a series of derivatives with hydroxyl and/or methoxyl substitutions
was synthesized and investigated, which provide the possible guidelines for future rational design
of potent BatatasinꢀDerived αꢀglucosidase inhibitors. In addition, as Batatasins are naturally
occurred within the tropical genus Dioscorea including D. opposita Thunb, D. dumetorum Pax and
D. composite Hemsl and these vegetables are widely consumed as botanical dietary supplements
in many areas of the world, this might be important for the role of these vegetables in dietary
recommendations to inhibit or retard carbohydrate digestion to a moderate extent.

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Acknowledgements
This work is supported by the grants from the National Technology R&D Program for the 12th
Fiveꢀyear Plan (No.2011BAI06B04), the National Natural Science Foundation of China and
Henan province (No.U1204304), the Natural Science Research Program of Henan Province of
China (No. 13A150053). College Science and technology innovation team program of Henan
Province (No. 14IRTSTHN030)

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Figure 1. Synthesis of Batatasins and their derivatives. Reagents and conditions: (i) PBr3,
CH2Cl2, rt, 2h; (ii) P(OEt)3, tetrabutylammonium bromide, 125 oC, 4h; (iii) NaH, THF, rt,
3h; (iv) Cyclohexane, I2, hν, 8h; (v) PdꢀC, H2, MeOH, room temprature, 12h
Figure 2. Inhibitory activities on αꢀGlucosidase (Values are the mean of at least three
independent determinations. NA means no inhibition)

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Figure 3. The comparison of IC50 values between the inhibitors and the control
Figure 4. The inhibitory mechanism of ꢀglucosidase with different concentrations of
compound 3 and 5

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Figure 5. LineweaverꢀBurk plot of PNP release by ꢀglucosidase in the presence of
compound 3 and 5 in phosphate buffer (pH 6.8) at 37 oC
Figure6. 3D Representation of the interaction of Batatasin I,III,IV and compound 5 in the
binding site predicted. Dotted red lines represent hydrogen bonds connecting hydroxyl and
amino groups with amino acid residues.

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Naturally occurred Batatasins and derivatives displayed remarkable inhibitory effects against
a-glucosidase, which provide promising chemical scaffolds for antidiabetic drugs development.