Cinnamic acid amides from Tribulus terrestris displaying uncompetitive α-glucosidase inhibition

Article abstract of DOI:10.1016/j.ejmech.2016.02.044

The α-glucosidase inhibitory potential of Tribulus terrestris extracts has been reported but as yet the active ingredients are unknown. This study attempted to isolate the responsible metabolites and elucidate their inhibition mechanism of α-glucosidase. By fractionating T. terristris extracts, three cinnamic acid amide derivatives (1-3) were ascertained to be active components against α-glucosidase. The lead structure, N-trans-coumaroyltyramine 1, showed significant inhibition of α-glucosidase (IC₅₀ Combining double low line 0.42 μM). Moreover, all active compounds displayed uncompetitive inhibition mechanisms that have rarely been reported for α-glucosidase inhibitors. This kinetic behavior was fully demonstrated by showing a decrease of both K_m and V_max, and K_ik/K_iv ratio ranging between 1.029 and 1.053. We progressed to study how chemical modifications to the lead structure 1 may impact inhibition. An α, β-unsaturation carbonyl group and hydroxyl group in A-ring of cinnamic acid amide emerged to be critical functionalities for α-glucosidase inhibition. The molecular modeling study revealed that the inhibitory activities are tightly related to π-π interaction as well as hydrogen bond interaction between enzyme and inhibitors.

Full text of DOI:10.1016/j.ejmech.2016.02.044

Accepted Manuscript
Cinnamic acid amides from Tribulus terrestris displaying uncompetitive α-glucosidase
inhibition
Yeong Hun Song, Dae Wook Kim, Marcus J. Curtis-Long, Chanin Park, Minky Son,
Jeong Yoon Kim, Heung Joo Yuk, Keun Woo Lee, Ki Hun Park
PII:
S0223-5234(16)30134-9
10.1016/j.ejmech.2016.02.044
EJMECH 8395
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 December 2015
Revised Date: 6 February 2016
Accepted Date: 17 February 2016
Please cite this article as: Y.H. Song, D.W. Kim, M.J. Curtis-Long, C. Park, M. Son, J.Y. Kim, H.J.
Yuk, K.W. Lee, K.H. Park, Cinnamic acid amides from Tribulus terrestris displaying uncompetitive
α-glucosidase inhibition, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.02.044.
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ACCEPTED MANUSCRIPT
Graphical abstract
Cinnamic acid amides from Tribulus terrestris displaying uncompetitive
α-glucosidase inhibition
Yeong Hun Songa, Dae Wook Kima, Marcus J. Curtis-Longb, Chanin Parkc, Minky Sonc,
Jeong Yoon Kima, Heung Joo Yuka, Keun Woo Leec,*, Ki Hun Parka,*

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Cinnamic acid amides from Tribulus terrestris displaying
uncompetitive α-glucosidase inhibition
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Yeong Hun Songa, Dae Wook Kima, Marcus J. Curtis-Longb, Chanin Parkc, Minky Sonc,
Jeong Yoon Kima, Heung Joo Yuka, Keun Woo Leec,*, Ki Hun Parka,*
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a Division of Applied Life Science (BK21 plus), IALS, Gyeongsang National University, Jinju
660-701, Republic of Korea
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Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York
14853, United States
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Division of Applied Life Science (BK21 plus), PMBBRC, RINS, Gyeongsang National
University, Jinju 660-701, Republic of Korea
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* Corresponding author. Tel.: +82-55-772-1965; fax: +82-55-772-1969 (K. H. Park)
Tel.: +82-55-772-1360; fax: +82-55-772-1359 (K. W. Lee)
E-mail addresses: khpark@gnu.ac.kr (K.H. Park), kwlee@gnu.ac.kr (K.W. Lee)

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Abstract
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The α-glucosidase inhibitory potential of T. terrestris extracts has been reported but as yet
the active ingredients are unknown. This study attempted to isolate the responsible
metabolites and elucidate their inhibition mechanism of α-glucosidase. By fractionating T.
terristris extracts, three cinnamic acid amide derivatives (1-3) were ascertained to be active
components against α-glucosidase. The lead structure, N-trans-coumaroyltyramine 1, showed
significant inhibition of α-glucosidase (IC50 = 0.42 ꢀM). Moreover, all active compounds
displayed uncompetitive inhibition mechanisms that have rarely been reported for α-
glucosidase inhibitors. This kinetic behavior was fully demonstrated by showing a decrease
of both Km and Vmax, and Kik/Kiv ratio ranging between 1.029 – 1.053. We progressed to study
how chemical modifications to the lead structure 1 may impact inhibition. An α, β-
unsaturation carbonyl group and hydroxyl group in A-ring of cinnamic acid amide emerged
to be critical functionalities for α-glucosidase inhibition. The molecular modeling study
revealed that the inhibitory activities are tightly related to π-π interaction as well as hydrogen
bond interaction between enzyme and inhibitors.
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Key words: Tribulus terrestris, α-glucosidase, uncompetitive inhibition, cinnamic acid amide,
molecular docking
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1. Introduction
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Tribulus terrestris Linn is a renowned saponin rich plant also called Gokhru, which
belongs to the family Zygophyllaceae and is widely distributed thought India and the
southern part of China. Phytochemical studies have estimated that this plant also contains
various bioactive metabolites such as flavonoids [1], alkaloids [2], lignanamides [3] and
cinnamic acid amides [4] in addition to saponins [5]. Since it is related with a diverse array of
health promoting constituents, T. terrestris has been used as a traditional medicine to treat
edema, rheumatism, menorrhagia, eye trouble and sexual dysfunction related disorders [6-8].
Various components of this plant have been proven to have cytotoxic and hyperlipidemic
effects, as well as papain like protease (PLpro) inhibition [9,10]. Based on clinical studies,
several pharmaceutical preparations and food supplements derived from this plant are
commercially available on the global market. Lamba et al. reported ethanolic extract of T.
terrestis showed significant inhibition of α-glucosidase as well as antidiabetic effects in-vivo
[11]. It was also reported that saponin fraction inhibited the activity of α-glucosidase in small
intestines in rats and retarded the increase in postprandial blood glucose level in rats [12].
α-Glucosidase (EC 3.2.1.20, α-D-glucoside glucohydrolase) is exo-acting enzyme that
plays essential roles in carbohydrate quality control, processing and metabolism [13]. In
particular α-glucose plays essential role in biological recognition since it is situated on the
oligosaccharide chains of N-linked glycoproteins in the endoplasmic reticulum and on the
outer periphery of the cell [14]. Inhibition of α-glucosidase has a profound effect on the
glycan structure which consequently effects the maturation, transport and secretion and thus
has the power to alter cell-cell or cell-virus recognition process [15-17]. Glycosidase is also a
key player in sugar metabolism: by retarding the cleavage of complex carbohydrates,
postprandial glucose absorption in vivo can be attenuated, thus regulating blood sugar levels

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[18]. Therefore, glucosidase inhibition is applicable to the treatment of numerous diseases
including cancer, HIV and diabetes mellitus type-2 [19-21].
The aim of the present work was to investigate the α-glucosidase inhibitory components
of T. terrestris and evaluate their inhibition mechanism. Having achieved these goals, we
proceeded to elucidate the SAR of the best inhibitors to reveal the critical features for enzyme
inhibition. We also assessed how the cinnamic acid amides bound the target enzyme using in
silico docking in a bid to understand the unusual uncompetitive behavior shown by the
inhibitors.
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2. Results and discussion
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2.1. α-Glucosidase inhibitory cinnamic acid amides
Lamba et al. reported that ethanol extracts of T. terrestris inhibited α-glucosidase activity
significantly [11]. However the study focused only on the extracts of T. terrestris and did not
elucidate any compounds responsible for this activity. We tested the extracts from three
different polar solvents (EtOAc, MeOH, water) for their enzymatic inhibitory activities
against α-glucosidase. The enzyme was assayed according to a standard literature procedure
by following the hydrolysis of nitrophenyl glycoside spectrophotometrically. The methanol
extract was determined to be the richest source of α-glucosidase inhibitors as it gave the
strongest inhibition (10 ꢀg/ml, 87 %). Purification of the methanol extract over silica gel,
sephadex LH-20, and octadecyl-funtionalized silica gel yielded six cinnamic acid amides.
Isolated compounds (1-6) were identified as N-trans-coumaroyltyramine [22] (1), N-trans-
caffeoyltyramine [23] (2), N-trans-feruloyltyramine [22] (3), terrestriamide [24] (4) N-trans-
feruloyloctopamine [22] (5) and terrestrimine [10] (6) through analysis of spectroscopic data
and comparison with previous data.

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The biological activities of the isolated cinnamic acid amides (1-6) were assessed against
α-glucosidase. All isolated cinnamic acid amides (1-6) inhibited α-glucosidase in a
concentration dependent manner with IC50s ranging between 0.4 ~ 225.8 ꢀM. Compound (1)
was found to be the most potent inhibitor with an IC50 of 0.42 ꢀM.
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The potency of these inhibitors was affected significantly by subtle changes to the 1-ethyl
4-hydroxybenzene moiety within the cinnamic acid amide (Figure 1). For instance,
compound 3 (R2=R3=H, IC50 = 10.6 ꢀM) was 20-fold more potent than compound 5 that
bears a hydroxylated ethyl group (R2=H, R3=OH, IC50 = 187.8 ꢀM). A similar phenomenon
can be observed when comparing compound 4 (R2=H, R3=oxo, IC50 = 61.5 ꢀM) and 6
(R2=OH, R3=oxo, IC50 = 225.8 ꢀM). It appears that better inhibition is observed when there
is an only free hydroxyl group in the A-ring. This can be seen by comparing compound 1
(R1=H, IC50 = 0.42 ꢀM) with 3,4-dihydroxy analogue 2 (R1=OH, IC50 = 1.86 ꢀM) and 3-
methoxy-4-hydroxy analogue 3 (R1=OMe, IC50 = 10.6 ꢀM). This outcome is contrary to the
general expectation that inhibitors bearing a greater number of hydroxyl groups are more
potent because they can better mimic sugars.
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To determine the specificity of compounds 1-6 for α-glucosidase, their inhibitory
activities against the related enzymes β-glucosidase, α-galactosidase, α-mannosidase and α-
amylase were determined. None of the inhibitors showed and activity against any of these
hydrolase at concentrations up to 200 ꢀM. The inhibition of compound 1 is demonstrated in
Figure 2 representatively.
2.2. Chemical modification of lead compound 1
Since compound 1 was the most potent inhibitor we set out to determine the structural
requirements for the inhibitory activity against α-glucosidase by undertaking further SAR.
Four target sites were varied; (1) α, β-double bond; (2) 4-hydroxyl group within the B ring; (3)

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the 5-hydroxyl group on B ring; (4) the 4-hydroxyl group on ring A. Amide derivatives (1-1e)
were easily obtained through the coupling reaction of suitable acids and ethylbenzene
derivatives using BOP (experimental section). For example, compound 1 was prepared from
p-coumaric acid and tyramine using BOP in DMF.
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Synthesized compounds 1a, 1b, and 1c, which possess 4'-hydroxy, 4'-hydrogen, 4'-
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fluorine and 4', 5'-dihydroxy, respectively, were similar in potency to compound 1 (Figure 3).
This result indicates that there is a large degree of tolerance in the B ring. Compound 1d
which did not possess a hydroxyl function on the A ring was a very poor inhibitor (IC50
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215 ꢀM). This implies that a H-bond donor is important in this position. The α, β-unsaturated
carbonyl group also played a critical role in determining α-glucosidase inhibition potency.
Thus, compound 1 was 30-fold more effective than compound 1e (IC50 = 19.03 ꢀM). This
effect is presumably related to the enhanced conformational rigidity of 1 relative to 1e.
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2.3. Enzyme kinetic analysis
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All inhibitors manifested a similar relationship between enzyme activity and
concentration. The relevant data for the most potent inhibitor, compound 1, is illustrated in
Figure 4 as example. Plots of residual enzyme activity versus enzyme concentration at
different concentrations of compound 1 gave a family of straight lines with a common y-axis
intercept, indicating that 1 is a reversible inhibitor. Augmenting the concentration of
compound 1 resulted in a reduction of the slopes of the lines. All active compounds showed
uncompetitive inhibition behavior.
Uncompetitive inhibitors of α-glucosidase have rarely been reported and the structural
features have not been systematically investigated [25]. We therefore undertook a detailed
kinetic analysis of the inhibition wherein the enzyme inhibition behaviors of cinnamic acid

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amide derivatives were modeled using double-reciprocal plots. This analysis showed that
both Km and Vmax calculated from Lineweaver-Burk plots decreased as inhibitor concentration
increased; as can be seen directly from the graph, families of 1/V versus 1/[S] regression line
displayed nearly identical slopes, as expected for uncompetitive inhibition behavior (Figure
4B) [26]. As shown Figure 4C, this same pattern was seen when data plotted according to
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Dixon (1/V vs [I]). Ki's were calculated using Cornish-Bowden plot (equation 3) yielding
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values between 0.29 and 62.1 ꢀM (Figure 4D). To further confirm uncompetitive behavior,
the results were applied to Yang's method [27]. According to this procedure; Km and Vmax are
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plotted against the inhibitor concentration. The new kinetic constant Kik can be fit to equation
1, while Kiv can be fit equation 2. From the result of the fit, Kik/Kiv ratio were between 1.029
and 1.053 that is further consistent with uncompetitive behavior [28]. This finding maybe
important because uncompetitive inhibitors are to be regarded as superior for drug
development when compared to competitive and noncompetitive inhibitors and are expected
to display better in-vivo efficacy [25].
2.4. Molecular docking experiments
To investigate relationships between the inhibitory activities and binding conformations
of cinnamic acid amides on α-glucosidase, a homology model structure of α-glucosidase was
constructed (Supplementary material) and compounds 1, 1a, and 1e, which act as
uncompetitive inhibitors, were docked into the active site of α-glucosidase. Our molecular
docking results showed that all the three compounds were bound to the entrance part of the
active site, thereby preventing the release of the substrate (Figure 5). A-ring of the
compounds was surrounded by Thr215, Ala216, Gly217, Leu218, His245, Glu276, Val277,
and Ala278, and the hydroxyl group of A-ring formed two hydrogen bonds (H-bonds) with

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Gly217 and Glu276, in all the systems. The H-bonds were stably maintained at the average
distances of about 0.2 nm during the last 100 ps of the simulation (Figure 6A and B). The
amide groups of 1 and 1a had H-bonds to the reducing end of the maltose, whereas the bond
was not found for 1e. The H-bond distances for the 1 and 1a were converged at 0.22 nm
(Figure 6C). B-ring of 1 interacts with Phe300, Thr307, Ser308, Pro309, and Arg312 (Figure
5A), and one H-bond was formed between the hydroxyl group of the ring and Ser308 (Figure
6D). However, due to the absence of the hydroxyl group, the B-ring of 1a could not make any
interaction with Thr307, Ser308, and Pro309 (Figure 5B). The hydroxyl group of B-ring of 1e
was not able to form a H-bond with Ser308, hence it seems that the double bond between A-
ring and amide group (α,β-unsaturated carbonyl group) in 1 and 1a contributes to stabilize the
binding. Although the three compounds were bound to the entrance of active site at a similar
manner, the average numbers of H-bonds were different with the value of 3.79, 2.72, and 1.95
for 1, 1a, and 1e, respectively. It is found that the numbers of H-bonds are well aligned with
the order of the inhibitory activities for the compounds against α-glucosidase. In addition, we
suggest that π-π interaction between the α,β-unsaturated carbonyl group of the compounds
and Phe157 would also contribute to stabilize binding of the compounds to α-glucosidase
(Figure 7). The average plane to plane distances between the conjugated π-electron cloud,
which is extended from A-ring to the amide group of the compounds, and the benzene ring of
Phe157 were 0.40 nm, 0.46 nm, and 0.52 nm for 1, 1a, and 1e, respectively (Figure 7A). And
the interaction potential energies between the compounds and Phe157 were -27.51, -22.18,
and -17.78 kJ/mol, respectively (Figure 7B). The results clearly show that the distances and
energy values are well suited with the inhibitory activities. We think that the double bond
plays an important role on formation of π-π interaction which seems to help the inhibitors for
binding more stable (Figure 7C).
Therefore, it can be concluded that our molecular modeling studies successfully provided

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a possible structural explanation for the difference in the inhibitory activities of the three
compounds against α-glucosidase and also contributed to understand the mechanism and
structural insight for the inhibitor and protein binding in molecular level.
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3. Conclusion
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In conclusion, we have undertaken a thorough investigation into the α-glucosidase
inhibition of the important medicinal plant, T. terrestris. The principal components were
identificated as cinnamic acid amides that showed uncompetitive behavior that was fully
demonstrated with kinetic parameters Vmax, Km, Kik and Kiv. The most active inhibitor (1)
showed significant inhibition with IC50 = 0.42 ꢀM. Our SAR including some synthesized
derivatives of 1 indicated that an α, β-unsaturation carbonyl group and a hydroxyl group in
A-ring were critical functions. In the molecular modeling studies, detailed analyses for the
molecular interactions were consistent with this conclusion. This analysis also suggested that
number of hydrogen bonds and strength of π-π interactions between the compound and
protein are strongly correlated with inhibitory activities.
4. Materials and Methods
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4.1. General apparatus and Chemicals
Organic solvents used for isolation were of first grade and the stock solution and buffers
were prepared with milli Q water. Analytical grade methanol, acetonitrile and acetic acid for
HPLC were purchased from J.T. Baker (Phillipsburg, NJ, USA). Column chromatography
was carried out using silica gel (230-400 mesh, Merck), RP-18 (ODS-A, 12 nm, S-150 ꢀM,
YMC), and Sephadex LH-20 (Amersham Biosciences). Enzymatic assays were carried out on
a SpectraMax M3Multi-Mode Microplate Reader (Molecular device, USA). 1H and 13C NMR,

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as well as 2D NMR data, were obtained on a Bruker AM 500 (1H NMR at 500MHz, 13C
NMR at 125 MHz) spectrometer (Bruker, Karlsruhe, Germany) in CD3OD or DMSO-D6 with
TMS as internal standard. EIMS, HREIMS and FABMS, HRFABMS were obtained on a
JEOL JMS-700 mass spectrometer (JEOL, Tokyo, Japan). Infra-red (IR) spectra were
recorded on
a
Varian 640-IR (Varian, Inc. USA) infrared Fourier transform
spectrophotometer (KBr). Melting points (mp) were measured on a Thomas Scientific
Capillary Melting Point Apparatus and are uncorrected. Qualitative analyses were made using
an Agilent 1100 liquid chromatography (Agilent Technologies, PaloAlto, CA, USA). Reagent
grade chemicals were purchased from Sigma Chemical Co. (St. Louis, USA). The fruits of T.
terrestris [imported from China, as permitted by Korea Food and Drug Administration
(KFDA)] were purchased from a local market.
4.2. Extraction and Isolation
The dried fruits (2.5 kg) of T. terrestris were extracted using methanol (10 L × 3) at room
temperature to give crude extract (312g, 12.5%). The methanol extract (312 g) was washed
with n-hexane (3 L × 3, with sonication) to remove oils to give subextract (118 g). A part of
the extract (10 g) was fractionated (3 times) on reversed-phase (RP) silica gel column (340 g,
C18) using MPLC (Puriflash 450, Interchim, Montlucon, France) with a linear gradient of 0-
90 % CH3OH/H2O and a 40 ml/min flow rate to afford seven fractions (A-G). Fraction C (1.3
g), E (2.8 g) and F (1.2 g) were grouped together and fractionated via MPLC using a silica
gel column (200 g) with elution using a gradient of increasing CH3OH (0-15 %) in CHCl3 to
give subfractions A-H. Subfraction B (190 mg), enriched with 1 was further chromatography
over a C18 column (15 g) eluting with a H2O/MeOH gradient (30-60 % MeOH) to give afford
compound 1 (30 mg). Subfraction C (460 mg), enriched with 3 and 4 was further
chromatographed over a C18 column (30 g) eluting with a H2O/MeOH gradient (40-60 %

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MeOH) to afford compound 3 (41 mg) and 4 (32 mg). Subfractions D-E (625 mg), enriched
with 2 and 6, were further chromatographed over sephadex LH-20 (CH3OH) to give
subfractions E1-E7. Subsequent separation of subfraction E3 (130 mg) by RP-MPLC using
CH3OH-H2O (40:60) as mobile phase afforded compound 2 (25 mg). Subfraction E4 (108 mg)
was chromatographed over a C18 column (15 g) using CH3OH-H2O (50:50) to give
compound 6 (15 mg). Subfraction F (225 mg) was purified using Sephadex LH-20 column
chromatography, eluting with CH3OH to afford compound 5 (26 mg). All isolated
compounds were identified by comparison to previously reported spectroscopic data
(Supplementary data) [10, 22-24].
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4.2.1. N-trans-Coumaroyltyramine (1)
White powder; mp 255–257 °C; EIMS, m/z 283 [M]+; HREIMS, m/z 283.1209 (calcd for
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C17H17O3 283.1208); IR (KBr) 3432, 3300, 3175, 3023, 2940, 1660 cm-1; 1H-NMR (CD3OD,
500 MHz) δ: 7.36 (1H, d, J = 15.7 Hz), 7.30 (2H, d, J = 8.6 Hz), 6.96 (2H, d, J = 8.4 Hz),
6.69 (2H, d, J = 8.6 Hz), 6.62 (2H, d, J = 8.4 Hz) 6.30 (1H, d, J = 15.7 Hz), 3.37 (2H, t, J =
7.4 Hz), 2.66 (2H, t, J = 7.5 Hz).
4.2.2. N-trans-Caffeoyltyramine (2)
White powder; mp 215–217 °C; EIMS, m/z 299 [M]+; HREIMS, m/z 299.1155 (calcd for
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C17H17NO4 299.1158); IR (KBr) 3361, 3193, 3030, 3024, 2955, 1650 cm-1; H-NMR
(CD3OD, 500 MHz) δ: 7.41 (1H, d, J = 15.7 Hz), 7.07 (2H, d, J = 8.4 Hz), 7.02 (1H, d, J =
1.7 Hz), 6.92 (1H, dd, J = 8.2, 1.8 Hz), 6.79 (1H, d, J = 8.1 Hz), 6.74 (2H, d, J = 8.4 Hz),
6.37 (1H, d, J = 15.6 Hz), 3.47 (2H, t, J = 7.6 Hz), 2.76 (2H, t, J = 7.5 Hz).
4.2.3. N-trans-Feruloyloctopamine (3)
White powder; mp 145–147 °C; EIMS, m/z 313 [M]+; HREIMS, m/z 313.1309 (calcd for
C18H19NO4 313.1314); IR (KBr) 3300, 3015, 2930, 1650 cm-1; 1H-NMR (CD3OD, 500 MHz)

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δ: 7.47 (1H, d, J = 15.7 Hz), 7.12 (1H, d, J = 1.7 Hz), 7.07 (2H, d, J = 8.4 Hz), 7.04 (1H, dd,
J = 8.2, 1.8 Hz), 6.82 (1H, d, J = 8.2 Hz), 6.75 (2H, d, J = 8.4 Hz), 6.44 (1H, d, J = 15.7Hz),
3.85(3H, s, OCH3), 3.50 (2H, t, J = 7.2 Hz), 2.78 (2H, t, J = 7.3 Hz).
4.2.4. Terrestriamide (4)
Yellowish powder; mp 185–187 °C; EIMS, m/z 327 [M]+; HREIMS, m/z 327.1103 (calcd
1
for C18H17NO5 327.1107); IR (KBr) 3310, 2932, 2857, 1666, 1644 cm-1; H-NMR (DMSO-
d6, 500 MHz) δ: 7.91 (2H, d, J = 8.7 Hz), 7.37 (1H, d, J = 15.7 Hz), 7.17 (1H, d, J = 1.4 Hz),
7.04 (1H, dd, J = 8.2, 1.5 Hz), 6.89 (2H, d, J = 8.7 Hz), 6.82 (1H, d, J = 8.2 Hz), 6.68 (1H, d,
J = 15.7 Hz), 4.66(2H, d, J = 5.5 Hz), 3.88 (3H, s, OCH3).
4.2.5. N-trans-Feruloyloctopamine (5)
Yellowish powder; mp 196–198 °C; FAB-MS, m/z 330.2 [M+H]+; HRFAB-MS, m/z
330.1343 (calcd for C18H20NO5 330.1341, corresponding to C18H19NO5); [α]D20-18 (c 0.1,
CH3OH); IR (KBr) 3520, 3390, 3264, 2927, 1651 cm-1; 1H-NMR (CD3OD, 500 MHz) δ: 7.48
(1H, d, J = 15.7Hz), 7.25 (2H, d, J = 8.5 Hz), 7.13 (1H, d, J = 1.6 Hz), 7.05 (1H, dd, J = 8.5,
1.8 Hz), 6.82 (1H, s), 6.80 (2H, d, J = 8.6 Hz), 6.49 (1H, d, J = 15.7 Hz), 4.76 (1H, m), 3.89
(3H, s, OCH3), 3.58 (1H, dd, J = 13.6, 4.9Hz), 3.49 (1H, dd, J = 13.6, 7.9Hz).
4.2.6. Terrestrimine (6)
Yellowish powder; mp 172–175 °C; FAB-MS, m/z 344.2 [M+H]+; HRFAB-MS, m/z
344.1143 (calcd for C18H18NO6 344.1134, corresponding to C18H17NO6); IR (KBr) 3320,
3280, 2927, 2853, 1691, 1650 cm-1; 1H-NMR (CD3OD, 500 MHz) δ: 7.98 (2H, d, J = 8.8 Hz),
7.55 (1H, d, J = 15.7 Hz), 7.16 (1H, d, J = 1.6 Hz), 7.07 (1H, dd, J = 8.2, 1.7 Hz), 6.88 (2H, d,
J = 8.8 Hz), 6.82 (1H, d, J = 8.2 Hz), 6.58 (1H, s), 6.51 (1H, d, J = 15.6 Hz) 3.89 (3H, s,
OCH3).

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4.3. General procedure for cinnamic acid amides (1-1d)
BOP (Castro's reagent [29], 0.25 mmol) was added to a mixture solution of acid (0.20
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mmol) and amine (0.20 mmol) in dry DMF (20 ml) under a nitrogen atmosphere at room
temperature. To this mixture, trimethylamine (0.40 mmol) was added drop wise with
vigorous stirring. The stirring was continued for 12 h. The reaction mixture was quenched
with water (100 ml) and then extracted with ethylacetate (3 × 50 ml). Organic layer was
wished with brine and dried over anhydrous Na2SO4. The solvent was evaporated and residue
was subjected to silica gel chromatography using CHCl3/CH3OH (10:1) as eluent to give
corresponding amides (1-1d) as 30 ~ 80% yield.
4.3.1. (E)-3-(4-hydroxyphenyl)-N-phenylethyl acrylamide (1a)
Yellowish powder; mp 143–146 °C; EIMS, m/z 267 [M]+; HREIMS, m/z 267.1259 (calcd
1
for C17H17NO2 267.1259); IR (KBr) 3423, 3140, 3015, 2951, 1655 cm-1; H-NMR (CD3OD,
300 MHz) δ: 7.48 (1H, d, J = 15.7 Hz), 7.42 (2H, d, J = 8.6 Hz), 7.24 (2H, m), 7.04 (2H, m),
6.99 (1H, br), 6.81 (2H, d, J = 8.6 Hz), 6.41 (1H, d, J = 15.7 Hz), 3.51 (2H, t, J = 7.3 Hz),
2.84 (2H, t, J = 7.3 Hz).
4.3.2. (E)-N-(4-fluorophenethyl)-3-(4-hydroxyphenyl) acrylamide (1b)
White powder; mp 176–179 °C; EIMS, m/z 285 [M]+; HREIMS, m/z 285.1164 (calcd for
1
C17H16FNO2 285.1165); IR (KBr) 3398, 3150, 3015, 2930, 1660 cm-1; H-NMR (CD3OD,
300 MHz) δ: 7.36 (1H, d, J = 15.7 Hz), 7.29 (2H, d, J = 8.5 Hz), 7.19 (2H, m), 7.12 (2H, m),
6.69 (2H, d, J = 8.6 Hz), 6.29 (1H, d, J = 15.7 Hz), 3.42 (2H, t, J = 7.2 Hz), 2.76 (2H, t, J =
7.3 Hz).
4.3.3. (E)-N-(3, 4-dihydroxyphenethyl)-3-(4-hydroxyphenyl) acrylamide (1c)

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White powder; mp 163–166 °C; EIMS, m/z 299 [M]+; HREIMS, m/z 299.1157 (calcd for
C17H17NO4 299.1158); IR (KBr) 3403, 3360, 2925, 1651 cm-1; 1H-NMR (CD3OD, 300 MHz)
δ: 7.48 (1H, d, J = 15.7 Hz), 7.42 (2H, d, J = 8.6 Hz), 6.8 (2H, d, J = 8.6 Hz), 6.72 (2H, m),
6.58 (1H, dd, J = 7.9, 2.0 Hz), 6.42 (1H, d, J = 15.7 Hz), 3.48 (2H, t, J = 7.0 Hz), 2.73 (2H, t,
J = 7.1 Hz).
4.3.4. N-(4-hydroxyphenethyl) cinnamamide (1d)
White powder; mp 190–193 °C; EIMS, m/z 267 [M]+; HREIMS, m/z 267.1256 (calcd for
C17H17NO2 267.1259); IR (KBr) 3425, 3270, 3020, 2925, 1660 cm-1; 1H-NMR (CD3OD, 300
MHz) δ: 7.50 (3H, m), 7.35 (3H, m), 7.05 (2H, m), 6.71 (2H, m), 6.56 (1H, d, J = 15.7 Hz),
3.46 (2H, t, J = 7.1 Hz), 2.75 (2H, t, J = 7.1 Hz).
4.3.5. N-(4-hydroxyphenethyl)-3-(4-hydroxyphenyl) propanamide (1e)
White powder; mp 161–165 °C; EIMS, m/z 285 [M]+; HREIMS, m/z 285.1366 (calcd for
C17H19NO3 285.1365); IR (KBr) 3405, 3202, 3020, 2943, 1610 cm-1; 1H-NMR (CD3OD, 300
MHz) δ: 6.94 (4H, dd, J = 8.4, 1.7 Hz), 6.68 (4H, dd, J = 8.5, 1.8 Hz), 3.27 (2H, m), 2.76 (2H,
t, J = 7.5 Hz), 2.59 (2H, t, J = 7.5 Hz), 2.36 (2H, t, J = 7.6 Hz).
4.4. Assay of α-glucosidase inhibitory activity
α-Glucosidase inhibitory activity was calculated using literature experimental procedures
with some modifications [30,31]. The substrate, p-nitrophenyl-α-D-glucopyranoside (pNPG),
was dissolved to a concentration of 1.2 mM in potassium buffer [50 mM potassium
phosphate (pH 6.8)]. Liberated p-nitrophenol was immediately quantified spectrometrically
on a Spectra Max M3 Multi-Mode Microplate reader (Molecular Devices, CA, USA) at 37 °C,
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405 nm for 15 min. The α-glucosidase stock solution (EC 3.2.1.20. from Baker's Yeast) was
0.45 units in 20 ꢀl of phosphate buffer. The compounds were dissolved in DMSO and diluted

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to the corresponding concentration. The assay was carried out as follows: 40 ꢀl of substrate
(final concentration 0.24 mM was added to 10 ꢀl of sample in phosphate buffer (130 ꢀl) in 96
well plate and then enzyme (20 ꢀl) was added. The inhibitory effects of the tested compounds
were expressed as the concentrations that inhibited 50 % of enzyme activity (IC50). The
percent inhibition ratio (percent) was calculated according to the following equation: %
inhibition = [(rate of control reaction ‒ rate of sample reaction) / rate of control reaction] ×
100.
4.5. Enzyme inhibition kinetic
The enzyme inhibition modality was investigated in experiments using incremental
concentration (0.125 ~ 0.5 mΜ) of substrate and by testing the compound at different
concentrations as indicated. The data were analyzed using nonlinear regression program,
Sigma Plot (SPCC Inc., Chicago, IL, USA) to find the individual parameters for each curve.
The Michaelis-Menten constant Km and maximal velocity Vmax were determined using the
Lineweaver-Burk plot. The inhibition constant (Ki) was determined according to equations (3)
derived from Dixon and Cornish-Bowden [32]. Kik and Kiv rate constants were calculated
according to equation (1-2) suggested by Yang et al [28]. Linear regression analysis were
performed with Excel.
ꢁ ꢂ
I
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Km = Km,0 × ꢀ1 +
Vm = Vm,0 × ꢀ1 +
ꢃ
(1)
(2)
Kik
ꢁ ꢂ
I
ꢃ
Kiv
s
Km
V
i
=
ꢀ1+ ꢃ + Vs ꢀ1+ i ꢃ (3)
v
Ki
K'i
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359
4.6. Homology modeling

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A protein BLAST search was executed to find homologues of the target sequence in the
protein data bank (PDB) database with PSI-BLAST algorithm. A sequence alignment
between query and template was carried out using ClustalW2 [33] available on the EMBL-
EBI website with default parameters. A model structure was built based on a result of the
sequence alignment using Build Homology Models protocol implemented in Discovery studio
(DS) 4.0 software. The stereo chemical quality of the model structure was assessed based on
Ramachandran plot and QMEAN score provided by PROCHECK [34] and SWISS-MODEL
server [35], respectively.
4.7. Molecular docking calculation
Molecular docking calculations of a substrate (maltose) and three uncompetitive
inhibitors (compound 1, 1a, and 1e) to α-glucosidase-maltose complex structure were
performed by GOLD Suite 5.2.2 (the Cambridge Crystallographic Data Center, UK) applying
the default values for the genetic algorithm (GA) [36]. All atoms within 15 Å from the center
of the active site were defined as a docking pocket. The number of docking runs was set to 50
for each inhibitor.
4.8. Molecular dynamics simulation
All MD simulations were carried out using GROMACS 5.0.2 package in conjunction
with the all-atom AMBER03 force field [37]. Topologies for the substrate and the inhibitors
were generated by ACPYPE scrip [38]. The systems were enclosed in a dodecahedron box
with periodic boundary conditions and were explicitly solvated with TIP3P water molecules
[39]. The net charge of the systems was neutralized by randomly replacing water molecules
with sodium counter ions. The pair list was updated every 20 fs with a cut-off of 1.0 nm. Van
der Waals (VDW) interactions were calculated with a cut-off of 1.0 nm and long-range

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dispersion corrections for energy and pressure were applied to account for the truncation of
VDW interaction. Electrostatic interactions were computed with a cut-off of 1.0 nm and the
particle-mesh Ewald (PME) method [40] was used to treat long-range electrostatic interaction
with a Fourier grid spacing of 0.12 nm and a fourth-order interpolation. The energy
minimization for each system was performed using a steepest descent algorithm until the
maximum force was less than 1,000 kJ mol-1 nm-1 on any atom. The systems were then
equilibrated in two phases, during which position restraints were placed on all heavy atoms of
the solute. The first phase of equilibration was accomplished under a canonical NVT
ensemble for 100 ps and temperature was controlled at 300 K by Berendsen thermostat with a
coupling time constant of 0.1 ps [41]. The second phase in an isothermal-isobaric NPT
ensemble were conducted for 100 ps using Nose-Hoover thermostat [42] with time constant
of 2.0 ps and Parrinello-Rahman barostat [43] with time constant of 5.0 ps to maintain
temperature and pressure at 300 K and 1 bar, respectively. The production MD runs were
subsequently executed for 1 ns under the same NPT ensemble without any restraint. All bond
lengths were constrained to equilibrium distances using P-LINCS algorithm [44]. The time
step for integration was set at 0.2 fs.
4.9. Statistical analysis
All measurements were made in at least triplicate. The results were subject to variance
analysis using Sigma plot. Differences were considered significant at p < 0.05.
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Acknowledgments
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This research was supported by the Ministry of Agriculture, Food and Rural Affairs (No.
315032-04-1-SB010) and Next-Generation BioGreen 21 program (SSAC, No. PJ01107001),

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Rural Development Administration, Republic of Korea. All students were supported by a
scholarship from the BK21 plus program.
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Table
Table 1. Inhibitory effects of cinnamic amide derivatives on α-glucosidase activity.
α-glucosidase
Compound
IC50a (µM)
0.42 ± 0.01
1.86 ± 0.04
10.62 ± 0.8
61.54 ± 1.2
187.84 ± 3.4
225.83 ± 5.6
0.31 ± 0.02
1.22 ± 0.03
1.63 ± 0.06
215.25 ± 4.8
19.03 ± 0.9
21.26 ± 0.8
Kinetic mode (Kib, µM)
Uncompetitive (0.35)
Uncompetitive (1.77)
Uncompetitive (11.32)
Uncompetitive (62.1)
NT c
1
2
3
4
5
NT c
6
Uncompetitive (0.29)
Uncompetitive (1.38)
Uncompetitive (1.74)
NT c
1a
1b
1c
1d
1e
DNJ
Uncompetitive (18.21)
NT c
a All compounds were examined in a set of experiments repeated three times(The ± values
were SEM); IC50 values of compounds represent the concentration that caused 50% enzyme
activity loss.b Values of inhibition constant. c NT is not tested

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Table 2. Effect of different concentrations of compound 1 on Vmax, Km, and the Kik to Kiv
ratio using pNPG as substrate. Vmax and Km values were calculated according to Lineweaver-
Burk from the data shown in Figure 4B. Kik/Kiv ratio was calculated according to Yang et al.
Inhibitor (1)
Substrate (pNPG)
[I] µM
Vmax
Km
Kik/Kiv
0
0.093
0.051
0.036
0.022
0.013
176.557
94.851
64.713
35.103
20.927
//
0.19
0.39
0.78
1.56
1.029
1.037
1.053
1.034

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FIGURE CAPTIONS
Figure 1. Cinnamic acid amides from the fruits of T. terrestris.
Figure 2. Selectivity of compound 1 with related hydrolases. Inhibitors were tested on
glycosidases (α-glucosidase, β-glucosidase, α-amylase, α-galactosidase, and α-mannosidase).
Data represent the results of three independent experiments performed with triplicates of each
sample.
Figure 3. Analogues of coumaroyltyramine 1
Figure 4. Enzymatic kinetics for compound 1 using pNPG as substrate. (A) Relationship of
the catalytic activity of α-glucosidase and concentration of compound 1. (B) Lineweaver-
Burk plot (1/V vs 1/ [S]). (C) Dixon plot (1/V vs [I]). (D) Cornish-Bowden plot ([S]/V vs [I]).
Figure 5. Representations of binding modes for three uncompetitive inhibitors after MD
simulation. Binding conformations of (A) compound 1, (B) compound 1a, and (C) compound
1e in the active site of α-glucosidase. Hydrogen bonds are shown by orange dashed lines.
Residues involved in hydrogen bond and π-π interactions are labeled in orange and magenta,
respectively.
Figure 6. Hydrogen bond interactions between compounds and α-glucosidase-maltose
complex. Interatomic distances between; (A) compound:O7 and Gly217:H, (B)
compound:H5 and Glu276:O, (C) compound:H8 and maltose:O5', (D) compound:H17 and
Ser308:O. The average distances for the last 100 ps is indicated by black lines.
Figure 7. π-π interaction between compounds and Phe157. (A) Interaction distances between
planes of α,β-unsaturated carbonyl group of compounds and benzene ring of Phe157. (B)
Interaction potential energies between compounds and Phe157. The average distances and
energies for the last 100 ps are indicated by black lines. (C) Representations of molecular
interaction between compounds and Phe157.

ACCEPTED MANUSCRIPT
OH
O
R2
B
R1
N
A
H
R3
HO
1 R1 = H
R2 = H R3 = H
R2 = H R3 = H
2 R1 = OH
3 R1 = OCH3 R2 = H R3 = H
4 R1 = OCH3 R2 = H R3= O (oxo)
5 R1 = OCH3 R2 = H R3 = OH
6 R1 = OCH3 R2 = OH R3= O (oxo)
Figure 1.

ACCEPTED MANUSCRIPT
α-Glucosidase
β-Glucosidase
α-Amylase
α-Galactosidase
α-Mannosidase
100
80
60
40
20
0
- 8
- 7
- 6
- 5
- 4
Log [1] / M
Figure 2.

ACCEPTED MANUSCRIPT
R2
O
B
N
H
R3
A
R1
1
R1 = OH R2 = OH R3 = H
1a R1 = OH R2 = H
1b R1 = OH R2 = F
R3 = H
R3 = H
1c R1 = OH R2 = OH R3 = OH
1d R1 = H
R2 = OH R3 = H
OH
O
N
H
HO
1e
Figure 3.