Food Research International 54 (2013) 1376–1382

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Food Research International

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Phenylpropanoid glycoside inhibition of pepsin, trypsin and

α-chymotrypsin enzyme activity in Kudingcha leaves from
Ligustrum purpurascens
Xuli Wu a,⁎, WenPu Wang a, Tian Zhu a, Ting Liang a, FangQi Lu a, Weiyi He a, Haiping Zhang b, Zhigang Liu a,
ShaoHeng He c, Kaiping Gao a, Zhendan He a,⁎
a
School of Medicine, Shenzhen University, Shenzhen, Guangdong Province 518060, PR China
b
School of Life Science, Xiamen University, Xiamen, Fujian Province 361005, PR China
c
First Affiliated Hospital, Liaoning Medical University, Jingzhou, Liaoning Province 121001, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Phenylpropanoid glycosides (PPGs) are important ingredients in Kudingcha bitter tea from Ligustrum
Received 14 August 2013 purpurascens, possessing many beneficial properties. Here, we investigated the effect of PPGs on the activity of
Accepted 9 October 2013 the digestive enzymes pepsin, trypsin and α-chymotrypsin. Furthermore, we explored the interactions between
PPGs and digestive enzymes by a multi-spectroscopic method and docking studies. PPGs could inhibit digestive
Keywords:
enzyme activities with a non-competitive pattern of inhibition. PPGs may have a binding effect on these digestive
Phenylpropanoid glycoside
Inhibitor
enzymes, changing the polarity and the structure of enzymes, which results in decreased enzyme activity. This
Pepsin description of the effects of PPG inactivation of pepsin, trypsin and α-chymotrypsin helps reveal the potential
Trypsin anti-nutritional property of PPGs.
α-Chymotrypsin Crown Copyright © 2013 Published by Elsevier Ltd. All rights reserved.
Interaction

1. Introduction
Kudingcha (bitter tea), a well known Chinese transitional drinking tea,
has long been used in folk medicine as a diuretic and slimming agent, and
for the treatment of hypertension, sorethroat as well as inflammation.
Chinese Kudingcha is divided into 2 groups: small-leaved and large-
leaved Kudingcha. Large-leaved Kudingcha includes the Ilex kudingcha
and Ilex latifolia species. Ligustrum purpurascens, belonging to the
oleaceous plant group, is a kind of original material of small-leaved
Kudingcha. L. purpurascens is a small tree growing in southwestern
China, Vietnam, India, Cambodia and Burma (Qiu, Miao, & Chang, 1992).
The leaves of L. purpurascens have shown antioxidant activity and have
contributed to health (Filip & Ferraro, 2003; He et al., 2003; Heck &
Mejia, 2007).
L. purpurascens is rich in glycosides (Wu et al., 2011). The
phenylpropanoid glycosides (PPGs) are important ingredients in
L. purpurascens (He, Liu, & Yang, 1992; He et al., 2003) (Fig. 1) and
have anti-tumor, anti-inflammatory, antibacterial, and antiviral activities
(Lau, He, Dong, Fung, & But, 2002; Ohno, Inoue, Ogihara, & Saracoglu,
2002; Song et al., 2012; Zhu et al., 2009). Acteoside, osmanthuside,
ligupurpuroside A, ligupurpuroside B, and ligupurpuroside C are the
major constituents of PPGs, and they have show strong antioxidant
activities (He et al., 1992).
Most of the polyphenolic compounds from medicinal herbs can
strongly interact with globular proteins (Charlton et al., 2002). A number
of studies showed that some polyphenolic compounds can inhibit
digestive enzymes, which may be due to their interaction (Koo & Noh,
2007; Siebert, Troukhanova, & Lynn, 1996). So, the polyphenolic com-
pounds from Kudingcha might be involved in inhibiting the activity of
digestive enzymes. With the increasing consumption of Kudingcha, an
anti-nutritional problem linked to the Kudingcha has attracted concern.
In a previous study, we isolated PPGs from the leaves of
L. purpurascens (Song et al., 2012). In this study, we investigated the
effect of PPGs in Kudingcha from L. purpurascens on inhibiting the
activity of the typical digestive enzymes pepsin, trypsin and α-
chymotrypsin in vitro. We used a multi-spectroscopic method and
docking studies to help explore the anti-nutritional property of PPGs.
2. Materials and methods
2.1. Materials

Porcine pepsin, bovine trypsin, bovine α-chymotrypsin and casein

⁎ Corresponding authors at: School of Medicine, Shenzhen University Nanhai Ave 3688,
were from Sigma Chemicals (St. Louis, MO), and phenylpropanoid
Shenzhen, Guangdong 518060, PR China. Tel.: +86 755 86671909; fax: +86 755 86671906. glycosides were prepared in our laboratory. All aqueous solutions were
E-mail addresses: wxl@szu.edu.cn (X. Wu), hezhendan@gmail.com (Z. He). prepared with newly double-distilled water.

0963-9969/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2013.10.020
 X. Wu et al. / Food Research International 54 (2013) 1376–1382 1377

A 90%
80% Pepsin
Trypsin
70% α - chymotrypsin

60%

Inhibition%
50%
40%
30%
20%
10%
0%
0 :1 0. 1 :1 0. 2 :1 0. 4 :1 0. 6 :1 0. 8 :1 1 :1 1. 2 :1
Ratio of PPGs Enzyme

B 6

5 Control
Fig. 1. General structure of phenylpropanoid glycosides (PPGs). 1mg/mL
4
3mg/mL
2.2. Enzyme activity assay 3

1/[V]
2
Enzyme activity was determined with casein used as a substrate as
described (Wang, 2005). In brief, the working solution of pepsin was 1
prepared with 0.02 M lactic acid buffer (pH 3.0), and the working
solutions of trypsin and α-chymotrypsin were prepared with 0.05 M 0
-0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25
phosphate buffered saline (pH 7.5). Enzyme activity assay involved a -1
reaction medium (3 mL) containing 5 mg enzyme and 10 mg casein in
the presence and absence of PPGs. PPG solution was added to the -2
reaction mixtures just before the reaction (PPGs:enzyme at a mass 1/[S]
ratio of 0:1, 0.1:1, 0.2:1, 0.4:1, 0.6:1, 0.8:1 and 1:1). An amount of
5 mL 0.4 M sodium carbonate solution and 1 mL Folin reagent were
C 5
added to the reaction mixture and kept at 40 °C for 20 min. The 4 Control
hydrolysis of casein to tyrosine was monitored at 680 nm. Control
1mg/mL
enzyme activity was determined without PPGs. One enzyme unit is 3
made up of 1 g enzyme that releases 1 μg tyrosine per minute. All 2
3mg/mL

samples were analyzed in triplicate. The inhibition (%) of enzyme

1/[V]

activity was calculated as follows: 1

activity control−activity test 0

Inhibition % ¼ =  100: -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25
activity control -1

-2
2.3. Michael's constant and inhibitory patterns of PPGs on digestive enzyme -3
activity 1/[S]
To determine the inhibitory pattern of PPGs, enzyme activities were D 16
analyzed at different casein concentrations (5–20 mg/mL) and in the
14 Control
presence of 0, 1 and 3mg/mL PPGs. Lineweaver–Burk plots were created
by plotting the reciprocal of the enzyme reaction velocity (1/V) against 12 1mg/mL
the reciprocal of the substrate concentration (1/[S]). The maximum 10 3mg/mL
reaction velocity (Vmax) and the Michael's constant (Km) were 8
1/[V]

acquired from the reciprocal of the intercept with the 1/V axis and
6
from the negative reciprocal of the intercept with the 1/[S] axis,
respectively. All samples were analyzed in triplicate. 4
2
2.4. Fluorescence spectroscopy 0
-0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25
-2
The interaction between PPGs and enzymes was quantified by
fluorimetric titration as follows: 1.0 × 10−5 M pepsin solution was -4
prepared with 0.02 M HCl (pH 2.0); 1.0 × 10−5 M trypsin and α- 1/[S]
chymotrypsin solutions were prepared with 0.05 M KH2PO4 (pH 7.5). A Fig. 2. Inhibition of enzymes pepsin, trypsin and α-chymotrypsin by PPGs (A); Lineweaver–
1.0 × 10−3 M PPG solution was prepared with 0.02 M HCl (pH 2.0) and Burk plot of the reaction of pepsin (C), trypsin (B) and α-chymotrypsin (D) in the presence of
transferred to a 10 mL flask, treated with pepsin (1.0 mL), and PPGs.
1378 X. Wu et al. / Food Research International 54 (2013) 1376–1382

supplemented with 0.02 M HCl (pH 2.0) to a final volume of 10 mL. A
1.0 × 10−3 M PPG solution was prepared with 0.05 M KH2PO4 (pH 7.5)
and transferred to a 10 mL flask, treated with trypsin and α-
chymotrypsin (1.0 mL) and supplemented with 0.05 M KH2PO4
(pH 7.5) to a final volume of 10 mL. All samples were incubated at
fixed temperatures (17 °C, 27 °C and 37 °C) for 1 h.
The fluorescence spectra were recorded on a Hitachi-850 spectro-
fluorometer (Hitachi Co., Japan) in a 1-cm quartz cell at excitation
280 nm. In a temperature-controlled environment (17 °C, 27 °C and
37 °C), the excitation and emission bandwidth value was 5 nm, and the
emission spectra were from 300 to 450 nm.
Fluorescence quenching was analyzed by use of the modified Stern–
Volmer equation (Lakowicz, 1999): lg(F0 − F) ∕ F = lgKa + nlg[Q], where
n is the number of binding sites per enzyme, Ka is the Stern–Volmer
quenching constant, and F0 and F are the relative fluorescence intensities
in the absence and presence of the quencher, respectively. From the
intercept and slope with the plots of lg(F0 − F) ∕ F against lg[Q], we
calculated the Ka and n values (Wu, Wu, et al., 2011; Wu et al., 2013).

A 3500 1.0

l g[ (F0- F) /F]
0.5
3000
a 0.0
Flourescence intensity

2500 y = 1. 2381x + 5. 7547

R2 = 0 . 9 9 6 2
-0.5
k
2000 -1.0
-5. 0 -4. 8 -4. 6 -4. 4 -4. 2 -4. 0
1500 l g[ Q]

1000

500

0
300 320 340 360 380 400 420 440
Wavelength(nm)

B 4500
1.0
4000
l g[ (F0- F) /F]

0.5
Flourescence intensity

3500
0.0
3000 a
y = 1. 3743x + 6. 1197
-0.5 R2 = 0 . 9 9 6 1
2500
k -1.0
2000 -5. 0 -4. 8 -4. 6 -4. 4 -4. 2 -4. 0
1500 l g[ Q]

1000
500
0
300 320 340 360 380 400 420 440
Wavelength(nm)

C 6000
1.5
l g[ (F0- F) /F]

1.0
5000
0.5
Flourescence intensity

0.0
4000 a
y = 1. 5647x + 7. 0858
-0.5 R2 = 0 . 9 9 3

3000 -1.0
k -5. 0 -4. 8 -4. 6 -4. 4 -4. 2 -4. 0
l g[ Q]
2000

1000

0
300 320 340 360 380 400 420 440
Wavelength(nm)
Fig. 3. The quenching effect of PPGs on enzyme fluorescence intensity at 37 °C, λex = 280 nm. (A) Pepsin: (a-k) pepsin was 1.0 × 10−5 M, with PPG concentration increased from 0.0 to
10.0 × 10−4 M. (B) Trypsin: (a-k) trypsin was 1.0 × 10−5 M, with PPG concentration increased from 0.0 to 10.0 × 10−4 M. (C) α-Chymotrypsin: (a-k) α-chymotrypsin was 1.0 × 10−5 M,
with PPG concentration increased from 0.0 to 10.0 × 10−4 M.
 X. Wu et al. / Food Research International 54 (2013) 1376–1382 1379

Table 1
Thermodynamic variables for phenylpropanoid glycosides (PPGs) binding to pepsin, trypsin and α-chymotrypsin.
Δ Δ Δ
Temperature Ka n G H S
(L mol−1) (kJ mol−1) (kJ mol−1) (J mol−1 K−1)

Pepsin 17 °C 6.16 × 104 1.17 −26.59 121.30 485.30

27 °C 1.18 × 105 1.20 −29.14
37 °C 5.68 × 105 1.24 −34.15
Trypsin 17 °C 1.03 × 105 1.08 −28.79 281.34 795.15
27 °C 3.87 × 105 1.13 −32.09
37 °C 1.32 × 106 1.37 −36.32
α-Chymotrypsin 17 °C 6.78 × 105 1.31 −33.49 182.05 699.60
27 °C 1.16 × 106 1.38 −34.82
37 °C 1.21 × 107 1.56 −42.05

Considering that temperature does not vary significantly, the
enthalpy change (ΔH) can be considered a constant. From the binding
constants at different temperatures, the ΔH and the free energy change
(ΔG) can be calculated from the van't Hoff equation: lnK2 ∕ K1 =
(1 ∕ T1 − 1 ∕ T2)ΔH ∕ R, where T is the experiment temperature, K is
the binding constant at the corresponding T, and R is the gas constant.
Then the free energy of interaction can be calculated from the following
equation: ΔG = −RTlnK = ΔH − TΔS.
2.5. Circular dichroism (CD) measurements
A 1.0 × 10−7 M pepsin solution was prepared with 0.02 M HCL
(pH 2.0); 1.0 × 10−7 M trypsin and α-chymotrypsin solutions were
prepared with 0.05 M KH2PO4 (pH 7.5). The enzyme spectra were
recorded at PPG concentrations of 0.0, 0.5, 1.0, and 2.0 × 10−7 M at
37 °C with a Jasco-810 spectrophotometer (JASCO, Tokyo) in cells of
1.0-mm path length. The spectra were measured from 190 to 250 nm,
3 times for each spectrum. CD spectroscopy data were uploaded to
DICHROWEB (online server for protein secondary structure analyses)
to determine the secondary structure.
2.6. Docking studies
Acteoside is the major active component and predominant PPG of
L. purpurascens (He et al., 2011). So, we selected acteoside as a
representation of PPGs for docking studies with pepsin, trypsin and α-
chymotrypsin by using the MOE software. The porcine pepsin, bovine
trypsin and bovine α-chymotrypsin structures were downloaded from
the Brookhaven Protein Data Bank [accession nos. 5PEP (porcine pepsin),
2PTN (bovine trypsin) and 2CHA (bovine α-chymotrypsin)]. The 3-D
structure of acteoside was created from PM3 semi-empirical calculations
by use of the Chem3D Ultra 6.0 software. All enzymes were considered
rigid and acteoside was considered flexible during docking. The docked
complexes were optimized according to the fit within the receptor
pocket for discrete and continuum electrostatics, Van der Waals
interaction, hydrogen bonding, hydrophobicity and entropy. The ranking
of the generated solutions was executed by the scoring function of the
MOE software. UCSF Chimera software was used for visualization.
and Km values remained stable. Thus, the inhibition of pepsin, trypsin
and α-chymotrypsin by PPGs was non-competitive.
3.2. Fluorescence spectra
Tryptophan (Trp) fluorescence may change depending on the
impact of the interaction between a protein and another molecule
(Tayeh, Rungassamy, & Albani, 2009). Porcine pepsin has 2 Trp residues,
bovine trypsin has 4 Trp residues, and bovine α-chymotrypsin has 8 Trp
residues. Therefore, the interactions between PPGs with pepsin, trypsin
and α-chymotrypsin can be studied by fluorescence quenching of the
intrinsic Trp fluorescence in these enzymes.
The fluorescence intensity of pepsin, trypsin and α-chymotrypsin
gradually decreased with the addition of PPGs, so PPGs interacted
with these enzymes (Fig. 3, no data shown at 17 °C and 27 °C). Fig. 3
shows red shifts of the wavelength maxima of pepsin from 342 to
356 nm, trypsin from 342 to 354 nm, and α-chymotrypsin from 334 to
348 nm with the addition of PPGs, so the binding of PPGs to these
enzymes increased the polar environment around the Trp residues of
the proteins. The finding was related to more exposure of Trp residues
and an unfolded protein structure (Kanakis et al., 2011; Tayeh et al.,
2009; Wu et al., 2012).
Scatchard plots can be used to calculate the binding constant Ka and
the number of binding sites per protein n. The binding variables are
shown in Table 1. The binding constant Ka values for the interaction
between PPGs and pepsin were calculated as 6.16 × 104, 1.18 × 105,
and 5.68 × 105 M at 17, 27 and 37 °C, respectively, with an average n
value of 1.20. The Ka values for the interaction between PPGs and
trypsin were 1.03 × 105, 3.87 × 105 and 1.32 × 106 M at 17, 27 and
37 °C, respectively, with an average n value of 1.19. The Ka values for
the interaction between PPGs and α-chymotrypsin were 6.78 × 105,
1.16 × 106 and 1.21 × 107 M at 17, 27 and 37 °C, respectively, with an
average n value of 1.42. As compared with other tea polyphenol–protein
interactions with binding constant Ka from 1.0 × 104 to 1.0 × 105 M
(Kanakis et al., 2011; Wu et al., 2013; Zerrin, Elif, & Yasar, 2010), the
Ka values between PPGs and our enzymes represent comparatively
strong ligand–protein interactions.

3. Results and discussion

3.1. Inhibition of enzyme activity by PPGs Table 2

Secondary structure analysis of the native enzymes and their interaction with PPGs by
The PPG activity inhibition values were 51.0%, 76.8% and 67.8% for circular dichroism.
PPGs:pepsin, PPGs:trypsin and PPGs:α-chymotrypsin at a mass ratio Secondary structural elements α-Helix (%) β-Sheet (%) Random coil (%)
of 1.2:1 (Fig. 2A). IC50 values for PPGs inhibiting pepsin, trypsin and
Native pepsin 22.4 30.1 47.5
α-chymotrypsin activity were 0.68, 0.38 and 0.42 mg/mL, respectively. Pepsin–PPGs (1:2) 20.8 26.9 54.3
To characterize the inhibitory pattern of digestive enzymes by PPGs, Native trypsin 13.9 28.1 58.0
we varied the PPG and substrate concentrations to investigate the Trypsin–PPGs (1:2) 11.4 26.5 62.1
reaction kinetics. Lineweaver–Burk plots in Fig. 2B–D show that with Native α-chymotrypsin 58.0 13.9 28.1
α-Chymotrypsin–PPGs (1:2) 57.2 11.0 31.8
increasing PPG concentration, the Vmax values for enzymes decreased
1380 X. Wu et al. / Food Research International 54 (2013) 1376–1382

3.3. Binding mode and thermodynamic variables
Electrostatic interactions, van der Waals interactions, hydrogen
bonds and hydrophobic forces are the major interactions between
micromolecules and biomolecules. The model of interaction can be
defined according to the ΔH and ΔS data. We found positive ΔH and ΔS
values, so the hydrophobic interaction may play a major role in the
interactions between PPGs and the digestive enzymes we examined.
The negative ΔG values suggested that the binding of PPGs with the
digestive enzymes was spontaneous.

Fig. 4. Molecular docking of lipases with acteoside. Best-docked conformations of lipase–acteoside complexes; acteoside is shown in white. 2-D illustrations of the amino acid residues
interacting with acteoside were created by use of LigX in MOE; acteoside is represented as a chemical formula, and the residues are in a circular shape. Complexes of A, pepsin; B, trypsin;
and C, α-chymotrypsin.
 X. Wu et al. / Food Research International 54 (2013) 1376–1382
3.4. CD spectra
The pepsin–PPG, trypsin–PPG and α-chymotrypsin–PPG complexes
were characterized by CD. With PPG treatment, changes in spectra were
observed in pepsin, trypsin and α-chymotrypsin. We determined
quantitatively the secondary structures by using the SELCON3 method
in DICHROWEB (Table 2). After PPG binding, α-helical and β-sheet
contents decreased and the unordered structure content increased.
For example, the proportions for free pepsin were α-helical 22.4%, β-
sheet 30.1% and unordered structure 47.5%. In pepsin–PPG complexes,
the proportions for α-helical (free protein) decreased to 20.8% and β-
sheet (free protein) decreased to 26.9% with increased proportion of
unordered structure (free protein) to 54.3%. The decrease in α-helical
and β-sheet contents and increase in the unordered structure content
suggest that the binding effect of PPGs on these digestive enzymes
caused the unfolded protein structure, which is consistent with our
fluorescence spectroscopy analysis.
3.5. Docking studies
To substantiate the results from spectroscopy experiments, we
performed docking studies to determine the preferred binding site of
acteoside on pepsin, trypsin and α-chymotrypsin. The stereoviews of
acteoside–enzyme complexes are in Fig. 4. In the acteoside–pepsin
complex, acteoside is found near Asn37, Ser35, Gly34, Asp32, Tyr75,
Ile120, Ile30, Phe117, Phe111, Gly76, Glu13, Thr77, Thr12, Ser219,
Gly217, Thr218, Asp215, Tyr 189 and Ile128, and the hydrogen bondings
are between acteoside and Asp32, Glu13, Thr77 and Thr218 of pepsin. In
the acteoside–trypsin complex, acteoside is surrounded by Trp215,
Thr98, Gly236, Ser195, Asn97, Gln175, Asn97, Gln175, Asp189,
Gln192, Ser 217, Leu99, Cys 220, and His57, and the hydrogen bondings
are between acteoside and Ser190 and Ser214 of trypsin. In the
acteoside–α-chymotrypsin complex, acteoside is near Gly193, Ser214,
Asp194, Ser195, His57, Cys58, Phe41, His40, Phe39, His40, Phe39,
Gly142, Cys191 and Gly216, and the hydrogen bondings are between
acteoside and Gly193 and Ser195 of α-chymotrypsin. The hydrogen
bondings played an important role in increasing the binding affinity of
acteoside to the enzymes. The catalytic sites of porcine pepsin are
Asp32 and Asp215 (Sielecki, Fedorov, Boodhoo, Andreeva, & James,
1990). The catalytic sites of bovine trypsin and α-chymotrypsin are
residues Ser195, His57 and Asp102 (Pratt, Voet, & Voet, 2008). From
our docking results, acteoside might bind Asp32 and Asp 215 of pepsin
and Ser195 and His57 of trypsin and α-chymotrypsin, which might
affect the combination of these enzymes and the substrate.
Acteoside comprises 25% of total PPGs and demonstrates the highest
antioxidation property and activity in L. purpurascens (He et al., 2011;
Song et al., 2012). It is the most representative of the PPGs in
L. purpurascens. Thus, docking studies can help better understand the
interactions between PPGs and digestive enzymes.
Polyphenols from plants may interact with proteins. Some poly-
phenols have a strong ability to interact with digestive enzymes on the
basis of interactions between polyphenols and the protein, which reduces
food digestibility (Rohn, Rawel, & Kroll, 2002; Sung & Sang, 2007;
Thielecke & Boschmann, 2009). To our knowledge, this study is the first
to describe PPGs inactivating pepsin, trypsin and α-chymotrypsin. As
well, we described the interactions between PPGs and these digestive
enzymes.
Most studies suggest that the interaction between polyphenols and
protein is non-covalent (Murray, Williamson, Lilley, & Haslam, 1994;
Siebert et al., 1996; Wu, Wu et al., 2011; Wu et al., 2013). The non-
covalent interactions between proteins and phenolic compounds are
hydrophobic in nature and may stabilize with hydrogen binding
(Murray et al., 1994). The PPG structure contains many hydroxyl and
galloyl groups. From the results of fluorescence spectra and docking
studies, the PPG–enzyme binding phenomenon may be achieved by
hydrophobic interactions of the interior hydrophobic groups of
1381
enzymes with PPGs and then by the formation of hydrogen bonds
between polar groups (OH, SH and NH groups) of the enzyme with
the OH groups of PPGs.
The enzyme activity depends on its specific conformation; that is, the
enzyme's specific conformation change leads to its activity change. The
specific conformation of the enzyme relies on the covalent and non-
covalent interactions of enzyme amino acid residues. The internal non-
covalent interactions of the amino acid residues in the enzyme may be
changed if a certain compound is added. Non-covalent interactions
between small molecules and enzymes may alter the conformation of
the enzymes (Han et al., 2008; Zhu, Wang, Sun, Liu, & Wang, 2010). In
this work, we determined the effect of PPGs on pepsin, trypsin and α-
chymotrypsin activities and the change in protein conformations. PPGs
from L. purpurascens considerably decreased the catalytic activity of
pepsin, trypsin and α-chymotrypsin. Thus, PPGs may have a binding
effect on these digestive enzymes; changing the polarity and structure
of enzymes results in decreasing the enzyme activity. Furthermore, the
reduction in enzyme activity might be due to the increasing in-
accessibility of the substrate to the enzyme by the attachment of PPGs.
Therefore, the decrease in digestive enzyme activities was caused not
only by unfolded protein but also might be due to physical binding of
PPGs. The pattern of PPGs inhibiting these digestive enzymes is non-
competitive, which supports our hypothesis.
Most of the consumed polyphenolic compounds of food intake
remain in the gastrointestinal tract (Lapidot, Harel, Granit, & Kanner,
1998). So PPGs in the gastrointestinal tract may have complexing
abilities with digestive enzymes, which leads to reduced digestive
enzyme activity in the body. Small-leaved Kudingcha bitter tea is
considered a safe natural beverage rich in PPGs. However, its PPGs
might inhibit digestive enzyme activity. Our findings are similar to
those for other plant polyphenols. Some polyphenols, such as catechin,
gallotannin and tannic acid, play an important role in enzyme activity
inhibition (Shi, He, & Haslam, 1994; Siebert et al., 1996).
4. Conclusion
In conclusion, PPGs from small-leaved Kudingcha bitter tea might
have inhibitory effects on digestive enzymes, which may be due to the
interaction between PPGs and the enzymes. Because of the great health
potential of PPGs in small-leaved Kudingcha, knowledge of the effects of
PPGs on the digestive enzymes will help achieve the maximum use of
PPGs and minimize the anti-nutritional effects. However, the effects of
PPG in animal experiments need further examination.
Acknowledgments
This study was supported in part by the Natural Science Funding
of Shenzhen University (no. 801-00035911), the Natural Science
Funding of Guangdong Province (no. S2012010008514 and no.
S2013010016791) and the Shenzhen Funding for Technology Develop-
ment Project (no. CXZZ20130320165017541, no. SW201110010, and
no. JCYJ20130326110246234).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.foodres.2013.10.020.
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