An assessment of the interaction for three Chrysanthemum indicum flavonoids and α‐amylase by surface plasmon resonance

Abstract This study evaluated the interaction of Chrysanthemum indicum (CI) flavonoids (luteolin, acacetin, and buddleoside) with α‐amylase. Surface plasmon resonance (SPR) assay showed their equilibrium dissociation constants (KD) are 1.9695 ± 0.12, 2.9240 ± 0.20, and 3.2966 ± 0.08 mM at pH 6.0, respectively. Furthermore, their binding affinities were influenced by KCl, MgCl2, and CaCl2. Enzymatic kinetic studies revealed that three flavonoids exhibited noncompetitive α‐amylase inhibitory activity. The inhibitory sequence is luteolin > acacetin > buddleoside, which was in accordance with the results of binding affinity from SPR. 1,1‐diphenyl‐2‐picryl hydrazyl radical assay demonstrated that antioxidant activities of three flavonoids were inhibited significantly with α‐amylase. Meanwhile, the study reveals that hydroxyl on C′‐4, C′‐5, and C‐7 of flavonoids play an important role on the interaction of three flavonoids with α‐amylase. Also, SPR could be used as sensor for rapid screening inhibitors of α‐amylase and provide useful information for the application of C. indicum flavonoids in food and pharmaceutical area.

Acarbose, commercial available α-amylase inhibitor, is typical therapeutic agent used to control postprandial glucose concentration. However, it has been reported to cause some gastrointestinal side effects, such as diarrhea, flatulence, and abdominal pain (Shah, Khalil, Ul-Haq, & Panichayupakaranant, 2017;Yang, He, & Lu, 2014). Compared with the synthetic drugs, the natural molecules from plant have become a more acceptable alternative for treating T2D. Flavonoids are a class of natural small molecules with broad biological activity (Shen, Xu, & Lu, 2012;Tomás-Barberán & Andrés-Lacueva, 2012). Recently, they have received much attention for their inhibitory activity against α-amylase and relatively low toxicity to animals (Cao & Chen, 2012;Lu et al., 2017). Further, a series of studies have demonstrated that the structure and concentration of flavonoids and structure of α-amylase may greatly influence the extent of the flavonoids/α-amylase interaction (Cao & Chen, 2012;Lo et al., 2008;Wang, Du, & Song, 2010). So, flavonoids have been considered as a good source for screening of α-amylase inhibitor.
Chrysanthemum indicum (CI) is a kind of herbaceous plant. Its flowers have been used for several centuries as a traditional Chinese medicine to treat various infectious diseases, immune-related disorders, and eye diseases (Cheng, Li, & Hu, 2005;Zhu, Yang, Yang, Yang, & Zhou, 2005). Flavonoids are important bioactive components in the flowers of CI, including buddleoside, acacetin, and luteolin (Wang et al., 2000). The content of these flavonoids compounds has been used as the quality standard of CI. To the best of our knowledge, the interaction between CI flavonoids and α-amylase has not been clearly demonstrated in detail.
Surface plasmon resonance (SPR) is considered one of the most powerful techniques for evaluating the affinity kinetics of molecular interaction, which allow accurate estimation of distinct association/dissociation rate constants and equilibrium parameters in different reaction models without labels (Tan et al., 2014;Tiwari et al., 2014). In this study, the binding kinetics of CI flavonoids (buddleoside, acacetin, and luteolin) and α-amylase were monitored in vitro, and the effects of the external factor on their binding affinities were also analyzed using SPR biosensor. On this basis, the inhibitions of three flavonoids on α-amylase activity were examined, and a reasonable inhibiting mode was proposed.
Furthermore, we studied whether the antioxidant activity of these active constituents can be affected during the interaction with α-amylase by 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical assay. The difference of the interaction between the three flavonoids and α-amylase was analyzed based on the molecular structures of three flavonoids (Figure 1). The obtained results may be able to provide useful information for the more effective application of CI in food and pharmaceutical area.

| Apparatus
A commercial BI-2000 SPR instrument (Biosensing Instrument Inc.) was used for all SPR experiments in this study. The bare Au sensor chip was obtained from Biosensing Instrument Inc. The preparation of Au sensor chip can be referred to our previous published paper (Liu et al., 2014). A flow delivery system incorporated in the BI-SPR platform pumped samples onto the SPR sensor chip at a flow rate of 10 μl/min. The 0.01 M PBS (pH = 6.0) buffer was used as the running buffer. The BI-SPR 2000 control software (version 2.2.0.) was used to perform instrument operation and data processing. The Varioskan Flash (Multiskan GO 1510, Thermo Fisher Scientific) was used for the α-amylase inhibitory activity and DPPH radical assays.

| SPR measurement of three flavonoids and α-amylase interactions
Binding assay of three flavonoids to α-amylase was carried out using the SPR sensor. The immobilization of α-amylase on the chip surface was performed using a standard amine coupling procedure as described previously (Liu, Luo, Li, She, & Gao, 2017). The acceptable immobilization level of the α-amylase (referred to as bound and final α-amylase responses) was about 300 mDeg. After the stable baseline was obtained, different concentrations of flavonoids (50-800 μM) were injected over the chip surface coated with α-amylase, respectively. The SPR angle was monitored until the baseline stabilization.
To enable reuse of the SPR chip, the chip surface could be regenerated using 2 mM NaOH after each measurement. Regeneration parameters were based on the strength of interaction between the analyte and α-amylase. The chip surface was rinsed by PBS between each step. All the experiments were repeated three times, and kinetic parameters (k a , k d ) were deduced by nonlinear fitting of the primary sensorgram data based on the 1:1 Langmuir-binding model using the BI-SPR 2000 control software (version 2.2.0.). The model has been widely used in protein-ligand binding analysis and can be calculated through the following formula (Gombau et al., 2019;Islam, Shen, Gurgel, Rojas, & Carbonell, 2014;Lee, Jeong, Jones, & Kim, 2011): where R is the SPR signal at time t, and C is the concentration of the analyte. R max is the maximum analyte binding capacity in SPR signal. k a is the association rate constant and k d is the dissociation rate constant. (1)

| Effect of pH and salt on the interaction between three flavonoids and α-amylase
The effect of pH on the interaction between three flavonoids and α-amylase was carried out within the pH range (3-9) based on the method described in the above experiment. As is known to all,

| Effect of three flavonoids on α-amylase activity
The changes of α-amylase activity after adding different concentrations of the three flavonoids were investigated according to previously reported method with a slight modification (Zengin, 2016).
In brief, 0.05 ml α-amylase (300 mM in PBS buffer, pH = 6.0) was incubated with 0.5 ml of each of the three flavonoids at various concentrations (20, 40 and 80 μM) for 10 min at 37°C, respectively.
Then, 2 ml of starch solution (0.1 M in PBS buffer, pH = 6.0) was added to the above mixture. After incubation for 10 min at 37°C, 0.5 ml of 0.01 M iodine-potassium iodide solution was added to start the reaction. Finally, PBS (pH = 6.0) was added to give a final volume of 8 ml. Thereafter, the assay was carried out by measuring the absorbance at 560 nm using the Microplate Spectrophotometer. All experiments were performed in triplicates, and the inhibitory percentage of α-amylase activity was calculated through the following formula (Shah et al., 2017): where A 0 is the absorbance without flavonoids, and A is the absorbance with flavonoids.
To further explore the inhibitory type of three flavonoids on α-amylase, kinetic analysis was carried out by using Lineweaver-Burk plots. Starch was used as substrate, and the inhibition kinetics Chemical structures of buddleoside, acacetin, luteolin, and acarbose 2.6 | Effect of α-amylase on antioxidant activity of three flavonoids The DPPH assay was performed to assess effect of α-amylase on antioxidant activity of three flavonoids as following procedure (Kim et al., 2014). 2 mM DPPH solution was prepared by dissolving 0.0787 g of DPPH in 100 ml of anhydrous ethanol and stored at −20°C. α-Amylase (300 μM) and three flavonoids (2.5-125 μM) were prepared with PBS (pH = 6.0) and ethanol, respectively.
Then, samples were incubated for 10 min at room temperature.
Absorbance at 517 nm was measured in the spectrophotometer.
DPPH radical scavenging activity was calculated as follows : in which A C is the absorbance of the DPPH (100 μl DPPH and 100 μl ethanol), A i is the absorbance of the DPPH and sample (100 μl DPPH, 50 μl flavonoids, and 50 μl ethanol, or 100 μl DPPH, 50 μl flavonoids, 10 μl α-amylase, and 40 μl ethano1), and A j is the absorbance of the blank sample (50 μl flavonoids and 150 μl ethanol). All experiments were carried out in triplicate, and the results were expressed as mean ± RSD.
IC 50 values were obtained based on plotting the percentage of DPPH radical scavenging activity against the flavonoids concentration.

| Statistical analysis
The data were expressed as means ± relative standard deviation (n = 3). Statistical analysis was compared using a one-way analysis of variance in SPSS 18.0 (SPSS, Chicago, IL, USA), with p < .05 being considered statistically significant.

| Interaction of three flavonoids and α-amylase
The SPR sensorgrams in Figure 2  SPR response at the end of dissociation and that at the beginning of association is denoted as Δθ, which can be used to compare the binding ability of different substances to α-amylase. The Figure 2d shows the Δθ of the interaction for each flavonoid (200 μM) and acarbose (200 μM) with α-amylase, respectively.
As shown in Figure 2 and Table 1, the binding affinity of flavonoids and acarbose for α-amylase is acarbose > luteolin > acacetin > buddleoside. This result suggests that the binding affinity was affected by the number and position of hydroxyl group (Figure 1).
The interaction of these analytes with α-amylase may be achieved by hydrophobic interactions in nature and then stabilized by hydrogen bonds Lu et al., 2017). This is usually enhanced with increasing the number and reactivity of hydroxyl group (Li, Yang, Gao, Zhang, & Wu, 2011;Wang et al., 2010). As shown in Figure Lo et al., 2008). Furthermore, the binding affinity of acacetin with α-amylase (K D : 2.924 ± 0.2 mM) is greater than that of the buddleoside with α-amylase (K D : 3.2966 ± 0.08 mM), indicating that hydroxyl group on position C-7 of A-ring is very important for the binding of the flavonoids with α-amylase. After the hydroxyl group is substituted by a glycoside, steric hindrance may take place, which weakens the binding interaction between buddleoside and α-amylase (Cao & Chen, 2012;Li et al., 2009). Based on the above results and analysis, it is clearly demonstrated that the SPR sensor may provide more information to evaluate the interaction of the flavonoids with α-amylase.

| Effect of pH and salt on the interaction between three flavonoids and α-amylase
The interaction of between three flavonoids and α-amylase was studied at different pH using SPR (Figure 3a). At the chosen pH range, the binding affinity of three flavonoids and α-amylase is strongest at pH 6. This may be ascribed to the α-amylase isoelectric point (5.04) effect . When the pH value deviates from the isoelectric point, the associated structure of α-amylase will change.

| Effect of three flavonoids on α-amylase activity
The experimental results in Figure 4 demonstrate that luteolin, acacetin, and buddleoside can dose dependently inhibit α-amylase activity. At concentration of 80 μM, three flavonoids markedly inhibited α-amylase activity ranging from 6.76% to 21.29%. The ability of inhibition is luteolin > acacetin > buddleoside, which was in accordance with the results of binding affinity from SPR experiments (Table 1). These results further demonstrate that the free hydroxyl groups in B-ring and A-ring (red in Figure 1) are important for the interaction of three flavonoids with α-amylase. There are several works showed that the hydroxylation on positions C-3′ and C-4′ of B-ring of flavonoids remarkably improved the inhibition for α-amylase (Lo et al., 2008;Wang et al., 2010). Other scholars demonstrated that flavonoids without one hydroxyl group on any of positions 5, 6, or 7 of A-ring showed no inhibition for digestive enzyme (Cao & Chen, 2012;Gao, Nishioka, Kawabata, & Kasai, 2004). Besides, hydroxyl group on position C-7 of A-ring is very important for the binding of  (Table 2). There are several works showed that the main inhibition mode determined for polyphenolic compounds-digestive enzymes is noncompetitive (Martinez-Gonzalez et al., 2017;Yang & Kong, 2016). The result of a noncompetitive inhibition of flavonoid-α-amylase was supported by the analysis of tea polyphenols-pancreatic α-amylase system (Yang & Kong, 2016).

| Effect of α-amylase on antioxidant activity of flavonoids
DPPH radical assay is a rapid, simple, and stable method for the determination of antioxidant capacity of flavonoids. The effect of α-amylase on antioxidant activity of flavonoids was assessed using DPPH radical assay.  Table 3), which was in accordance with the results of binding affinity and α-amylase inhibitory activity (Tables 1 and 2). This result illustrated the antioxidant activity of three flavonoids is closely related to its hydroxyl groups.

| CON CLUS ION
To conclude, a low-cost, simple, sensitive, and label-free method was successfully applied to investigate real-time interactions of the luteolin, acacetin, and buddleoside with α-amylase, and the influence of external factors (pH, KCl, MgCl 2 , and CaCl 2 ) on the interaction.
The affinity order is luteolin > acacetin > buddleoside. In addition, the binding of three flavonoids with α-amylase can not only inhibit α-amylase activity with noncompetitive mode, but also decrease the antioxidant activities of three flavonoids. Furthermore, the results reveal the significance of hydroxyl on C′-4, C′-5 of B-ring, and C-7 of A-ring of three flavonoids for binding with α-amylase. Besides, the glycosylation of hydroxyl group on flavonoids weakened the binding interaction between flavonoids and α-amylase. These results provide scientific support for the proper use of luteolin, acacetin, and buddleoside as potential inhibitors of the α-amylase.

ACK N OWLED G M ENT
This study was supported by the National Natural Science Foundation of China (No. 31671931 and 31601551) and "1515" talent cultivation plan of Hunan Agricultural University.

CO N FLI C T O F I NTE R E S T
The authors notify that there are no conflicts of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing. Values are mean ± RSD (n = 3). Mean values followed by different letters are significantly different (p < .05).