Analysis of Xanthine Oxidase Inhibitors from Clerodendranthus spicatus with Xanthine Oxidase Immobilized Silica Coated Fe 3 O 4 Nanoparticles

In this study, xanthine oxidase immobilized silica coated Fe3O4 nanoparticles (Fe3O4@SiO2-XO) were successfully prepared and characterized by transmission electron microscope, X-ray powder diffraction, Fourier transform infrared spectroscopy and vibrating sample magnetometer. The average diameter of the Fe3O4 nanoparticles was about 300 nm to 350 nm with a shell thickness of 60 nm. The maximum saturation magnetization of the Fe3O4@SiO2-XO nanoparticles was 44.9 emu/g, which ensured the separation from the medium within one minute by using an ordinary magnet. A xanthine oxidase (XO) inhibitor screening method using Fe3O4@SiO2-XO nanoparticles was established and utilized in the extract of Clerodendranthus spicatus. Under the optimized conditions, two compounds were screened out and identified as gardenin B and eupatorin. The half maximal inhibitory concentration (IC50) values of these two compounds were 1.488 μg/mL and 11.197 μg/mL, respectively. The interactions between these two compounds and XO were investigated by the fluorescence spectroscopic method. The results suggested that the quenching effects of gardenin B and eupatorin were due to a static quenching mechanism. Furthermore, gardenin B showed stronger binding capacity than that of eupatorin. In conclusion, this screening method exhibited efficiency and reusability in screening, identification and analysis of enzyme inhibitors from complex mixtures.


Introduction
Natural products were fruitful sources of bioactive compounds for drug discovery [1].However, unclear function mechanisms, complex composition, and tiring separation and purification procedures often hinder the research of natural products [2].Thus, the research on active compounds in natural products would benefit from more efficient and novel screening technologies.Up to now, many screening methods based on solid phase extraction, membrane filtration, and hyphenated instruments were developed [3][4][5].In recent decades, magnetic nanoparticles gained great interests in biological and medicinal fields.Many kinds of magnetic nanoparticles were applied as the catalysts, the carriers, the supports and so on [6,7].Unlike the other nanomaterials, well dispersed magnetic nanoparticles in solution could be immediately separated from medium by an external magnet [8].High magnetic response, high stability and high surface area made magnetic nanoparticles become ideal solid supports for enzyme immobilization [9].Enzyme modified magnetic nanoparticles could be used as agents in the screening of active and bioactive compounds from natural products.Because of the advantages in low sample consumption, easy operation and rapid screening, this kind of screening strategy was widely applied [10][11][12].
Xanthine oxidase (XO) is a critical cytosolic enzyme which is widely distributed in mammalian tissues.XO could catalyze the oxidation of hypoxanthine to xanthine and further catalyze the oxidation of xanthine to uric acid [13].High concentrations of uric acid would result in the hyperuricemia and gout [14].Accordingly, the inhibition of XO could reduce the production of uric acid and become a of therapeutic method for treating hyperuricemia and gout.Allopurinol is an XO inhibitor and is widely clinical applied in related treatments [15].However, many adverse reactions to allopurinol, like allergic reactions, nephropathy, and hepatitis, have been observed and have limited the usage of allopurinol [16].Hence, more potential XO inhibitors from natural products with fewer adverse effects and higher therapeutic activity are needed.
Clerodendranthus spicatus (Thunb.) is a plant species and a popular folk medicine in China.It was widely distributed in southern China, including in Fujian, Yunnan and Hainan provinces [17].As a traditional Chinese medicine, Clerodendranthus spicatus has been used in the treatment of urinary lithiasis, rheumatism, acute nephritis, chronic nephritis, and cystitis [18].So far, previous documented reports on the chemical composition of Clerodendranthus spicatus showed the existence of plenty of diterpenes, flavonoids, triterpenes and phenolic acids [19].Water extract of Clerodendranthus spicatus was often investigated because water soluble phenolic acids were considered to be responsible for those activities [20,21].However, more systematical research on Clerodendranthus spicatus was still necessary since many valuable active compounds in Clerodendranthus spicatus were not clear.
In this study, xanthine oxidase immobilized silica coated Fe 3 O 4 nanoparticles (Fe 3 O 4 @SiO 2 -XO) were successfully prepared and characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD), transmission electron microscope (TEM), and vibrating sample magnetometer (VSM).Previous research in our lab demonstrated that extract of Clerodendranthus spicatus typically inhibited the activity of XO.Consequently, a XO inhibitor screening method using Fe 3 O 4 @SiO 2 -XO nanoparticles was established and utilized in the extract of Clerodendranthus spicatus based on our previous research.According to our proposed screening method, two compounds were screened out and identified as gardenin B and eupatorin.The inhibitions on XO of gardenin B and eupatorin were further verified and the interactions between these two compounds and XO were investigated by fluorescence spectroscopic methods.

Characterization of Fe 3 O 4 @SiO 2 -XO Nanoparticles
The morphologies of Fe 3 O 4 , silica coated Fe 3 O 4 nanoparticles (Fe 3 O 4 @SiO 2 ) and Fe 3 O 4 @SiO 2 -XO nanoparticles were characterized by TEM to visually observe the morphologies and the changes of nanoparticles during the preparation.TEM images of these nanoparticles are shown in Figure 1. Figure 1a showes the morphology of Fe 3 O 4 nanoparticles.It could be seen that the Fe 3 O 4 nanoparticles had a round shape and were well dispersed.The average diameter of Fe 3 O 4 nanoparticles was about 300 nm to 350 nm. Figure 1b shows the morphology of Fe 3 O 4 @SiO 2 nanoparticles.It could be seen that a light grey coating layer existed on the surface of the Fe 3 O 4 nanoparticles and that the shell thickness of the silica layer was about 60 nm.After the amino functionalization and enzyme immobilization, the change in shape of the final Fe 3 O 4 @SiO 2 -XO nanoparticles was small.The surface of the Fe 3 O 4 @SiO 2 -XO nanoparticles showed some roughness, which might have been caused by the amino groups and the enzyme molecules.The structures of synthesized Fe3O4 and Fe3O4@SiO2 nanoparticles were analyzed by XRD and the results are shown in Figure 2.For Fe3O4 nanoparticles, the characteristic peaks were marked by corresponding indices (111), ( 220), (311), (400), ( 422), ( 511), ( 440) and (533) in accordance with the standard Fe3O4 crystal XRD card.For Fe3O4@SiO2 nanoparticles, the characteristic peaks were similar to that of Fe3O4 nanoparticles except for the peak around 24°.The peak around 24° could be attributed to the silica [22].Based on these analyses, the coating of silica could be considered successful.To confirm the silica coating and amino groups on the surface of Fe3O4 nanoparticles, FT-IR analysis was also performed.As shown in Figure 3a, the FT-IR spectrum of Fe3O4 nanoparticles showed a characteristic Fe-O vibration band at 588 cm −1 [23].Figure 3b shows the FT-IR spectrum of Fe3O4@SiO2 nanoparticles.Compared with naked Fe3O4 nanoparticles, the feature peak in the spectrum of Fe3O4@SiO2 nanoparticles at 796 cm −1 and those around 1085 cm -1 belonged to the Si-O-Si stretching vibration, the Si-O-H stretching vibration and the Si-O vibration, respectively [24].After the amino functionalization, the FT-IR spectrum of amino Fe3O4@SiO2 nanoparticles showed all of the peaks mentioned above.Moreover, the enhanced wide peak at 3400 cm −1 could be attributed to the N-H stretching vibration [23].These results indicated that the silica coating and the following amino functionalization on the surface of Fe3O4 nanoparticles were successful.220), (311), (400), ( 422), ( 511), ( 440) and (533) in accordance with the standard Fe 3 O 4 crystal XRD card.For Fe 3 O 4 @SiO 2 nanoparticles, the characteristic peaks were similar to that of Fe 3 O 4 nanoparticles except for the peak around 24 • .The peak around 24 • could be attributed to the silica [22].Based on these analyses, the coating of silica could be considered successful.The structures of synthesized Fe3O4 and Fe3O4@SiO2 nanoparticles were analyzed by XRD and the results are shown in Figure 2.For Fe3O4 nanoparticles, the characteristic peaks were marked by corresponding indices (111), ( 220), (311), (400), ( 422), ( 511), ( 440) and (533) in accordance with the standard Fe3O4 crystal XRD card.For Fe3O4@SiO2 nanoparticles, the characteristic peaks were similar to that of Fe3O4 nanoparticles except for the peak around 24°.The peak around 24° could be attributed to the silica [22].Based on these analyses, the coating of silica could be considered successful.To confirm the silica coating and amino groups on the surface of Fe3O4 nanoparticles, FT-IR analysis was also performed.As shown in Figure 3a, the FT-IR spectrum of Fe3O4 nanoparticles showed a characteristic Fe-O vibration band at 588 cm −1 [23].Figure 3b shows the FT-IR spectrum of Fe3O4@SiO2 nanoparticles.Compared with naked Fe3O4 nanoparticles, the feature peak in the spectrum of Fe3O4@SiO2 nanoparticles at 796 cm −1 and those around 1085 cm -1 belonged to the Si-O-Si stretching vibration, the Si-O-H stretching vibration and the Si-O vibration, respectively [24].After the amino functionalization, the FT-IR spectrum of amino Fe3O4@SiO2 nanoparticles showed all of the peaks mentioned above.Moreover, the enhanced wide peak at 3400 cm −1 could be attributed to the N-H stretching vibration [23].These results indicated that the silica coating and the following amino functionalization on the surface of Fe3O4 nanoparticles were successful.To confirm the silica coating and amino groups on the surface of Fe 3 O 4 nanoparticles, FT-IR analysis was also performed.As shown in Figure 3a, the FT-IR spectrum of Fe 3 O 4 nanoparticles showed a characteristic Fe-O vibration band at 588 cm −1 [23].Figure 3b shows the FT-IR spectrum of Fe 3 O 4 @SiO 2 nanoparticles.Compared with naked Fe 3 O 4 nanoparticles, the feature peak in the spectrum of Fe 3 O 4 @SiO 2 nanoparticles at 796 cm −1 and those around 1085 cm -1 belonged to the Si-O-Si stretching vibration, the Si-O-H stretching vibration and the Si-O vibration, respectively [24].After the amino functionalization, the FT-IR spectrum of amino Fe 3 O 4 @SiO 2 nanoparticles showed all of the peaks mentioned above.Moreover, the enhanced wide peak at 3400 cm −1 could be attributed to the N-H stretching vibration [23].These results indicated that the silica coating and the following amino functionalization on the surface of Fe 3 O 4 nanoparticles were successful.In order to evaluate the magnetic properties of prepared nanoparticles, the Fe3O4, Fe3O4@SiO2 and Fe3O4@SiO2-XO nanoparticles were characterized by VSM.The magnetization curves of these nanoparticles are shown in Figure 4.It can be seen that the maximum saturation magnetization of Fe3O4 nanoparticles was 68.9 emu/g.After the coating of silica and the immobilization of XO, the maximum saturation magnetizations of Fe3O4@SiO2 and Fe3O4@SiO2-XO nanoparticles dropped to 48.8 emu/g and 44.9 emu/g, respectively [25].Although the maximum saturation magnetization of Fe3O4@SiO2-XO nanoparticles decreased, the materials could be separated from the reaction medium within one minute.Hence, the magnetic response property of the prepared Fe3O4@SiO2-XO nanoparticles was acceptable for the isolation and related applications.

Optimization of Screening Conditions
In order to ensure the efficiency of screening and to give sufficient reaction time, the screening time from 0 min to 180 min of Fe3O4@SiO2-XO nanoparticles with extract was investigated.When Fe3O4@SiO2-XO nanoparticles were incubated with extract for a certain time, the Fe3O4@SiO2-XO nanoparticles were retained by a magnet and washed with buffer solution.Finally, the nanoparticles were eluted by a methanol solution and the absorbance of the elution was detected by Ultraviolet-visible spectroscopy (UV-VIS) Spectrophotometer at 254 nm.Because the elution contained screened compounds and those compounds showed absorbance at 254 nm, higher In order to evaluate the magnetic properties of prepared nanoparticles, the Fe 3 O 4 , Fe 3 O 4 @SiO 2 and Fe 3 O 4 @SiO 2 -XO nanoparticles were characterized by VSM.The magnetization curves of these nanoparticles are shown in Figure 4.It can be seen that the maximum saturation magnetization of Fe 3 O 4 nanoparticles was 68.9 emu/g.After the coating of silica and the immobilization of XO, the maximum saturation magnetizations of Fe 3 O 4 @SiO 2 and Fe 3 O 4 @SiO 2 -XO nanoparticles dropped to 48.8 emu/g and 44.9 emu/g, respectively [25].Although the maximum saturation magnetization of Fe 3 O 4 @SiO 2 -XO nanoparticles decreased, the materials could be separated from the reaction medium within one minute.Hence, the magnetic response property of the prepared Fe 3 O 4 @SiO 2 -XO nanoparticles was acceptable for the isolation and related applications.In order to evaluate the magnetic properties of prepared nanoparticles, the Fe3O4, Fe3O4@SiO2 and Fe3O4@SiO2-XO nanoparticles were characterized by VSM.The magnetization curves of these nanoparticles are shown in Figure 4.It can be seen that the maximum saturation magnetization of Fe3O4 nanoparticles was 68.9 emu/g.After the coating of silica and the immobilization of XO, the maximum saturation magnetizations of Fe3O4@SiO2 and Fe3O4@SiO2-XO nanoparticles dropped to 48.8 emu/g and 44.9 emu/g, respectively [25].Although the maximum saturation magnetization of Fe3O4@SiO2-XO nanoparticles decreased, the materials could be separated from the reaction medium within one minute.Hence, the magnetic response property of the prepared Fe3O4@SiO2-XO nanoparticles was acceptable for the isolation and related applications.

Optimization of Screening Conditions
In order to ensure the efficiency of screening and to give sufficient reaction time, the screening time from 0 min to 180 min of Fe3O4@SiO2-XO nanoparticles with extract was investigated.When Fe3O4@SiO2-XO nanoparticles were incubated with extract for a certain time, the Fe3O4@SiO2-XO nanoparticles were retained by a magnet and washed with buffer solution.Finally, the nanoparticles were eluted by a methanol solution and the absorbance of the elution was detected by Ultraviolet-visible spectroscopy (UV-VIS) Spectrophotometer at 254 nm.Because the elution contained screened compounds and those compounds showed absorbance at 254 nm, higher

Optimization of Screening Conditions
In order to ensure the efficiency of screening and to give sufficient reaction time, the screening time from 0 min to 180 min of Fe 3 O 4 @SiO 2 -XO nanoparticles with extract was investigated.When Fe 3 O 4 @SiO 2 -XO nanoparticles were incubated with extract for a certain time, the Fe 3 O 4 @SiO 2 -XO nanoparticles were retained by a magnet and washed with buffer solution.Finally, the nanoparticles were eluted by a methanol solution and the absorbance of the elution was detected by Ultraviolet-visible spectroscopy (UV-VIS) Spectrophotometer at 254 nm.Because the elution contained screened compounds and those compounds showed absorbance at 254 nm, higher absorbance of the elution at 254 nm meant that a higher concentration of compounds were screened out and, thus that the screening was closer to complete.Therefore, the screening capacity was expressed as the absorbance at 254 nm in a relative form (%) with the maximal value as 100%.As shown in Figure 5a, after 90 min screening, the screening capacity reached maximum.Hence, 90 min was enough for the screening of Fe 3 O 4 @SiO 2 -XO nanoparticles with extract.
Reuse of magnetic nanoparticles could apparently reduce experimental cost and improve efficiency.The reusability of Fe 3 O 4 @SiO 2 -XO nanoparticles was investigated and shown in Figure 5b.After one round of screening, the Fe 3 O 4 @SiO 2 -XO nanoparticles were washed with buffer solution and then transferred into another extract solution to start a new round of screening.The elution of each round was monitored by UV-VIS Spectrophotometer at 254 nm and the screening capacity was expressed as the absorbance at 254 nm in a relative form (%) with the maximal value as 100%.As a result, the screening capacity of Fe 3 O 4 @SiO 2 -XO nanoparticles after five rounds screening retained 93% of its initial capacity.As a support for enzyme, Fe 3 O 4 @SiO 2 nanoparticles could effectively protect the enzyme activity and increase the practicality of this method.
absorbance of the elution at 254 nm meant that a higher concentration of compounds were screened out and, thus that the screening was closer to complete.Therefore, the screening capacity was expressed as the absorbance at 254 nm in a relative form (%) with the maximal value as 100%.As shown in Figure 5a, after 90 min screening, the screening capacity reached maximum.Hence, 90 min was enough for the screening of Fe3O4@SiO2-XO nanoparticles with extract.
Reuse of magnetic nanoparticles could apparently reduce experimental cost and improve efficiency.The reusability of Fe3O4@SiO2-XO nanoparticles was investigated and shown in Figure 5b.After one round of screening, the Fe3O4@SiO2-XO nanoparticles were washed with buffer solution and then transferred into another extract solution to start a new round of screening.The elution of each round was monitored by UV-VIS Spectrophotometer at 254 nm and the screening capacity was expressed as the absorbance at 254 nm in a relative form (%) with the maximal value as 100%.As a result, the screening capacity of Fe3O4@SiO2-XO nanoparticles after five rounds screening retained 93% of its initial capacity.As a support for enzyme, Fe3O4@SiO2 nanoparticles could effectively protect the enzyme activity and increase the practicality of this method.

Screening of XO Inhibitors from Clerodendranthus spicatus
Our previous studies indicated that the extract of Clerodendranthus spicatus showed inhibition activities on XO.In this experiment, the inhibitory activity of the extract of Clerodendranthus spicatus was also tested to ensure the smooth implementation of this work.The results showed that the extract of Clerodendranthus spicatus exhibited inhibition on XO with the IC50 value at 147.0 μg/mL, which indicated there were plenty of potential XO inhibitors in the extract.Therefore, the analysis of XO inhibitors from extract of Clerodendranthus spicatus was meaningful.
The chromatograms of Clerodendranthus spicatus in aqueous and eluent solutions after screening using Fe3O4@SiO2-XO nanoparticles are shown in Figure 6.In order to obtain the optimum chromatograms for analysis, various gradient elution modes, several flow rates and column temperatures were evaluated.Finally, acceptable separation was achieved within of 60 min analysis.The chromatogram of Clerodendranthus spicatus extract can be found in Figure 6a.It can be seen that most of the peaks in the sample reached baseline separation and that the peak shape presented well.
When Fe3O4@SiO2-XO nanoparticles were incubated with extract of Clerodendranthus spicatus, the potential XO inhibitors would bind to Fe3O4@SiO2-XO nanoparticles based on affinity interaction.Meanwhile, the other components in the extract would not have any specific or chemical interaction with the Fe3O4@SiO2-XO nanoparticles.Finally, those bound potential XO inhibitors would be eluted by an organic solution and analyzed for further experiments.It can be seen in Figure 6b that two peaks appeared in the chromatogram of the eluent after screening with active Fe3O4@SiO2-XO nanoparticles; they are marked with numbers in Figure 6.Deactivated Fe3O4@SiO2-XO nanoparticles, after high temperature process, were also used in screening to exclude nonspecific binding between active compounds and Fe3O4@SiO2-XO nanoparticles, and thus confirm the accuracy of the screening results.Figure 6c shows the

Screening of XO Inhibitors from Clerodendranthus spicatus
Our previous studies indicated that the extract of Clerodendranthus spicatus showed inhibition activities on XO.In this experiment, the inhibitory activity of the extract of Clerodendranthus spicatus was also tested to ensure the smooth implementation of this work.The results showed that the extract of Clerodendranthus spicatus exhibited inhibition on XO with the IC 50 value at 147.0 µg/mL, which indicated there were plenty of potential XO inhibitors in the extract.Therefore, the analysis of XO inhibitors from extract of Clerodendranthus spicatus was meaningful.
The chromatograms of Clerodendranthus spicatus in aqueous and eluent solutions after screening using Fe 3 O 4 @SiO 2 -XO nanoparticles are shown in Figure 6.In order to obtain the optimum chromatograms for analysis, various gradient elution modes, several flow rates and column temperatures were evaluated.Finally, acceptable separation was achieved within of 60 min analysis.The chromatogram of Clerodendranthus spicatus extract can be found in Figure 6a.It can be seen that most of the peaks in the sample reached baseline separation and that the peak shape presented well.
When Fe 3 O 4 @SiO 2 -XO nanoparticles were incubated with extract of Clerodendranthus spicatus, the potential XO inhibitors would bind to Fe 3 O 4 @SiO 2 -XO nanoparticles based on affinity interaction.Meanwhile, the other components in the extract would not have any specific or chemical interaction with the Fe 3 O 4 @SiO 2 -XO nanoparticles.Finally, those bound potential XO inhibitors would be eluted by an organic solution and analyzed for further experiments.It can be seen in Figure 6b that two peaks appeared in the chromatogram of the eluent after screening with active Fe 3 O 4 @SiO 2 -XO nanoparticles; they are marked with numbers in Figure 6.
Deactivated Fe 3 O 4 @SiO 2 -XO nanoparticles, after high temperature process, were also used in screening to exclude nonspecific binding between active compounds and Fe 3 O 4 @SiO 2 -XO nanoparticles, and thus confirm the accuracy of the screening results.Figure 6c shows the chromatogram of eluent after screening with deactivated Fe 3 O 4 @SiO 2 -XO nanoparticles.Obviously, no peak was observed in the chromatogram at the same retention time of peak 1 and 2. Therefore, it could be ensured that the two compounds marked with numbers, which needed further identification and analysis, were specifically bound to Fe 3 O 4 @SiO 2 -XO nanoparticles.
chromatogram of eluent after screening with deactivated Fe3O4@SiO2-XO nanoparticles.Obviously, no peak was observed in the chromatogram at the same retention time of peak 1 and 2. Therefore, it could be ensured that the two compounds marked with numbers, which needed further identification and analysis, were specifically bound to Fe3O4@SiO2-XO nanoparticles.

Identification of Screened Potential XO Inhibitors
In order to confirm the structures of the two screened potential XO inhibitors, High Performance Liquid Chromatography-Mass Spectrum (HPLC-MS) analysis was conducted and the related MS and UV data are shown in Table 1.

Identification of Screened Potential XO Inhibitors
In order to confirm the structures of the two screened potential XO inhibitors, High Performance Liquid Chromatography-Mass Spectrum (HPLC-MS) analysis was conducted and the related MS and UV data are shown in Table 1.Compound 1 exhibited two absorbance bands at 280 and 330 nm.In the negative ion mode of MS analysis, compound 1 showed the deprotonated molecular ion [M − H] − at 357 m/z and [2M − H] − at 715 m/z in the MS spectrum.According to the references, compound 1 was identified as gardenin B [26].Compound 2 showed UV absorbance peaks at 250 nm, 275 nm and 340 nm.Meanwhile, the deprotonated molecular ion [M − H] − at 343 m/z and [2M − H] − at 687 m/z could be confirmed in the MS spectrum of compound 2 with the negative ion mode.In the positive ion mode, the protonated molecular ion [M + H] + was found at 345 m/z, and [2M + Na]+ at 711 m/z.Therefore, the molecular weight of compound 2 could be considered as 344.Compound 2 was identified as eupatorin because the UV and MS data were in agreement with previous reports [27].The standard gardenin B and eupatorin with high purities were also analysed by HPLC and the chromagtograms are shown in Figure 7.It can be seen that the retention time of the two standards were in accordance with those in the extract.

Inhibition on XO of Screened Potential XO Inhibitors
Since the screened potential XO inhibitors were identified as gardenin B and eupatorin, the XO inhibitory activities of them were evaluated.As a result, gardenin B and eupatorin exhibited inhibition on XO with IC50 values of 1.488 μg/mL and 11.197 μg/mL, respectively.According to reported literatures, eupatorin showed inhibition on XO [28].The experiment results showed that both of these compounds could actually inhibit XO and they could prove the effectiveness of this screening method.

Fluorescent Quenching of Screened Compounds on XO
Since protein exhibits fluorescence spectrum light, fluorescent quenching could be used to investigate the binding between active compounds and protein.The fluorescence emission spectrum of XO and the spectra with the addition of gardenin B and eupatorin, respectively, are shown in Figure 8. XO showed a fluorescence emission peak at 337 nm with the excitation wavelength at 280 nm.Different levels of fluorescence quenchings could be observed when increasing concentrations of gardenin B and eupatorin were added.Therefore, the Stern-Volmer plots for XO fluorescence quenching by gardenin B and eupatorin were further investigated.The plots are shown in Figure 9 and the binding parameters are summarized in Table 2.The Stern-Volmer plots of gardenin B and eupatorin were linear and the fitting degrees were 0.9988 and 0.9957, respectively.The satisfactory results reflected that the calculations of Kq and Ksv were suitable.The linear Stern-Volmer plots indicated a single way of quenching, which meant that either

Inhibition on XO of Screened Potential XO Inhibitors
Since the screened potential XO inhibitors were identified as gardenin B and eupatorin, the XO inhibitory activities of them were evaluated.As a result, gardenin B and eupatorin exhibited inhibition on XO with IC 50 values of 1.488 µg/mL and 11.197 µg/mL, respectively.According to reported literatures, eupatorin showed inhibition on XO [28].The experiment results showed that both of these compounds could actually inhibit XO and they could prove the effectiveness of this screening method.

Fluorescent Quenching of Screened Compounds on XO
Since protein exhibits fluorescence spectrum light, fluorescent quenching could be used to investigate the binding between active compounds and protein.The fluorescence emission spectrum of XO and the spectra with the addition of gardenin B and eupatorin, respectively, are shown in Figure 8. XO showed a fluorescence emission peak at 337 nm with the excitation wavelength at 280 nm.Different levels of fluorescence quenchings could be observed when increasing concentrations of gardenin B and eupatorin were added.Therefore, the Stern-Volmer plots for XO fluorescence quenching by gardenin B and eupatorin were further investigated.

Inhibition on XO of Screened Potential XO Inhibitors
Since the screened potential XO inhibitors were identified as gardenin B and eupatorin, the XO inhibitory activities of them were evaluated.As a result, gardenin B and eupatorin exhibited inhibition on XO with IC50 values of 1.488 μg/mL and 11.197 μg/mL, respectively.According to reported literatures, eupatorin showed inhibition on XO [28].The experiment results showed that both of these compounds could actually inhibit XO and they could prove the effectiveness of this screening method.

Fluorescent Quenching of Screened Compounds on XO
Since protein exhibits fluorescence spectrum light, fluorescent quenching could be used to investigate the binding between active compounds and protein.The fluorescence emission spectrum of XO and the spectra with the addition of gardenin B and eupatorin, respectively, are shown in Figure 8. XO showed a fluorescence emission peak at 337 nm with the excitation wavelength at 280 nm.Different levels of fluorescence quenchings could be observed when increasing concentrations of gardenin B and eupatorin were added.Therefore, the Stern-Volmer plots for XO fluorescence quenching by gardenin B and eupatorin were further investigated.The plots are shown in Figure 9 and the binding parameters are summarized in Table 2.The Stern-Volmer plots of gardenin B and eupatorin were linear and the fitting degrees were 0.9988 and 0.9957, respectively.The satisfactory results reflected that the calculations of Kq and Ksv were suitable.The linear Stern-Volmer plots indicated a single way of quenching, which meant that either The plots are shown in Figure 9 and the binding parameters are summarized in Table 2.The Stern-Volmer plots of gardenin B and eupatorin were linear and the fitting degrees were 0.9988 and 0.9957, respectively.The satisfactory results reflected that the calculations of K q and K sv were suitable.The linear Stern-Volmer plots indicated a single way of quenching, which meant that either dynamic quenching or static quenching played a key role.The value of K q reflected the efficiency of quenching and the accessibility of fluorophores.Based on previous research, if K q is near 1.0 × 10 10 L/mol/s, the quenching can be considered as dynamic quenching.Meanwhile, if K q is greater than 2.0 × 10 10 L/mol/s, the quenching can be considered as static quenching [29].In this study, the K q values of gardenin B and eupatorin were 600-fold (1.2194 × 10 13 L/mol/s) and 500-fold (1.0097 × 10 13 L/mol/s) greater than 2.0 × 10 10 L/mol/s, respectively.This suggested that the quenching effects of gardenin B and eupatorin belong to static quenching mechanisms.L/mol/s, the quenching can be considered as dynamic quenching.Meanwhile, if Kq is greater than 2.0 × 10 10 L/mol/s, the quenching can be considered as static quenching [29].In this study, the Kq values of gardenin B and eupatorin were 600-fold (1.2194 × 10 13 L/mol/s) and 500-fold (1.0097 × 10 13 L/mol/s) greater than 2.0 × 10 10 L/mol/s, respectively.This suggested that the quenching effects of gardenin B and eupatorin belong to static quenching mechanisms.

The Binding Constants and the Number of Binding Sites
For static quenching, the number of binding sites per protein molecule (n) and binding constants (Ka) could be calculated according to Equation (3).The plots of log[(F0 − F)/F] versus log[Q] for gardenin B and eupatorin are shown in Figure 10 and the calculations of slope and intercept are shown in Table 2.The double-logarithm curves were obviously linear and the fitting degrees were 0.9950 and 0.9962, indicating that the calculations were acceptable for this study [30].The corresponding calculated Ka (n) values between gardenin B and eupatorin and XO were 2.636 × 10 6 (1.1124) and 4.612 × 10 3 (0.9534) at 300.15 K, respectively.Accordingly, gardenin B showed stronger binding capacity than eupatorin.

Table 2.
The affinity constants of gardenin B and eupatorin for XO.Abbreviations: K q , K av , R 2 , n, number of binding sites per protein molecule.

The Binding Constants and the Number of Binding Sites
For static quenching, the number of binding sites per protein molecule (n) and binding constants (K a ) could be calculated according to Equation (3).The plots of log[(F 0 − F)/F] versus log[Q] for gardenin B and eupatorin are shown in Figure 10 and the calculations of slope and intercept are shown in Table 2.The double-logarithm curves were obviously linear and the fitting degrees were 0.9950 and 0.9962, indicating that the calculations were acceptable for this study [30].The corresponding calculated K a (n) values between gardenin B and eupatorin and XO were 2.636 × 10 6 (1.1124) and 4.612 × 10 3 (0.9534) at 300.15 K, respectively.Accordingly, gardenin B showed stronger binding capacity than eupatorin.L/mol/s, the quenching can be considered as dynamic quenching.Meanwhile, if Kq is greater than 2.0 × 10 10 L/mol/s, the quenching can be considered as static quenching [29].In this study, the Kq values of gardenin B and eupatorin were 600-fold (1.2194 × 10 13 L/mol/s) and 500-fold (1.0097 × 10 13 L/mol/s) greater than 2.0 × 10 10 L/mol/s, respectively.This suggested that the quenching effects of gardenin B and eupatorin belong to static quenching mechanisms.

The Binding Constants and the Number of Binding Sites
For static quenching, the number of binding sites per protein molecule (n) and binding constants (Ka) could be calculated according to Equation (3).The plots of log[(F0 − F)/F] versus log[Q] for gardenin B and eupatorin are shown in Figure 10 and the calculations of slope and intercept are shown in Table 2.The double-logarithm curves were obviously linear and the fitting degrees were 0.9950 and 0.9962, indicating that the calculations were acceptable for this study [30].The corresponding calculated Ka (n) values between gardenin B and eupatorin and XO were 2.636 × 10 6 (1.1124) and 4.612 × 10 3 (0.9534) at 300.15 K, respectively.Accordingly, gardenin B showed stronger binding capacity than eupatorin.

Preparation of Ethanol Extract of Clerodendranthus spicatus
20.0 g of powdered Clerodendranthus spicatus were dissolved in 200 mL of 90% (v/v) ethanol solution and extracted three times (each for 2 min) in a microwave oven with the power of 60%.After the reaction, the solvent was cooled down at room temperature and removed using vacuum rotary evaporation to obtain 2.46 g of deep green residues.Finally, the residues were dissolved in 50 mL of water and filtered through a 0.45 µm membrane.The final extract was stored at 4 • C for further experiments.

Preparation of Fe 3 O 4 @SiO 2 -XO Nanoparticles
The Fe 3 O 4 nanoparticles were prepared according to our previous publications [31].1.35 g of FeCl 3 •6H 2 O, 3.60 g of sodium acetate and 1.00 g of polyethylene glycol 6000 were dissolved in 40 mL of ethylene glycol.The mixture was mechanical stirred under ultrasonic at room temperature for several minutes in order to acquire a homogeneous solution and the solution was poured in a Teflon-lined, stainless steel autoclave.The autoclave was heated at 180 • C for 6 h.The obtained black Fe 3 O 4 nanoparticles were separated using a magnet and washed with ethanol three times and stored in 40 mL of ethanol.
For the preparation of the silica shell, 200 mg of Fe 3 O 4 nanoparticles were dispersed in the mixture of 475 mL of ethanol, 140 mL of water and 15 mL of ammonia aqueous solution (28 wt.%).Under the mechanical stirring of 900 r/min, 6 mL of tetraethyl orthosilicate was added dropwise into the mixture.The silica coating reaction was performed at 25 • C for 8 h.The obtained Fe 3 O 4 @SiO 2 nanoparticles were retained from the mixture by an ordinary magnet and washed with ethanol three times.Then the Fe 3 O 4 @SiO 2 nanoparticles were dispersed in 200 mL of ethanol followed by the addition of 10 mL of 3-aminopropyltriethoxysilane, dropwise, at 25 • C to complete the amino modification of Fe 3 O 4 @SiO 2 nanoparticles.After mechanical stirring of 900 r/min for 6 h, the prepared amino Fe 3 O 4 @SiO 2 nanoparticles were washed with ethanol and water three times, and stored at 4 • C in water.
For the immobilization of XO on amino Fe 3 O 4 @SiO 2 nanoparticles, 2 mL of glutaraldehyde solution (5% w/v) were added into 20 mg of the previously obtained amino Fe 3 O 4 @SiO 2 nanoparticles.After being shaken in a thermostatic oscillator at 25 • C for 1 h, the activated Fe 3 O 4 @SiO 2 nanoparticles were retained and then washed with water three times.Then, they were transferred into a tube containing 1 mL of XO solution (1.0 mg/mL in phosphate buffered saline) and shaken in thermostatic oscillator at 25 • C for 1 h.At last, the prepared Fe 3 O 4 @SiO 2 -XO nanoparticles were collected by magnet, washed with a buffer solution three times and dispersed in a buffer at 4 • C for further use.

Characterization of Fe 3 O 4 @SiO 2 -XO Nanoparticles
TEM was utilized to confirm the surface morphology of materials, using a Tecnai-G20 transmission electron microscope (FEI, Hillsboro, OR, USA).The XRD could confirm the crystalline structure of Fe 3 O 4 and Fe 3 O 4 @SiO 2 nanoparticles using powder X-ray Diffractometer (Rigaku RINT 2500, Rigaku Corporation, Tokyo, Japan) with Cu/Kα radiation at 30 mA and 40 kV.FT-IR spectra were observed by a Nicolet avatar 360 FT-IR spectrophotometer (Thermo Fisher Nicolet, Orlando, FL, USA).Magnetic properties of the prepared materials were measured at room temperature on a vibration sample magnetometer VSM7407 (Lake Shore, Westerville, OH, USA).

XO Activity and Inhibition Test
The activity test of XO was similar to other publications, with some modifications [32].Firstly, 1 mL of water and 1 mL of xanthine (1.0 mg/mL) were mixed in the cuvette.Then, 20 µL of XO solution (1.0 mg/mL) was added into the cuvette and the absorbance at 295 nm was continuously monitored by an UV-VIS Spectrophotometer.The increase in absorbance at 295 nm in 40 s was recorded as the control.For the inhibition test, 1 mL of test samples with different concentrations were firstly mixed with 20 µL of XO solution (1 mg/mL) for one minute in the cuvette and then 1 mL of xanthine (1.0 mg/mL) was added.The increase in absorbance at 295 nm in 40 s was recorded by a UV-VIS Spectrophotometer.The inhibition of XO activity was calculated according to the following equation.
where ∆A and ∆A 0 are the absorbance increases of sample and control solution.The inhibition was expressed as the concentration of sample needed to inhibit 50% of the enzymatic activity (IC 50 ).All the assays were performed in triplicate.

Screening of XO Inhibitors from Clerodendranthus spicatus
The screening of XO inhibitors from Clerodendranthus spicatus was conducted using Fe 3 O 4 @SiO 2 -XO nanoparticles according to our previous reports, with some modifications [33].The first incubation step was conducted by mixing 50 mg of Fe 3 O 4 @SiO 2 -XO nanoparticles and 2.0 mL of extract of Clerodendranthus spicatus in the tube, and then shaking the tube in a thermostatic oscillator at 25 • C for 90 min.Then, the Fe 3 O 4 @SiO 2 -XO nanoparticles were magnetic separated from the mixture by using an ordinary magnet and washed with water three times in order to remove unspecific binding.Finally, 2.0 mL of methanol solution were added into Fe 3 O 4 @SiO 2 -XO nanoparticles and the mixture was shaking for 5 min.The Fe 3 O 4 @SiO 2 -XO nanoparticles were retained by magnet and the methanol solution was filtered through a 0.45 µm membrane and stored at 4 • C in refrigerator before the usage.In order to confirm that the binding was specific, a control experiment was done by using deactivated Fe 3 O 4 @SiO 2 -XO nanoparticles after high temperature processing as a substitution under the same screening procedures.

HPLC Analysis of Samples
HPLC separation was conducted for the qualitative analysis of the components in extract of Clerodendranthus spicatus.The HPLC analysis was completed on an Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, CA, USA).The Waters Xbridge™ C 18 reverse phase column (250 mm × 4.6 mm i.d., 5 µm) (Waters, Milford, MA, USA) was used as separation column.A gradient elution program was used with solvent A (water containing 0.1% (v/v) acetic acid) and solvent B (acetonitrile containing 0.1% (v/v) acetic acid) as follows: 0-10 min, 10% B; 10-50 min, 10-40% B; and 50-60 min, 40-70% B. The column temperature was maintained at 25 • C and the flow rate was 0.8 mL/min.5 µL of sample after filtration by a 0.45 µm membrane was injected and the chromatogram was recorded at 254 nm with scan wavelength from 190 to 400 nm.

Identification of the Screened Compounds
The screened potential XO inhibitors from extract of Clerodendranthus spicatus were subsequently analyzed by HPLC-MS to identify the chemical structures.The HPLC conditions were the same as the afore-mentioned HPLC analysis conditions.HPLC-MS analysis was completed on the Agilent 6460 Triple Quadrupole LC-MS system (Agilent Technologies Inc., Santa Clara, CA, USA).An Electron Spray Ionization (ESI) interface was used in both positive and negative ionization mode.Full-scan mode from 100 to 1000 m/z was used as the mass detection mode.

XO Fluorescence Quenching
The fluorescence spectra of XO and the effects of samples on XO were performed using a Hitachi F-7000 fluorometer (Tokyo, Japan).The fluorescence quenching effect was evaluated to determine the binding constants between compounds and XO based on the procedures reported by previous literatures with some modifications [34].2.0 mL of XO solution (1.0 µmol/L in phosphate buffered saline, pH 7.4) was poured into a fluorescence quartz cuvette and the fluorescence emission spectra was recorded as blank from 300 nm to 450 nm under an excitation wavelength at 280 nm.Then, 10 µL of sample (0.1 mmol/L in dimethyl sulfoxide for gardenin B and 1.0 mmol/L in DMSO for eupatorin) was added dropwise into the cuvette containing XO solution.The fluorescence emission spectra under the same condition and the fluorescence intensities at 337 nm were recorded.Each test was performed in triplicate.The fluorescence quenching of XO with gardenin B and eupatorin was described using the following Stern-Volmer equation [35]: where F 0 is the fluorescence intensity of XO at 337 nm, F is the fluorescence intensity at 337 nm in the presence of the sample, [Q] is the corresponding sample concentration, K q and K sv are the quenching constants, and τ 0 is the average lifetime (6.2 ns).The relationship between fluorescence quenching intensity at 337 nm and sample concentration was described by the binding constant formula: Log 10 (F 0 − F)/log 10 K a + nlog 10 [Q] (3) where F 0 is the fluorescence intensity of XO at 337 nm, F is the fluorescence intensity at 337 nm in the presence of the sample, [Q] is the corresponding sample concentration, K a is the binding constant, and n is the number of binding sites per XO molecule.All the samples showed no emission spectra in the scanned range under excitation.

Conclusions
In this study, xanthine oxidase immobilized silica coated Fe 3 O 4 nanoparticles were successfully prepared and characterized.The average diameter of Fe 3 O 4 nanoparticles was about 300 nm to 350 nm and the thickness of the silica shell was 60 nm.The maximum saturation magnetization of Fe 3 O 4 @SiO 2 -XO nanoparticles was 44.9 emu/g, which ensures their separation from medium within one minute using an ordinary magnet.93% of the initial screening capacity of Fe 3 O 4 @SiO 2 -XO nanoparticles could be retained after five rounds of screening.The XO inhibitor screening method using Fe 3 O 4 @SiO 2 -XO nanoparticles was established and utilized in the extract of Clerodendranthus spicatus.Two compounds were screened out and identified as gardenin B and eupatorin.The IC 50 values of these two compounds were verified.These two compounds could quench the XO fluorescence spectrum.The quenching activity of both compounds belonged to static quenching mechanism and gardenin B showed stronger binding capacity than eupatorin.In conclusion, this screening method showed efficiency and reusability in the rapid screening and identification of enzyme inhibitors from complex mixtures.
Compound 1 exhibited two absorbance bands at 280 and 330 nm.In the negative ion mode of MS analysis, compound 1 showed the deprotonated molecular ion [M − H] − at 357 m/z and [2M − H] − at 715 m/z in the MS spectrum.According to the references, compound 1 was identified as gardenin B [26].Compound 2 showed UV absorbance peaks at 250 nm, 275 nm and 340 nm.Meanwhile, the deprotonated molecular ion [M − H] − at 343 m/z and [2M − H] − at 687 m/z could be confirmed in the MS spectrum of compound 2 with the negative ion mode.In the positive ion mode, the protonated molecular ion [M + H] + was found at 345 m/z, and [2M + Na] + at 711 m/z.Therefore, the molecular weight of compound 2 could be considered as 344.Compound 2 was identified as eupatorin because the UV and MS data were in agreement with previous reports[27].The standard gardenin B and eupatorin with high purities were also analysed by HPLC and the chromagtograms are shown in Figure7.It can be seen that the retention time of the two standards were in accordance with those in the extract.
Appl.Sci.2018, 8, x FOR PEER REVIEW 8 of 13 dynamic quenching or static quenching played a key role.The value of Kq reflected the efficiency of quenching and the accessibility of fluorophores.Based on previous research, if Kq is near 1.0 × 10 10
Appl.Sci.2018, 8, x FOR PEER REVIEW 8 of 13 dynamic quenching or static quenching played a key role.The value of Kq reflected the efficiency of quenching and the accessibility of fluorophores.Based on previous research, if Kq is near 1.0 × 10 10

Table 1 .
The identification, retention time, MS and UV data of screened compounds.

Table 1 .
The identification, retention time, MS and UV data of screened compounds.

Table 2 .
The affinity constants of gardenin B and eupatorin for XO.Abbreviations: Kq, Kav, R 2 , n, number of binding sites per protein molecule.

Table 2 .
The affinity constants of gardenin B and eupatorin for XO.Abbreviations: Kq, Kav, R 2 , n, number of binding sites per protein molecule.