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An immobilization enzyme for screening lipase inhibitors from Tibetan medicines
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An immobilization enzyme for screening lipase inhibitors from Tibetan medicines Jia Liu ConceptualizationMethodologyValidationFormal analysisInvestigationWriting-Original Draft , Run-Tian Ma VisualizationInvestigationWriting-Review & Editing , Yan-Ping Shi SupervisionProject administrationFunding acquisition PII: DOI: Reference:
S0021-9673(19)31143-4 https://doi.org/10.1016/j.chroma.2019.460711 CHROMA 460711
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Journal of Chromatography A
Received date: Revised date: Accepted date:
26 September 2019 14 November 2019 14 November 2019
Please cite this article as: Jia Liu ConceptualizationMethodologyValidationFormal analysisInvestigationWriting-Origi Run-Tian Ma VisualizationInvestigationWriting-Review & Editing , Yan-Ping Shi SupervisionProject administrationF An immobilization enzyme for screening lipase inhibitors from Tibetan medicines, Journal of Chromatography A (2019), doi: https://doi.org/10.1016/j.chroma.2019.460711
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Highlights
Fe3O4@TiO2 nanoparticle was fabricated as magnetic support of immobilized lipase.
A capillary electrophoresis method was established for screening lipase inhibitors.
Oxytropis falcate and its compounds were firstly reported as lipase inhibitors.
1
An immobilization enzyme for screening lipase inhibitors from Tibetan medicines
Jia Liu a,b, Run-Tian Ma a, Yan-Ping Shi a
a
CAS Key Laboratory of Chemistry of Northwestern Plant Resources, Key
Laboratory for Natural Medicines of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Lanzhou 730000, P. R. China b
University of Chinese Academy of Sciences, Beijing 100049, P. R. China
Corresponding author. E-mail address: [email protected] (R.-T. Ma). Corresponding author. E-mail address: [email protected] (Y.-P. Shi). 2
ABSTRACT With the increasing demand for lipase inhibitors and new drugs used in the clinical treatment of obesity,
it is of great significance to screen lipase inhibitors from
traditional Chinese medicines (TCMs) via capillary electrophoresis. In this work, Fe3O4@TiO2 nanoparticles was fabricated by solvothermal method and employed as an improved magnetic support to immobilize lipase through electrostatic interaction. By the method of transmission electron microscopy, fourier transform infrared spectroscopy and X-ray diffraction, the magnetic nanoparticles were characterized. The immobilized enzyme possessed advantages of a wider range for pH and temperature endurance, better storage stability and reusability. The kinetics performances of the immobilized lipase were studied. When p-Nitrophenyl palmitate (pNPP) was used as enzyme substrate, the Michaelis-Menten constant was calculated to be 2.51 mM and its inhibition constant for Orlistat was ascertained to be 13.41 μM. Ultimately, the established method was applied to lipase inhibitors screening from 6 Tibetan medicines with lipase inhibitory activity and Oxytropis falcate Bunge was screened out for its supreme lipase inhibitory activity. 11 compounds in the Oxytropis falcate Bunge were further screened, five compounds exhibited similar inhibitory activity to Orlistat, and
one compound (kaempferol) presented better inhibitory
activity than Orlistat, which is the most commonly used drugs to treat obesity in clinic. This work not only developed a method for new anti-obesity drugs discovery, but also provided inspiration for exploring new medicinal value of the TCMs.
Keywords: immobilized lipase, Fe3O4@TiO2 nanoparticles, lipase inhibitors screening, Tibetan medicines, Oxytropis falcate Bunge
1. Introduction 3
Over the past decades, obesity rates have been dramatically increased. Today, it is estimated that about one third of the world's population are overweight. Based on the previous trend, it has been estimated that the obese population in the United States would reach 85% of the total population in 2030 [1]. About 3.40 million of adults died of obesity each year [2]. Thus, obesity is recognized as a global chronic disease. While the energy intake is much higher than energy expenditure, the accumulation of fats leads to the occurrence of obesity. When the value of body mass index (BMI) exceeds 30, it would be diagnosed as an obese patient [3]. Obesity also causes relevant diseases, such as hyperlipidemia, arteriosclerosis, type 2 diabetes, cardiovascular disease and hypertension [4]. Therefore, the treatment of obesity must be highly valued by obese patients. Generally, fats could be decomposed by lipase, thus it is of great significance for the development of lipase inhibitors as anti-obesity drugs. Currently, Orlistat is recognized as one of the most commonly used drugs to treat obesity in clinic. It is an irreversible lipase inhibitor which blocks the absorption and reabsorption of the dietary fat [5]. However, this drug may cause several adverse effects, such as fatty diarrhea, stool urgency, fecal incontinence, allergies and so on [6-8]. Therefore, it is urgent to explore more lipase inhibitors as anti-obesity drugs, which is similiar to Orlistat with better safety for clinical treatment of obesity. At present, it has been reported that traditional Chinese medicines (TCMs) have been extensively used to relieve obesity [9, 10]. For example, Magnoliae cortex [11], Lotus leaf [12], Cortex Mori Radicis [13] and Scutellaria baicalensis [14] were all reported to possess anti-obesity activity. Tibetan medicines is a very important branch 4
of TCMs, which has special effects in the treatment of cardiovascular disease, hepatobiliary disease, commonly respiratory diseases, frequently-occurring diseases and various incurable diseases [15]. However, there were extremely few reports about Tibetan medicines with weight-loss effect. Thus, it is worth exploring the rich treasure of Tibetan medicines which possess anti-obesity activity. With the development of enzyme inhibitors screening technologies, various technologies emerged, including high performance liquid chromatograph-mass spectrometry (HPLC-MS) [12, 16], ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) [17], colorimetric assay [18] and photometric bioassay [19]. Capillary electrophoresis (CE)-based enzyme inhibitors screening method has also been employed as a powerful tool to screen enzyme inhibitors [20-25], owing to its significant advantages of excellent reusability, low cost, good operational stability, convenient purification procedures and the prolonged survival time of enzyme [21, 26, 27]. A great deal of researches demonstrated that the immobilized enzyme has a wider range for pH and temperature endurance than free enzyme, immobilization support
and the
is an important part in the CE-based enzyme inhibitors
screening method. Magnetic supports have been extensively applied to immobilize the enzyme [28, 29]. However, conventional magnetic supports always have smooth surface and no wrinkles, which could not provide as sufficient sites as possible for enzyme immobilization [30-33]. Titanium dioxide with rough surface possesses the properties of high specific surface areas, nontoxicity, low-cost and wide ranges of pH 5
and temperature [34-40]. Thus, combining titanium dioxide with magnetic support would significantly enhance the enzyme immobilization efficiency. In this work, coupled with CE, a strategy of immobilization enzyme was designed for lipase inhibitors screening. The solvothermal approach was chosen to prepare magnetic support, since the synthesized Fe3O4@TiO2 nanoparticles possessed appropriate size and excellent dispersity. Through electrostatic interaction, lipase was immobilized on the magnetic support. p-Nitrophenyl palmitate (pNPP) was used as enzyme substrate, at the wavelength of 405 nm, the kinetics was studied. For further study, six classic Tibetan medicines were chosen for lipase inhibitor screening. It was surprising to find that Oxytropis falcate Bunge possessed superior lipase inhibitory effect. On the basis of this, 11 compounds in Oxytropis falcate Bunge were screened and it was found that the inhibitory effects of five compounds exhibited similar inhibitory activity to Orlistat, and
one compound (kaempferol) presented better
inhibitory activity than Orlistat. This work not only improved the development of magnetic supports in enzyme inhibitors screening, but also provided inspiration for new anti-obesity drugs discovery from TCMs.
2. Materials and methods 2.1 Chemicals Candida rugosa lipase (CRL, EC 3.1.1.3) from candida rugosa was purchased from Sigma Chemical Co. (St. Louis, MO, USA). p-Nitrophenyl palmitate (pNPP) and titanium (IV) butoxide was purchased from Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China). Orlistat was purchased from Macklin Biochemical Co., Ltd 6
(Shanghai, China). Ferric chloride hexahydrate (FeCl3·6H2O), anhydrous sodium acetate (NaAc), sodium dihydrogen phosphate (NaH2PO4), sodium tetraborate decahydrate (Na2B4O7·10H2O), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were obtained from Tianjin Kermel Chemical Reagent Co., Ltd (Tianjin, China). Tri (hydroxymethyl) aminomethane was purchased from Beijing Yili Fine Chemicals Co., Ltd. (Beijing, China). Ethylene glycol, polyethylene glycol (PEG) and ethanol were purchased from Li’anlong Bohua Pharmaceutical Chemical Co., Ltd (Tianjin, China). All Tibetan medicines, Arctium lappa L (AL), Zingiber officinale Rosc (ZO), Oxytropis falcate Bunge (OF), Cortex Moutan (CM), Alpinia katsumadai Hayata (AK) and Glycyrrhiza uralensis Fisch (GU) were purchased from a local Hui Ren Tang drug store (Lanzhou, China). The compounds, 2',4'-dihydroxychalone (compound 1), 7-hydroxy-3',4'-methylenedioxyisoflavone (compound 2), dihydroxy-5'-methoxyisoflavone
(compound
3),
7,3'-
5,7,2'-trihydroxy-4'-
methoxyisoflavone (compound 4), 5,2',4'-trihydroxy-7-methoxyisoflavone (compound 5), 5,7-dihydroxydihydroflavone (compound 6), apigenin (compound 7), isorhamnetin (compound 8), luteolin (compound 9), kaempferol (compound 10) and genistein (compound 11) from Oxytropis falcate Bunge were isolated by our laboratory. A series of incubation buffer with different pH value (4.0-10.0) were obtained by dissolving NaH2PO4 in ultrapure water with the concentration of 20 mM and its pH value was adjusted by Na2HPO4, combined with NaOH or HCl. The background electrolyte (BGE) was obtained by dissolving Na2B4O7·10H2O in ultrapure water with the concentration of 20 mM and its pH value was adjusted to 9.0 with 1.0 M HCl. The 7
stock solution of lipase was prepared in 20 mM of phosphate buffer. The solution of substrate (pNPP) was prepared in Tris-HCl with certain concentrations of 5 mM. The solutions of inhibitor (Orlistat) and compounds from Oxytropis falcate Bunge were prepared in anhydrous methanol solution with certain concentrations of 100 mM. All the stock solutions were stored at -20 °C and diluted with phosphate buffer before use each day. The obtained Tibetan medicines were air-dried and crushed with a pulverizer. Powders (4.5 g) were firstly immersed in 100 mL of methanol for 7 days and then the supernatant was collected. Subsequently, the residue was dispersed in 80 mL of methanol for 7 days, and the supernatant was collected. Finally, the residue was immersed in 50 mL of methanol for 7 days and the supernatant was collected. All the supernatant were mixed together, filtered through a piece of filter paper and the filtrate was evaporated to dryness with rotary evaporator. Finally, the residues were dispersed in 4.5 mL of methanol for future use. 2.2 CE conditions An Agilent 7100 capillary electrophoresis system (Waldbronn, Germany) equipped with a diode array detector (DAD) system was used for separating and detecting the enzymatic reaction mixture. Data analysis and instrumental runs were operated via an Agilent CE Chemstation (Rev. B 04.02). A piece of uncoated fused silica capillary (Ruifeng Chromatographic Device Co., Ltd, Hebei, China) with a dimension of 50 µm i.d.×33 cm (24.5 cm to the detection window) was employed. The measurements of pH values for all the buffers were operated via a Sartourius PB8
10 pH meter (Sartorius, Germany). Ultrapure water used in this experiment was produced by a Milli-Q water system (Shanghai Laikie Instrument Co., Ltd, China). The cartridge temperature was set as 20oC and the detection wavelength was set as 405 nm with a bandwidth of 30 nm. The electrophoresis was operated at 20 kV. The capillary condition was initialized by rinsing with methanol for 5 min, ultra-pure water for 5 min, 1 M NaOH for 30 min and ultrapure water for 5 min. At the beginning of each day, the capillary was rinsed with 1 M NaOH for 15 min and ultrapure water for 10 min. Between runs, the capillary was rinsed with 1 M NaOH for 1 min, ultrapure water for 1 min and BGE for 1.5 min. At the end of each day, the capillary was conditioned with 1 M NaOH for 15 min and ultrapure water for 10 min to ensure a clean environment of the capillary inner wall. 2.3 Magnetic Fe3O4 nanoparticles preparation Magnetic Fe3O4 nanoparticles were synthesized by solvothermal method [41]. Briefly, FeCl3·6H2O (1.35 g) was dispersed in ethylene glycol (40 mL), forming orange solution. Subsequently, NaAc (3.6 g) and PEG (1.0 g) were added into the above solution and the reaction was proceeded by vigorously magnetic stirring for 30 min. The obtained solution was then transferred into a Teflon-lined stainless-steel autoclave and heated to 200oC for 24 h. When cooled to room temperature, the resultant black products were washed several times with ultrapure water, ethanol and dried in a vacuum drying oven at 60oC for 8 h. 2.4 Fe3O4@TiO2 nanoparticles preparation For Fe3O4@TiO2 preparation [42], Fe3O4 nanoparticles (NPs) (80 mg) was 9
thoroughly dispersed in a mixture of N,N-dimethylformamide (20 mL) and isopropyl alcohol (60 mL) under the condition of ultrasonic vibration for 30 min. Subsequently, titanium butoxide (4 mL) was added into the above obtained suspension drop by drop and the reaction was proceeded for 30 min at room temperature. The obtained mixture was transferred into a Teflon-lined stainless-steel autoclave and heated at 200℃ for 24 h. When cooled to room temperature, the synthesized product was successively washed several times with ultrapure water, ethanol, lyophilized and stored at 4℃ for future use. The preparation procedure of the Fe3O4@TiO2/CRL nanoparticles was shown in Fig. 1. 2.5 Enzyme immobilization The lipase was successfully immobilized on the synthetic Fe3O4@TiO2 NPs through the following step. Briefly, Fe3O4@TiO2 NPs were homogenously dispersed in 0.1 M HCl solution to activate surface hydroxyl groups of Fe3O4 @TiO2 NPs. Then the supernatant of the above suspension was discarded by applying external magnet. After rinsing with ultrapure water until neutral, the resultant magnetic Fe 3O4 @TiO2 NPs were stored in phosphate buffer at 4℃ for future use. In order to determine the optimum conditions for enzyme immobilization, the immobilization pH (3, 4, 5, 6, 7), immobilization time (1, 2, 3, 4, 5 h) and immobilization enzyme concentration (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 mg/mL) were investigated, respectively. 5 mg of Fe3O4@TiO2 NPs were uniformly dispersed in 1 mL of phosphate buffer, adding 1 mL of lipase solution into the suspension under the vigorous shaking. After shaking for a period of time at room 10
temperature, the mixture was separated magnetically, rinsing several times with phosphate buffer, dispersed in 2 mL of phosphate buffer and stored at 4℃ for following study. 2.6 Characterization According to the fourier-transform infrared (FT-IR) spectra, the chemical composition of synthetized Fe3O4 NPs and Fe3O4@TiO2 NPs were characterized with a Nexus 870 Fourier Transform Infrared Spectrometer (Thermo Fisher, USA), and the wavenumbers were ranged from 500 cm-1 to 4000 cm-1. Through an X’pert PRO Xray diffraction (PANalytical B.V., Netherlands) with Ni-filtered Cu Kα radiation over the angular range from 8° to 90°, the crystal structures of the magnetic NPs were characterized. The morphological features of the pure Fe3O4 and Fe3O4@TiO2 NPs were obtained through a transmission electron microscope (TEM) (FEITECNAI G2TF20, USA). The particle size distributions of the magnetic NPs was determined through the Malvern Zeta sizer Nano ZS (Malvern Instruments, UK) working on the dynamic light scattering (DLS) platform. The magnetic properties were characterized by vibrating sample magnetometer (VSM) (Lakeshore, USA). 2.7 Immobilized enzyme kinetics study The obtained immobilized enzyme was dispersed into a homogeneous solution under the condition of ultrasonication. Subsequently, 150 μL of the above suspension was transferred into 1.5 mL Eppendorf tube. The supernatant was discarded by applying external magnet, and adding 300 μL of substrate solution into the above suspension. The final concentration of the substrate was 0.4, 0.6, 0.8, 1.0, 1.2 mM, 11
respectively. The reaction mixture was incubated at 60oC for 15 min and the reaction was terminated by magnetically separating the immobilized enzyme nanoparticles from the mixture. Finally, the supernatant was injected into CE for further analysis. 2.8 Inhibition study and inhibitors screening Inhibition study was investigated by calculating Km of the immobilized lipase. Km, Michaelis-Menten constant [22], which illustrates the affinity of enzyme for substrate, was calculated using the Lineweaver-Burk plot:
Where S represents the concentration of the substrate and v means the peak areas of the product pNP. Under the optimum conditions, the Km of the immobilized lipase was measured by analyzing different concentrations of pNPP ranging from 0.4 mM to 1.2 mM. Each substrate concentration was operated in triplicated. For lipase inhibitor screening, 150 μL of Fe3O4@TiO2/CRL was mixed with 300 μL of substrate solution following 100 μL of Tibetan medicine extract was added and incubated at 60oC. 15 min later, the immobilized enzyme NPs was separated and the supernatant was analyzed by CE. The lipase inhibitor screening for compounds were operated using the same procedure above mentioned, except compounds were substitute for Tibetan medicines extracts.
3. Results and discussion 3.1 Characterization TEM images obtained for Fe3O4 and Fe3O4@TiO2 NPs were shown in Fig. 2. As 12
can been seen, the spherical Fe3O4 NPs (Fig. 2a) showed excellent dispersibility and its Energy Dispersive X-Ray Spectroscopy (EDS) data was listed in Fig. S1a. After the solvothermal reaction between Fe3O4 NPs and titanium butoxide, the Ti element emerged in Fig. S1b, in addition to the Fe and O element, proving the successful preparation of TiO2 on the surface of Fe3O4 NPs. In Fig. 2b, the classic core-shell structure of Fe3O4@TiO2 NPs formed and their surface became rough (Fig. 2b, inset), which could provide sufficient binding sites for lipase. Meanwhile, the dispersibility of magnetic NPs still maintained, which was also beneficial to lipase immobilization. The DLS characterization of the magnetic NPs was applied to illustrate the existence of TiO2 shell. According to Fig. 2c, the diameter of Fe3O4 NPs was about 350.7 nm. After the modification of TiO2, the diameter of Fe3O4@TiO2 NPs (Fig. 2d) was about 386.4 nm, which demonstrated the successful preparation of TiO2 and its diameter was about 17.9 nm. The magnetic NPs were characterized by FT-IR spectra, in order to determine their chemical composition. The characteristic peak of Fe-O group was observed around 577 cm-1 (Fig. 3a), which confirmed the successful formation of Fe3O4. The adsorption peak at 443 cm-1 in Fig.3b was assigned to stretching vibration of Ti-O bonds, revealing the porous TiO2 shells had been coated on the surface of Fe3O4. Furthermore, XRD analysis was also applied to characterize the magnetic NPs. For Fe3O4 (Fig. 3b), six characteristic diffraction peaks ((220), (311), (400), (422), (511) and (440)) were observed, which corresponded to the standard pattern for Fe3O4 (JCPDS #65-3107), further confirming the cubic spinel structure of Fe3O4 13
NPs.
Meanwhile, almost similar XRD pattern of Fe3O4@TiO2 (Fig. 3b) NPs was obtained as compared to Fe3O4 NPs, indicating the formation of the TiO2 shell would not change the crystal structure of Fe3O4 NPs. Furthermore, the magnetic properties of magnetic NPs were characterized by VSM. The analysis result (Fig. S2) demonstrated that the magnetic NPs were superparamagnetism and could be conveniently separated from the reaction matrix. 3.2 Optimization of the immobilization conditions In order to attain the immobilized lipase with high relative activity, the effect of the immobilization pH, immobilization time and immobilization lipase concentration were investigated. Fig. 4a showed the influence of pH on the relative activity of the immobilized lipase. In this set of experiments, the immobilization pH value ranging from 3.0 to 7.0 was investigated (immobilization time 2 h, immobilization lipase concentration 1.4 mg/mL, enzymatic reaction time 15 min, enzymatic reaction temperature 60 oC). As indicated in Fig. 4a, the highest relative activity was obtained at the pH value of 4.0, thus the optimum immobilization pH was set to be 4.0. The influence of immobilization time, ranging from 1 h to 5 h (immobilization pH 4.0, immobilization lipase concentration 1.4 mg/mL, enzymatic reaction time 15 min, enzymatic reaction temperature 60oC), on the relative activity were also investigated. As indicated in Fig. 4b, the relative activity tended to increase from 77.8% to 100% with increasing immobilization time from 1 h to 2 h, indicating that lipase molecules have been successfully immobilized on the supports. However, the relative activity was declined 14
to 80.3% with prolonging duration time to 5 h. This is mostly due to excess lipase immobilized on the support led to the inaccessibility of the substrate to the active sites of lipase [43]. Based on the results, it could be ensured that the best immobilization time was 2 h. In Fig. 4c, different immobilization lipase concentrations ranging from 0.2 mg/mL to 2.0 mg/mL (initial lipase concentration) was employed for lipase immobilization on the magnetic support (immobilization pH 4.0, immobilization time 2h, enzymatic reaction time 15 min, enzymatic reaction temperature 60℃). As illustrated in Fig. 4c, the best immobilization lipase concentration of 1.4 mg/mL was employed. With the lipase concentration increased from 0.2 mg/mL to 1.4 mg/mL, the relative activity of the immobilized lipase was enhanced from 39.3% to 100%. However, increasing the lipase concentration beyond 1.4 mg/mL caused the relative activity to decrease. With the immobilization enzyme loaded excessively, the surface of the magnetic support was saturated, thus leading to an intermolecular steric hindrance among the immobilized lipase molecules and further limiting the diffusion of the substrate to the lipase molecules [43, 44]. Based on the above results, it could be concluded that the best immobilization lipase concentration was 1.4 mg/mL. 3.3 Optimization of the enzymatic reaction conditions The influence of enzymatic reaction time on the free and immobilized lipase activities were investigated in Fig. 5a. The relative activity of the immobilized lipase tended to increase from 40.4% to 100% with prolonging reaction time from 5 min to 15 min. And it tended to decrease beyond 15 min, which was mostly due to the lipase 15
deactivation with a longer reaction time. At the enzymatic reaction time of 15 min, both the free and immobilized lipase showed the highest relative activity and the relative activity of immobilized lipase was higher than that of the free lipase in the studied reaction time. Taking the highest reaction efficiency into consideration, the enzymatic reaction time was fixed at 15 min. The effects of temperature on the free and immobilized lipase relative activity were also measured, as illustrated in Fig. 5b. The relative activity of the immobilized lipase was enhanced with a rise in the temperature from 40 oC to 60oC, reaching the highest enzymatic activity at 60oC. With the temperature increased beyond 60oC, the relative activity of the immobilized lipase tended to decrease. Besides, there was a same trend for the relative activity of the free lipase. Meanwhile, the relative activity of the immobilized lipase was always higher than that of the free lipase at the same conditions, which could be attributed to the stabilized backbone of the immobilized lipase molecules. As a consequence, the optimum temperature for the enzymatic reaction was set as 60oC. 3.4 Reusability and storage stability of the immobilized lipase Reusability of the immobilized enzyme is an important factor for cost-effective usage of lipase in the large-scale application. Thus, the reusability of the immobilized lipase was studied under the optimum reaction conditions. After the first usage, the immobilized lipase was separated by applying an external magnet and followed by washing with phosphate buffer solution for the subsequent consecutive usages. As indicated in Fig. S3, the results showed about 49.6% of its initial activity maintained 16
after 5 successive reuses of the immobilized lipase. The reason for such a decrease in the relative activity was the denaturation of lipase. The storage stability of the immobilized lipase is also a crucial factor for the practical applications. The storage stabilities of immobilized and free lipase were tested by measuring the enzyme activity after a storage period of 30 days at 4℃, respectively. As shown in Fig. S4, the immobilized lipase lost about 22.4% of its initial activity after 30 days storage. However, the free lipase lost about 64.7% of its initial activity. Based on the above results, the immobilized lipase showed better storage stability and in accordance with the literature [45]. 3.5 Kinetics study of the immobilized lipase As Fig. S5 demonstrated the linearity between the concentration of pNPP and the corrected peak areas, velocity was represented by the corrected peak areas in this work. According to the Michaelis-Menten model, the Lineweaver-Burk plot was 1/v = (0.005662 ± 0.0001901) (1/[S]) + (0.00226 ± 0.000298) with a determination coefficient of 0.9983 (Fig. 6a). And Km of the immobilized lipase was calculated to be 2.51 mM and 9.96 mM for the free lipase (Fig. S6), indicating that the immobilized lipase had a higher affinity for pNPP than that of the free lipase, which demonstrated that the immobilized lipase obtained better catalytic activity. Enzyme inhibitor affects the binding of the enzyme to the substrate, resulting in the reduction or denaturation of enzyme activity. From the inhibition kinetics of the enzyme, the type of action of the inhibitor, the inhibition constant of the inhibitor and the inhibition rate could be determined. In this work, Orlistat was employed as a 17
model inhibitor and its inhibition performance was studied. In the presence of different concentrations of Orlistat, the Lineweaver-Burk plot for the immobilized lipase was shown in Fig. 6b-d. With an increase of Orlistat concentration, the value of vertical axis intercept (1/Vmax) increased and the value of horizontal axis intercept (1/Km) remained unchanged. Taking above results into consideration, Orlistat is a noncompetitive inhibitor, which was consistent with the literature [46, 47]. And its value of Ki, inhibition constant, was calculated to be 13.41 μM by secondary plot method (Fig. S7), The half maximal inhibitory concentration (IC50) is also an important parameter to evaluate the inhibitory ability of the inhibitor. Under a condition of 0.5 mM substrate solution, a range of different concentrations of Orlistat were added to obtain the inhibition plot (Fig. 7). By plotting the inhibition rate versus the longarithm of Orlistat concentration, IC50 was determined as 4.37 mM, which was in accordance with the literature [14, 17]. The inhibition rate was calculated as follows: Inhibition (%) = (1-x/blank)×100
Eq. (1)
Where x and blank represent the peak areas of pNP measured with and without inhibitor, respectively. Z’ factor, a characteristic parameter which is used to evaluate the quality and accuracy of a drug screening method [48]. When the Z’ factor is in the range of 0.5 to 1, a screening method is accurate and feasible. In our work, Z’ factor was determined as 0.95 (n=9), demonstrating that the inhibitor screening method is excellent. The above results illustrated that the established method was feasible for the following 18
lipase inhibitor screening. 3.6 Inhibitors screening from Tibetan medicines Under the optimum conditions, the immobilized lipase was employed to screen lipase inhibitors from 6 Tibetan medicines, AL, ZO, OF, CM, AK and GU, and their inhibitions were calculated according to the Eq. (1). According to the above screening results (Fig. 8), AL, ZO, CM, AK and GU showed relatively weak inhibitory activities against lipase even at the concentration of 100 mg/mL, indicating these Tibetan medicines might not suitable for the treatment of obesity. However, Oxytropis falcate Bunge provided 54.0% inhibition at the concentration of 50 mg/mL, which was superior to the other 5 Tibetan medicines. When the concentration was increased to 100 mg/mL, its inhibition (68.6%) was obviously higher than those of 5 Tibetan medicines, exhibiting the strongest inhibition performance against lipase. Thus, Oxytropis falcate Bunge might be a potential drug for the treatment of obesity. To further explore the lipase inhibitory activity of Oxytropis falcate Bunge, 11 compounds from Oxytropis falcate Bunge isolated by our laboratory were screened, and the results were listed in Fig. 9. The detailed information of the 11 compounds from Oxytropis falcate Bunge was shown in the Fig. S8. When the concentrations of the tested compounds were 50 μM, compounds 1, 2, 3, 6 possessed similar lipase inhibitory activities to Orlistat. The lipase inhibitory activities of compounds 5 and 7 were 20% higher than that of Orlistat, and compound 10 (kaempferol) was almost 30% higher than that of Orlistat. When the concentrations of the tested compounds 19
increased to 100 μM, compounds 1, 2, 5, 6, 7, 8, 9 possessed similar lipase inhibitory activities to Orlistat and compound 10 was obviously superior to Orlistat. These results demonstrated that compounds 1, 2, 5, 6, 7 might be potential drugs for obesity treatment. Surprisingly, compound 10 had the best inhibitory activity against lipase among these compounds and Orlistat. Thus, compound 10 might be a new pharmaceutical molecule for obesity treatment. Meanwhile, an amazing medicinal value of anti-obesity for Oxytropis falcate Bunge was discovered in this work, and anti-obesity function was different from the anti-inflammatory and analgesic reported in the literatures [49, 50]. From the perspective of inhibitors screening, it could be concluded that compounds 1, 2, 5, 6, 7, 10 might be elected as candidates for antiobesity drugs and their inhibitory mechanisms need to be researched. In addition, the successful screening of these potential lipase inhibitors also confirmed the feasibility of the established method. Thus, this method is expected to be applied to other inhibitors screening.
4. Conclusion An immobilized enzyme method for screening lipase inhibitors coupled with CE from Tibetan medicines was realized in this study. Fe3O4@TiO2 nanoparticles was prepared and employed as the magnetic support to provide sufficient binding sites for lipase. Through electrostatic interaction, the lipase was immobilized on the surface of magnetic support and its surface morphology, chemical composition, crystal structure, kinetics study, reusability and storage stability were systematically investigated. The kinetics performances of the immobilized lipase were carefully studied. The prepared 20
Fe3O4@TiO2/CRL was successfully applied to screen lipase inhibitors from Tibetan medicines, and a platform for screening lipase inhibitors from TCMs was successfully developed. Oxytropis falcate Bunge, which was a classic Tibetan medicine with significant anti-inflammatory effect, was screened out for its supreme inhibitory activity. Furthermore, the inhibitory activity of the 11 compounds in Oxytropis falcate Bunge were analyzed, compounds 1, 2, 5, 6, 7 exhibited similar inhibitory activity to Orlistat, and compound 10 (kaempferol) presented better inhibitory activity than Orlistat.
In our opinion, this work could not only establish a method of
immobilization lipase for the screening lipase inhibitors from Tibetan medicines, but also provide a step forward the development of weight-loss drugs.
Declarations of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos. 21775153, 21804135 and 21974145), and the Scholar Program of West Light Project of the Chinese Academy of Sciences.
Author Contributions Section Jia Liu: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing-Original Draft. Run-Tian Ma: Visualization, Investigation, Writing-Review & Editing. Yan-Ping Shi: Supervision, Project administration, Funding acquisition. 21
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Figure captions
Figure 1. The schematic of Fe3O4@TiO2/CRL nanoparticles preparation.
28
Figure 2. TEM images ((a) and (b)) and DLS graphs ((c) and (d)) of Fe 3O4 and Fe3O4@TiO2 nanoparticles.
Figure 3. FT-IR spectra of (a) Fe3O4@TiO2, Fe3O4 nanoparticles and XRD spectra of (b) Fe 3O4, Fe3O4@TiO2 nanoparticles.
29
Figure 4. Influence of (a) immobilization pH, (b) immobilization time and (c) immobilization enzyme concentration on the relative activity of the immobilized lipase.
Figure 5. Influence of (a) enzymatic reaction time and (b) temperature on the relative activity of the immobilized lipase.
Figure 6. Lineweaver-Burk plot of lipase in the presence of the inhibitor Orlistat. The concentrations of Orlistat: (a) 0 μM; (b) 10 μM; (c) 30 μM; (d) 50 μM. 30
Figure 7. Inhibition plot of Orlistat to immobilized lipase.
Figure 8. Results of lipase inhibitors screening from 6 kinds of Tibetan medicines.
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Figure 9. Results of lipase inhibitors screening from Oxytropis falcate Bunge.
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An immobilization enzyme for screening lipase inhibitors from Tibetan medicines Jia Liu ConceptualizationMethodologyValidationFormal analysisInvestigationWriting-Original Draft , Run-Tian Ma VisualizationInvestigationWriting-Review & Editing , Yan-Ping Shi SupervisionProject administrationFunding acquisition PII: DOI: Reference:
S0021-9673(19)31143-4 https://doi.org/10.1016/j.chroma.2019.460711 CHROMA 460711
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
26 September 2019 14 November 2019 14 November 2019
Please cite this article as: Jia Liu ConceptualizationMethodologyValidationFormal analysisInvestigationWriting-Origi Run-Tian Ma VisualizationInvestigationWriting-Review & Editing , Yan-Ping Shi SupervisionProject administrationF An immobilization enzyme for screening lipase inhibitors from Tibetan medicines, Journal of Chromatography A (2019), doi: https://doi.org/10.1016/j.chroma.2019.460711
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Highlights
Fe3O4@TiO2 nanoparticle was fabricated as magnetic support of immobilized lipase.
A capillary electrophoresis method was established for screening lipase inhibitors.
Oxytropis falcate and its compounds were firstly reported as lipase inhibitors.
1
An immobilization enzyme for screening lipase inhibitors from Tibetan medicines
Jia Liu a,b, Run-Tian Ma a, Yan-Ping Shi a
a
CAS Key Laboratory of Chemistry of Northwestern Plant Resources, Key
Laboratory for Natural Medicines of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Lanzhou 730000, P. R. China b
University of Chinese Academy of Sciences, Beijing 100049, P. R. China
Corresponding author. E-mail address: [email protected] (R.-T. Ma). Corresponding author. E-mail address: [email protected] (Y.-P. Shi). 2
ABSTRACT With the increasing demand for lipase inhibitors and new drugs used in the clinical treatment of obesity,
it is of great significance to screen lipase inhibitors from
traditional Chinese medicines (TCMs) via capillary electrophoresis. In this work, Fe3O4@TiO2 nanoparticles was fabricated by solvothermal method and employed as an improved magnetic support to immobilize lipase through electrostatic interaction. By the method of transmission electron microscopy, fourier transform infrared spectroscopy and X-ray diffraction, the magnetic nanoparticles were characterized. The immobilized enzyme possessed advantages of a wider range for pH and temperature endurance, better storage stability and reusability. The kinetics performances of the immobilized lipase were studied. When p-Nitrophenyl palmitate (pNPP) was used as enzyme substrate, the Michaelis-Menten constant was calculated to be 2.51 mM and its inhibition constant for Orlistat was ascertained to be 13.41 μM. Ultimately, the established method was applied to lipase inhibitors screening from 6 Tibetan medicines with lipase inhibitory activity and Oxytropis falcate Bunge was screened out for its supreme lipase inhibitory activity. 11 compounds in the Oxytropis falcate Bunge were further screened, five compounds exhibited similar inhibitory activity to Orlistat, and
one compound (kaempferol) presented better inhibitory
activity than Orlistat, which is the most commonly used drugs to treat obesity in clinic. This work not only developed a method for new anti-obesity drugs discovery, but also provided inspiration for exploring new medicinal value of the TCMs.
Keywords: immobilized lipase, Fe3O4@TiO2 nanoparticles, lipase inhibitors screening, Tibetan medicines, Oxytropis falcate Bunge
1. Introduction 3
Over the past decades, obesity rates have been dramatically increased. Today, it is estimated that about one third of the world's population are overweight. Based on the previous trend, it has been estimated that the obese population in the United States would reach 85% of the total population in 2030 [1]. About 3.40 million of adults died of obesity each year [2]. Thus, obesity is recognized as a global chronic disease. While the energy intake is much higher than energy expenditure, the accumulation of fats leads to the occurrence of obesity. When the value of body mass index (BMI) exceeds 30, it would be diagnosed as an obese patient [3]. Obesity also causes relevant diseases, such as hyperlipidemia, arteriosclerosis, type 2 diabetes, cardiovascular disease and hypertension [4]. Therefore, the treatment of obesity must be highly valued by obese patients. Generally, fats could be decomposed by lipase, thus it is of great significance for the development of lipase inhibitors as anti-obesity drugs. Currently, Orlistat is recognized as one of the most commonly used drugs to treat obesity in clinic. It is an irreversible lipase inhibitor which blocks the absorption and reabsorption of the dietary fat [5]. However, this drug may cause several adverse effects, such as fatty diarrhea, stool urgency, fecal incontinence, allergies and so on [6-8]. Therefore, it is urgent to explore more lipase inhibitors as anti-obesity drugs, which is similiar to Orlistat with better safety for clinical treatment of obesity. At present, it has been reported that traditional Chinese medicines (TCMs) have been extensively used to relieve obesity [9, 10]. For example, Magnoliae cortex [11], Lotus leaf [12], Cortex Mori Radicis [13] and Scutellaria baicalensis [14] were all reported to possess anti-obesity activity. Tibetan medicines is a very important branch 4
of TCMs, which has special effects in the treatment of cardiovascular disease, hepatobiliary disease, commonly respiratory diseases, frequently-occurring diseases and various incurable diseases [15]. However, there were extremely few reports about Tibetan medicines with weight-loss effect. Thus, it is worth exploring the rich treasure of Tibetan medicines which possess anti-obesity activity. With the development of enzyme inhibitors screening technologies, various technologies emerged, including high performance liquid chromatograph-mass spectrometry (HPLC-MS) [12, 16], ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) [17], colorimetric assay [18] and photometric bioassay [19]. Capillary electrophoresis (CE)-based enzyme inhibitors screening method has also been employed as a powerful tool to screen enzyme inhibitors [20-25], owing to its significant advantages of excellent reusability, low cost, good operational stability, convenient purification procedures and the prolonged survival time of enzyme [21, 26, 27]. A great deal of researches demonstrated that the immobilized enzyme has a wider range for pH and temperature endurance than free enzyme, immobilization support
and the
is an important part in the CE-based enzyme inhibitors
screening method. Magnetic supports have been extensively applied to immobilize the enzyme [28, 29]. However, conventional magnetic supports always have smooth surface and no wrinkles, which could not provide as sufficient sites as possible for enzyme immobilization [30-33]. Titanium dioxide with rough surface possesses the properties of high specific surface areas, nontoxicity, low-cost and wide ranges of pH 5
and temperature [34-40]. Thus, combining titanium dioxide with magnetic support would significantly enhance the enzyme immobilization efficiency. In this work, coupled with CE, a strategy of immobilization enzyme was designed for lipase inhibitors screening. The solvothermal approach was chosen to prepare magnetic support, since the synthesized Fe3O4@TiO2 nanoparticles possessed appropriate size and excellent dispersity. Through electrostatic interaction, lipase was immobilized on the magnetic support. p-Nitrophenyl palmitate (pNPP) was used as enzyme substrate, at the wavelength of 405 nm, the kinetics was studied. For further study, six classic Tibetan medicines were chosen for lipase inhibitor screening. It was surprising to find that Oxytropis falcate Bunge possessed superior lipase inhibitory effect. On the basis of this, 11 compounds in Oxytropis falcate Bunge were screened and it was found that the inhibitory effects of five compounds exhibited similar inhibitory activity to Orlistat, and
one compound (kaempferol) presented better
inhibitory activity than Orlistat. This work not only improved the development of magnetic supports in enzyme inhibitors screening, but also provided inspiration for new anti-obesity drugs discovery from TCMs.
2. Materials and methods 2.1 Chemicals Candida rugosa lipase (CRL, EC 3.1.1.3) from candida rugosa was purchased from Sigma Chemical Co. (St. Louis, MO, USA). p-Nitrophenyl palmitate (pNPP) and titanium (IV) butoxide was purchased from Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China). Orlistat was purchased from Macklin Biochemical Co., Ltd 6
(Shanghai, China). Ferric chloride hexahydrate (FeCl3·6H2O), anhydrous sodium acetate (NaAc), sodium dihydrogen phosphate (NaH2PO4), sodium tetraborate decahydrate (Na2B4O7·10H2O), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were obtained from Tianjin Kermel Chemical Reagent Co., Ltd (Tianjin, China). Tri (hydroxymethyl) aminomethane was purchased from Beijing Yili Fine Chemicals Co., Ltd. (Beijing, China). Ethylene glycol, polyethylene glycol (PEG) and ethanol were purchased from Li’anlong Bohua Pharmaceutical Chemical Co., Ltd (Tianjin, China). All Tibetan medicines, Arctium lappa L (AL), Zingiber officinale Rosc (ZO), Oxytropis falcate Bunge (OF), Cortex Moutan (CM), Alpinia katsumadai Hayata (AK) and Glycyrrhiza uralensis Fisch (GU) were purchased from a local Hui Ren Tang drug store (Lanzhou, China). The compounds, 2',4'-dihydroxychalone (compound 1), 7-hydroxy-3',4'-methylenedioxyisoflavone (compound 2), dihydroxy-5'-methoxyisoflavone
(compound
3),
7,3'-
5,7,2'-trihydroxy-4'-
methoxyisoflavone (compound 4), 5,2',4'-trihydroxy-7-methoxyisoflavone (compound 5), 5,7-dihydroxydihydroflavone (compound 6), apigenin (compound 7), isorhamnetin (compound 8), luteolin (compound 9), kaempferol (compound 10) and genistein (compound 11) from Oxytropis falcate Bunge were isolated by our laboratory. A series of incubation buffer with different pH value (4.0-10.0) were obtained by dissolving NaH2PO4 in ultrapure water with the concentration of 20 mM and its pH value was adjusted by Na2HPO4, combined with NaOH or HCl. The background electrolyte (BGE) was obtained by dissolving Na2B4O7·10H2O in ultrapure water with the concentration of 20 mM and its pH value was adjusted to 9.0 with 1.0 M HCl. The 7
stock solution of lipase was prepared in 20 mM of phosphate buffer. The solution of substrate (pNPP) was prepared in Tris-HCl with certain concentrations of 5 mM. The solutions of inhibitor (Orlistat) and compounds from Oxytropis falcate Bunge were prepared in anhydrous methanol solution with certain concentrations of 100 mM. All the stock solutions were stored at -20 °C and diluted with phosphate buffer before use each day. The obtained Tibetan medicines were air-dried and crushed with a pulverizer. Powders (4.5 g) were firstly immersed in 100 mL of methanol for 7 days and then the supernatant was collected. Subsequently, the residue was dispersed in 80 mL of methanol for 7 days, and the supernatant was collected. Finally, the residue was immersed in 50 mL of methanol for 7 days and the supernatant was collected. All the supernatant were mixed together, filtered through a piece of filter paper and the filtrate was evaporated to dryness with rotary evaporator. Finally, the residues were dispersed in 4.5 mL of methanol for future use. 2.2 CE conditions An Agilent 7100 capillary electrophoresis system (Waldbronn, Germany) equipped with a diode array detector (DAD) system was used for separating and detecting the enzymatic reaction mixture. Data analysis and instrumental runs were operated via an Agilent CE Chemstation (Rev. B 04.02). A piece of uncoated fused silica capillary (Ruifeng Chromatographic Device Co., Ltd, Hebei, China) with a dimension of 50 µm i.d.×33 cm (24.5 cm to the detection window) was employed. The measurements of pH values for all the buffers were operated via a Sartourius PB8
10 pH meter (Sartorius, Germany). Ultrapure water used in this experiment was produced by a Milli-Q water system (Shanghai Laikie Instrument Co., Ltd, China). The cartridge temperature was set as 20oC and the detection wavelength was set as 405 nm with a bandwidth of 30 nm. The electrophoresis was operated at 20 kV. The capillary condition was initialized by rinsing with methanol for 5 min, ultra-pure water for 5 min, 1 M NaOH for 30 min and ultrapure water for 5 min. At the beginning of each day, the capillary was rinsed with 1 M NaOH for 15 min and ultrapure water for 10 min. Between runs, the capillary was rinsed with 1 M NaOH for 1 min, ultrapure water for 1 min and BGE for 1.5 min. At the end of each day, the capillary was conditioned with 1 M NaOH for 15 min and ultrapure water for 10 min to ensure a clean environment of the capillary inner wall. 2.3 Magnetic Fe3O4 nanoparticles preparation Magnetic Fe3O4 nanoparticles were synthesized by solvothermal method [41]. Briefly, FeCl3·6H2O (1.35 g) was dispersed in ethylene glycol (40 mL), forming orange solution. Subsequently, NaAc (3.6 g) and PEG (1.0 g) were added into the above solution and the reaction was proceeded by vigorously magnetic stirring for 30 min. The obtained solution was then transferred into a Teflon-lined stainless-steel autoclave and heated to 200oC for 24 h. When cooled to room temperature, the resultant black products were washed several times with ultrapure water, ethanol and dried in a vacuum drying oven at 60oC for 8 h. 2.4 Fe3O4@TiO2 nanoparticles preparation For Fe3O4@TiO2 preparation [42], Fe3O4 nanoparticles (NPs) (80 mg) was 9
thoroughly dispersed in a mixture of N,N-dimethylformamide (20 mL) and isopropyl alcohol (60 mL) under the condition of ultrasonic vibration for 30 min. Subsequently, titanium butoxide (4 mL) was added into the above obtained suspension drop by drop and the reaction was proceeded for 30 min at room temperature. The obtained mixture was transferred into a Teflon-lined stainless-steel autoclave and heated at 200℃ for 24 h. When cooled to room temperature, the synthesized product was successively washed several times with ultrapure water, ethanol, lyophilized and stored at 4℃ for future use. The preparation procedure of the Fe3O4@TiO2/CRL nanoparticles was shown in Fig. 1. 2.5 Enzyme immobilization The lipase was successfully immobilized on the synthetic Fe3O4@TiO2 NPs through the following step. Briefly, Fe3O4@TiO2 NPs were homogenously dispersed in 0.1 M HCl solution to activate surface hydroxyl groups of Fe3O4 @TiO2 NPs. Then the supernatant of the above suspension was discarded by applying external magnet. After rinsing with ultrapure water until neutral, the resultant magnetic Fe 3O4 @TiO2 NPs were stored in phosphate buffer at 4℃ for future use. In order to determine the optimum conditions for enzyme immobilization, the immobilization pH (3, 4, 5, 6, 7), immobilization time (1, 2, 3, 4, 5 h) and immobilization enzyme concentration (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 mg/mL) were investigated, respectively. 5 mg of Fe3O4@TiO2 NPs were uniformly dispersed in 1 mL of phosphate buffer, adding 1 mL of lipase solution into the suspension under the vigorous shaking. After shaking for a period of time at room 10
temperature, the mixture was separated magnetically, rinsing several times with phosphate buffer, dispersed in 2 mL of phosphate buffer and stored at 4℃ for following study. 2.6 Characterization According to the fourier-transform infrared (FT-IR) spectra, the chemical composition of synthetized Fe3O4 NPs and Fe3O4@TiO2 NPs were characterized with a Nexus 870 Fourier Transform Infrared Spectrometer (Thermo Fisher, USA), and the wavenumbers were ranged from 500 cm-1 to 4000 cm-1. Through an X’pert PRO Xray diffraction (PANalytical B.V., Netherlands) with Ni-filtered Cu Kα radiation over the angular range from 8° to 90°, the crystal structures of the magnetic NPs were characterized. The morphological features of the pure Fe3O4 and Fe3O4@TiO2 NPs were obtained through a transmission electron microscope (TEM) (FEITECNAI G2TF20, USA). The particle size distributions of the magnetic NPs was determined through the Malvern Zeta sizer Nano ZS (Malvern Instruments, UK) working on the dynamic light scattering (DLS) platform. The magnetic properties were characterized by vibrating sample magnetometer (VSM) (Lakeshore, USA). 2.7 Immobilized enzyme kinetics study The obtained immobilized enzyme was dispersed into a homogeneous solution under the condition of ultrasonication. Subsequently, 150 μL of the above suspension was transferred into 1.5 mL Eppendorf tube. The supernatant was discarded by applying external magnet, and adding 300 μL of substrate solution into the above suspension. The final concentration of the substrate was 0.4, 0.6, 0.8, 1.0, 1.2 mM, 11
respectively. The reaction mixture was incubated at 60oC for 15 min and the reaction was terminated by magnetically separating the immobilized enzyme nanoparticles from the mixture. Finally, the supernatant was injected into CE for further analysis. 2.8 Inhibition study and inhibitors screening Inhibition study was investigated by calculating Km of the immobilized lipase. Km, Michaelis-Menten constant [22], which illustrates the affinity of enzyme for substrate, was calculated using the Lineweaver-Burk plot:
Where S represents the concentration of the substrate and v means the peak areas of the product pNP. Under the optimum conditions, the Km of the immobilized lipase was measured by analyzing different concentrations of pNPP ranging from 0.4 mM to 1.2 mM. Each substrate concentration was operated in triplicated. For lipase inhibitor screening, 150 μL of Fe3O4@TiO2/CRL was mixed with 300 μL of substrate solution following 100 μL of Tibetan medicine extract was added and incubated at 60oC. 15 min later, the immobilized enzyme NPs was separated and the supernatant was analyzed by CE. The lipase inhibitor screening for compounds were operated using the same procedure above mentioned, except compounds were substitute for Tibetan medicines extracts.
3. Results and discussion 3.1 Characterization TEM images obtained for Fe3O4 and Fe3O4@TiO2 NPs were shown in Fig. 2. As 12
can been seen, the spherical Fe3O4 NPs (Fig. 2a) showed excellent dispersibility and its Energy Dispersive X-Ray Spectroscopy (EDS) data was listed in Fig. S1a. After the solvothermal reaction between Fe3O4 NPs and titanium butoxide, the Ti element emerged in Fig. S1b, in addition to the Fe and O element, proving the successful preparation of TiO2 on the surface of Fe3O4 NPs. In Fig. 2b, the classic core-shell structure of Fe3O4@TiO2 NPs formed and their surface became rough (Fig. 2b, inset), which could provide sufficient binding sites for lipase. Meanwhile, the dispersibility of magnetic NPs still maintained, which was also beneficial to lipase immobilization. The DLS characterization of the magnetic NPs was applied to illustrate the existence of TiO2 shell. According to Fig. 2c, the diameter of Fe3O4 NPs was about 350.7 nm. After the modification of TiO2, the diameter of Fe3O4@TiO2 NPs (Fig. 2d) was about 386.4 nm, which demonstrated the successful preparation of TiO2 and its diameter was about 17.9 nm. The magnetic NPs were characterized by FT-IR spectra, in order to determine their chemical composition. The characteristic peak of Fe-O group was observed around 577 cm-1 (Fig. 3a), which confirmed the successful formation of Fe3O4. The adsorption peak at 443 cm-1 in Fig.3b was assigned to stretching vibration of Ti-O bonds, revealing the porous TiO2 shells had been coated on the surface of Fe3O4. Furthermore, XRD analysis was also applied to characterize the magnetic NPs. For Fe3O4 (Fig. 3b), six characteristic diffraction peaks ((220), (311), (400), (422), (511) and (440)) were observed, which corresponded to the standard pattern for Fe3O4 (JCPDS #65-3107), further confirming the cubic spinel structure of Fe3O4 13
NPs.
Meanwhile, almost similar XRD pattern of Fe3O4@TiO2 (Fig. 3b) NPs was obtained as compared to Fe3O4 NPs, indicating the formation of the TiO2 shell would not change the crystal structure of Fe3O4 NPs. Furthermore, the magnetic properties of magnetic NPs were characterized by VSM. The analysis result (Fig. S2) demonstrated that the magnetic NPs were superparamagnetism and could be conveniently separated from the reaction matrix. 3.2 Optimization of the immobilization conditions In order to attain the immobilized lipase with high relative activity, the effect of the immobilization pH, immobilization time and immobilization lipase concentration were investigated. Fig. 4a showed the influence of pH on the relative activity of the immobilized lipase. In this set of experiments, the immobilization pH value ranging from 3.0 to 7.0 was investigated (immobilization time 2 h, immobilization lipase concentration 1.4 mg/mL, enzymatic reaction time 15 min, enzymatic reaction temperature 60 oC). As indicated in Fig. 4a, the highest relative activity was obtained at the pH value of 4.0, thus the optimum immobilization pH was set to be 4.0. The influence of immobilization time, ranging from 1 h to 5 h (immobilization pH 4.0, immobilization lipase concentration 1.4 mg/mL, enzymatic reaction time 15 min, enzymatic reaction temperature 60oC), on the relative activity were also investigated. As indicated in Fig. 4b, the relative activity tended to increase from 77.8% to 100% with increasing immobilization time from 1 h to 2 h, indicating that lipase molecules have been successfully immobilized on the supports. However, the relative activity was declined 14
to 80.3% with prolonging duration time to 5 h. This is mostly due to excess lipase immobilized on the support led to the inaccessibility of the substrate to the active sites of lipase [43]. Based on the results, it could be ensured that the best immobilization time was 2 h. In Fig. 4c, different immobilization lipase concentrations ranging from 0.2 mg/mL to 2.0 mg/mL (initial lipase concentration) was employed for lipase immobilization on the magnetic support (immobilization pH 4.0, immobilization time 2h, enzymatic reaction time 15 min, enzymatic reaction temperature 60℃). As illustrated in Fig. 4c, the best immobilization lipase concentration of 1.4 mg/mL was employed. With the lipase concentration increased from 0.2 mg/mL to 1.4 mg/mL, the relative activity of the immobilized lipase was enhanced from 39.3% to 100%. However, increasing the lipase concentration beyond 1.4 mg/mL caused the relative activity to decrease. With the immobilization enzyme loaded excessively, the surface of the magnetic support was saturated, thus leading to an intermolecular steric hindrance among the immobilized lipase molecules and further limiting the diffusion of the substrate to the lipase molecules [43, 44]. Based on the above results, it could be concluded that the best immobilization lipase concentration was 1.4 mg/mL. 3.3 Optimization of the enzymatic reaction conditions The influence of enzymatic reaction time on the free and immobilized lipase activities were investigated in Fig. 5a. The relative activity of the immobilized lipase tended to increase from 40.4% to 100% with prolonging reaction time from 5 min to 15 min. And it tended to decrease beyond 15 min, which was mostly due to the lipase 15
deactivation with a longer reaction time. At the enzymatic reaction time of 15 min, both the free and immobilized lipase showed the highest relative activity and the relative activity of immobilized lipase was higher than that of the free lipase in the studied reaction time. Taking the highest reaction efficiency into consideration, the enzymatic reaction time was fixed at 15 min. The effects of temperature on the free and immobilized lipase relative activity were also measured, as illustrated in Fig. 5b. The relative activity of the immobilized lipase was enhanced with a rise in the temperature from 40 oC to 60oC, reaching the highest enzymatic activity at 60oC. With the temperature increased beyond 60oC, the relative activity of the immobilized lipase tended to decrease. Besides, there was a same trend for the relative activity of the free lipase. Meanwhile, the relative activity of the immobilized lipase was always higher than that of the free lipase at the same conditions, which could be attributed to the stabilized backbone of the immobilized lipase molecules. As a consequence, the optimum temperature for the enzymatic reaction was set as 60oC. 3.4 Reusability and storage stability of the immobilized lipase Reusability of the immobilized enzyme is an important factor for cost-effective usage of lipase in the large-scale application. Thus, the reusability of the immobilized lipase was studied under the optimum reaction conditions. After the first usage, the immobilized lipase was separated by applying an external magnet and followed by washing with phosphate buffer solution for the subsequent consecutive usages. As indicated in Fig. S3, the results showed about 49.6% of its initial activity maintained 16
after 5 successive reuses of the immobilized lipase. The reason for such a decrease in the relative activity was the denaturation of lipase. The storage stability of the immobilized lipase is also a crucial factor for the practical applications. The storage stabilities of immobilized and free lipase were tested by measuring the enzyme activity after a storage period of 30 days at 4℃, respectively. As shown in Fig. S4, the immobilized lipase lost about 22.4% of its initial activity after 30 days storage. However, the free lipase lost about 64.7% of its initial activity. Based on the above results, the immobilized lipase showed better storage stability and in accordance with the literature [45]. 3.5 Kinetics study of the immobilized lipase As Fig. S5 demonstrated the linearity between the concentration of pNPP and the corrected peak areas, velocity was represented by the corrected peak areas in this work. According to the Michaelis-Menten model, the Lineweaver-Burk plot was 1/v = (0.005662 ± 0.0001901) (1/[S]) + (0.00226 ± 0.000298) with a determination coefficient of 0.9983 (Fig. 6a). And Km of the immobilized lipase was calculated to be 2.51 mM and 9.96 mM for the free lipase (Fig. S6), indicating that the immobilized lipase had a higher affinity for pNPP than that of the free lipase, which demonstrated that the immobilized lipase obtained better catalytic activity. Enzyme inhibitor affects the binding of the enzyme to the substrate, resulting in the reduction or denaturation of enzyme activity. From the inhibition kinetics of the enzyme, the type of action of the inhibitor, the inhibition constant of the inhibitor and the inhibition rate could be determined. In this work, Orlistat was employed as a 17
model inhibitor and its inhibition performance was studied. In the presence of different concentrations of Orlistat, the Lineweaver-Burk plot for the immobilized lipase was shown in Fig. 6b-d. With an increase of Orlistat concentration, the value of vertical axis intercept (1/Vmax) increased and the value of horizontal axis intercept (1/Km) remained unchanged. Taking above results into consideration, Orlistat is a noncompetitive inhibitor, which was consistent with the literature [46, 47]. And its value of Ki, inhibition constant, was calculated to be 13.41 μM by secondary plot method (Fig. S7), The half maximal inhibitory concentration (IC50) is also an important parameter to evaluate the inhibitory ability of the inhibitor. Under a condition of 0.5 mM substrate solution, a range of different concentrations of Orlistat were added to obtain the inhibition plot (Fig. 7). By plotting the inhibition rate versus the longarithm of Orlistat concentration, IC50 was determined as 4.37 mM, which was in accordance with the literature [14, 17]. The inhibition rate was calculated as follows: Inhibition (%) = (1-x/blank)×100
Eq. (1)
Where x and blank represent the peak areas of pNP measured with and without inhibitor, respectively. Z’ factor, a characteristic parameter which is used to evaluate the quality and accuracy of a drug screening method [48]. When the Z’ factor is in the range of 0.5 to 1, a screening method is accurate and feasible. In our work, Z’ factor was determined as 0.95 (n=9), demonstrating that the inhibitor screening method is excellent. The above results illustrated that the established method was feasible for the following 18
lipase inhibitor screening. 3.6 Inhibitors screening from Tibetan medicines Under the optimum conditions, the immobilized lipase was employed to screen lipase inhibitors from 6 Tibetan medicines, AL, ZO, OF, CM, AK and GU, and their inhibitions were calculated according to the Eq. (1). According to the above screening results (Fig. 8), AL, ZO, CM, AK and GU showed relatively weak inhibitory activities against lipase even at the concentration of 100 mg/mL, indicating these Tibetan medicines might not suitable for the treatment of obesity. However, Oxytropis falcate Bunge provided 54.0% inhibition at the concentration of 50 mg/mL, which was superior to the other 5 Tibetan medicines. When the concentration was increased to 100 mg/mL, its inhibition (68.6%) was obviously higher than those of 5 Tibetan medicines, exhibiting the strongest inhibition performance against lipase. Thus, Oxytropis falcate Bunge might be a potential drug for the treatment of obesity. To further explore the lipase inhibitory activity of Oxytropis falcate Bunge, 11 compounds from Oxytropis falcate Bunge isolated by our laboratory were screened, and the results were listed in Fig. 9. The detailed information of the 11 compounds from Oxytropis falcate Bunge was shown in the Fig. S8. When the concentrations of the tested compounds were 50 μM, compounds 1, 2, 3, 6 possessed similar lipase inhibitory activities to Orlistat. The lipase inhibitory activities of compounds 5 and 7 were 20% higher than that of Orlistat, and compound 10 (kaempferol) was almost 30% higher than that of Orlistat. When the concentrations of the tested compounds 19
increased to 100 μM, compounds 1, 2, 5, 6, 7, 8, 9 possessed similar lipase inhibitory activities to Orlistat and compound 10 was obviously superior to Orlistat. These results demonstrated that compounds 1, 2, 5, 6, 7 might be potential drugs for obesity treatment. Surprisingly, compound 10 had the best inhibitory activity against lipase among these compounds and Orlistat. Thus, compound 10 might be a new pharmaceutical molecule for obesity treatment. Meanwhile, an amazing medicinal value of anti-obesity for Oxytropis falcate Bunge was discovered in this work, and anti-obesity function was different from the anti-inflammatory and analgesic reported in the literatures [49, 50]. From the perspective of inhibitors screening, it could be concluded that compounds 1, 2, 5, 6, 7, 10 might be elected as candidates for antiobesity drugs and their inhibitory mechanisms need to be researched. In addition, the successful screening of these potential lipase inhibitors also confirmed the feasibility of the established method. Thus, this method is expected to be applied to other inhibitors screening.
4. Conclusion An immobilized enzyme method for screening lipase inhibitors coupled with CE from Tibetan medicines was realized in this study. Fe3O4@TiO2 nanoparticles was prepared and employed as the magnetic support to provide sufficient binding sites for lipase. Through electrostatic interaction, the lipase was immobilized on the surface of magnetic support and its surface morphology, chemical composition, crystal structure, kinetics study, reusability and storage stability were systematically investigated. The kinetics performances of the immobilized lipase were carefully studied. The prepared 20
Fe3O4@TiO2/CRL was successfully applied to screen lipase inhibitors from Tibetan medicines, and a platform for screening lipase inhibitors from TCMs was successfully developed. Oxytropis falcate Bunge, which was a classic Tibetan medicine with significant anti-inflammatory effect, was screened out for its supreme inhibitory activity. Furthermore, the inhibitory activity of the 11 compounds in Oxytropis falcate Bunge were analyzed, compounds 1, 2, 5, 6, 7 exhibited similar inhibitory activity to Orlistat, and compound 10 (kaempferol) presented better inhibitory activity than Orlistat.
In our opinion, this work could not only establish a method of
immobilization lipase for the screening lipase inhibitors from Tibetan medicines, but also provide a step forward the development of weight-loss drugs.
Declarations of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos. 21775153, 21804135 and 21974145), and the Scholar Program of West Light Project of the Chinese Academy of Sciences.
Author Contributions Section Jia Liu: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing-Original Draft. Run-Tian Ma: Visualization, Investigation, Writing-Review & Editing. Yan-Ping Shi: Supervision, Project administration, Funding acquisition. 21
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Figure captions
Figure 1. The schematic of Fe3O4@TiO2/CRL nanoparticles preparation.
28
Figure 2. TEM images ((a) and (b)) and DLS graphs ((c) and (d)) of Fe 3O4 and Fe3O4@TiO2 nanoparticles.
Figure 3. FT-IR spectra of (a) Fe3O4@TiO2, Fe3O4 nanoparticles and XRD spectra of (b) Fe 3O4, Fe3O4@TiO2 nanoparticles.
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Figure 4. Influence of (a) immobilization pH, (b) immobilization time and (c) immobilization enzyme concentration on the relative activity of the immobilized lipase.
Figure 5. Influence of (a) enzymatic reaction time and (b) temperature on the relative activity of the immobilized lipase.
Figure 6. Lineweaver-Burk plot of lipase in the presence of the inhibitor Orlistat. The concentrations of Orlistat: (a) 0 μM; (b) 10 μM; (c) 30 μM; (d) 50 μM. 30
Figure 7. Inhibition plot of Orlistat to immobilized lipase.
Figure 8. Results of lipase inhibitors screening from 6 kinds of Tibetan medicines.
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Figure 9. Results of lipase inhibitors screening from Oxytropis falcate Bunge.
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