Yi Tao*ab,
Yanhui Jiangab,
Weidong Liab and
Baochang Caiab
aSchool of Pharmacy, Nanjing University of Chinese Medicine, Xianlin Campus, 138 Xianlin Avenue, Nanjing 210023, PR China. E-mail: taoyi1985812@126.com; Fax: +86-25-86798281; Tel: +86-25-86798281
bJiangsu Key Laboratory of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing, 210023, PR China
First published on 11th October 2016
A very convenient, sensitive and precise solid-phase extraction approach was established for extract and analysis of acetylcholinesterase binders from crude extract of Corydalis yanhusuo. This approach was based on the retention of binders by acetylcholinesterase immobilized zeolite. The retained acetylcholinesterase binders were eluted and analyzed by ultra-high performance liquid chromatography and quadrupole-time-of-flight mass spectrometry. The powder X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier transform infrared (FT-IR) spectroscopy techniques were employed for the characterization of acetylcholinesterase immobilized zeolite. Some experimental conditions such as incubation temperature, time, buffer pH and ion strength, which may affect binding capability, were investigated by using coptisine as a model inhibitor. The optimal incubation conditions were as follows: wash times: 4, wash solvent: 50% methanol–water, incubation time: 20 min, temperature: 37 °C, ion strength: 10 mM, pH: 7.4. The proposed approach was successfully applied for the extraction of acetylcholinesterase binders from crude extract of Corydalis yanhusuo. These binders were further validated by acetylcholinesterase inhibitory assay. Fourteen acetylcholinesterase inhibitors were identified, and ten of which, including dehydrocorydaline, allocryptopine, corydaline, dehydroglaucine, protopine, tetrahydrocoptisine, tetrahydropalmatine, corynoline, tetrahydrocolumbamne and tetrahydroberberine, were reported for the first time. In addition, the merits and shortcomings of zeolite based solid-phase extraction approach were compared with that of magnetic nanoparticles based solid-phase extraction approach.
Corydalis yanhusuo, which is a folk medicine distributed in southeast of China, is employed in traditional Chinese medicines as an analgesic agent for treating spastic pain, abdominal pain, menstrual pain, and pain due to injuries.3 The major constituents of Corydalis yanhusuo were protoberberine-type alkaloids and tetrahydroprotoberberine-type alkaloids.4 Coptisine, one of the major alkaloids, exhibited significantly inhibitory effects against AChE. Moreover, quaternary ammonium alkaloids of many medicinal plants are important plant metabolites with significant effects on inhibition of neuromuscular transmission.5 Therefore, it was hypothesized that other AChE inhibitory agents may exist in the plant.
Zeolites are metastable crystalline aluminosilicate molecular sieves with uniform pores of molecular dimensions that are widely applied in catalysis,6–8 separations,9 and adsorption.10 Adsorption is one of the simplest methods to immobilize enzyme and presents the additional advantage of being a “soft” and inexpensive method. Main advantages of using zeolites as support for enzyme immobilization11 include (i) the materials typically exhibit high internal surface areas, allowing them to physically adsorb significant amounts of enzymes, (ii) generating a strong but reversible immobilization that enables us to recover expensive enzymes,12,13 and (iii) providing a possibility to selective separation of immobilized enzymes from a reaction mixture.
In this study, a new approach using AChE immobilized zeolite to solid-phase extraction of AChE binders from crude extract of Corydalis yanhusuo was established. A flowchart of the proposed approach is shown in Fig. 1. First, zeolites were immersed into AChE solution and adsorbed AChE onto their surface. Second, the crude extract of Corydalis yanhusuo was incubated with AChE immobilized zeolite for an appropriate period. Subsequently, the unbound compounds and nonspecific binding compounds were separated and abandoned, whereas the specific bound compounds were dissociated from AChE immobilized zeolite using organic denaturing reagents. The specific bound compounds were analyzed by ultra-high performance liquid chromatography and quadrupole-time-of-flight mass spectrometry and their activities were validated using AChE inhibitory assay. In addition, the merits and shortcomings of zeolite based solid-phase extraction approach were compared with that of magnetic nanoparticles based solid-phase extraction approach.
![]() | ||
Fig. 1 Schematic diagram of the acetylcholinesterase immobilized zeolite-based solid-phase extraction approach. |
Three parameters were investigated in the current study to optimize the adsorption procedure. First, experiments with different kinds of zeolites (i.e. zeolite Y, zeolite ZSM-5 and zeolite beta) and a constant amount of AChE (5 mL, 2 mg mL−1) were performed to study the amount of protein efficiently immobilized on the zeolites. Samples of the supernatants were withdrawn and the amount of entrapped protein on the zeolites was calculated from the protein mass balance among the initial and final AChE solutions. Second, PBS solutions with different pH values (5.8, 6.2, 6.8, 7.2 and 8.0) were investigated. Third, different temperatures (25, 30, 37 and 45 °C) were used to study the influence of temperature on the adsorption efficiency. Analysis of variance was used to assess the results.
Recovery of AChE from adsorbents under certain conditions is an important issue. Therefore, desorption behavior of AChE from zeolites was also performed. Prior to the desorption experiment, AChE solution and zeolites were incubated at room temperature, because the adsorption equilibrium of the AChE on zeolites was sufficiently reached around 45 min under this experimental condition. The mixtures were centrifuged to obtain zeolite-adsorbed fractions which include zeolites and adsorbed AChE. Then, zeolite-adsorbed fractions were washed three times with fresh buffer to exclude non-specific and weakly adsorbed AChE. Desorption of AChE adsorbed on zeolites was performed by addition of 0.2 M NaOH solutions to the zeolite-adsorbed fraction. The mixture of NaOH solution and zeolite-adsorbed fraction was incubated for 45 min at 37 °C. Supernatant was obtained by flash spin down at 12000 rpm for several seconds. Concentration of desorbed protein in supernatant was determined by Bradford method. Meanwhile, the pH of desorbed protein solution was adjusted to 7.0 by adding HCl solution. Then, the protein solution was freeze-dried to obtain protein powder. The activity of the desorbed protein powder was compared with that of original protein powder.
The conjugation of AChE onto amine-terminated magnetic nanoparticles was performed by a typical glutaraldehyde activation procedure. 25 μL amine-terminated magnetic nanoparticles were suspended in 120 μL 1% glutaraldehyde for 1 h. The amine-terminated magnetic nanoparticles were recovered by magnetic separation and washed three times with phosphate buffer solution to remove excess glutaraldehyde. Then, the amine-terminated magnetic nanoparticles was suspended in AChE solution (1.0 mg mL−1, dissolved in phosphate buffer solution) and shaken for 2 h at 25 °C, after which, the AChE conjugated magnetic nanoparticles was recovered by magnetic separation and thoroughly rinsed with phosphate buffer solution three times to remove unbound AChE. Experiments with varying amounts of magnetic nanoparticles (25, 50, 75, 100, 125, 150 and 175 μL) and a constant amount of AChE (100 μL, 1 mg mL−1) were performed to study the amount of protein efficiently immobilized on the magnetic nanoparticles. The supernatant and three wash solutions were kept in order to determine AChE loading efficiency by Bradford method. The AChE conjugated magnetic nanoparticles were dispersed in phosphate buffer solution and stored at 4 °C for further experiments.
Coptisine exhibited AChE inhibitory activity and was a kind of major alkaloids.14 Alkaloids were the major constituents of Corydalis yanhusuo. The same kind of compounds shared the same characteristics. Therefore, coptisine was selected as a model compound to optimize the extraction condition. Six parameters were investigated. First, different wash times (one to five times) and different denature solvents including acetonitrile–water and methanol–water, each with five proportions (10, 30, 50, 70 and 90%, v/v), were investigated. Samples of the washed supernatants were withdrawn and analyzed by HPLC. Second, the effect of gradient pH (5.7, 6.2, 6.8, 7.4 and 8.0) of PBS was studied. Third, different incubation time (5, 10, 20, 30 and 40 min) were investigated. Fourth, different ion strengths of the incubation buffer PBS (10, 50, 100, 200, 500 and 1000 mM) were studied. Finally, gradient incubation temperatures (20, 25, 30, 37 and 45 °C) were investigated.
The specificity of the method was determined by analyzing AChE non-binder (caffeic acid) for interference at the retention times of the AChE binder (coptisine). Specificity was assessed by comparing the peak of coptisine to that in a solution spiked with analyte at 0.1 mg mL−1 for coptisine and caffeic acid. The AChE immobilized zeolites based solid-phase extraction was carried out as described above. The calibration curves were constructed by plotting the peak area of coptisine to its gradient concentrations (0.002–1 mg mL−1) on the basis of linear regression model. Lower limit of quantification (LLOQ) was defined as the lowest analytical concentration on the calibration curve, which generated S/N ratios of about 10. The accuracy and precision of the method were evaluated by repeated analyses of QC samples (0.016 mg mL−1 for coptisine) on three consecutive days. The measured precision was expressed as relative standard deviation (R.S.D.), and the accuracy was expressed as relative error (R.E.). Matrix effects were investigated on crude extract of Corydalis yanhusuo, by calculating the ratio of the peak area of coptisine in the presence of crude extract of Corydalis yanhusuo to the peak area in absence of crude extract of Corydalis yanhusuo at three different QC concentrations (low, medium, and high).
AChE immobilized zeolites based solid-phase extraction was performed according to the above procedure. Briefly, 20 μL aliquots of crude extract of Corydalis yanhusuo was incubated with 0.025 mg AChE immobilized zeolites in a total volume of 200 μL PBS buffer (10 mM, pH 7.4). After incubation at 37 °C for 20 min, AChE immobilized zeolites were washed four times with 200 μL of the PBS buffer and the supernatants were abandoned. The AChE immobilized zeolites were subsequently incubated in 20 μL of 50% (v/v) methanol/water for 10 min to dissociate specific bound compounds and the eluate was collected and analyzed via UPLC-Q-TOF/MS. Denatured AChE immobilized zeolites and blank zeolites were used as control to exclude the nonspecific binding.
AChE conjugated magnetic nanoparticles based solid-phase extraction was performed as below. The reaction mixtures of the crude extract of Corydalis yanhusuo (20 μL) and AChE conjugated magnetic nanoparticles (0.045 mg) were prepared in phosphate buffer solution to reach a final volume of 200 μL, and incubated for 20 min at 37 °C using water bath. The AChE conjugated magnetic nanoparticles were washed four times with 200 μL of the buffer. Finally, the magnetic nanoparticles were subsequently incubated in 20 μL of 50% (v/v) methanol/water for 10 min to disclose specific bound compounds. The eluate was collected and retained for analysis via UPLC-Q-TOF-MS.
TEM image of magnetic nanoparticles revealed an average diameter of 300 nm (see Fig. S4†). The close-up of the AChE conjugated magnetic nanoparticles was present in Fig. S4B.† The red arrows clearly shows the presence of thin gray film on the surface of the AChE conjugated magnetic nanoparticles, indicating AChE was successfully conjugated onto the surface of magnetic nanoparticles. The main peaks of the X-ray diffraction of the magnetic nanoparticles, such as 2.9706, 2.5322, 2.1008, 1.7122, 1.6146 and 1.4837, matched well with the standard magnetite Fe3O4 XRD spectrum (see Fig. S5†). Magnetization curves of blank magnetic nanoparticles (black) and AChE conjugated magnetic nanoparticles (red) were shown in Fig. S6.† Maximum saturation magnetization (50.2 emu g−1) of AChE conjugated magnetic nanoparticles is a little less than that (79.4 emu g−1) of blank magnetic nanoparticles due to the nonmagnetic AChE on the surface. The amounts of magnetic nanoparticles were varied to determine the optimal ratio for AChE binding efficacy with a constant amount of AChE. As shown in Fig. S7,† with increasing the amount of Fe3O4 added at a constant tyrosinase amount of 100 μg, the amount of tyrosinase bound gradually increased and then reached a plateau (∼99%) when the amount of Fe3O4 added was above 175 μL. The results suggested that a ratio of 175 μL Fe3O4 per 100 μg tyrosinase was sufficient for immobilization.
The effect of pH values of the incubation buffer was also investigated. The variation in buffer pH from 5.7 to 7.4 increased the percentage of adsorbed AChE from 86.7% to 91.8%. The difference was statistically significant (see Fig. S8B†). The influence of temperatures on the adsorption was studied and the result was presented in Fig. S8C.† The highest percentage of adsorbed AChE was achieved when the incubation temperature was set to 26 °C. In summary, the optimal conditions of AChE adsorption were as follows: adsorption support: zeolite beta; incubation pH: 6.2; incubation temperature: 26 °C.
Desorption of AChE from zeolites was performed by addition of 0.2 M NaOH solutions to the AChE adsorbed zeolite. The mixture of NaOH solution and zeolite-adsorbed fraction was incubated for 45 min at 37 °C. Supernatant was obtained by flash spin down at 12000 rpm for several seconds. Concentration of desorbed AChE in supernatant was determined by Bradford method. The time-desorption ratio curve was shown in Fig. S9.† About 90% desorption was achieved after 15 min's desorption. Further increase the incubation time didn't lead to the increase of desorption ratio. Meanwhile, the activity of recovered protein powder was comparable with that of original protein powder by incubation with the substrate.
The type of possible interactions between zeolites and AChE are complicated. Since the zeolite structures have microporous pores which are too small with respect to the kinetic diameter of the AChE, the adsorption occurs solely on the external crystalline surface. They could consist in acid–base reactions between the amino groups of the protein and the surface hydroxyl groups of the zeolites (silanol groups) or van der Waals or electrostatic interactions. The schematic illustration of the possible mechanism of adsorption and desorption behavior of the AChE on zeolite beta were shown in Fig. S10.† AChE was comprised of basic amino and acidic carboxyl functional groups as well as a characteristic side chain. The amino and carboxyl groups give the enzyme unusual electrolytic properties. At high pH, the carboxyl group tends to be dissociated, giving the compound a negative charge. At low pH, the amino group, as well as the overall molecule, becomes positively charged. At an interim pH known as the isoelectric point, enzyme in solution has a net charge of zero. The isoelectric point of AChE was about 7.5. As shown in Fig. S8,† the optimal pH for adsorption was determined to be 6.2, which was below the isoelectric point of AChE. The net charge of AChE was positive. It may substitute the positive charged H+ sites in the H-type zeolite beta. After immersed into NaOH solution, the positive charged AChE was neutralized and dissociated from the zeolite. Na+ ion may take the place of AChE on zeolite. In summary, electrostatic interactions play the pivotal role in the adsorption and desorption behavior of AChE onto the zeolite.
The pH of a solution can have several effects on the structure and activity of enzymes. Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis. In general, enzyme has a pH optimum. The isoelectric point of AChE was 7.5 and the pKa value of coptisine was 10.7. AChE was negative charged within the pH range 5.7–7.5, while coptisine was positive charged within the pH range 7.5–8.0. The electrostatic attraction between AChE and coptisine increased when the pH gradually increased. As was shown in Fig. S12A,† the largest binding degree of coptisine was achieved at pH around 7.4.
The length of incubation time is a factor affecting the binding degree. As was shown in Fig. S12B,† the optimum incubation time was about 20 min. Further increase of the incubation time led to unfavorable binding, indicating that the active site of AChE had already been saturated with coptisine after 20 min' incubation. Therefore, 20 min was selected as the length of incubation time.
The total ion concentration in solution will affect important properties such as the distribution of charge on exterior surfaces of enzyme, in addition to the reactivity of the catalytically active groups. As was shown in Fig. S12C,† the increasing in the PBS concentration from 10 mM to 1000 mM led to the decrease in the bound ratio of coptisine. This phenomenon may be explained by the fact that the surface charge of AChE was neutralized by excessive PBS, which led to a significant decrease in the electrostatic interaction between coptisine and AChE. Thus, PBS concentration of 10 mM was chosen in this experiment.
Enzymes are catalysts and act to reduce the amount of activation energy required for a reaction to occur. Raising the temperature will reduce the amount of required activation energy allows for the reaction to occur, but also may denature an enzyme so that it is no longer functional. Lowering the temperature will reduce the rate of the reaction so low as to make it seem non-functional. In general, enzyme has a temperature optimum. As was shown in Fig. S12D,† the optimum temperature was about 37 °C due to the largest binding degree at this temperature.
Collectively, the optimal AChE immobilized zeolite based solid-phase extraction conditions were as follows: pH value: 7.4, incubation time: 20 min, ion strength: 10 mM, temperature: 37 °C.
The calibration curve, linear range, LLOD, LLOQ, and repeatability of coptisine were performed using the developed approach. Reasonable correlation coefficient values (r2 = 0.9977) showed good correlations between coptisine concentrations and its peak area within the ranges tested. The LLOD and LLOQ for coptisine were determined as 0.293 μg mL−1 and 0.488 μg mL−1, respectively. The repeatability present as RSD (n = 6) was 0.99%. The overall intra- and inter-day variations (RSD) of coptisine were 0.98% and 3.79%, respectively. The developed approach showed good accuracy with the recoveries ranging from 103% and 105% (see Table 1). As shown in Fig. S14,† the activity of AChE immobilized zeolite retained 89% after ten cycles. Reusability can decrease the test costs and enhance the experimental efficiency. Collectively, the results demonstrated that the method was sensitive, precise, and accurate enough for zeolites based solid-phase extraction of AChE binders.
Sample | Initial (μM) | Added concentration (μM) | Found concentration (μM) | Recovery (%) | RSD (%) |
---|---|---|---|---|---|
Crude extract | 166.47 | 195.12 | 366.55 | 103 | 2.2 |
780.47 | 974.99 | 104 | 0.9 | ||
1560.49 | 1811.96 | 105 | 0.7 |
No. | tR (min) | MS2 | Formula | ESI-MS(+) | Identification | Structure type | |
---|---|---|---|---|---|---|---|
Measured mass [M]+ or [M + H]+ | Error (ppm) | ||||||
a Compared to standard compounds, I: benzylisoquinoline alkaloid; II: protopine-type alkaloids; III: protoberberine-type alkaloids; IV: tetrahydroprotoberberine-type alkaloids; V: aporphine-alkaloids; VI: sphingolipid. | |||||||
1 | 3.261 | 177.0549, 145.0285 | C17H23NO4 | 307.1771 | −2.3 | Unknown | — |
2 | 5.674 | 271.1339, 269.1186, 237.0911, 209.0966, 175.0750, 143.0491, 107.0495 | C19H23NO3 | 314.1760 | 3.0 | Armepavine | I |
3 | 5.902 | 178.0869, 163.0628 | C19H21NO4 | 328.1554 | 3.2 | Scoulerine | IV |
4a | 7.314 | 178.0867, 163.0627 | C20H23NO4 | 342.1710 | 3.0 | Tetrahydrocolumbamine | IV |
5 | 7.504 | 341.1634, 192.1023, 177.0787 | C21H25NO4 | 356.1866 | 2.7 | Glaucine | V |
6 | 7.911 | 341.1638, 192.1026, 177.0790 | C21H25NO4 | 356.1865 | 2.4 | Glaucine isomer | V |
7a | 8.448 | 336.1249, 275.0716, 247.0764, 206.0820, 189.0787, 188.0712, 149.0600 | C20H19NO5 | 354.1348 | 3.4 | Protopine | II |
8 | 8.933 | 341.1641, 192.1026, 178.0867, 163.0633, 151.0765 | C21H25NO4 | 356.1867 | 3.0 | Isocorybulbine | IV |
9a | 9.463 | 352.1561, 290.0953, 206.0820, 189.0789, 188.0714 | C21H23NO5 | 370.1662 | 3.5 | Allocryptopine | II |
10a | 9.722 | 325.1447, 310.1208, 295.0983, 294.1255, 279.1023, 192.1015, 176.0708, 165.0910 | C21H25NO4 | 356.1868 | 3.3 | Tetrahydropalmatine | IV |
11a | 10.240 | 292.0977, 277.0747, 262.0874 | C19H14NO4 | 320.0928 | 3.3 | Coptisine | III |
12a | 11.410 | 354.1716, 192.1020, 165.0912 | C22H27NO4 | 370.2026 | 3.6 | Corydaline | IV |
13 | 11.706 | 337.1326, 336.1245, 322.1094, 308.1298, 294.1142, 293.1063 | C21H22NO4 | 352.1555 | 3.3 | Dehydrocorybulbine | III |
14a | 12.782 | 337.1325, 336.1239, 322.1090, 308.1293, 294.1138, 292.0958 | C21H22NO4 | 352.1558 | 4.2 | Palmatine | III |
15a | 13.078 | 321.1017, 320.0936, 306.0775, 304.0988, 292.0984, 278.0823 | C20H18NO4 | 336.1243 | 3.8 | Berberine | III |
16a | 14.133 | 350.1406, 336.1252, 322.1454, 308.1300, 306.1150 | C22H24NO4 | 366.1714 | 3.9 | Dehydrocorydaline | III |
17 | 18.223 | 337.0968, 322.0737, 306.0781 | C20H18NO5 | 352.1199 | 5.5 | Berberastine | III |
18 | 23.192 | 256.2649, 106.0869, 102.0920, 88.0766, 70.0667 | C16H35NO2 | 274.2752 | 4.2 | Hexadecasphinganine | VI |
19 | 23.340 | 300.2914, 256.2638, 102.0919, 88.0764, 70.0665 | C18H39NO3 | 318.3020 | 5.4 | Phytosphingosine | VI |
20 | 24.184 | 425.2209, 379.2231, 249.1446 | C22H28O10 | 453.1699 | 0.6 | Unknown | — |
21 | 24.455 | 284.2956, 106.0865, 88.0762 | C18H39NO2 | 302.3064 | 3.5 | Sphinganine | VI |
22 | 25.386 | 212.2378, 91.0544, 58.0670 | C21H38N | 304.3010 | 3.7 | Unknown | — |
23 | 25.765 | — | C27H36O7 | 473.2534 | 0.0 | Unknown | — |
24 | 27.431 | — | C25H28O7 | 441.1913 | 1.2 | Unknown | — |
25 | 30.538 | 321.3172, 303.3060, 163.1484, 149.1325, 135.1170, 97.1012, 83.0863 | C22H44NO | 338.3433 | 3.0 | Unknown | — |
The developed AChE immobilized zeolites based solid-phase extraction approach was applied to the crude extract of Corydalis yanhusuo and the results were shown (see Fig. 3B–D). Compound 1–8, 10–12, 14, 16 and 17 were the AChE binders identified from crude extract of Corydalis yanhusuo. The AChE conjugated magnetic nanoparticles based solid-phase extraction approach was also used to extract binders from the crude extract of Corydalis yanhusuo and the results were displayed in Fig. 4. Compounds 4, 6–12, 14 and 16 were identified as AChE binders. A Venn diagram was generated for demonstrating commonly and exclusively detected AChE binders by using the two solid-phase extraction approaches (see Fig. S17†). Nine compounds, including tetrahydrocolumbamine, glaucine isomer, protopine, isocorybulbine, tetrahydropalmatine, coptisine, corydaline, palmatine, dehydrocorydaline, were the common AChE binders identified.
Compound 2 showed the [M + H]+ ion at m/z 314 and produced characteristic ions at m/z 107 and 206 in MS2 spectrum, which were formed by inductive cleavage and α-cleavage at the nitrogen of the tyramine moiety. Compared with the literature,16 compound 2 was tentatively deduced as armepavine. Aporphine alkaloids do not undergo the RDA reaction and are thus easily distinguished from the protopine and tetrahydroprotopine alkaloids. The retro-Diels–Alder (RDA) reaction is a characteristic fragmentation pathway of the tetrahydroprotoberberine. Compound 4 displayed [M + H]+ ion at m/z 342 and yielded the predominant product ions at m/z 178 and 163, corresponding to the RDA C-ring opening and loss of a methyl group (see Fig. S18A†). Compounds 3 and 4 gave the same product ions at m/z 178 and 163, but compound 3 showed an decrease of 14 Da of the corresponding [M]+ compared to compound 4, Meanwhile, compound 3 had an earlier retention time. Compared with the literature,16 compounds 3 and 4 were tentatively identified as scoulerine and tetrahydrocolumbamine. Compound 5 showed [M + H]+ ion at m/z 356 and gave the predominant product ions at m/z 192 and 177, which resulted from RDA C-ring opening and loss of a methyl group (see Fig. S18B†). Compound 5 was tentatively identified as glaucine. Compounds 6, 8 and 5 shared the same [M + H]+ ion at m/z 356 and produced the same fragment ions at m/z 341 and 192 (see Fig. S19A†). Considering the retention time of the three compounds, compound 6 and 8 were plausibly deduced as glaucine isomer and isocorybulbine.17
Common characteristic product ions of protopine-type alkaloids were yielded by various dissociation processes such as retro-Diels–Alder (RDA) fragmentation, dehydration, and successive dissociation of substitute groups in MS2 spectrum.17 Compounds 7 and 9 showed the [M + H]+ ions at m/z 354 and 370, respectively (see Fig. S18C†). Compared with the standards, compounds 7 and 9 were characterized as protopine and allocryptopine, respectively. Two compounds shared common characteristic ions at m/z 206, 189, and 188, which were produced by retro-Diels–Alder (RDA) fragmentations and the loss of H2O and hydroxyl group, respectively. The neutral loss of H2O led to product ions at m/z 336 of compound 7 and m/z 352 of compound 9 in MS2 spectrum. Compound 10 showed [M + H]+ ion at m/z 356 and produced characteristic ions at m/z 325, 310, 295, 279 in MS2 spectrum (see Fig. S19B†), which corresponded to the loss of a methoxy radical, one methyl radicals, two methyl radicals and a methoxy radical. Based on the above information, compound 10 was plausibly assigned as tetrahydropalmatine.
The characteristic product ions of protoberberine-type alkaloids were yielded by the successive cleavage of substituted groups of methyl, CO radical on the A- and D-rings.17 No RDA fragmentation occurs due to that it was difficult to open the ring. Compound 12 displayed [M + H]+ ion at m/z 370 and yielded the fragment ions at m/z 354, 192 and 165, which resulted from loss of CH2 group, RDA C-ring opening and rearranged RDA reactions. Thus, compound 12 was deduced as corydaline.18 Compounds 11 and 14–16 were identified as coptisine, palmatine, berberine and dehydrocorydaline and validated with reference standards. For instance, compound 16 showed [M]+ ions at m/z 354 and yielded fragment ions at m/z 350, 336, 322, and 308, corresponding to [M − CH3 − H]+, [M − 2CH3]+, [M − CH3 − H − CO]+, and [M − 2CH3 − CO]+. Compared with the literature,19 compound 16 was unambiguously deduced as dehydrocorydaline. Compounds 13 and 14 shared the same [M]+ ion at m/z 354 and produced the same fragment ions at m/z 336, 322, and 308 (see Fig. S19C†). Compounds 13 and 14 were structural isomers. Compared with the standard, compound 14 was unambiguously identified as palmatine. Compound 13 was tentatively deduced as dehydrocorybulbine in comparison with the literature.19
The assay results of other compounds were presented in Table 3. Palmatine chloride, exhibited the best AChE inhibitory activity with an IC50 value of 0.64 μM. Coptisine showed a comparable inhibitory effect with that of galantamine with an IC50 value of 4.40 μM. The AChE inhibitory activities of other alkaloids were decreased in the following order: dehydrocorydaline, berberine, columbamine, tetrahydroberberine, allocryptopine, dehydroglaucine, protopine, tetrahydrocolumbamine, tetrahydro-palmatine, tetrahydrocoptisine, corynoline and corydaline. The AChE inhibitory effect of columbamine, coptisine, palmatine and berberine had been reported before.14 The AChE inhibitory effects of dehydrocorydaline, allocryptopine, corydaline dehydro-glaucine, protopine, tetrahydrocoptisine, tetrahydropalmatine, corynoline, tetrahydro-columbamne and tetrahydroberberine were reported for the first time.
No. | Compound | IC50 (μM) ± SD |
---|---|---|
4 | Tetrahydrocolumbamine | 136.20 ± 2.77 |
7 | Protopine | 128.55 ± 3.91 |
9 | Allocryptopine | 104.48 ± 2.02 |
10 | Tetrahydropalmatine | 137.16 ± 3.19 |
11 | Coptisine | 4.40 ± 0.12 |
12 | Corydaline | 241.61 ± 21.37 |
14 | Palmatine chloride | 0.64 ± 0.00 |
15 | Berberine | 26.60 ± 0.50 |
16 | Dehydrocorydaline | 10.13 ± 0.07 |
Columbamine | 47.14 ± 0.57 | |
Tetrahydrocoptisine | 167.27 ± 4.58 | |
Corynoline | 198.67 ± 3.24 | |
Dehydroglaucine | 119.84 ± 3.12 | |
Tetrahydroberberine | 60.95 ± 3.54 | |
Control | Galantamine | 3.48 ± 0.02 |
Method | Zeolite based SPE method | Magnetic beads based SPE method |
---|---|---|
Enzyme immobilization mode | Adsorption | Covalent bonding |
Protein–material amount ratio (mg mg−1) | 1.76![]() ![]() |
0.57![]() ![]() |
Activity of the enzyme | Function | Function |
Reusability | Yes | Yes |
Regeneration | Yes (desorption) | No |
Environmentally friendly | Yes | No |
Cost | Cheap | Cheap |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra24585d |
This journal is © The Royal Society of Chemistry 2016 |