YAN Luyun, HU Weiping, WU Xiangrong, ZHANG Bowen, ZHU Jinhua*, ZHAO Dongbao,2

(1. Henan International Joint Laboratory of Medicinal Plants Utilization,College of Chemistry and Chemical Engineering,Henan University, Kaifeng 475004,Henan, China; 2.Center for Multi-Omics Research,School of Life Science,Henan University,Kaifeng 475004,Henan,China)

Abstract: It is challenging to establish an efficient method for the preparative isolation and purification of organic acids from L. japonica leaves. In this work, we used ZIF-8 as sorbent for selective extraction of organic acids from L. japonica leaves and studied the adsorption mechanism. The main parameters such as the amount of absorbent, adsorption time, extraction temperature and desorption conditions were evaluated. Under the optimum condition, the components enriched on ZIF-8 were found mainly to be organic acids (neochlorogenic acid, chlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C). The recoveries of ZIF-8 for compounds 1-6 at three spiked levels (0.5, 5, 10 mg·L-1) in the ethyl acetate phase were in the range 70.7%-107.7%, with RSDs between 0.89% and 7.57%. Furthermore, we proposed that the adsorption of ZIF-8 for organic acids were mainly attributed to surface adsorption, electrostatic and hydrogen bond interactions. These results demonstrated the feasibility of MOFs in selective extraction of active components from complex medicinal plants.

Keywords: L. japonica leaves; organic acids; ZIF-8; selective extraction

LonicerajaponicaThunb is a common medicinal plant, which plays an significant role in traditional Chinese medicine formula for treating various diseases, including arthritis, diabetes mellitus, fever, infections, sores and swelling[1-5]. Our research have proved that the composition such as organic acids, flavonoids, iridoids, alkanes, olefins and sterols in flower bud, leaves and stem parts ofLonicerajaponicaThunb were not so much difference[6]. Furthermore, the production ofLonicerajaponicaleaves was five times larger than that of flowers, they can be used as an alternative medicinal resource to the flowers of the plant[7]. Many studies have indicated that the extracts of leaves have antioxidant and tyrosinase-inhibitory properties and might be a potential source of preservatives for use in the food or pharmaceutical industries. Therefore, it is urgent to establish an efficient method for the preparative isolation and purification of active compounds fromL.japonicaleaves. Till now, many conventional separation methods (e.g., silica gel, polyamide column chromatography, macroporous resin, HSCCC and Sephadex LH-20) have been used for separating compounds fromL.japonicaleaves[1-2,7]. However, due to the structural similarity and their instability, there are many disadvantages of these methods such as irreversible sample adsorption, complicated multiple steps requirement and large solvent consumption. Therefore, it is quite challenging not only for the development of materials science but also for the discovery of new drugs or leading compounds to explore novel sorbents which have good selectivity and large capacity to extract and separate of active components from medicinal plants.

Metal-organic frameworks (MOFs) are an emerging class of hybrid inorganic organic microporous crystalline materials self-assembled from metal ions with organic linkers via coordination bonds[8]. Due to their unique properties, such as high surface area, good thermal stability, uniform structured nanoscale cavities, availability of in-pore functionality and outer-surface modification, MOF materials have great potential to be candidates in gas storage[9-10], chromatography[11-13], separation science[14-15], drug delivery[16], catalysis[17]and sensors[18]. Due to the exceptional prospects of MOFs in adsorption and the significant roles of active components in complex medicinal plants, application of MOFs in selective extraction and separation of active components from complex medicinal plants have been a promising topic. Several research groups reported the use of MOF materials for the separation and adsorption of compounds from natural plants. For example, PU/GO/BA-MOF was applied for selective adsorption and effective separation of cis-diol containing luteolin (LTL) from peanut shell coarse extract[19]. CUI et al.[20]reported their primary attempt of zeolitic imidazolate framework-8 (ZIF-8) as a model MOF for the selective extraction of a flavonoid named 3,4-dihydroxy-8,9-methylenedioxypterocarpan from a traditional medicinal plantCaraganaJubata.

ZIFs belong to a subclass of MOFs with zeolite-type topologies, constructing from tetrahedral metal ions bridged by imidazolate. ZIFs are not only exhibit high porosity and large surface areas like other MOFs, but also show exceptional thermal and chemical stability in water and organic solvents, making it promising to be a suitable candidate for adsorption and extraction. ZIF-8 is one of the widely studied and applied ZIFs, which is formed from zinc ions and 2-methylimidazole (Hmim) linkers with sodalite (SOD) topology[21]. In addition, organic acids are the typical components inL.japonicaleaves, including chlorogenic acid, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, neochlorogenic acid and caffeic acid, featuring many significant pharmacological activities such as anti-inflammatory, antioxidant, antibacterial, antiviral[22-25]. In the present work, we reported the selective adsorption of organic acids extracted fromL.japonicaleaves using ZIF-8, and a adsorption mechanism based on theoretical study using DFT (Density Functional Theory) and Zeta potential investigation were proposed.

1 Materials and methods

1.1 Chemicals and reagents

All chemicals and reagents were at least of analytical grade. Ultrapure water (18.2 MΩ·cm-1) purified with a Milli-Q purification system (Millipore, Bedford, MA, USA) was used throughout all experiments. Zinc nitrate hexahydrate (99%), acetic acid and 2-methylimidazole (98%) were bought from Aladdin Chemistry Co., Ltd (Shanghai, China). Methanol, petroleum ether,n-butanol, dichloromethane (DCM), and ethyl acetate were purchased from Concord Technology Co., Ltd. (Tianjin, China). HPLC grade acetonitrile were obtained from Merck KGaA (Darmstadt, Germany), Neochlorogenic acid, Chlorogenic acid, Caffeic acid, Isochlorogenic acid A, Isochlorogenic acid B, Isochlorogenic acid C were purchased from Cdmust Biotechnology Co., Ltd (Chengdu, China).

1.2 Instruments and HPLC conditions

The X-ray powder diffraction (XRD) measurement was recorded on a Bruker D8 Advance X-ray diffractometer with Cu Kαradiation. Infrared absorption spectrum was performed using infrared fourier transform spectrometer (Bruker, VERTEX 70). The surface areas of the materials were calculated according to the Brunauer-Emmett-Teller (BET) method obtained from nitrogen adsorption-desorption isotherms (ASAP 2020, Micromeritics, USA). The thermal gravimetric analysis (TGA) was performed on a DMA861e thermal gravimetric analyzer (Mettler Toledo, Switzerland) from room temperature to 800 ℃ at a ramp rate of 10 ℃·min-1. JEM 2100 transmission electron microscopy (TEM) have been adopted as an effective tool to characterize the microstructure and morphology of the nanoparticles. X-ray photoelectron spectroscopy (XPS) measurements were performed on an ESCALAB 250XI (Thermo Fisher Scientific Ltd. USA).

The separation and determination were performed using HPLC system (Agilent 1260 series, Agilent Technologies, Calif., USA) coupled to a diode array detector (DAD). The analytes were separated by COSMOSIL 5 C18-PAQ column (4.6×250 mm, 5 μm). The temperature was kept at 25 ℃ and the injection volume was 10 μL, and the flow rate was 1 mL·min-1. The mobile phase consisted of water containing 5% (volume fraction acetic acid (A) and acetonitrile (B). The gradient elution program was as follows: 0-10 min = 12%-20% B, 10-30 min = 20%-40% B, 30-35 min = 40%-12% B. The DAD detector wavelength was set at 327 nm.

1.3 Synthesis of ZIF-8

ZIF-8 was synthesized by a modified version of a rapid room temperature method[26]. Typically, a solution of Zn(NO3)2·6H2O (2.933 g, 9.87 mmol) in methanol (200 mL) was rapidly poured into a solution of 2-methylimidazole (6.489 g, 79.04 mmol) in methanol (200 mL) under magnetic stirring. The mixture was slowly turned turbid and after 1 h the nanocrystals were separated from the milky dispersion by centrifugation, washed with fresh methanol, and dried at 60 ℃.

1.4 Preparation of ethyl acetate extract of L. japonica leaves and standard solution

L.japonicaleaves were collected from Henan University campus, Kaifeng, China during January 2019. The air-driedL.japonicaleaves were smashed and then extracted with 80% methanol at 80 ℃. The methanol extract was suspended in water and partitioned successively with petroleum ether, DCM, ethyl acetate, andn-butanol. The ethyl acetate phase ofL.japonicaleaves was prepared for further use.

The stock standard solution of neochlorogenic acid (6 mg), chlorogenic acid (13 mg), caffeic acid (7.5 mg) and isochlorogenic acid (A,B,C) (14 mg, respectively) were prepared in methanol in the same 50 mL volumetric flask. The other concentration of mixed standard solution was prepared by diluting of the stock solution with methanol. The standard solution was selected as the model matrix for extraction.

1.5 Extraction of e-a extract and standard solution on ZIF-8

The whole procedure was showed in Fig.1. Initially, the standard solution (diluted 10 times) or the ethyl acetate extract sample (0.2 g·L-1) was prepared in centrifuge tubes. The adsorption and desorption processes are all completed at room temperature. For the extraction, 6 mg of ZIF-8 were mixed with 3 mL ethyl acetate extract sample. Subsequently, the mixture was vortexed 30 s at 3 000 r/min and then was decanted into a 3.0 mL empty polypropylene tube which was equipped with a frit and a 0.45 μm filter membrane on the bottom. Thereafter, the polypropylene tube was connected with SPE vacuum manifold and the ZIF-8 sorbent was intercepted into the tube. Then the collected ZIF-8 were desorbed with 3 mL PBS solution under ultrasonication for 30 min. The tube connected with SPE vacuum manifold again. Finally, the desorption solution was filtered through 0.22 μm filter and 10 μL was injected into HPLC system for further analysis.

Fig.1 Schematic illustration for selective extraction of organic acids from L. japonica leaves using ZIF-8 as the sorbent

2 Results and discussion

2.1 Characterization of ZIF-8

The synthesized ZIF-8 was characterized with XRD, FTIR, BET, TGA (Fig.2) and TEM (Fig.3). The spectrum of ZIF-8 nanocrystals (Fig.2a) was characterized by the high intensity peaks including (011), (002), (112), (022), (013), (222) ,(233) and (134) which is completely consistent with the previous work, which also demonstrate the high crystalline of ZIF-8 nanoparticles[27]. The functional groups of ZIF-8 were identified using FTIR spectroscopy. In fact, the peak at 424 cm-1corresponds to stretching bend of Zn-N. The peaks located at 500-1 350 and 1 350-1 500 cm-1, corresponding to plane bending and stretching of the imidazole ring, respectively. The peaks corresponding to C=N stretching appeared at 1 583 cm-1, aromatic and aliphatic C-H stretching located at 2 913 and 3 123 cm-1, respectively (Fig.2b). The specific surface area and pore volume of the synthesized ZIF-8 were calculated from N2adsorption-desorption experiments (Fig.2c). The Brunauer-Emmett-Teller surface area, total pore volume and pore width of ZIF-8 were 1609 m2·g-1, 0.99 cm3·g-1and 1.076 nm, respectively. The TGA curve shows that the synthesized ZIF-8 is thermally stable up to 450 ℃ (Fig.2d). The TEM image exhibits the synthesized ZIF-8 is hexagonal crystals and the particle size was about 25 nm (Fig.3).

Fig.2 Characterization of the synthesized ZIF-8: (a) XRD patterns; (b) FT-IR spectra; (c) N2 adsorption-desorption isotherms; (d)TGA curve

Fig.3 TEM images of the synthesized ZIF-8

2.2 Optimization of extraction process

The relevant experimental parameters such as desorption condition, adsorbent dosage, extraction time, and extraction temperature were investigated to achieve high extraction efficiencies by the ZIF-8 nanoadsorbent. In order to further analyze the enrichment ability of ZIF-8 to organic acids, we prepared standard solution of compounds1-6and discussed the optimum conditions. All experiments were performed in triplicate and the results were used for optimization.

2.2.1 Effects of desorption condition

In the process of adsorption and desorption experiments, it is very important to select a solvent which can effectively elute the target analyte from the adsorbent. Five common solvents, namely ethyl alcohol, acetonitrile, ethyl acetate, methanol and PBS were tested. The results of HPLC analysis showed that PBS buffer solution had the highest desorption ability, while other organic solvents could not achieve good desorption effect (Fig.4a). Firstly, the hydrogen bond between macromolecules of organic acid and adsorbent ZIF-8 was greatly weakened by the phosphate contained in PBS buffer solution. Subsequently, we studied the effect of pH on the desorption efficiency of organic acids by ZIF-8. Fig.4b showed the results found in a pH range from 3 to 8, as can be seen that the desorption efficiency begins to decrease rapidly when the pH was higher than 6. Therefore, PBS with pH=5.8 was chosen as eluent. These results can be explained by the intermolecular forces and electrostatic interactions. In addition, through Zeta potential analysis, we found that organic acid biomacromolecules have negative charges, while ZIF-8 has positive charges. Under PBS (pH=5.8) organic acid biomacromolecules take negative charges, thus weakening the electrostatic interaction with adsorbent ZIF-8.

2.2.2 Effects of adsorption condition

The effect of adsorbent dosage is among the key factors that influence the adsorption efficiency what makes the study of this parameter is a major necessity. To optimize the amount of sorbent for extraction procedure, different amount of ZIF-8 ranging from 1.0 to 15.0 mg were studied. Fig.4c showed that the extraction efficiencies of the analytes (Neochlorogenic acid, Chlorogenic acid, Caffeic acid, Isochlorogenic acid A, Isochlorogenic acid B, Isochlorogenic acid C) all increased with increasing adsorbent amount from 1 to 6 mg, and then reached a plateau. Therefore, the optimum amount of adsorbent is 6 mg.

The optimum time for target extraction was determined by varying the extraction time from 0.2 to 3.5 min. Fig.4d showed that when the time was increased from 0.2 to 3.5 min, there was no significant improvement in the extraction efficiency. Therefore, 0.5 min was sufficient for complete extraction.

The extraction temperature also affects the adsorption efficiency. Fig.4e showed that the extraction efficiencies is maximum when the temperature is 25 ℃, presumably because the ZIF-8 was synthesized at room temperature.

Fig.4 Effects of (a) desorption solvent, (b) pH of PBS, (c) amount of absorbent, (d) adsorption time, and (e) extraction temperature on efficiencies of organic acids extractions. The peaks 1-6 were neochlorogenic acid, chlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C in turn

2.3 Evaluation of method performance

The proposed method was evaluated under the optimum conditions by performing a series of experiments to determine the linear range, correlation coefficient, LOD, and precision. The obtained calibration data are summarized in Table 1. Good linearity was achieved in different linear range for compounds1-6with the correlation coefficients (R2)≥0.996 7. The limits of detection (LODs) for the samples were determined at the signal-to-noise ratios of 3 times. The RSDs for compounds1-6were <4.1%. Obviously, the proposed method had high sensitivity and reproducibility and could be used to analyze real samples containing the organic acids.

Table 1 Regression equations, Correlation coefficient (R2), Linear range of compounds 1-6

2.4 Selective extraction of compounds 1-6

The ethyl acetate phase ofL.japonicaleaves was analyzed by HPLC separation using the optimized mobile phase, and its components were found to be complex (Fig.5a). The ethyl acetate phase ofL.japonicaleaves was enriched and extracted by ZIF-8, and the remaining solution was detected by HPLC. It was found that most of compounds1-6decreased, but other components remained (Fig.5b). Then, the adsorbed ZIF-8 was eluted by PBS solution. The components enriched on ZIF-8 were found to be mainly compounds1-6by HPLC analysis (Fig.5c). According to the previous studies of our group and through standard addition experiments, we determined that compounds1-6were neochlorogenic acid, chlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C in turn (Fig.6). Through calculating the peak areas of compounds1-6in the ethyl acetate phase before adsorption and desorption solution, recoveries of ZIF-8 for compounds1-6are 137.1%, 103.4%, 89.5%, 169.6%, 134.2% and 108.7%，respectively.

Fig.5 HPLC chromatograms of the ethyl acetate extract of L. japonica leaf before adsorption (a) and after adsorption (b) with ZIF-8, and the desorption solution of ZIF-8 after adsorption (c). The peaks identification was the same as in Fig.4 (a)

Fig.6 Optimized 3D structures of compounds 1-6

Table 2 showed the recoveries of ZIF-8 for compounds1-6at three spiked levels (0.5, 5, 10 mg·L-1) in the ethyl acetate phase were in the range 70.7%-107.7%, with RSDs between 0.89% and 7.57%. Furthermore, the table showed that with the increase of spiked levels, the recoveries of the compounds1-3(neochlorogenic acid, chlorogenic acid, caffeic acid) decreases gradually. The phenomenon likely resulted from the composition of Isochlorogenic acid A, B, C are much higher than other compounds. Also, another reason is that the components of the ethyl acetate phase ofL.japonicaleavesare very complex. In summary, ZIF-8 is more capable of enriching and separating organic acids in ethyl acetate phase ofL.japonicaleaves than other compounds.

Table 2 Recoveries and relative standard deviations (RSDs, n=5) of compounds 1-6 in the ethyl acetate extract at three different spiked levels

2.5 Adsorption mechanism study

To explain the adsorption mechanism of ZIF-8 for compounds1-6(1-Neochlorogenic acid, 2-Chlorogenic acid, 3-Caffeic acid, 4-Isochlorogenic acid B, 5-Isochlorogenic acid A, 6-Isochlorogenic acid C), the physicochemical properties of compounds1-6, including molecular weight, pKaand logKow, were obtained from the SciFinder scholar database. Also, the 3D structures of compounds1-6were optimized using Gauss View 5.0. In addition, the molecular dimensions of compounds1-6are about 1.505 nm×0.760 nm×0.506 nm, 1.565 nm×0.968 nm×0.564 nm, 1.003 nm×0.588 nm×0.436 nm, 1.732 nm×1.406 nm×1.307 nm, 1.522 nm×1.005 nm×1.958 nm, and 1.170 nm×1.337 nm×1.529 nm, respectively (Table 3). However, the pore width of ZIF-8 is 1.076 nm based on N2adsorption-desorption experiments (Fig.2c). These results indicated that only caffeic acid is smaller than that of the ZIF-8 pore and other compounds are much larger than the ZIF-8. Therefore, the selectivity of ZIF-8 for caffeic acid resulted from the intra-pore adsorption and other compounds resulted from the surface adsorption.

Table 3 Physicochemical properties of compounds 1-6

To further reveal the adsorption mechanism of ZIF-8 for compounds1-6, we tested the Zeta potential of ZIF-8 and organic acids in methanol solution (Fig.7). It is obvious from this figure that ZIF-8 have positive surface charges, while the mixed standard solution have more negative surface charges and a small amount of positive charges. Therefore, the selectivity of ZIF-8 for caffeic acid resulted from the electrostatic interaction.

Fig.7 Zeta potential of ZIF-8 and organic acids

The X-ray photoelectron spectroscopy (XPS) experiments were further applied to elucidate the adsorption mechanism (Fig.8).There were no obvious changes of the bonding energies for Zn 2p1/2and 2p3/2peaks of ZIF-8 before and after adsorption of organic acids, suggesting the Zn metal center in ZIF-8 should not be the binding sites for organic acids. However, the N1 s peaks at 400.95 eV for ZIF-8 were shifted to 398.99 eV after adsorption of organic acids, revealing the N sites on ZIF-8 played significant roles for adsorption of organic acids. Therefore, we speculate that the N…H…O hydrogen bond interactions between the two N atoms on ZIF-8 and two OH on organic acids result in the good selectivity of ZIF-8 for organic acids.

Fig.8 XPS spectra of (a) Zn and (b) N for ZIF-8 before and adsorption of organic acids

3 Conclusions

In this work, we have reported the feasibility of ZIF-8 to selectively extract of the organic acids (neochlorogenic acid, chlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C) out from the complex medicinal plants extract ofL.japonicaleaves. Good linear ranges, precision and satisfactory recoveries were achieved under the optimum conditions. In addition, we elucidated the adsorption mechanism of ZIF-8 for organic acids from surface adsorption, electrostatic and the hydrogen bond interactions. Such method makes us possible to rapidly and simply enrich a class of components from complex matrices which can be hardly achieved on the separation procedures using traditional methods. The follow-up works should pay more attentions to the post-modification of MOFs in extraction and separation of pharmacological active components from medicinal plants.