Phytochemical content and antioxidant activity of different anatomical parts of Corchorus olitorius and C. capsularis during different phenological stages.

Different parts of Corchorus olitorius and C. capsularis possess different antioxidant compounds. This study investigated the phytochemical components and antioxidant capacities of ultrasound-assisted extraction of different plant parts of both species using spectrophotometry at various phenological stages. Results also indicate that leaves, stems and roots of C. olitorius at various growth stages showed higher phytochemical components and antioxidant potential compared to C. capsularis. The phytochemical components from roots to leaves in C. olitorius including total polyphenol 0.97-11.11 mg GAE/g DW, total flavonoid 0.99-7.78 mg QE/g DW and total tannin 4.02-26.89 TA E/g DW, whereas C. capsularis total polyphenol 1.04-7.93 mg GAE/g DW, total flavonoid 0.77-5.5.92 mg QE/g DW and total tannin content 3.17-22.73 TA E/g DW. C. olitorius produced overall 22.23%, 13.61%, 12.24% higher total polyphenol, total flavonoid and total tannin, respectively compare to C. capsularis. Different parts extract also significantly affected antioxidant capacities including DPPH, ABTS, and FRAP activity with values of 22.03-79.46% inhibition, 10.84-104.10 µmol TE/g DW, and 10.84-104.10 µmol Fe2+/g DW respectively for C. olitorius, while C. capsularis demonstrated 14.03-70.97% of DPPH inhibition, 9.16-95.60 µmol TE/g DW of ABTS and 5.31-71.82 µmol Fe2+/g DW of FRAP activity. Moreover, leaves of the flowering stage, young stems and aged roots of both species displayed a higher content of phytochemical and antioxidant activities than other growth stages. A positive correlation between the phytochemical and antioxidant potential indicated that phenolic constituents solely affected antioxidant activity. Thus, this study established that the plant's parts and phenological growth stages significantly influence the concentration of phytoconstituents and antioxidant activities, and determine the harvesting stages of the different organs of C. olitorius and C. capsularis for considerable medicinal importance as folk and industry.

Fabrication of Ce-doped Hollow NiCo2 O4 Nanoprisms with Heterointerface from MOF-Engaged Strategy for Oxygen Evolution Reaction.

The reasonable design and controlled synthesis of efficient and hollow nanocatalysts with plentiful heterointerface and fully exposed active sites to accelerate the electron transfer and mass transfer process for oxygen evolution reaction (OER) is highly desirable for water splitting by electrolysis. Herein, a metal-organic framework (MOF)-engaged strategy is developed to prepare Ce-doped hollow mesoporous NiCo2 O4 nanoprisms (NiCo2 O4 /CeO2 HNPs) for enhanced OER. Due to the advanced synthesis strategy generating a large number of interfaces between NiCo2 O4 and CeO2 , as well as modulated electrons of the active center by the synergistic action of multi-metals, the obtained catalyst exhibits excellent OER performance with a small overpotential of 290 mV at current density (J) of 10 mA cm-2 . Spinel/Perovskite hollow nanoprisms synthesized by a similar way demonstrates the versatility of our strategy. This work may provide new insights into the development of rare earth-doped hollow polymetallic spinel oxide catalysts.

Optimizing elicitation strategy of salicylic acid for flavonoid and phenolic production of fed-batch cultured Oplopanax elatus adventitious roots.

Oplopanax elatus is a valuable medicinal plant, but its plant resource is lacking. Adventitious root (AR) culture of O. elatus is an effective way for the production of plant materials. Salicylic acid (SA) exerts enhancement effect on metabolite synthesis in some plant cell/organ culture systems. To clarify the elicitation effect of SA on fed-batch cultured O. elatus ARs, this study investigated the effects of SA concentration, and elicitation time and duration. Results showed that flavonoid and phenolic contents, and antioxidant enzyme activity obviously increased when the fed-batch cultured ARs were treated with 100 µM SA for 4 days starting on day 35. Under this elicitation condition, total flavonoid and phenolic contents reached 387 rutin mg/g DW and 128 gallic acid mg/g DW, respectively, which were significantly (p < 0.05) higher than those in the SA-untreated control. In addition, DPPH scavenging and ABTS+ scavenging rates, and Fe2+ chelating rate also greatly increased after SA treatment, and their EC50 values were 0.0117, 0.61, and 3.34 mg/L, respectively, indicating the high antioxidant activity. The findings of the present study revealed that SA could be used as an elicitor to improve the flavonoid and phenolic production in fed-batch O. elatus AR culture.

A Comprehensive Review of C. capsularis and C. olitorius: A Source of Nutrition, Essential Phytoconstituents and Pharmacological Activities.

Plant bioactive compounds have gained global significance in terms of both medicinal and economic ramifications due to being easily accessible and are believed to be effective with fewer side effects. Growing relevant clinical and scientific evidence has become an important criterion for accepting traditional health claims of medicinal plants and also supports the traditional uses of Corchorus as folk medicine. C. capsularis and C. olitorius have broad applications ranging from textile to biocomposite, and young leaves and shoots are used as healthy vegetables and have long been used as traditional remedies for fever, ascites, algesia, liver disorders, piles, and tumors in many cultures. This review systematically summarized and emphasized the nutritional attributes, mostly available bioactive compounds, and biological and potential pharmaceutical properties of C. capsularis and C. olitorius, disclosed to users and non-users. Results suggest that various phytochemicals such as cardiac glycosides, phenols, flavonoids, sterols, lipids, and fatty acids were found or analytically identified in different plant parts (leaf, stem, seed, and root), and many of them are responsible for pharmacological properties and their antitumor, anticancer, antioxidant, antinociceptive, anti-inflammatory, analgesic, antipyretic, antiviral, antibacterial, anticonvulsant, antidiabetic and antiobesity, and cardiovascular properties help to prevent and cure many chronic diseases. In addition to their use in traditional food and medicine, their leaves have also been developed for skin care products, and some other possible uses are described. From this review, it is clear that the isolated compounds of both species have great potential to prevent and treat various diseases and be used as functional foods. In conclusion, this comprehensive review establishes a significant reference base for future research into various medical and functional food applications.

Magnetic molecularly imprinting polymers, reduced graphene oxide, and zeolitic imidazolate frameworks modified electrochemical sensor for the selective and sensitive detection of catechin.

A glassy carbon electrode (GCE) was modified with magnetic molecularly imprinted polymers (mMIPs) using catechin as a template, reduced graphene oxide (rGO), and zeolitic imidazolate frameworks-8 (ZIF-8) for the sensitive detection of catechin (mMIPs/rGO-ZIF-8/GCE). The prepared rGO, ZIF-8, and mMIPs exhibited typical structures and properties determined by various characterizations. The mMIPs showed good selectivity for catechin among several structural analogs. The mMIPs/rGO-ZIF-8/GCE showed a higher maximum peak current for catechin than that of a single component modified GCE. After the optimization of the material ratio, coating amounts, pH, and scan rate, the mMIPs/rGO-ZIF-8/GCE exhibited good selectivity, good linearity, and a low detection limit (LOD) for catechin. The linear range was 0.01 nmol/L-10 µmol/L and the LOD was 0.003 nmol/L (S/N = 3). The relative standard deviations for reproducibility and stability tests (n = 6) were 5.2% and 6.1%, respectively. A recovery between 99.1 and 101.3% was obtained in the detection of catechin in spiked samples. Based on these findings, the proposed mMIPs/rGO-ZIF-8/GCE could be developed further, and future research could be conducted on alternate fabrication strategies and methods to create more portable and practical electrochemical sensors. Graphical Abstract.

A semi-supervised approach for extracting TCM clinical terms based on feature words.

BACKGROUND: A semi-supervised model is proposed for extracting clinical terms of Traditional Chinese Medicine using feature words. METHODS: The extraction model is based on BiLSTM-CRF and combined with semi-supervised learning and feature word set, which reduces the cost of manual annotation and leverage extraction results. RESULTS: Experiment results show that the proposed model improves the extraction of five types of TCM clinical terms, including traditional Chinese medicine, symptoms, patterns, diseases and formulas. The best F1-value of the experiment reaches 78.70% on the test dataset. CONCLUSIONS: This method can reduce the cost of manual labeling and improve the result in the NER research of TCM clinical terms.

Screening and Evaluation of Xanthine Oxidase Inhibitors from Gnetum parvifolium in China.

As a traditional natural medicine for treating many kinds of diseases, Gnetum parvifolium showed apparent inhibition on xanthine oxidase (XO). In this study, ultrafiltration combined with liquid chromatography-mass spectrometry (LC-MS) is used for the screening of XO inhibitors from Gnetum parvifolium. Their antioxidation, XO inhibition, and enzymic kinetic parameters are also determined. Finally, piceatannol (1), rhaponiticin (2), resveratrol (3), and isorhapontigenin (4) are screened out and identified as XO inhibitors from the extract of Gnetum parvifolium. Four inhibitors show better inhibition than allopurinol and good radical scavenging abilities. However, the antioxidant activities are weaker than ascorbic acid. The kinetic parameters illustrate the inhibition mode of XO by piceatannol is competitive type, while the inhibition modes for rhaponiticin, resveratrol and isorhapontigenin are uncompetitive types. In order to evaluate the difference among samples obtained in China, the amounts of four inhibitors and related activities in 20 samples are assessed and analyzed by partial least squares analysis. The results indicate piceatannol contribute the highest coefficients in three kinds of activities. Based on these findings, more comprehensive research on pharmaceutical and biochemical activities of these four XO inhibitors could be conducted in future.

In vitro effect of flaxseed oil and α-linolenic acid against the toxicity of lipopolysaccharide (LPS) to human umbilical vein endothelial cells.

Consumption of α-linolenic acid (ALA)-rich flaxseed oil is a possible way to supplement ω-3 polyunsaturated fatty acids, but the beneficial effects to cardiovascular systems are still controversial. In this study, human umbilical vein endothelial cells (HUVECs) treated with 100 ng/mL lipopolysaccharide (LPS) were used as the in vitro model and the potential beneficial effects of well-characterized flaxseed oil and ALA were studied. Exposure of HUVECs to LPS for 24 h significantly promoted inflammatory response as release of interleukin 6 (IL-6), soluble intercellular cell adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) as well as adhesion of THP-1 monocytes, but did not induce cytotoxicity or oxidative stress. ALA, but not flaxseed oil, significantly reduced LPS-induced release of sICAM-1 and sVCAM-1, without effect on THP-1 adhesion. No radical scavenging activity was observed after flaxseed oil or ALA exposure. Rather, ALA at high concentrations promoted intracellular superoxide associated with damages to lysosomes, which was not observed in flaxseed oil exposed cells. These results indicated that ALA at high concentrations could inhibit inflammatory responses in LPS-treated HUVECs in vitro but might also promote a modest effect in cytotoxicity and oxidative stress.

The effects of baicalein or baicalin on the colloidal stability of ZnO nanoparticles (NPs) and toxicity of NPs to Caco-2 cells.

Recent study suggested that the presence of phytochemicals in food could interact with nanoparticles (NPs) and consequently reduce the toxicity of NPs, which has been attributed to the antioxidant properties of phytochemicals. In this study, we investigated the interactions between ZnO NPs and two flavonoids baicalein (Ba) or baicalin (Bn) as well as the influence of the interactions on the toxicity of ZnO NPs to Caco-2 cells. The antioxidant properties of Ba and Bn were confirmed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, with Ba being stronger. However, the presence of Ba or Bn did not significantly affect cytotoxicity, intracellular superoxide or release of inflammatory cytokines of Caco-2 cells after ZnO NP exposure. When Ba was present, the cellular viability of Caco-2 cells after exposure to ZnO NPs was slightly increased, associated with a modest decrease of intracellular Zn ions, but these effects were not statistically different. Ba was more effective than Bn at changing the hydrodynamic sizes, Zeta potential and UV-Vis spectra of ZnO NPs, which indicated that Ba might increase the colloidal stability of NPs. Taken together, the results of the present study indicated that the anti-oxidative phytochemical Ba might only modestly protected Caco-2 cells from the exposure to ZnO NPs associated with an insignificant reduction of the accumulation of intracellular Zn ions. These results also indicated that when assessing the combined effects of NPs and phytochemicals to cells lining gastrointestinal tract, it might be necessary to evaluate the changes of colloidal stability of NPs altered by phytochemicals.

Antibacterial Activity of Polyphenols: Structure-Activity Relationship and Influence of Hyperglycemic Condition.

Polyphenols are plant-derived natural products with well-documented health benefits to human beings, such as antibacterial activities. However, the antibacterial activities of polyphenols under hyperglycemic conditions have been rarely studied, which could be relevant to their antibacterial efficacy in disease conditions, such as in diabetic patients. Herein, the antibacterial activities of 38 polyphenols under mimicked hyperglycemic conditions were evaluated. The structure-antibacterial activity relationships of polyphenols were also tested and analyzed. The presence of glucose apparently promoted the growth of the bacterial strains tested in this study. The OD600 values of tested bacteria strains increased from 1.09-fold to 1.49-fold by adding 800 mg/dL glucose. The polyphenols showed structurally dependent antibacterial activities, which were significantly impaired under the hyperglycemic conditions. The results from this study indicated that high blood glucose might promote bacterial infection, and the hyperglycemic conditions resulting from diabetes were likely to suppress the antibacterial benefits of polyphenols.

Screening and separation of α-amylase inhibitors from Solanum nigrum with amylase-functionalized magnetic graphene oxide combined with high-speed counter-current chromatography.

A screening method using α-amylase-functionalized magnetic graphene oxide combined with high-speed counter-current chromatography was proposed and utilized to screen and separate α-amylase inhibitors from extract of Solanum nigrum. The α-amylase-functionalized magnetic graphene oxide was characterized and found to demonstrate satisfactory structure, magnetic response (24.5 emu/g), and reusability (retained 90% of initial activity after five cycles). The conditions for the screening with α-amylase functionalized magnetic graphene oxide were optimized and set at pH 7.0 and 25°C. As a result, two potent flavonoid compounds, apigenin-7-O-glucuronide (1) and astragalin (2), were separated and collected through high-speed counter-current chromatography and subjected to high-performance liquid chromatography analysis with purity higher than 90% (according to HPLC data), which were identified as α-amylase inhibitors. These results suggested that utilization of α-amylase functionalized magnetic graphene oxide in the rapid screening and isolation bioactive compounds from complex natural products is a feasible and environmentally friendly method.

Rapid Screening and Identification of BSA Bound Ligands from Radix astragali Using BSA Immobilized Magnetic Nanoparticles Coupled with HPLC-MS.

Radix astragali is widely used either as a single herb or as a collection of herbs in a complex prescription in China. In this study, bovine serum albumin functionalized magnetic nanoparticles (BSA-MN) coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS) were used to screen and identify bound ligands from the n-butanol part of a Radix astragali extract. The prepared BSA-MN showed sufficient magnetic response for the separation with an ordinary magnet and satisfied reusability. Fundamental parameters affecting the preparation of BSA-MN and the screening efficiency were studied and optimized. Under the optimum conditions, four bound ligands were screened out from the n-butanol part of a Radix astragali extract and identified as genistin (1), calycosin-7-O-ß-d-glucoside (2), ononin (3) and formononetin (4). This effective method could be widely applied for rapid screening and identification of active compounds from complex mixtures without the need for preparative isolation.

Simultaneous determination of six synthetic phenolic antioxidants in edible oils using dispersive liquid-liquid microextraction followed by high-performance liquid chromatography with diode array detection.

A simple, rapid, organic-solvent- and sample-saving pretreatment technique, called dispersive liquid-liquid microextraction, was developed for the determination of six synthetic phenolic antioxidants from edible oils before high-performance liquid chromatography with diode array detection. The entire procedure was composed of a two-step microextraction and a centrifugal process and could be finished in about 5 min, only consuming only 25 mg of sample and 1 mL of the organic solvent for each extraction. The influences of several important parameters on the microextraction efficiency were thoroughly investigated. Recovery assays for oil samples were spiked at three concentration levels, 50, 100 and 200 mg/kg, and provided recoveries in the 86.3-102.5% range with a relative standard deviation below 3.5%. The intra-day and inter-day precisions for the analysis were less than 3.8%. The proposed method was successfully applied for the determination of synthetic phenolic antioxidants in different oil samples, and satisfactory results were obtained. Thus, the developed method represents a viable alternative for the quality control of synthetic phenolic antioxidant concentrations in edible oils.

Analysis of α-amylase inhibitor from corni fructus by coupling magnetic cross-linked enzyme aggregates of α-amylase with HPLC-MS.

As a carrier-free immobilization strategy, magnetic cross-linked enzyme aggregates (MCLEAs) showed improved enzyme activity, stability and magnetic response. In this study, MCLEAs of α-amylase (MCLEAs-amylase) was prepared under optimized conditions and characterized with scanning electron microscope and vibrating sample magnetometer. The prepared MCLEAs-amylase showed an amorphous structure and the saturation magnetization was 33.5emu/g, which was sufficient for magnetic separation. Then MCLEAs-amylase coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS) was utilized to screen and identify α-amylase inhibitors from ethyl acetate extract of corni fructus. The experiment conditions were optimized. At the optimum conditions (incubation time: 10min, pH: 7.0 and temperature: 20°C), querciturone was successfully screened and identified with weak non-specific binding. The screening result was verified by inhibition assays and the IC50 value of querciturone was 22.5µg/mL. This method provided a rapid way to screen active compounds from natural products.

[Flavonoids of Echinps latifolius suppress Wnt signaling in adjuvant arthritis rats].

The role of flavonoids of Echinps latifolius (FELT) in Wnt signaling was investigated in adjuvant arthritis (AA) rats. The therapeutic effects of FELT on AA rats were detected by rat arthritis score and MTT. The effect of FELT gavage treatment on the Wnt signaling key gene ß-catenin, C-myc and cyclin D1 in synovium from AA rats was detected by Real-time qPCR, and the effects of FELT gavage treatment on the upstream negative regulation gene SFRP 1,2,4,5 in synovium from AA rats were detected by Real-time qPCR. The results showed that FELT gavage treatment significantly inhibited arthritis score and MTT values in AA rats, significantly inhibited the expression of the Wnt signaling gene ß-catenin, C-myc and cyclin D1, significantly up-regulated the expression of the up- stream negative regulation gene SFRP 1,2,4. FELT has a better therapeutic effect for AA rats.

Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high-performance liquid chromatography and high-speed countercurrent chromatography.

A simple, rapid, and effective assay based on ultrafiltration combined with high-performance liquid chromatography and high-speed countercurrent chromatography was developed for screening and purifying alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root extract. Experiments were carried out to optimize binding conditions including alcohol dehydrogenase concentration, incubation time, temperature, and pH. By comparing the chromatograms, three compounds were found possessing alcohol dehydrogenase binding activity in Glycyrrhiza uralensis root. Under the target-guidance of ultrafiltration combined with the high-performance liquid chromatography experiment, liquiritin (1), isoliquiritin (2), and liquiritigenin (3) were separated by high-speed countercurrent chromatography using ethyl acetate/methanol/water (5:1:4) as the solvent system. The alcohol dehydrogenase inhibitory activities of these three isolated compounds were assessed; compound 2 showed strongest inhibitory activity with an IC50 of 8.95 µM. The results of the present study indicated that the combinative method using ultrafiltration, high-performance liquid chromatography and high-speed countercurrent chromatography could be widely applied for the rapid screening and isolation of enzyme inhibitors from complex mixtures.

Integration of magnetic solid phase fishing and off-line two-dimensional high-performance liquid chromatography-diode array detector-mass spectrometry for screening and identification of human serum albumin binders from Radix Astragali.

Radix Astragali is one of the most popular traditional medicinal herb and healthy dietary supplement. Isoflavonoids and astragalosides are the main bioactive ingredients. However, the systematic bioactive component analysis is inadequate so far. Then a facile method based on Fe3O4@SiO2-human serum albumin (Fe3O4@SiO2-HSA) magnetic solid phase fishing integrated with two-dimensional high-performance liquid chromatography-diode array detector-mass spectrometry (2D HPLC-DAD-MS(n)) was developed to fish out and identify HSA binders from Radix Astragali. The immobilized HSA displayed a high stability with 96.2% retained after ten consecutive cycles. 2D HPLC system (size exclusion chromatography×reversed phase chromatography, SEC×RP) were developed and optimised. Forty-seven bioactive compounds including thirty-four isoflavonoids and thirteen astragalosides were screened and identified or tentatively deduced based on their retention time, ultraviolet (UV), accurate molecular weight and diagnostic fragment ions. The results indicated that the integrated method could be widely applied for systematical fishing and identification of bioactive compounds, especially for low-abundance and overlapped compounds, from complex mixtures.

Selective fishing and analysis of xanthine oxidase binders from two Fabaceae species by coupling enzyme functionalized core-shell magnetic nanoparticles with HPLC-MS.

Xanthine oxidase (XOD) immobilized core-shell magnetic silica (Fe3O4@SiO2-XOD) nanoparticles coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS) was developed to fish out and analyze XOD binders from two Fabaceae species, Puerariae lobata flower and Glycyrrhiza uralensis root. The prepared Fe3O4@SiO2-XOD nanoparticles exhibited good specificity for XOD binders, better dispersion in aqueous solution and reusability than those of Fe3O4-XOD nanoparticles. The amount of XOD immobilized onto Fe3O4@SiO2 nanoparticles was 339.9µg/mg and the activity of Fe3O4@SiO2-XOD nanoparticles remained 95% after ten times usage. The optimum conditions of selective fishing were optimized, and finally incubating pH was set at 7, incubating temperature at 25°C and adsorption time at 30min. Twelve XOD binders were successfully identified from ethyl acetate extract of P. lobata flower and G. uralensis root. The developed method provides a rapid, purposeful and effective way to identify active compounds from natural complex mixtures.

Analysis of tyrosinase binders from Glycyrrhiza uralensis root: evaluation and comparison of tyrosinase immobilized magnetic fishing-HPLC and reverse ultrafiltration-HPLC.

Tyrosinase inhibitors play an important role in cosmetic products, food supplements and medicinal industries. In this study, a new facile method based on tyrosinase immobilized magnetic fishing (IMF) coupled with high performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS) was developed to screen and identify tyrosinase binders from Glycyrrhiza uralensis root. Experiments were carried out to select optimal immobilization and incubation conditions. The activity of immobilized tyrosinase retained 76.3% after ten consecutive cycles, and remained over 95% when stored at 4°C for about two months. Eleven tyrosinase binders, including original compounds and transformation products were successfully screened and characterized from G. uralensis root, while dihydrodaidzein and pratensein were reported for the first time. Compared with ultrafiltration-HPLC assay, IMF-HPLC displayed many attractive advantages, including convenient solid-liquid separation ability, good stability, high reusability and activity, which indicated that IMF-HPLC had a broad applicability. The results indicated that IMF-HPLC method is a facile, effective and reproductive way to screen and identify active components from complex mixtures.

Separation and purification of bovine serum albumin binders from Fructus polygoni orientalis using off-line two-dimensional complexation high-speed counter-current chromatography target-guided by ligand fishing.

In this study, off-line two-dimensional (2D) complexation high speed counter-current chromatography (HSCCC) was developed for the separation of bovine serum albumin (BSA) binders from the ethyl acetate extract of Fructus polygoni orientalis. Target-guided strategy of BSA functionalized iron oxide magnetic nanoparticles coupled with high performance liquid chromatography-tandem mass spectrometry ((BSA-Fe3O4 MNPs)-HPLC-MS/MS) experiment was proposed. In the orthogonal separation system, a Normal-Phase HSCCC with 0.01mol/L copper ion as complexation agent in the aqueous phase was employed for the first dimension and Recycling HSCCC, Reverse-Phase HSCCC with 0.1mol/L copper ion were used for the second dimension in parallel. Including two pairs of cis-trans isomers, seven BSA binders including 3,5,7-Trihydroxychromone (1), taxifolin (2), N-cis-paprazine (3), N-cis-feruloyltyramine (4), N-trans-paprazine (5), N-trans-feruloyltyramine (6) and an unidentified compound (7) were obtained. The purities of these seven compounds were all over 95.0% as determined by HPLC. The complexation HSCCC behaviors of seven compounds were also investigated by studying their relationship with copper ion. Results showed that the combinative method using (BSA-Fe3O4 MNPs)-HPLC and HSCCC is a quick, efficient, and reproductive technique to isolate potentially bioactive compounds from the complex mixture system of natural products. And the usage of off-line 2D-HSCCC and introduction of chelating metal ion into solvent system are effective ways to implement HSCCC separations in complex samples.