Artificial antibody-antigen-directed immobilization of α-amylase to hydrolyze starch for cascade reduction of 2-nitro-4-methylphenol to 2-amino-4-methylphenol.

Nitrophenol is a hazardous substance that poses a threat to the environment and human health, and its treatment has attracted widespread attention. The purpose of this study is to establish an environmentally friendly α-amylase system for the hydrolysis of starch to reduce nitrophenol to aminophenol through cascade reactions. The α-amylase system was obtained through artificial antibody-antigen-directed immobilization, including the synthesis of artificial antibodies, synthesis of artificial antigens, and affinity assembly. In this process, catechol and protocatechuic aldehyde were used to prepare artificial antibodies and artificial antigens respectively through polymerization and Schiff base reactions. Then, artificial antibodies captured the catechol in the artificial antigen structure to form immobilized α-amylases. Compared with free α-amylase, the immobilized α-amylase showed a good reusability and excellent regenerative ability. Subsequently, the immobilized α-amylase were used in the reaction of catalyzing starch hydrolysis to synthesize 2-amino-4-methylphenol, and the yield of 2-amino-4-methylphenol was 58.88 ± 0.19 %. After 5 consecutive catalytic reactions, a yield of 47.61 ± 1.27 % can still be achieved.

Artificial antibody-antigen-directed immobilization of lipase for consecutive catalytic synthesis of ester: Benzyl acetate case study.

A strategy based on artificial antibody-antigen recognition was proposed for the specific directed immobilization of lipase. The artificial antibody was synthesized using catechol as a template, α-methacrylic acid as a functional monomer, and Fe3O4 as the matrix material. Lipase was modified with 3,4-dihydroxybenzaldehyde as an artificial antigen. The artificial antibody can specifically recognize catechol fragment in the enzyme structure to achieve the immobilization of lipase. The immobilization amount, yield, specific activity, and immobilized enzyme activity were 13.2 ± 0.2 mg/g, 78.9 ± 0.4 %, 7.9 ± 0.2 U/mgprotein, and 104.6 ± 1.7 U/gcarrier, respectively. Moreover, the immobilized lipase exhibited strong reusability and regeneration ability. Additionally, the immobilized lipase successfully catalyzed the synthesis of benzyl acetate and demonstrated robust continuous catalytic activity. These results fully demonstrate the feasibility of the proposed artificial antibody-antigen-directed immobilization of lipase.

Biomimetic immobilization of α-glucosidase inspired by antibody-antigen specific recognition for catalytic preparation of 4-methylumbelliferone.

Immobilization technology plays an important role in enhancing enzyme stability and environmental adaptability. Despite its rapid development, this technology still encounters many challenges such as enzyme leakage, difficulties in large-scale implementation, and limited reusability. Drawing inspiration from natural paired molecules, this study aimed to establish a method for immobilized α-glucosidase using artificial antibody-antigen interaction. The proposed method consists of three main parts: synthesis of artificial antibodies, synthesis of artificial antigens, and assembly of the artificial antibody-antigen complex. The critical step in this method involves selecting a pair of structurally similar compounds: catechol as a template for preparing artificial antibodies and protocatechualdehyde for modifying the enzyme to create the artificial antigens. By utilizing the same functional groups in these compounds, specific recognition of the antigen by the artificial antibody can be achieved, thereby immobilizing the enzymes. The results demonstrated that the immobilization amount, specific activity, and enzyme activity of the immobilized α-glucosidase were 25.09 ± 0.10 mg/g, 5.71 ± 0.17 U/mgprotein and 143.25 ± 1.71 U/gcarrier, respectively. The immobilized α-glucosidase not only exhibited excellent reusability but also demonstrated remarkable performance in catalyzing the hydrolysis of 4-methylumbelliferyl-α-D-glucopyranoside.

Optimization of liquid fermentation conditions for Coprinus comatus to enhance antioxidant activity.

Coprinus comatus is an edible and medicinal fungus. In this study, the antioxidant activity of the fermentation product of C. comatus was investigated through optimization of fermentation process. The results indicated that the fermentation product of C. comatus had obvious scavenging ability for 2,2'-Azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) free radical. The EC50 of the n-butanol extract from the fermentation product on ABTS·+ was 0.65 ± 0.02 mg/mL. On this basis, the liquid fermentation conditions of C. comatus were optimized through single factor and response surface optimization experiments according to the scavenging ability of ABTS·+ to improve the antioxidant capacity of the fermentation product. The results showed that when the 14% of C. comatus was fermented in a culture medium with a C/N ratio of 48:1 for 6 days, the ABTS·+ scavenging ability was the strongest, and the EC50 of n-butanol extract was 0.57 ± 0.01 mg/mL, which was 12.31% higher than the initial activity. This study laid the foundation for the development of C. comatus.

Near-infrared photodynamic and photothermal co-therapy based on organic small molecular dyes.

Near-infrared (NIR) organic small molecule dyes (OSMDs) are effective photothermal agents for photothermal therapy (PTT) due to their advantages of low cost and toxicity, good biodegradation, and strong NIR absorption over a wide wavelength range. Nevertheless, OSMDs have limited applicability in PTT due to their low photothermal conversion efficiency and inadequate destruction of tumor regions that are nonirradiated by NIR light. However, they can also act as photosensitizers (PSs) to produce reactive oxygen species (ROS), which can be further eradicated by using ROS-related therapies to address the above limitations of PTT. In this review, the synergistic mechanism, composition, and properties of photodynamic therapy (PDT)-PTT nanoplatforms were comprehensively discussed. In addition, some specific strategies for further improving the combined PTT and PDT based on OSMDs for cancer to completely eradicate cancer cells were outlined. These strategies include performing image-guided co-therapy, enhancing tumor infiltration, increasing H2O2 or O2 in the tumor microenvironment, and loading anticancer drugs onto nanoplatforms to enable combined therapy with phototherapy and chemotherapy. Meanwhile, the intriguing prospects and challenges of this treatment modality were also summarized with a focus on the future trends of its clinical application.

Ellagic acid from pomegranate peel: Consecutive countercurrent chromatographic separation and antioxidant effect.

Ellagic acid is one of the most representative natural antioxidants, and is rich in pomegranate peel. In this study, a consecutive countercurrent chromatographic (CCC) separation method was established to improve the preparative efficiency of ellagic acid from pomegranate peel. By optimizing the solvent system, sample size and flow rate, 280 mg of ellagic acid was obtained from 5 g of crude sample from pomegranate peel by CCC after six consecutive injections. Moreover, the values of EC50 for ellagic acid in scavenging ABTS·+ and DPPH· were 4.59 ± 0.07 and 10.54 ± 0.07 µg/ml, respectively, indicating a strong antioxidant activity. This study not only established a high-throughput method for the preparation of ellagic acid, but also provided a successful example for the development of and research on other natural antioxidants.

Fermentation of Robinia pseudoacacia flower for improving the antioxidation: optimized conditions, active composition, mechanism, and biotransformation process.

Robinia pseudoacacia flower is a natural product with many biological activities, including antioxidation. To further develop its antioxidation, the extract was fermented by Aspergillus niger FFCC 3112 in the medium with carbon to nitrogen ratio of 1.4:1 and initial pH of 4.2 for 3.5 days to form the best antioxidant activity of the fermentation product by strain screening, single factor optimization, and response surface methodology. Further analysis, isolation and activity determination showed that a main chemical component, kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-ß-D-galactopyranosyl-7-O-α-L-rhamnopyranoside, in the extract was completely hydrolyzed to kaempferol-7-O-α-L-rhamnopyranoside and kaempferol with better antioxidant activity through biotransformation, which was the basis for improving the antioxidant activity of fermentation products. Moreover, the mechanism of antioxidant and the contribution of phenolic hydroxyl groups were investigated by density functional theory. The result indicated that the antioxidant capacity of kaempferol-7-O-α-L-rhamnopyranoside and kaempferol increased with the increase of solvent polarity. In high-polarity solvents, they mainly scavenge free radicals through single electron transfer followed by proton transfer.

Elution-extrusion counter-current chromatographic separation and theoretical mechanism of antioxidant from Robinia pseudoacacia flower.

Robinia pseudoacacia flowers have attracted much attention because of numerous bioactivities. In this study, its extract showed the potential scavenging ability for 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) and 1,1-diphenyl-2-picrylhydrazyl free radicals. Under the guidance of antioxidant activity, the antioxidant extract was enriched by liquid-liquid extraction. The partition coefficients of the two main components in antioxidant extracts differed greatly, so in this study, elution-extrusion counter-current chromatography with the solvent system of n-hexane-ethyl acetate-methanol-water (2.5:5:2.5:5, v/v) was used to enhance the separation efficiency, and the two main components were successfully obtained. Among them, kaempferol showed strong antioxidant activity, which can be responsible for the activity of the extract. In order to deeply understand the antioxidant mechanism of kaempferol, the thermodynamics, frontier molecular orbital, and kinetics of scavenging free radicals were investigated by density functional theory. The results showed that 4'-OH in kaempferol was the most active group, which can scavenge free radicals by hydrogen atom transfer in non-polar solvents and activate 3-OH to generate double hydrogen atom transfer in the gas phase. But in polar solvents, it was more inclined to clear radicals through single electron transfer and proton transfer. The kinetic result showed that kaempferol needed 9.17 kcal/mol of activation energy to scavenge free radicals.

Preparation of Isoquercetin From Toona sinensis by Liquid-Liquid-Refining Extraction And Consecutive Counter-Current Chromatography.

A high-speed counter-current chromatographic (HSCCC) method using ethyl acetate-water as solvent system was established to separate isoquercetin from Toona sinensis. In an HSCCC single separation, the sample sizes of ethanol extract were optimized from 203 to 1200 mg. The results showed that the yield of the target compound increased from 4 to 26 mg, and the corresponding purity decreased from 93.30 to 81.82%. To further improve the yield and purity, liquid-liquid-refining extraction was introduced to pretreat the ethanol extract and enrich the target compound. The ethanol extract was extracted with n-hexane-ethyl acetate-method-water (1:5:1:5, v/v) and ethyl acetate-water in turn to remove the low-polarity and high-polarity impurities and obtain the crude sample. Under the similar conditions, 85.25 mg of the target compound with the purity of 95.12% was separated from 240 mg of the crude sample. Subsequently, a consecutive HSCCC was developed to obtain 257 mg of the target compound from 720 mg of crude sample, which was equivalent to 14.4 g of ethanol extract. This method improved the purity of the target compound, but more importantly, the sample size can reach 12 times of the maximum sample size of the ethanol extract in a single run.

Immobilized enzyme for screening and identification of anti-diabetic components from natural products by ligand fishing.

Diabetes is a chronic metabolic disease caused by insufficient insulin secretion and insulin resistance. Natural product is one of the most important resources for anti-diabetic drug. However, due to the extremely complex composition, this research is facing great challenges. After the advent of ligand fishing technology based on enzyme immobilization, the efficiency of screening anti-diabetic components has been greatly improved. In order to provide critical knowledge for future research in this field, the application progress of immobilized enzyme in screening anti-diabetic components from complex natural extracts in recent years was reviewed comprehensively, including novel preparation technologies and strategies of immobilized enzyme and its outstanding application prospect in many aspects. The basic principles and preparation steps of immobilized enzyme were briefly described, including entrapment, physical adsorption, covalent binding, affinity immobilization, multienzyme system and carrier-free immobilization. New formatted immobilized enzymes with different carriers, hollow fibers, magnetic materials, microreactors, metal organic frameworks, etc., were widely used to screen anti-diabetic compositions from various natural products, such as Ginkgo biloba, Morus alba, lotus leaves, Pueraria lobata, Prunella vulgaris, and Magnolia cortex. Furthermore, the challenges and future prospects in this field were put forward in this review.

Fermentation of Agaricus bisporus for antioxidant activity: response surface optimization, chemical components, and mechanism.

Agaricus bisporus is one of the most widely cultivated edible mushrooms in the world. The chemical components of A. bisporus have a wide range of biological activities. In order to deeply understand the antioxidant properties of A. bisporus, this study conducted an investigation on the components of A. bisporus fermentation. Through the single factor experiment and response surface optimization, it was found that when the C/N ratio was 45:1, the inoculum concentration was 10%, and the fermentation time was 7 d, the n-butanol extract of the fermentation product had the strongest scavenging capacity for free radical generated through 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS·+). The concentration for 50% of the maximal effect (EC50) was 0.33 ± 0.01 mg/mL. Moreover, in order to identify the two main components, the elution-extrusion counter-current chromatography (EECCC) was employed for separation, where 5,5'-oxy-dimethyl-bis(2-furfuraldehyde) and 5-(butoxymethyl) furfural were obtained. The antioxidant activity of 5,5'-oxy-dimethyl-bis(2-furfuraldehyde) (EC50 = 0.26 ± 0.01 mg/mL) was superior to that of 5-butylmethyl furfural (EC50 = 1.52 ± 0.02 mg/mL), indicating that 5,5'-oxy-dimethyl-bis(2-furfuraldehyde) was the main antioxidant in the fermentation products. The thermodynamic parameters and frontier molecular orbitals of 5,5'-oxy-dimethyl-bis (2-furanaldehyde) was evaluated by density functional theory (DFT). The result indicated 5,5'-oxy-dimethyl-bis(2-furanaldehyde) scavenged free radicals in polar media through single electron transfer followed by proton transfer (SET-PT).

Dual-target affinity analysis and separation of α-amylase and α-glucosidase inhibitors from Morus alba leaves using a magnetic bifunctional immobilized enzyme system.

Morus alba leaves are a natural product with great antidiabetic potential. However, the therapeutic efficacy of natural products is usually achieved through the interaction of active compounds with specific targets. Among them, active compounds with multi-target therapeutic functions are more effective than single-target enzymes. In this study, a bienzyme system was constructed by co-immobilizing α-amylase and α-glucosidase onto Fe3 O4 for affinity screening of dual-target active components in the complex extract from M. alba leaves. As a result, a potential active compound was selectively screened by ligand fishing, separated by high-speed countercurrent chromatography using a solvent system of ethyl acetate-n-butanol-water (3:2:5, v/v), and identified as rutin. In addition, the result of molecular docking showed that rutin could interact with the active center of α-amylase and α-glucosidase through multiple hydrogen bonds, van der Waals forces, etc. to play an inhibitory role. These results demonstrate the effectiveness of the polydopamine magnetically immobilized bienzyme system for dual-target affinity screening of active substances. This study not only reveals the chemical basis of the antidiabetic activity of M. alba leaves from a dual-target perspective, but also promotes the progress of multitarget affinity screening.

A simple and effective method based on enzyme-ligand complex for affinity analysis of lipase inhibitor from Schisandra chinensis (Turcz.) Baill.

Schisandra chinensis (Turcz.) Baill has various biological activities including anti-obesity. Rapid analysis and screening of active compounds from natural extracts is one of the challenges faced by natural drug research. In order to analyze and screen lipase inhibitor from Schisandra chinensis extract, a method inspired by the specific binding of enzyme to ligand was developed and established. Through optimization of incubation conditions, such as time, temperature, and pH, the potential active compound was locked by comparing the change of the chemical components of the S. chinensis extract before and after incubation with lipase. Subsequently, the target compound was isolated by high-speed counter-current chromatography and was identified as 5-hydroxymethyl-2-furaldehyde. Moreover, in vitro activity determination confirmed that 5-hydroxymethyl-2-furaldehyde with an IC50 value of 284.78 ± 16.45 µg/mL interacted with the lipase through non-competitive inhibition. Furthermore, molecular docking further revealed that 5-hydroxymethyl-2-furaldehyde can be embedded into the active pocket of lipase via multiple hydrogen bonds and other interactions. This study not only screened a potential lipase inhibitor from S. chinensis through the newly developed method, but also can be used as a typical reference for the discovery of active components from functional foods and natural products.

Preparation of 5-hydroxymethylfurfural from Schisandra chinensis (Turcz.) Baill by high-speed counter-current chromatography: Comparison of conventional and consecutive separation.

Schisandra chinensis is a plant with high medicinal value, which contains many medicinal ingredients, including 5-hydroxymethylfurfural. In the present study, an efficient method based on high-speed counter-current chromatography was established for the preparation of 5-hydroxymethylfurfural from Schisandra chinensis. Petroleum ether-ethyl acetate-methanol-water (2:5:2:5, v/v) was selected as the solvent system for high-speed counter-current chromatography. In order to improve the yield of single separation, the sample size was continuously optimized and improved. The results showed that 1,250 mg was the most suitable sample size, and 41 mg of the target compound with 97% purity was obtained in a single run. To further improve the yield, consecutive high-speed counter-current chromatography was introduced and compared with the results of a high-speed counter-current chromatography single run. The results showed that although the purity was reduced to 92%, 430 mg of the target compound was obtained from 12.5 g of ethanol extract within 670 min after 10 consecutive injections. This indicated that consecutive separation not only increased the yield of the target compound, but also saved the separation time and greatly improved the separation efficiency of high-speed counter-current chromatography.

A strategy to process hundred-gram level complex sample using liquid-liquid-refining extraction and consecutive counter-current chromatography: Toona sinensis case study.

Large-scale preparation of target compounds from complex samples is facing great challenges. In the present study, an efficient strategy for large-scale preparation of target compound was proposed and successfully applied in the separation of active components from Toona sinensis. The pretreatment technology of liquid-liquid refining extraction (LLRE) combined with consecutive high-speed counter-current chromatography (HSCCC) was used to process hundred grams of extractions. Firstly, two phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (5:5:5:5, v/v) and (2:5:2:5, v/v) were used to remove low polar and high polar impurities from 100 g crude extracts of T. sinensis, respectively, and 9.25 g of crude sample was obtained. And then, n-hexane-ethyl acetate-methanol-water (2.5:5:2.5:5, v/v) was used as the solvent system for HSCCC separation. The isocratic elution mode with max loading and consecutive injections mode were investigated to obtain more target compound. As a result, ethyl gallate with purity of 97% was successfully separated by 5 times consecutive counter-current chromatography. The separation was repeated once. Finally, ethyl gallate (3.73 g) was isolated from 9.25 g of crude sample (100 g crude extracts). The results demonstrated that the yield increased from 0.26 g/h/L of untreated crude extract to 0.93 g/h/L of LLRE pre-treated sample for single injection, and further increased to 1.62 g/h/L for 5 consecutive injections mode with the present method.

An efficient method based on an inhibitor-enzyme complex to screen an active compound against lipase from Toona sinensis.

As a popular vegetable, Toona sinensis has a wide range of bioactivities including lipase inhibitory activity. In the present study, an efficient and rapid method using a ligand-enzyme complex was established for screening of an active compound against lipase from Toona sinensis. The ethyl acetate extract of Toona sinensis showed good lipase inhibitory activity. After incubation with lipase, one of the compounds in the extract decreased significantly while comparing the HPLC chromatograms before and after incubation, which indicated that it may be the active compound bound to lipase. Then, the compound was isolated using a Sephadex LH-20 column and identified as 1,2,3,4,6-penta-O-galloyl-ß-D-glucose. The in vitro activity test showed that the compound had good inhibitory activity against lipase, and its IC50 value was 118.8 ± 1.53 µg mL-1. The kinetic experiments indicated that 1,2,3,4,6-penta-O-galloyl-ß-D-glucose inhibited lipase through mixed competitive and non-competitive inhibitions. Further docking results showed that the target compound could bind to the active site of lipase stably through seven hydrogen bonds, resulting in a docking energy of -8.31 kcal mol-1. The proposed method can not only screen the lipase inhibitors from Toona sinensis quickly and effectively, but also provide an effective way for the rapid screening of active substances in natural food and plants.

Rapid screening and separation of active compounds against α-amylase from Toona sinensis by ligand fishing and high-speed counter-current chromatography.

In the present study, an efficient method based on ligand fishing and high-speed counter-current chromatography (HSCCC) was established to screen, enrich and separate the active components with the α-amylase inhibitory activity from a traditional dish Toona sinensis. The active components were screened from T. sinensis by ligand fishing using the magnetic immobilized α-amylase prepared through solvothermal and crosslinking methods. HSCCC was used to separate the target compound according to the K value. As a result, a potential active compound 1,2,3,4,6-penta-O-galloyl-ß-d-glucose and a non-target compound quercetin-3-O-α-L-rhamnopyranoside were separated and identified. In-vitro experiments indicated that 1,2,3,4,6-penta-O-galloyl-ß-d-glucose had the activity against α-amylase and the IC50 value was 93.49 ± 0.80 µg/mL which was higher than that of the non-target compound. The result further confirmed the molecular fishing effect of magnetic immobilized α-amylase. The present study can not only find and separate the hypoglycemic substances in T. sinensis quickly and effectively, but also can provide a new approach for the study of natural active components.

Enzyme reaction-guided identification of active components from the flowers of Sophora japonica var. violacea.

The flower of S. japonica is a favorite food and used as traditional medicine. In the present study, a facile and effective method based on the changes in the composition before and after the enzyme reaction was established to screen the active compounds from complex natural products. The separation of an active compound from the ethanolic extracts of Sophora japonica var. violacea, which exhibited the α-amylase inhibitory activity is presented as an example. The analysis of HPLC showed that one component was reduced by 25% after the enzyme reaction. The potential active compound was isolated via LH-20 gel permeation chromatography and identified as kaempferol 3-O-α-l-rhamnopyranosyl-(1 → 6)-ß-d-galactopyranosyl-7-O-α-l-rhamnopyranoside by 1H and 13C NMR. The in vitro test indicated that the compound had the α-amylase inhibitory activity, and the IC50 was 88.56 ± 0.60 µg mL-1. The molecular docking study of this compound showed that the compound enfolded in the active sites of α-amylase completely and interacted with the amino acid residues through hydrogen bonds, van der Waals force and hydrophobic interactions.

Optimization of fungi co-fermentation for improving anthraquinone contents and antioxidant activity using artificial neural networks.

The fermentation products of edible fungi are rich in anthraquinones and have a variety of activities, including the antioxidant activity. Because of the large number of combinations, it is very difficult to obtain the optimal multi-strains co-fermentation to improve the yield of anthraquinone. In the present study, an intelligent model based on artificial neural networks (ANNs) using backpropagation (BP) and radial basis function (RBF) algorithms was developed and validated to predict the anthraquinone contents in 136 two fungi and 680 three fungi co-fermented products. After experimental validation of the anthraquinone contents, the mean absolute error and the mean bias error of the results from RBF ANN were lower than those from BP ANN. The results indicated that the anthraquinone contents in A. bisporus, C. comatus and H. erinaceus co-fermentation product was the highest (2.11%). Furthermore, this co-fermentation product showed strong antioxidant activity.

A strategy based on liquid-liquid-refining extraction and high-speed counter-current chromatography for the bioassay-guided separation of active compound from Taraxacum mongolicum.

The research of natural active substances is facing the problems of low separation efficiency and active component loss due to the complex composition of natural extracts. In this study, a strategy based on liquid-liquid-refining extraction and high-speed counter-current chromatography was established to solve this problem. Separation of an active compound with the α-amylase inhibitory activity from Taraxacum mongolicum Hand. -Mazz. was presented as an example. The ethyl acetate extract (FA) from T. mongolicum exhibited the potential effect on α-amylase and was divided into 8 fractions (FB-FI) by liquid-liquid-refining extraction. The results showed that the activity of FE was higher than the others. According to the results of liquid-liquid-refining extraction, a two-phase solvent system with a slightly higher polarity was selected to separate the fraction by HSCCC, and 110 mg of compound was separated from 900 mg FA using the model of consecutive separation. The compound was identified as luteolin by 1H NMR and 13C NMR. The IC50 of luteolin against α-amylase was 42.33±0.82 µg/mL. Then, molecular docking was introduced to study the relationship between the activity and the structure. The results showed that luteolin enfolded in the catalytic site of α-amylase through hydrogen bonds, van der Waals force and hydrophobic interaction, thus inhibiting the activity of the enzyme.