Target Screen of Anti-Hyperuricemia Compounds from Cortex Fraxini In Vivo Based on ABCG2 and Bioaffinity Ultrafiltration Mass Spectrometry.

The ATP-binding cassette (ABC) transporter ABCG2 is a significant urate transporter with a high capacity, and it plays a crucial role in the development of hyperuricemia and gout. Therefore, it has the potential to be targeted for therapeutic interventions. Cortex Fraxini, a traditional Chinese medicine (TCM), has been found to possess anti-hyperuricemia properties. However, the specific constituents of Cortex Fraxini responsible for this effect are still unknown, particularly the compound that is responsible for reducing uric acid levels in vivo. In this study, we propose a target screening protocol utilizing bio-affinity ultrafiltration mass spectrometry (BA-UF-MS) to expediently ascertain ABCG2 ligands from the plasma of rats administered with Cortex Fraxini. Our screening protocol successfully identified fraxin as a potential ligand that interacts with ABCG2 when it functions as the target protein. Subsequent investigations substantiated fraxin as an activated ligand of ABCG2. These findings imply that fraxin exhibits promise as a drug candidate for the treatment of hyperuricemia. Furthermore, the utilization of BA-UF-MS demonstrates its efficacy as a valuable methodology for identifying hit compounds that exhibit binding affinity towards ABCG2 within TCMs.

A comparative study of three chemometrics methods combined with excitation-emission matrix fluorescence for quantification of the bioactive compounds aesculin and aesculetin in Cortex Fraxini.

Cortex Fraxini is an important traditional Chinese herbal medicine with various medical functions. Aesculin and aesculetin are the main effective components of Cortex Fraxini. The fluorescence signals of the two compounds have a high degree of overlap with each other, making quantitative analysis difficult with conventional analytical methods. In the present study, different chemometrics methods, including lasso regression (LAR), interval partial least squares (iPLS), and multidimensional partial least squares (N-PLS) methods, were employed and combined with excitation-emission matrix (EEM) fluorescence for the purpose of accurate quantification of aesculin and aesculetin in Cortex Fraxini samples. The most satisfactory results were obtained by using the N-PLS method based on the EEM spectra without scatterings, with correlation coefficient of calibration and prediction values higher than 0.9972 and 0.9962, respectively, root mean squared errors for calibration and prediction values lower than 0.0304 and 0.1165, respectively, and recovery values in the range of 83.32%-104.62%. The obtained credible models indicated that the N-PLS method combined with EEM spectra has the advantages of being green, low cost, and accurate and it is a good strategy for the determination of active compounds in complex samples. To further confirm the accuracy of the obtained results, the same samples were analyzed by the recognized ultra-performance liquid chromatography method.

Green and enhanced extraction of coumarins from Cortex Fraxini by ultrasound-assisted deep eutectic solvent extraction.

Green and enhanced extraction of bioactive ingredients from medicinal plants has become a hot research field, and deep eutectic solvents have been considered as a novel kind of sustainable solvents in the extraction process. In this study, hydrogen bond acceptor (choline chloride, etc.) and hydrogen bond donor (l-malic acid, etc.) were used to prepare different kinds of deep eutectic solvents to extract coumarins from Cortex Fraxini. The extraction conditions, including the composition and moisture content of deep eutectic solvents, extraction time, and liquid-solid ratio, were systematically optimized basing on the extraction yield of coumarins. To further investigate the extraction mechanism, Fourier transform infrared spectroscopy was performed, and the microstructures of Cortex Fraxini powders were observed before and after extraction using scanning electron microscope. Results showed that the novel ultrasound-assisted extraction with conditions of deep eutectic solvent containing betaine/glycerin (1:3), aqueous solution (20%), solid-liquid ratio (15 mg/mL), and extraction time (30 min) exhibited the best extraction yields for the four target coumarins and much better extraction efficiency than with conventional solvent extractions. This suggests that the new ultrasound-assisted deep eutectic solvent extraction could be used as a green and high-efficient approach for extraction of the main coumarins from Cortex Fraxini.

Bioactive ingredients obtained from Cortex Fraxini impair interactions between FAS and GPI.

The high expression of fatty acid synthase (FAS) in tumor cells is consistent with their elevated requirement for fatty acids for cell membrane synthesis and energy supply to support their almost unlimited proliferation. The expression levels of FAS in tumor cells are related to their proliferation, invasion, and metastasis. This study investigated the possible bioactive ingredients (fraxin, esculetin, scopolin et al.) of Cortex Fraxini and their effects on the interaction between specific proteins. We used microscale thermophoresis (MST) to show that our target protein, FAS (screened by combining transcriptome and network pharmacology), bound to the active compounds in Cortex Fraxini. It was found that FAS bound strongly to Glucose-6-phosphate isomerase (GPI), and that scopolin could affect this interaction by proteomics and MST. The results of this study demonstrate that the active compounds in Cortex Fraxini could play an anti-tumor role by binding to FAS and inhibiting the interactions between FAS and GPI to affect glucose and lipid metabolism, and that the protein pathway is a potential novel target for tumor treatment.

Comparative pharmacokinetic study of the main components of cortex fraxini after oral administration in normal and hyperuricemic rats.

Cortex Fraxini is an important traditional Chinese herbal medicine used for the treatment of gout and hyperuricemia. An efficient and rapid ultra-performance liquid chromatography mass spectrometry method was developed and validated for simultaneous quantitation of six coumarins (aesculin, fraxin, aesculetin, fraxetin, sopoletin and 7-hydroxycoumarin) in normal and hyperuricemic rats plasma after oral administration of Cortex Fraxini. The method could successfully be applied for pharmacokinetics studies. The pharmacokinetic behavior of six coumarins in normal and hyperuricemia rats plasma was determined. Results showed that, for some of analytes, the pharmacokinetic parameters (AUC0-t , AUC0-∞ , Cmax , Tmax and CL) were significantly different between normal and hyperuricemic rats. The different pharmacokinetic parameters might result from renal impairment or a change of metabolic enzymes in the pathological state. The pharmacokinetic study in pathological state could provide more useful information to guide the clinical use of traditional Chinese herbal medicine.

Protective Effect of Erqi Decoction on Intestinal Tract of Acute Radiation Intestinal Injury Rats and Its Mechanism / 广州中医药大学学报

Objective To investigate the protective effects of Erqi Decoction(EQD; mainly composed of Radix Aristolochiae Kaempferi, Radix Rhizoma Seu Flos Cypripedii, Cortex Fraxini, Cortex Phellodendri, Radix et Rhizoma Rhei) on the intestinal tract in rats with acute radiation intestinal injury and its mechanism. Methods Sixty SD rats were randomly divided into normal group, model group, EQD group and Baitouweng Decoction group (BD group), 15 rats in each group. The acute radiation enteritis model was established by exposing the whole abdomen to a total dose of 10 Gy of 6 MV higher-energy X-rays. EQD group and BD group were given intragastrical administration with corresponding medicine of EQD at the dose of 8.85 g·kg-1·d-1, BD at the dose of 4.69 g·kg-1·d-1 respectively, and the normal group and the model group were given intragastrical administration with the same volume of normal saline. The treatment lasted for 7 continuous days. After modeling, the morphological change of the proximal ileum tissue was observed under light microscope. Villus height, crypt depth, and thickness of the ileal mucosa and entire wall were measured by image analysis system. The myeloperoxidase (MPO) content in ileum tissue was determined by spectrophotometer, and the expression levels of caspase -3 and proliferating cell nuclear antigen (PCNA) in ileum tissue were determined by immunohistochemistry. Results EQD group and BD group had milder injuries of the ileal structure, and had higher villus height, crypt depth, and thickness of mucosa and entire wall than those in the model group (P 0.05). MPO content in EQD group and BD group was decreased(P0.05). Conclusion EQD has certain protective effects against radiation-induced intestinal damage, which mechanism is probably associated with relieving the local intestinal inflammatory reaction, accelerating intestinal epithelial cell proliferation, and inhibiting intestinal epithelial cell apoptosis.

(1)H NMR and MS based metabolomics study of the therapeutic effect of Cortex Fraxini on hyperuricemic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Cortex Fraxini (CF) is an important traditional Chinese herbal medicine used for the treatment of gout and hyperuricemia. AIM OF THE STUDY: The aim of this study was to evaluate the anti-hyperuricemic effect of CF on hyperuricemic rats and to investigate its mechanism of action. MATERIALS AND METHODS: Metabolomics based on NMR and MS was used to study the therapeutic effect of CF on hyperuricemic rats. Plasma determination of uric acid (UA) showed that CF treatment markedly improved the UA level. Subsequently, metabolomics analysis was conducted using samples of plasma, kidney and urine, and orthogonal partial least squares-discriminant analysis (OPLS-DA) combined with principal component analysis (PCA) were used to detect potential biomarkers. RESULTS: A total of 26 biomarkers were identified as being primarily involved in amino acid metabolism, lipid metabolism, purine metabolism, amino acid metabolism and carbohydrate metabolism, and hyperuricemia can disturb the balance of many of these metabolic pathways in vivo. CONCLUSIONS: The variations in biomarkers revealed the therapeutic mechanism of CF, and a number of these biomarkers are not only significant for early diagnosis but also for predicting hyperuricemia.

An approach of ionic liquids/lithium salts based microwave irradiation pretreatment followed by ultrasound-microwave synergistic extraction for two coumarins preparation from Cortex fraxini.

Ionic liquids/lithium salts solvent system was successfully introduced into the separation technique for the preparation of two coumarins (aesculin and aesculetin) from Cortex fraxini. Ionic liquids/lithium salts based microwave irradiation pretreatment followed by ultrasound-microwave synergy extraction (ILSMP-UMSE) procedure was developed and optimized for the sufficient extraction of these two analytes. Several variables which can potentially influence the extraction yields, including pretreatment time and temperature, [C4mim]Br concentration, LiAc content, ultrasound-microwave synergy extraction (UMSE) time, liquid-solid ratio, and UMSE power were optimized by Plackett-Burman design. Among seven variables, UMSE time, liquid-solid ratio, and UMSE power were the statistically significant variables and these three factors were further optimized by Box-Behnken design to predict optimal extraction conditions and find out operability ranges with maximum extraction yields. Under optimum operating conditions, ILSMP-UMSE showed higher extraction yields of two target compounds than those obtained by reference extraction solvents. Method validation studies also evidenced that ILSMP-UMSE is credible for the preparation of two coumarins from Cortex fraxini. This study is indicative of the proposed procedure that has huge application prospects for the preparation of natural products from plant materials.

Comparative Study on Cortex Fraxini Total Coumarin of Different Extraction Processes / 中国中医药信息杂志

Objective To study the effects of different extraction processes on extraction rate of cortex fraxini total coumarin and active constitute percentage of major coumarin;To establish a proper extraction process.Methods Orthogonal design method was applied to set comprehensive index cortex fraxini total coumarin extraction rate as inspecting index. Water and alcohol were used as solvent respectively to optimize the extraction process of cortex fraxini.Results Optimal water extraction process:cortex fraxini decoction pieces mixed with nine times of water, decocted for three times, 90 mins each time. The pasty fluid generating rate of cortex fraxini was 28.87%, total coumarin percentage was 19.26%, extraction rate was 5.56%, total percentage of Aesculin, Aesculetin, Fraxin, Fraxetin was 13.47%, when water was used as solvent. Optimal alcohol extraction process:cortex fraxini decoction pieces mixed with eight times of 75% ethyl alcohol, refluxed twice, two hours each time. The pasty fluid generating rate of cortex fraxini was 30.47%, total coumarin percentage was 21.72%, extraction rate was 6.62%, total percentage of Aesculin, Aesculetin, Fraxin, Fraxetin was 15.29%, when alcohol was used as solvent. It was found that using alcohol as solvent had a 5.54% higher pasty fluid generating rate, a 12.77% higher total coumarion percentage, a 19.06% higher total coumarin extraction rate, and a 13.51% higher percentage of total four coumarin constitutes than using water, with statistical significance. Conclusion Extraction process by using alcohol as solvent is better than using water. So the optimal and stable extraction process of cortex fraxini total coumarin is using 75% alcohol as solvent.

Multi-responses extraction optimization based on response surface methodology combined with polarity switching HPLC-MS/MS for the simultaneous quantitation of 11 compounds in Cortex Fraxini: application to four species of Cortex Fraxini and its 3 confusable species.

A novel polarity switching high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) approach combining optimization of extraction condition by response surface methodology (RSM) was developed for the simultaneous quantitative analysis of 11 compounds in Cortex Fraxini, a commonly used traditional Chinese medicine. The ultrasonic extraction conditions of the 11 analytes including sample quantity, methanol concentration and extraction time were simultaneously optimized with a Box-Behnken design (BBD) and Derringer's desirability function. Multiple-reaction monitoring (MRM) scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run of 16min. Quantitative parameters of the proposed method with respect to limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy and stability were evaluated under optimum conditions, and the results indicated that the method was sensitive, specific and reliable. The developed method was successfully applied to determine the investigated compounds in four species of Cortex Fraxini and three kinds of its confusable species, and significant differences were found between the official and confusable species.

Differentiation of Cortex Fraxini and its three kinds of confusable species / 中草药

Object To provide a scientific basis for identification and rational use of the herbal medicine by the pharmacognostic study on Cortex Fraxini. Methods Microscopic identification, TLC, HPLC were used. Results There were remarkable differences in the genuine product, confusing product and fakements of Cortex Fraxini in characteristics of microscopic identification, fluorescence, TLC and HPLC. Conclusion The scientific basis has been established and provided for differentiation of confusing product and fakements.

Determination of aesculin and aesculetin in periderm and leaves of Cortex Fraxini among different provenances / 中成药

Objective: To determine the contents of aesculin and aesculetin in periderms and leaves of Cortex Fraxini among differnet provenances. Methods: HPLC was used with acetonitrile-water(15∶85) as the mobile phase and detected wavelength at 348nm. C 18 column was adopted. Results: The calibration curves were linear. The value of correlation coefficient were 0.9992 and 0.9994, respectively. The average recovery of aesculin was 98.2% and aesculetin was 99.2%. RSD were 2.24% and 2.15%, respectively. The quality differences of Cortex Fraxini among different provenances were remarked. The quality of Cortex Fraxini from Shanxi province was the best. Conclusion: The method is applied in determination and analytics of content of Cortex Fraxini and is rapid, simple and easy to carry out. The method is with the feature of accuracy, repetition and stability.

Determination of Fraxetin A and Fraxetin B in Cortex Fraxini by HPLC / 中成药

The determination of fraxetin A and fraxetin B in Cortex Fraxini have been carried out by HPLC on the stationary phase:octodecyl-silicohydride bonded silica gel column. The mobile phase was methanol-water(24:76). The detection wavelength was at 348nm. There was a good linear relationship in a concentration range of 16?g/ml~176?g/ml. The correlation coefficients were 0. 9995 and 0. 9994,respectively. The recovery of the added sample were 99. 1 ?1. 5% (n=5) for fraxetin A and 99.8?2. 5%(n=5) for fraxetin B. This method showed 0. 8% of within-day accuracy and 6. 2% of between-day accuracy,involving the advantages of simplicity,quickness and accuracy.