[Rapid determination of aesculin and aesculetin in Fraxini Cortex by high performance liquid chromatography-ultraviolet at equal absorption wavelength].

Fraxini Cortex is a traditional Chinese herbal medicine that has been used for thousands of years to treat dampness-heat diarrhea, dysentery, red or white vaginal discharge, painful swelling or redness of the eyes, and nebula. It contains various chemical components, including coumarins, iridoids, phenolic acids, and flavonoids. Coumarins are important active ingredients in Fraxini Cortex and have antibacterial, anti-inflammatory, antioxidant, antitumor, and antiviral activities. Aesculin and aesculetin are two major coumarin components of Fraxini Cortex that are widely used in its quality evaluation. Previous HPLC methods for determination of aesculin and aesculetin present several limitations, such as long analysis times and high solvent and reference compound consumption. In this study, a rapid, eco-friendly and cost saving HPLC method for the determination of aesculin and aesculetin in Fraxini Cortex was established by using the core-shell column and equal absorption wavelength (EAW). Different factors influencing the extraction process, such as the extraction solvent, temperature, and time, were assessed to obtain the optimal extraction conditions. The results showed that Fraxini Cortex samples could be well extracted by ultrasonic extraction for 5 min with a 25% ethanol aqueous solution. A core-shell column was used, and different mobile phases and flow rates were investigated to obtain the best rapid-HPLC separation conditions. The optimized HPLC conditions were as follows: a Poroshell 120 EC-C18 column (50 mm×4.6 mm, 2.7 µm), acetonitrile-0.1% formic acid aqueous solution (6∶94, v/v) as the eluent, a flow rate of 1.5 mL/min, and a column temperature of 25 ℃. The EAW of aesculin and aesculetin was a key factor in their determination using a single reference compound. EAW selection was performed in two steps. First, the UV spectra of two equimolar concentrations of the reference compounds (aesculin and aesculetin) were compared to determine the EAW of the two analytes. The EAW results were then verified by the HPLC analysis of the reference compound solutions. The final EAW of aesculin and aesculetin was 341 nm. The determination of aesculin and aesculetin using only one reference compound (i. e., aesculin) was achieved by HPLC-UV at this EAW. The newly developed HPLC method revealed a good linear relationship between the two target analytes (r=1.0000). The limits of detection (LODs) and limits of quantification (LOQs) were 1.5 µmol/L and 3.0 µmol/L, respectively, and the average recoveries of aesculin and aesculetin were 99.0% and 97.5%. The stabilities of the sample solutions were examined, and the two analytes demonstrated good stability for 24 h. The contents of the target analytes in 10 batches of Fraxini Cortex were determined using the proposed EAW method and the classic external standard method (ESM), and comparable concentrations were obtained. The contents of aesculin and aesculetin in the 10 batches of Fraxini Cortex were 0.26%-2.80% and 0.11%-1.47%, respectively. A t-test was conducted to compare the results of the proposed EAW technique with those obtained via the method reported in the Chinese Pharmacopoeia, and no significant difference between the two assay methods was noted (P>0.05). Comparison of the newly established EAW method with those reported in the literature revealed that our method required only 10 min to complete and used as little as 0.5 mL of the solvent and only one standard. Therefore, the developed EAW method is a rapid, simple, eco-friendly, and cost-effective analytical method that is suitable for the determination of aesculin and aesculetin in Fraxini Cortex and its related products. The proposed technique is an improved method for determining aesculin and aesculetin and contributes to the enhancement of the quality evaluation of Fraxini Cortex.

New insights into the chemical profiling, cytotoxicity and bioactivity of four Bunium species.

Bunium species have been reported to be used both as food and in traditional medicines. The scientific community has attempted to probe into the pharmacological and chemical profiles of this genus. Nonetheless, many species have not been investigated fully to date. In this study, we determined the phenolic components, antimicrobial, antioxidant, and enzyme inhibitory activities of aerial parts of four Bunium species (B. sayai, B. pinnatifolium, B. brachyactis and B. macrocarpum). Results showed that B. microcarpum and B. pinnatifolium were strong antioxidants as evidenced in the DPPH, ABTS, CUPRAC, and FRAP assays. B. brachyactis was the most effective metal chelator, and displayed high enzyme inhibition against cholinesterase, tyrosinase, amylase, glucosidase, and lipase. The four species showed varied antimicrobial activity against each microorganism. Overall, they showed high activity against P. mirabilis and E. coli (MIC and MBC <1 mg mL-1). B. brachyactis was more effective against Aspergillus versicolor compared to the standard drug ketoconazole. B. brachyactis was also more effective than both ketoconazole and bifonazole against Trichoderma viride. B. sayai was more effective than ketoconazole in inhibiting A. fumigatus. B. sayai was most non-toxic to HEK 293 (cellular viability = 117%) and HepG2 (cellular viability = 104%). The highest level of TPC was observed in B. pinnatifolium (35.94 mg GAE g-1) while B. microcarpum possessed the highest TFC (39.21 mg RE g-1). Seventy four compounds were detected in B. microcarpum, 70 in B. brachyactis, 66 in B. sayai, and 51 in B. pinnatifolium. Quinic acid, chlorogenic acid, pantothenic acid, esculin, isoquercitrin, rutin, apigenin, and scopoletin were present in all the four species. This study showed that the four Bunium species are good sources of biologically active compounds with pharmaceutical and nutraceutical potential.

Research on the pharmacodynamics and mechanism of Fraxini Cortex on hyperuricemia based on the regulation of URAT1 and GLUT9.

Fraxini Cortex (also known as Qinpi, QP) has been used for the treatment of hyperuricemia with a significant difference on efficacy of QP from different regions. However, it`s still unknown whether proportion of components is the key and why same kind of herbs have different therapeutic effects. In this study, different sources of QP were collected from Shaanxi Qinpi extracts (SQPE), Henan Qinpi extracts (HQPE), Hebei Qinpi extracts (GQPE) provinces in China. Rat model of hyperuricemia with hypoxanthine combined with potassium oxonate were established to determine the levels of blood urea nitrogen (BUN), serum uric acid (SUA), urine uric acid (UUA) and creatinine (Cr). Hematoxylin-eosin staining (H&E) and Periodic Acid-Schiff staining (PAS) were performed for renal pathology while Western blot analysis and real-time PCR analysis for proteins and mRNA expression levels. High-performance liquid chromatograph (HPLC) was used for components and composition analysis. Our results demonstrated that QPE from different regions could alleviate hyperuricemia via increasing significantly the SCr and BUN levels whereas decreasing markedly UCr, SUA and UUA levels. Additionally, QPE could also improve the pathological changes of the kidneys. The protein and mRNA levels of urate reabsorption transporter 1 (URAT1) and glucose transporter 9 (GLUT9) were down-regulated by QPE treatment. SQPE hold a better activity on improving hyperuricemia and regulating URAT1 and GLUT9. HPLC analysis showed that the proportion of four components aesculin, aesculetin, fraxin, fraxetin were 9.002: 0.350: 8.980: 0.154 (SQPE); 0.526: 0.164: 7.938: 0.102 (HQPE); 12.022: 1.65: 0.878: 1.064 (GQPE). These data indicate that this proportion of effective components may be an important factor for efficacy of QP and had implications for the treatment of hyperuricemia.

Simultaneous determination of anemoside B4, phellodendrine, berberine, palmatine, obakunone, esculin, esculetin in rat plasma by UPLC-ESI-MS/MS and its application to a comparative pharmacokinetic study in normal and ulcerative colitis rats.

A sensitive and rapid ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS/MS) method was developed for the simultaneous analysis of anemoside B4, phellodendrine, berberine, palmatine, obakunone, esculin, esculetin, toosendanin (IS1 of anemoside B4), tetrahydropalmatine (IS2 of phellodendrine, berberine, palmatine and obakunone) and scopoletin (IS3 of esculin and esculetin) and to compare the pharmacokinetics of these active ingredients in normal and ulcerative colitis rats. After methanol deproteinization, solvents were evaporated at 40°C under a gentle stream of nitrogen. Chromatography was performed using a C18 column with a gradient elution of 0.1% aqueous formic acid and acetonitrile at 0.4ml/min. Detection and measurement were performed on a 4000 QTRAP UPLC-MS/MS system from AB Sciex in the multiple reaction monitoring (MRM) mode. Phellodendrine, berberine, palmatine, obakunone, esculin, esculetin, tetrahydropalmatine (IS2) and scopoletin (IS3) were monitored under positive ionization conditions. Anemoside B4, and toosendanin (IS1) were monitored under negative ionization conditions. The optimized mass transition ion-pairs (m/z) were 1221.1/750.7 for anemoside B4, 343.2/193.2 for phellodendrine, 337.1/321.0 for berberine, 353.0/336.9 for palmatine, 455.1/161.1 for obakunone, 341.2/179.2 for esculin, 179.1/123.0 for esculetin, 573.4/531.4 for toosendanin (IS1), 356.2/192.2 for tetrahydropalmatine (IS2) and 193.0/133.1 for scopoletin (IS3).

Purification of coumarin compounds from Cortex fraxinus by adsorption chromatography.

In this paper, a chromatographic method for isolation and purification of coumarin compounds from Cortex fraxinus was established by using Superose 12 as the separation media for the first time. The conditions for separation were optimized. Four kinds of coumarin compounds including aesuletin, aesculin, fraxetin and fraxin were obtained. The purity of these compounds were 98.5, 99.1, 97.9 and 97.3%, respectively, which were determined by HPLC area normalization method. The chemical structures of the separated compounds were identified according to (1)H and (13)C nuclear magnetic resonance data. The retention behavior of the separated coumarin compounds on Superose 12 was also discussed. The retention is based on a mixture of hydrogen bonding and hydrophobic interactions between the coumarin compounds and the residues of the cross-linking reagents used in the manufacturing process of Superose 12. The results of this paper indicate that Superose 12 is not only suitable for size-exclusion chromatography of proteins and other biological macromolecules but also for low-molecular-weight natural products.

[Quantitative method for simultaneous assay of four coumarins with one marker in Fraxini Cortex].

OBJECTIVE: To establish a new quantitative method for simultaneous determination of multi-coumarins in Fraxini Cortex by using one chemical reference substance, and validate its feasibilities. METHOD: The new quality evaluation method, quantitative analysis of multi-components by singer-marker (QAMS), was established and validated with Fraxini Cortex. Four main coumarins were selected as analytes to evaluate the quality and their relative correlation factors (RCF) were determined by HPLC-DAD. Within the linear range, the values of RCF at 340 nm of aesculin to asculetin, fraxin and fraxetin were 1.771, 0.799, 1.409, respectively. And the contents of aesculin in samples of Fraxini Cortex were authentically determined by the external standard method, and the contents of the three other coumarins were calculated by their RCF. The contents of these four coumarins in all samples were also determined by the external standard method. RESULT: Within a certain range, the RCF had a good reproducibility (RSD 2.5%-3.9%). Significant differences were not observed between the quantitative results of two methods. CONCLUSION: It is feasible and suitable to evaluate the quality of Fraxini Cortex and its Yinpian by QAMS.

Carbon nanotube/poly(ethylene-co-vinyl acetate) composite electrode for capillary electrophoretic determination of esculin and esculetin in Cortex Fraxini.

In this report, a novel carbon nanotube/poly(ethylene-co-vinyl acetate) (CNT/EVA) composite electrode was developed for the amperometric detection in CE. The composite electrode was fabricated by packing a mixture of CNTs and melted EVA in a piece of fused-silica capillary under heat. It was coupled with CE for the separation and detection of esculin and esculetin in Cortex Fraxini, a traditional Chinese medicine, to demonstrate its feasibility and performance. Esculin and esculetin have been well separated within 9 min in a 40 cm long capillary at a separation voltage of 12 kV using a 50 mM borate buffer (pH 9.2). The new CNT-based CE detector offered significantly lower detection potentials, yielded enhanced S/N characteristics, and exhibited high resistance to surface fouling and enhanced stability. It showed long-term stability and reproducibility with relative standard deviations of less than 5% for the peak current (n=15) and should also find a wide range of applications in other microfluidic analysis systems.

[Study on the extracting and dried process of Qinxiangzhixie enteric-coated tablet].

OBJECTIVE: To study the optimum extracting and dried process of Qinxiangzhixie enteric-coated tablet. METHODS: With the content of Aesculin and Aescaletin as factor, the optimum extraction process was selected with the orthogonal design. RESULTS: The optimum extraction process conditions were as: decocting for 2 times, with water of 8, 6 times of the drug mixture respectively 1.5 h, 1.0 h respectively. The optimum decompression dried process conditions was as follows :60 degrees C, -0.08 pa. CONCLUSIONS: The experimental results provide the basis for the ascertainment of extraction process of Qinxiang enteric-coated tablet.

Micelle-mediated extraction and cloud point preconcentration for the analysis of aesculin and aesculetin in Cortex fraxini by HPLC.

In this paper, a micelle-mediated extraction and cloud point preconcentration method was developed for the determination of less hydrophobic compounds aesculin and aesculetin in Cortex fraxini by HPLC. Non-ionic surfactant oligoethylene glycol monoalkyl ether (Genapol X-080) was employed as the extraction solvent. Various experimental conditions were investigated to optimize the extraction process. Under optimum conditions, i.e. 5% Genapol X-080 (w/v), pH 1.0, liquid/solid ratio of 400:1 (ml/g), ultrasonic-assisted extraction for 30 min, the extraction yield reached the highest value. For the preconcentration of aesculin and aesculetin by cloud point extraction (CPE), the solution was incubated in a thermostatic water bath at 55 degrees C for 30 min, and 20% NaCl (w/v) was added to the solution to facilitate the phase separation and increase the preconcentration factor during the CPE process. Compared with methanol, which was used in Chinese Pharmacopoeia (2005 edition) for the extraction of C. fraxini, the extraction efficiency of 5% Genapol X-080 reached higher value.

Analysis of ultraviolet absorption spectrum of Chinese herbal medicine-Cortex Fraxini by double ANN.

A fast, accurate and convenient method for the simultaneous determination of multi-component in the Chinese herbal medicine was proposed by using ultraviolet absorption spectrum. In this method, dummy components were added to training sample, and a double artificial neural network (DANN) that has the function of high self-revision and self-simulation was used. Effect of other interference components could be eliminated by adjusting concentration of dummy components. Therefore, the accuracy of concentration prediction for multi-component in the complicated Chinese herbal medicine was improved. It has been realized that two effective components of Cortex Fraxini, aesculin and aesculetin, were simultaneously determined, without any separation. The predicted accuracy was 92% within the permitted relative errors. The measurement precisions of the aesculin and aesculetin were 0.37% and 1.5%, respectively.

Non-aqueous capillary electrophoresis for separation and simultaneous determination of fraxin, esculin and esculetin in Cortex fraxini and its medicinal preparations.

A non-aqueous capillary electrophoresis method has been developed for the separation and simultaneous determination of fraxin, esculin and esculetin in Cortex fraxini and its preparation for the first time. Optimum separation of the analytes was obtained on a 47 cm x 75 microm i.d. fused-silica capillary using a non-aqueous buffer system of 60 mM sodium cholate, 20 mM ammonium acetate, 20% acetonitrile and 3% acetic acid at 20 kV and 292 K, respectively. The relative standard deviations (RSDs) of the migration times and the peak heights of the three analytes were in the range of 0.23-0.28 and 2.12-2.60%, respectively. Detection limits of fraxin, esculin and esculetin were 0.1557, 0.4073 and 0.5382 microg/mL, respectively. In the tested concentration range, good linear relationships (correlation coefficients 0.9995 for fraxin, 0.9999 for esculin and 0.9992 for esculetin) between peak heights and concentrations of the analytes were observed. This method has been successfully applied to simultaneous determination of the three bioactive components with the recoveries from 90.2 to 109.2% in the five samples.

[The sedative effect of the Kneipp hay sack and balneological preparations of hay (author's transl)]. / Zur sedativen Wirkung des Kneippschen Heupacks und balneologischer Heupräparate

A chromatographic analysis of the hay charge used for balneological and thermotherapy which contains coumarin, the hydroxycoumarins umbelliferone and esculin and also the furanocoumarin imperatorin. The sedative effect of the hay can be explained by the coumarin and the imperatorin; the absorption site is to be found in the respiratory tract.