Quick identification of xanthine oxidase inhibitor and antioxidant from Erycibe obtusifolia by a drug discovery platform composed of multiple mass spectrometric platforms and thin‐layer chromatography bioautography

As a final step of the purine metabolism process, xanthine oxidase catalyzes the oxidation of hypoxanthine and xanthine into uric acid. Our research has demonstrated that Erycibe obtusifolia has xanthine oxidase inhibitory properties. The purpose of this paper is to describe a new strategy based on a combination of multiple mass spectrometric platforms and thin‐layer chromatography bioautography for effectively screening the xanthine oxidase inhibitory and antioxidant properties of E. obtusifolia. This strategy was accomplished through the following steps. (i) Separate the extract of E. obtusifolia into fractions by an autopurification system controlled by liquid chromatography with mass spectrometry. (ii) Determine the active fractions of E. obtusifolia by thin‐layer chromatography bioautography. (iii) Identify the structure of the main active compounds with the information provided by direct analysis in real time mass spectrometry. (iv) Calculate the IC₅₀ value of each compound against xanthine oxidase using high‐performance liquid chromatography. Using the caulis of E. obtusifolia as the experimental material, seven target peaks were screened out as xanthine oxidase inhibitors or antioxidants. Our screening strategy allows for rapid analysis of small molecules with almost no sample preparation and can be completed within a week, making it a useful assay to identify unstable compounds and provide the empirical foundation for E. obtusifolia as a natural remedy for gout and oxidative‐stress‐related diseases.     

Screening,separation, and evaluation of xanthine oxidase inhibitors from Paeonia lactiflora using chromatography combined with a multi‐mode microplate reader

Natural products have become one of the most important resources for discovering novel xanthine oxidase inhibitors, which are commonly employed in the treatment of hyperuricemia and gout. However, to date, few reports exist regarding the use of monoterpene glycosides as xanthine oxidase inhibitors. Thus, we herein report the use of ultrafiltration coupled with liquid chromatography in the screening of monoterpene glycoside xanthine oxidase inhibitors from the extract of Paeonia lactiflora (P. lactiflora ), and both high‐performance counter‐current chromatography and medium‐pressure liquid chromatography were employed to separate the main constituents. Furthermore, the xanthine oxidase inhibitory activities and the mechanisms of inhibition of the isolated compounds were evaluated using a multi‐mode microplate reader by Molecular Devices. As a result, three monoterpene glycosides were separated by combined high‐performance counter‐current chromatography and medium‐pressure liquid chromatography in purities of 90.4, 98.0, and 86.3%, as determined by liquid chromatography. These three compounds were identified as albiflorin, paeoniflorin, and 1‐O‐β‐ᴅ‐glucopyranosyl‐8‐O‐benzoylpaeonisuffrone by electrospray ionization tandem mass spectrometry, and albiflorin and paeoniflorin were screened as potential xanthine oxidase inhibitors by ultrafiltration with liquid chromatography. The evaluation results of xanthine oxidase inhibitory activity corresponded with the screening results, as only albiflorin and paeoniflorin exhibited xanthine oxidase inhibitory activity.     

Chemical fingerprint of Ganmaoling granule by double‐wavelength ultra high performance liquid chromatography and ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A method incorporating double‐wavelength ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was developed for the investigation of the chemical fingerprint of Ganmaoling granule. The chromatographic separations were performed on an ACQUITY UPLC HSS C₁₈ column (2.1 × 50 mm, 1.8 μm) at 30°C using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. A total of 11 chemical constituents of Ganmaoling granule were identified from their molecular weight, UV spectra, tandem mass spectrometry data, and retention behavior by comparing the results with those of the reference standards or literature. And 25 peaks were selected as the common peaks for fingerprint analysis to evaluate the similarities among 25 batches of Ganmaoling granule. The results of principal component analysis and orthogonal projection to latent structures discriminant analysis showed that the important chemical markers that could distinguish the different batches were revealed as 4,5‐di‐O‐caffeoylquinic acid, 3,5‐di‐O‐caffeoylquinic acid, and 4‐O‐caffeoylquinic acid. This is the first report of the ultra high performance liquid chromatography chemical fingerprint and component identification of Ganmaoling granule, which could lay a foundation for further studies of Ganmaoling granule.     

α-Glucosidase inhibitors screening from Cyclocarya paliurus based on spectrum–effect relationship and UPLC–MS/MS

Cyclocarya paliurus is an edible and medicinal plant exhibiting significant hypoglycemic effect. However, its active components are still unclear and need further elucidation. In this research, the active components of the leaves of C. paliurus responsible for the α-glucosidase inhibitory activity were screened and identified based on a spectrum–effect relationship study in combination with ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) analysis. The 70% ethanol eluate fraction of the leaves of C. paliurus with the strongest α-glucosidase inhibitory activity was obtained after extraction and purification with macroporous resin. Their chromatographic fingerprints (15 batches) were established by UPLC analysis and 32 common peaks were specified by similarity analysis. Their IC₅₀ values for α-glucosidase inhibition were measured by an enzymatic reaction. Several multivariate statistical analysis methods including hierarchical cluster analysis, principal component analysis, partial least square analysis and gray relational analysis were applied to explore the spectrum–effect relationship between common peaks and IC₅₀ values, and the chromatographic peaks making a large contribution to efficacy were screened out. To further elucidate the active components of leaves of C. paliurus, the 70% ethanol eluate fraction was characterized by UPLC–MS/MS analysis, and 10 compounds were identified. This study provides a valuable reference for further research and development of hypoglycemic active components of C. paliurus.     

Screening of analgesic and anti‐inflammatory active component in Fructus Alpiniae zerumbet based on spectrum–effect relationship and GC–MS

Fructus Alpiniae zerumbet is widely used in Guizhou province as a miao folk herb with anti‐inflammatory, analgesic, protection against cardiovascular diseases, antihypertension and antioxidant activities. To further investigate the chemical material basis, the spectrum–effect relationship was established using gray relational analysis between the chromatographic fingerprint and its bioactivities. Herein, the fingerprints of essential oils from Fructus Alpiniae zerumbet (EOFAZ) from various sources were determined by gas chromatography mass spectrometry, and the analgesic and anti‐inflammatory bioactivities were investigated using the mouse model of acetic acid‐induced writhing test and dimethylbenzene‐induced mouse ear edema test. Finally, 17 common peaks were identified from nine batches of A. zerumbet, by comparison with the standard mass spectra in Nist2005, Wiley275 library. Meanwhile, the results showed significant analgesic and anti‐inflammatory effects in all of the different sources of EOFAZ. In particularly, peak 1 (α‐pipene), peak 3 (β‐pinene), peak 9 (camphor) and peak 16 (α‐cadinol) might be the main bioactive ingredients for analgesic and anti‐inflammatory activities. The model of the spectrum–effect relationships of EOFAZ was successfully discovered, which provided a novel platform for finding the bioactive components, a theoretical foundation for its further study and helping to establish quality control of Fructus A. zerumbet.     

Quality evaluation of Hypericum ascyron extract by two‐dimensional high‐performance liquid chromatography coupled with the colorimetric 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide method

In this paper, a heart‐cutting two‐dimensional high‐performance liquid chromatography coupled with the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) method was established for controlling the quality of different batches of Hypericum ascyron extract for the first time. In comparison with the common one‐dimensional fingerprint, the second‐dimensional fingerprint compiled additional spectral data and was hence more informative. The quality of H. ascyron extract was further evaluated by similarity measures and the same results were achieved, the correlation coefficients of the similarity of ten batches of H. ascyron extract were ＞0.99. Furthermore, we also evaluated the quality of the ten batches of H. ascyron extract by antibacterial activity. The result demonstrated that the quality of the ten batches of H. ascyron extract was not significantly different by MTT. Finally, we demonstrated that the second‐dimensional fingerprint coupled with the MTT method was a more powerful tool to characterize the quality of samples of batch to batch. Therefore the proposed method could be used to comprehensively conduct the quality control of traditional Chinese medicines.     

Fast analysis of triterpenoids in Ganoderma lucidum spores by ultra‐performance liquid chromatography coupled with triple quadrupole mass spectrometry

A rapid and reliable method was established for simultaneous determination of main triterpenoids in Ganoderma lucidum spores using ultra‐high‐performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC‐TQ‐MS). The established method was validated in terms of linearity, sensitivity, precision, accuracy and stability, and was successfully applied to determine the contents of 10 main triterpenoids in different batches of G. lucidum spores. The analysis results showed that moderate levels of triterpenoids were found in G. lucidum spores. In addition, a MS full scan with a daughter ion scan experiment was performed to identify the potential derivatives of triterpenoids present in G. lucidum spores. As a result, a total of 22 triterpenoids from different G. lucidum spores were unequivocally or tentatively identified via comparisons with authentic standards and literatures. This method provides both qualitative and quantitative results without the need for repetitive UPLC‐MS analyses, thereby increasing efficiency and productivity, making it suitable for high‐throughput applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Relationship between the UPLC‐Q‐TOF‐MS fingerprinted constituents from Daphne genkwa and their anti‐inflammatory,anti‐oxidant activities

Daphne genkwa Sieb.et Zucc. is a well‐known medicinal plant. This study was designed to apply the ultra‐high performance liquid chromatography system to establish a quality control method for D. genkwa. Data revealed that there were 15 common peaks in 10 batches of D. genkwa Sieb. Et Zucc. (Thymelaeaceae) from different provinces of China. On this basis, the fingerprint chromatogram was established to provide references for quality control. Afterwards, the chemical constitutions of these common peaks were analyzed using the UPLC‐Q‐TOF‐MS system and nine of them were identified. In addition, LPS‐stimulated RAW264.7 murine macrophages and DPPH assay were used to study the anti‐inflammatory and anti‐oxidation effects of D. genkwa . Then the fingerprint–efficacy relationships between UPLC fingerprints and pharmacodynamic data were studied with canonical correlation analysis. Analysis results indicated that the anti‐inflammatory and anti‐oxidation effects differed among the 10 D. genkwa samples owing to their inherent differences of chemical compositions. Taken together, this research established a fingerprint–efficacy relationship model of D. genkwa plant by combining the UPLC analytic technique and pharmacological research, which provided references for the detection of the principal components of traditional Chinese medicine on bioactivity.     

Development of a method using high‐performance liquid chromatographic fingerprint and multi‐ingredients quantitative analysis for the quality control of Yangxinshi Pian

A simple and reliable method of high‐performance liquid chromatography with diode array detection method was developed for fingerprint analysis and simultaneous determination of six compounds including puerarin, salvianolic acid B, berberine hydrochloride, palmatine chloride, dehydrocorydaline, and icariin in the Chinese medicine preparation Yangxinshi Pian. The separation was performed on an Agilent Eclipse XDB‐C₁₈ reserved‐phase column (250 mm × 4.6mm I.D., 5 μm) using gradient elution with 50 mmol/L monopotassium phosphate aqueous solution and methanol as mobile phase at a flow rate of 1.0 mL/min. The column operating temperature was set at 30°C, and the detection wavelength was 280 nm. The method was validated by linearity, precision, accuracy, stability, and recovery. For fingerprint analysis, 25 peaks were selected as the common peaks, and four kinds of similarities including cosine similarity (S), ratio of similarity (S′), projection content similarity (C), and content similarity (P) were applied to evaluate the quality consistency of different batches of Yangxinshi Pian. The results showed that the developed method was an efficient tool for quality evaluation of Yangxinshi Pian.     

Integrated evaluation of HPLC and UV fingerprints for the quality control of Danshen tablet by systematic quantified fingerprint method combined with antioxidant activity

Danshen tablet, which consists of Salviae Miltiorrhizae Radix et Rhizoma, Notoginseng Radix et Rhizoma and Borneolum syntheticum , has been widely used in the therapy of cardiovascular disease. The aim of this study was to develop comprehensive evaluation methods for the quality control of Danshen tablet. First, five‐wavelength fusion fingerprint was established to avoid one‐sidedness of a single wavelength. Then, the ultraviolet spectrum fingerprint was applied to reflect the information of unsaturated bond and conjugated system of chemical substances in Danshen tablet. The similarity analyses of these two fingerprints were performed by systematic quantified fingerprint method in terms of qualitative and quantitative aspects. After that, the evaluation results of high‐performance liquid chromatography and ultraviolet fingerprints were integrated by the mean algorithm, which could reduce the error caused by single method. The integrated evaluation results showed that 30 batches of samples were classified into seven grades. Finally, the fingerprint–efficacy relationship was established using an on‐line antioxidant system and partial least‐squares model to explore the connection between chemical components and antioxidant activities. The methods established in this paper were found suitable for the analysis of Danshen tablet.     

离心超滤质谱法筛选中药复方二妙丸中黄嘌呤氧化酶抑制剂

采用离心超滤质谱分析技术(UF-UPLC/Q-TOF/MS), 结合体外酶活性实验方法, 对中药复方二妙丸提取物中的黄嘌呤氧化酶抑制剂进行了筛选. 体外酶活性实验测得二妙丸水提液对黄嘌呤氧化酶的半数抑制浓度(IC₅₀)为(0.218±0.0034) mg/mL, 表明二妙丸具有较强的黄嘌呤氧化酶抑制活性. 进一步采用离心超滤质谱技术对二妙丸水提物中潜在的黄嘌呤氧化酶抑制剂进行筛选, 从中筛选并鉴定了9种具有潜在黄嘌呤氧化酶抑制活性的化合物, 为开发黄嘌呤氧化酶抑制剂及阐明二妙丸治疗痛风和高尿酸血症的作用机制提供了一定的依据.     

Spectrum–effect relationship study between HPLC fingerprints and antioxidant of honeysuckle extract

Honeysuckle (Lonicera japonica flos) is a well‐known agent of edible and medicinal value in China and its antioxidative activity makes a major contribution to its dual use. However, the compounds responsible for its antioxidative activity are still unknown. In this study, 10 batches of honeysuckle were collected from different origins in China. The fingerprints were established by HPLC technique to investigate the compounds and a 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activity assay was carried out to evaluate their antioxidant activity. partial least squares regression analysis was applied to set up the regression equation between DPPH radical scavenging activity and average peak area of common peaks of fingerprints. The results showed that peaks 10 (isochlorogenic acid B), 12 (isochlorogenic acid C), 11 (isochlorogenic acid A) and 9 (cynaroside) in the fingerprints were closely related to the antioxidant activity of 50% methanol extracts of honeysuckle. This study successfully established the spectrum–effect relationship between HPLC fingerprints and DPPH radical scavenging activity and provided a general model for exploring active components with a combination of chromatography and efficacy.     

Identification and evaluation of the chemical similarity of Yindan xinnaotong samples by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry fingerprinting

Yindan xinnaotong, a compound preparation used in traditional Chinese medicine, is composed of eight herbs: Ginkgo biloba leaf (yinxingye), Salvia miltiorrhizae (danshen), Herba gynostemmatis (jiaogulan), Erigerontis herba (dengzhanxixin), Allii sativi bulbus (dasuan), Notoginseng radixe rhizoma (sanqi), Crataegi fructus (shanzha), and Borneolum (tianranbingpian). Yindan xinnaotong is primarily used to treat cardiovascular and cerebrovascular diseases. However, to date, no scientific methods have been established to assess the quality of Yindan xinnaotong. Therefore, a combinatorial method was developed based on chemical constituent identification and fingerprint analysis to assess the consistency of Yindan xinnaotong quality. In this study, ultra high performance liquid chromatography coupled with time‐of‐flight mass spectrometry was used to identify the chemical components of Yindan xinnaotong soft capsules. Approximately 74 components were detected, of which 70, including flavonoids, ginkgolide, phenolic acid, diterpenoid tanshinones, and ginsenoside, were tentatively identified. A fingerprint analysis was also conducted to evaluate the uniformity of the quality of Yindan xinnaotong soft capsules. Ten batches of Yindan xinnaotong soft capsules were analyzed. All of the resulting chromatograms were imported into the “Similarity Evaluation System for Chromatographic Fingerprints of TCM” (Chinese Pharmacopoeia Commission, version 2004A). The similarity scores of common peaks from these samples ranged from 0.903–1.000, indicating that samples from different batches were highly correlated.     

Spectrum–effect relationship between HPLC fingerprints and antioxidant activity of Angelica sinensis

Angelica sinensis is one of the most important traditional Chinese medicines and has antioxidant activities that greatly contribute to its pharmacological action. However, the compounds responsible for its antioxidant activity remain unknown. In this study, the fingerprints of 10 batches of A. sinensis collected from different locations in China were established with HPLC to identify the common peaks. The antioxidant activities of these 10 batches were evaluated with 2,2-diphenyl-1-picrylhydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and ferric-reducing antioxidant power assays. The spectrum–effect relationship between HPLC fingerprints and antioxidant effect of A. sinensis was examined by the partial-least-square regression analysis and the variable importance in projection method. Results showed that the antioxidant effect of A. sinensis results from the synergistic effect of various compounds, and peaks X3 and X7–X18 were the main substances responsible for antioxidant efficacy. This study successfully identified the spectrum–effect relationship between HPLC fingerprints and the antioxidant effect of A. sinensis. This relationship can provide methods for establishing the quality standards for A. sinensis and developing new and effective products of A. sinensis based on its antioxidant ingredients.     

Quantitative Determination of the Chemical Profile of the Plant Material “Qiang-huo” by LC-ESI-MS-MS

The rhizomes of Notopterygium incisum Ting. ex H. T. Chang and N. forbesii Boiss. are recorded in the pharmacopoeia of the People’s Republic of China under the same name “Qiang-huo”. Valid quality control of Qiang-huo is desirable due to the fact that the wild natural sources of Qiang-huo have almost been exhausted and large regulated cultivation is developing. In the present paper, HPLC fingerprinting was developed to identify and distinguish both species in detail. The unique properties of the HPLC fingerprint were validated by analyzing 15 batches of N. incisum and 11 batches of N. forbesii. A standardized procedure involving liquid chromatography–electrospray ionization–multiple stage mass spectrometry (HPLC-ESI-MSⁿ) was developed to identify the fingerprint components, and a total of eight characteristic peaks were unequivocally identified. An average chromatogram from 15 batches of N. incisum from different geographic sources, considered to be the original and genuine herbal medicine, was first established as the standard fingerprint. The chemical profiles of 11 batches of N. forbesii samples were found to be variable. The HPLC method can differentiate N. incisum from N. forbesii by either the marker compounds notoptol (4) and p-hydroxypenethylanisate (8) or the amounts of nodakenin (1), notopterol (5), and 6′-O-trans-feruloylnodakenin (7). The HPLC fingerprint analysis is specific and may serve for quality identification and comprehensive evaluation of Qiang-huo.     

Fingerprint analysis and multi‐ingredient quantitative analysis for quality evaluation of Xiaoyanlidan tablets by ultra high performance liquid chromatography with diode array detection

A rapid and sensitive ultra high performance liquid chromatography method with diode array detection was developed for the fingerprint analysis and simultaneous determination of seven active compounds in Xiaoyanlidan (XYLD) tablets. The chromatographic separations were obtained on an Agilent Eclipse plus C₁₈ column (50 × 2.1 mm id, 1.8 μm) using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. Within 63 min, 36 peaks could be selected as the common peaks for fingerprint analysis to evaluate the similarities among several samples of XYLD tablets collected from different manufacturers. In quantitative analysis, seven compounds showed good regression (R > 0.9990) within test ranges and the recovery of the method was within the range of 95.9–104.3%. The method was successfully applied to the simultaneous determination of seven compounds in six batches of XYLD tablets. These results demonstrate that the combination of chromatographic fingerprint analysis and simultaneous multi‐ingredient quantification using the ultra high performance liquid chromatography method with diode array detection offers a rapid, efficient, and reliable approach for quality evaluation of XYLD tablets.     

Spectrum–effect relationship of antioxidant and anti-inflammatory activities of Laportea bulbifera based on multivariate statistical analysis

Laportea bulbifera, named Hong He Ma in Chinese, is a Chinese herbal medicine commonly used by the Miao nationality of China. In this study, 43 batches of L. bulbifera were collected from different origins in China. Ethanol, ethyl acetate and petroleum ether were used to prepare different extracts of the plant. UHPLC technique was used to establish the fingerprints, whereas DPPH assay and RAW264.7 inflammatory cell models were used to evaluate the antioxidant and anti-inflammatory activities, respectively. Moreover, the spectrum–effect relationship between relative peak area of common peaks and efficacy value was set up by multivariate statistical analysis. Furthermore, 10 batches were selected randomly for validation of those models. The results showed that ethyl acetate and petroleum ether extracts possess excellent antioxidant and anti-inflammatory activities, respectively. Peaks A₆ and A₇ demonstrated the greatest antioxidant activity, while peak A₁₇ showed the strongest anti-inflammatory activity. After a verified experiment, the result was obtained and illustrated that the spectrum–effect relationship which we established could reliably infer antioxidant and anti-inflammatory compounds of the Chinese herbal medicine.     

Fingerprint and multicomponent quantitative analysis for the quality evaluation of Sibiraea angustata leaves by HPLC-DAD and HPLC-ESI-MS/MS combined with chemometrics

Sibiraea angustata leaves, known as a traditional Tibetan medicine, have been specially used in the treatment of indigestion and obesity. In the study, a simple and sensitive high-performance liquid chromatography (HPLC) method with a diode array detector (DAD) was established to solve the problem of lacking quality standard of S. angustata leaves, including the fingerprint analysis and quantification of six characteristic components. The analytical method was validated for linearity, repeatability, stability, recovery, and specificity. Seventeen raw samples and 1 processed sample of S. angustata leaves were collected from different locations of China to establish the fingerprint. The chemometric methods, including similarity analysis (SA), principal component analysis (PCA), and hierarchical clustering analysis (HCA), were applied to distinguish the 18 batches of S. angustata samples. The results successfully sorted these samples into five clusters and kept in line with each other. According to the result of the fingerprint analysis, 21 peaks were extracted to be the common peaks and most of them were identified by mass spectrometry (MS) with electron-spray ionization (ESI) in the negative mode. Meanwhile, the loading plot of PCA further indicated that the peaks of neochlorogenic acid, chlorogenic acid, ferulic acid, rutin, hyperin, and isoquercitrin played a greater role in the discrimination among the 21 peaks. So the six components mentioned above were investigated as index constituents to evaluate the quality of S. angustata leaves from different locations. The study demonstrated that the developed new method was a beneficial approach for authentication and quality evaluation of S. angustata leaves.     

Monitoring the quality consistency of Fufang Danshen Pills using micellar electrokinetic chromatography fingerprint coupled with prediction of antioxidant activity and chemometrics

A fast micellar electrokinetic chromatography fingerprint method combined with quantification was developed and validated to evaluate the quality of Fufang Danshen Pills, a traditional Chinese Medicine, which has been used in the treatment of cardiovascular system diseases, in which the tetrahedron optimization method was first used to optimize the background electrolyte solution. Subsequently, the index of the fingerprint information amount of I was performed as an excellent objective indictor to investigate the experimental conditions. In addition, a systematical quantified fingerprint method was constructed for evaluating the quality consistency of 20 batches of test samples obtained from the same drug manufacturer. The fingerprint analysis combined with quantitative determination of two components showed that the quality consistency of the test samples was quite good within the same commercial brand. Furthermore, the partial least squares model analysis was used to explore the fingerprint–efficacy relationship between active components and antioxidant activity in vitro, which can be applied for the assessment of anti‐oxidant activity of Fufang Danshen pills and provide valuable medicinal information for quality control. The result illustrated that the present study provided a reliable and reasonable method for monitoring the quality consistency of Fufang Danshen pills.