Plasma pharmacochemistry combined with pharmacokinetics and pattern recognition analysis to screen potentially bioactive components from Daming capsule using ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry

Daming capsule is a traditional Chinese medicine for hyperlipidemia treatment. However, the vague understanding of the bioactive components of Daming capsule hampers its modernization and internationalization. This work first developed a high‐throughput, high‐resolution, and high‐sensitivity ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method for identifying the absorbed compounds and monitoring the pharmacokinetics of Daming capsule. A high‐throughput strategy integrating plasma pharmacochemistry, pharmacokinetics, and pattern recognition analysis was also established for screening the bioactive components of Daming capsule in vivo. The established strategy based on ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry was successfully applied to screen the bioactive components of Daming capsule. Up to 53 absorbed compounds were identified. Six anthraquinones with fast and high absorption, namely, emodin‐O‐glucoside, aurantio‐obtusin, aloe‐emodin, rhein, emodin, and chrysophanol, were screened as potentially bioactive components of Daming capsule. The plasma pharmacochemistry and pharmacokinetics of Daming capsule were reported for the first time. Notably, the high‐throughput and reliable strategy facilitated the screening and identification of bioactive components of traditional Chinese medicine, thereby providing novel insights into the research and development of new drugs.     

Multi‐component profiles through the blood–brain barrier in rat after oral administration of over‐the‐counter drug Keke capsule by ultra‐performance liquid chromatography/quadrupole‐ time‐of‐flight MSE method

Keke capsule as a traditional Chinese medicine formulation is used to relieve cough, for analgesia and to reduce bronchial asthma. The multi‐components are absorbed into the blood and brain after oral administration of Keke capsule, with no systematic investigation so far. A reliable and rapid UPLC–QTOF–MS^E combined with a data processing software platform was used to characterize the components of Keke capsule and simultaneously identify bioactive components in blood and brain tissues in rat after oral administration. Consequently, a total of 41 components of Keke capsule, including alkaloids, flavone, flavonols, triterpene, lignanoid, organic acids, glycosides and coumarin were identified. Twenty‐one components were found in plasma, including 18 prototypes and three metabolites; 15 components were found in brain tissues, including 10 prototypes and five metabolites. Alkaloids and flavonoids in Keke capsule were the main components which were absorbed into blood. The main alkaloids of Keke capsule can pass through the blood–brain barrier and show different distribution tendencies in brain tissues. The main components of keke capsule was simultaneously analyzed by throughput analysis, and the corresponding bioactive components were examined by blood‐brain barrier in the rat after oral administration of the capsule.     

The profiling and identification of chemical components,prototypes and metabolites of Run‐zao‐zhi‐yang capsule in rat plasma,urine and bile by an UPLC‐Q‐TOF/MSE‐based high‐throughput strategy

Run‐zao‐zhi‐yang (RZZY) capsule, a traditional Chinese medicine formula, is popularly used for the treatment of dermatitis and eczema. However, few studies have been carried out on RZZY and its metabolites. In this study, we developed a three‐step strategy to rapidly characterize the chemical constituents and metabolites of RZZY using ultra‐high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. A total of 41 chemical components were characterized from RZZY. Among these, there are 11 flavonoids, six alkaloids, six stilbene glycosides, five anthraquinones and 13 other compounds. In addition, 18 prototypes and 35 metabolites were detected in rat plasma, urine and bile. This study offers an applicable approach for high‐throughput profiling and identification of chemical components and metabolites derived from traditional Chinese medicine formula in vivo, and also provides essential data for exploring bioactive ingredients and action mechanisms of RZZY.     

Characterization and identification of the metabolites of Menthae Haplocalycis Herba water extracts in rat plasma,urine, and feces by ultra‐high performance liquid chromatography with linear ion trap‐Orbitrap mass spectrometry

Menthae Haplocalycis Herba has been utilized for food and medicinal purposes in China for thousands of years. It has various efficacies, including dispelling wind and heat and relieving sore throat. M. Haplocalycis Herba has been also widely used in food, cosmetics, spices, and other fields. Exploring the constituents and detecting the metabolites of M. Haplocalycis are of great significance to clarify the effective substances. However, the in vivo metabolites of M. Haplocalycis Herba water extract are still unclear. Herein, a sensitive and specific method, ultra‐high performance liquid chromatography with linear ion trap‐Orbitrap mass spectrometry, established in this assay was used to study the metabolism of M. Haplocalycis Herba water extract in rat plasma, urine, and feces. We characterized and identified 9, 50, and 34 metabolites in plasma, urine, and feces, respectively. Seven metabolic pathways, including phase Ⅰ (isomerization, demethylation, hydroxylation, and dehydration) and phase Ⅱ (sulfation and glucuronidation) were mainly involved in the metabolism. It is the first systematic study on the metabolism of M. Haplocalycis Herba water extract in vivo, which enrich current understanding of the metabolic behavior of M. Haplocalycis Herba water extract and provide a metabolic rationale for further in‐depth in vivo biotransformation and pharmacokinetic analysis.     

Comprehensive characterization and quantification of multiple components in Dan‐Huang‐Qu‐Yu capsule using a multivariate data processing approach based on microwave‐assisted extraction with UHPLC and Q Exactive quadrupole‐orbitrap high‐resolution mass spectrometry

Dan‐Huang‐Qu‐Yu capsule, a Chinese herbal medicine compound preparation, is widely used for chronic pelvic inflammatory disease. In this study, a rapid, selective, and sensitive microwave‐assisted extraction ultra‐high‐performance liquid chromatography‐Q Exactive quadrupole‐orbitrap high‐resolution mass spectrometry method was developed for analyzing its chemical compositions. A total of 85 compounds, including 22 flavonoids, 8 terpenoids, 5 quinones, 5 phthaleolactone, 23 organic acids, and 22 other compounds were identified from Dan‐Huang‐Qu‐Yu capsule. Among them, 35 major compounds were unambiguously detected by comparing them with reference standards and selected as quality control markers, which were simultaneously determined in Dan‐Huang‐Qu‐Yu capsule. The established method was successfully validated and applied for simultaneous determination of 35 bioactive compounds in Dan‐Huang‐Qu‐Yu capsule from ten sample batches. The quantitative data of the analytes were analyzed by principal component analysis for quality assessment of Dan‐Huang‐Qu‐Yu capsule. Six compounds (e.g., astragaloside IV, salvianolic acid B, ellagic acid, chlorogenic acid, N‐butylidenephthalide, and luteolin) were screened out and regarded as chemical markers for quality control of Dan‐Huang‐Qu‐Yu capsule. The established method has been proved to be a novel and useful tool for rapid research of Dan‐Huang‐Qu‐Yu capsule. This research will provide reference for the scientific research of traditional Chinese medicines.     

Quality assessment of Herba Leonuri based on the analysis of multiple components using normal‐ and reversed‐phase chromatographic methods

A novel, improved, and comprehensive method for quality evaluation and discrimination of Herba Leonuri has been developed and validated based on normal‐ and reversed‐phase chromatographic methods. To identify Herba Leonuri , normal‐ and reversed‐phase high‐performance thin‐layer chromatography fingerprints were obtained by comparing the colors and R _f values of the bands, and reversed‐phase high‐performance liquid chromatography fingerprints were obtained by using an Agilent Poroshell 120 SB‐C18 within 28 min. By similarity analysis and hierarchical clustering analysis, we show that there are similar chromatographic patterns in Herba Leonuri samples, but significant differences in counterfeits and variants. To quantify the bio‐active components of Herba Leonuri , reversed‐phase high‐performance liquid chromatography was performed to analyze syringate, leonurine, quercetin‐3‐O‐robiniaglycoside, hyperoside, rutin, isoquercitrin, wogonin, and genkwanin simultaneously by single standard to determine multi‐components method with rutin as internal standard. Meanwhile, normal‐phase high‐performance liquid chromatography was performed by using an Agilent ZORBAX HILIC Plus within 6 min to determine trigonelline and stachydrine using trigonelline as internal standard. Innovatively, among these compounds, bio‐active components of quercetin‐3‐O‐robiniaglycoside and trigonelline were first determined in Herba Leonuri . In general, the method integrating multi‐chromatographic analyses offered an efficient way for the standardization and identification of Herba Leonuri .     

Rapid analysis and characterization of multiple constituents of corn silk aqueous extract using ultra‐high‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

Corn silk is a well‐known traditional Chinese medicine that has been widely used for its antidiabetic, antioxidant, antihyperlipidemic, and other effects in China for thousands of years. Numerous studies have revealed that corn silk contains multiple bioactive constituents that are beneficial for human health. However, the constituents of corn silk in vivo remain ambiguous. In this study, high‐throughput ultra‐high‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry technology using multivariate statistical analysis was established to systematically investigate the constituents migrating into blood from corn silk aqueous extract. As a result, 76 compounds were identified, including caffeic acid and ten of its derivatives, (E)‐p‐coumaric acid and two of its derivatives, ferulic acid and four of its derivatives, and five flavones. Among the identified constituents, 21 constituents, including nine prototype components and 12 metabolites derived from eight components, were characterized in sequence. Based on the significance of the results, the applied approach was powerful for the accurate determination and rapid screening of bioactive components from corn silk aqueous extract. The obtained results are valuable for the in‐depth understanding and further pharmacological study of corn silk aqueous extract.     

Characterization of chemical constituents and absorbed components,screening the active components of gelanxinning capsule and an evaluation of therapeutic effects by ultra‐high performance liquid chromatography with quadrupole time of flight mass spectrometry

We revealed the potential biomarker and pathway of gelanxinning capsule on rat model with coronary heart disease, which aims to clarify holistic therapeutic effect and predict quality‐markers of gelanxinning capsule. Ultra‐high performance liquid chromatography coupled with mass spectrometry based on metabolomics technique was used to find the biomarkers and related metabolic pathways of coronary heart disease model, which evaluates the intervention effect of gelanxinning capsule. Using serum pharmacochemistry of traditional Chinese medicine and Pearson correlation analysis, effective ingredients in serum is analyzed to characterize the activity of gelanxinning capsule on coronary heart disease under valid state. A total of 20 biomarkers from coronary heart disease were identified and 12 of them were regulated by gelanxinning capsule treatment, which is mainly involved in sphingolipid metabolism and glycerophospholipid metabolism. With the high sensitivity liquid chromatography coupled with mass spectrometry technology, a total of 46 compounds from gelanxinning capsule were identified in vitro and 25 of them were absorbed in blood. The correlation analysis of serum biomarkers and absorbed components was used to find 11 compounds as quality‐markers to be responsible for the efficacy of gelanxinning capsule. This strategy was successfully applied to screening of potential mechanism and quality‐markers from herbal medicine.      

Identification of components and metabolites in plasma of type 2 diabetic rat after oral administration of Jiao‐Tai‐Wan using ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry

Jiao‐Tai‐Wan, which is composed of Coptis Rhizoma and Cinnamon Cortex, has been recently used to treat type 2 diabetes. Owing to lack of data on its prototypes and metabolites, elucidation of the pharmacological and clinically safe levels of this formula has been significantly hindered. To screen more potential bioactive components of Jiao‐Tai‐Wan, we identified its multiple prototypes and metabolites in the plasma of type 2 diabetic rats by ultra high performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry. A total of 47 compounds were identified in the plasma of type 2 diabetic rats, including 22 prototypes and 25 metabolites, with alkaloids constituting the majority of the absorbed prototype components. In addition, this is the first study to detect vanillic acid, gallic acid, chlorogenic acid, protocatechuic acid, 2‐hydroxycinnamic acid, 3‐hydroxycinnamic acid, 4‐hydroxycinnamic acid, and 2‐methoxy cinnamic acid after oral administration of Jiao‐Tai‐Wan. The prototypes from Jiao‐Tai‐Wan were extensively metabolized by demethylation, hydroxylation, and reduction in phase Ⅰ metabolic reactions and by methylation or conjugation of glucuronide or sulfate in phase Ⅱ reactions. This is the first systematic study on the components and metabolic profiles of Jiao‐Tai‐Wan in vivo. This study provides a useful chemical basis for further pharmacological research and clinical application of Jiao‐Tai‐Wan.     

Solidification of floating organic drop liquid‐phase microextraction cell fishing with gas chromatography–mass spectrometry for screening bioactive components from Amomum villosum Lour

In this study, a novel solidification of floating organic drop liquid‐phase microextraction cell fishing with gas chromatography–mass spectrometry (SFOD‐LPME‐CF‐GC‐MS) method was established and used to screen, isolate and analyze bioactive components from Amomum villosum Lour. extract. Through comparision of its effect on the models of normal cell and inflammatory cells, anti‐inflammatory active components of essential oil from A. villosum Lour. were readily screened, and the components obtained are in agreement with related pharmacological articles. SFOD‐LPME‐CF‐GC‐MS was used to analyze the interaction of A. villosum Lour. extracts with normal and lipopolysaccharide‐stimulated RAW264.7 macrophage cells. The effect of A. villosum Lour. essential oil extracts in the LPS‐stimulated RAW264.7 model were also assessed in terms of cytotoxicity and nitric oxide production as an indication of bioactivity. Three potentially bioactive components were identified, demonstrating that SFOD‐LPME‐CF‐GC‐MS can be used successfully in the drug‐screening process. This approach avoids the requirement for protein precipitation, but more importantly, generates a high concentration ratio, allowing analysis of trace components in traditional Chinese medicines. SFOD‐LPME‐CF‐GC‐MS is a simple, fast, effective and reliable method for the screening and analysis of bioactive components, and it can be extended to screen other bioactive components from TCMs. Copyright © 2014 John Wiley & Sons, Ltd.     

Pharmacokinetics study of multiple components absorbed in rat plasma after oral administration of Stemonae radix using ultra‐performance liquid‐chromatography/mass spectrometry with automated MetaboLynx software analysis

Stemonae radix (Stemona tuberosa Lour, Bai Bu) is a traditional Chinese medicinal (TCM) plant known for its antitussive and anti‐ectoparasitic activity; however, the in vivo pharmacokinetic of its multiple bioactive components remains unknown. In this article, UPLC‐Q‐TOF‐high‐definition mass spectrometry (HDMS) coupled with automated data analysis MetaboLynx? software together were first developed to screen the potentially bioactive components in the rat plasma after oral administration of Stemonae radix. Time course of the absorbed components of Stemonae radix was built to evaluate pharmacokinetic behaviors. This rapid automated analysis method was successfully applied for identification, screening, and monitoring of the 28 constituents absorbed and metabolized studies of Stemonae radix after oral administration to rats. The results showed that the ongoing changes of 28 constituents including eight parent compounds and 20 metabolites in vivo were observed to find biomarkers. From the angle of behavior in vivo, it suggested that croomine and tuberostemonine would be potential efficacy markers. This work also demonstrated that the pharmacokinetics‐based UPLC‐Q‐TOF‐HDMS can provide a reliable means of identifying and screening potentially bioactive components contributing to pharmacological effects of medicinal herbs, and to better clarify its action mechanism, further prospecting natural products in the search for new leads in drug discovery.     

Ultra‐high‐pressure‐assisted extraction of wedelolactone and isodemethylwedelolactone from Ecliptae Herba and purification by high‐speed counter‐current chromatography

Ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was employed to extract and purify wedelolactone and isodemethylwedelolactone from Ecliptae Herba. The operating conditions of ultra‐high‐pressure extraction were optimized using an orthogonal experimental design. The optimal conditions were 80% aqueous methanol solvent, 200 MPa pressure, 3 min extraction time and 1:20 (g/mL) solid–liquid ratio for extraction of wedelolactone and isodemethylwedelolactone. After extraction by ultra‐high pressure, the extraction solution was concentrated and subsequently extracted with ethyl acetate; a total of 2.1 g of crude sample was obtained from 100 g of Ecliptae Herba. A two‐phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (3:7:5:5, v/v) was used for high‐speed counter‐current chromatography separation, by which 23.5 mg wedelolactone, 6.8 mg isodemethylwedelolactone and 5.5 mg luteolin with purities >95% were purified from 300 mg crude sample in a one‐step separation. This research demonstrated that ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was an efficient technique for the extraction and purification of coumestans from plant material.     

HPLC‐Q‐TOF‐MS/MS for analysis of major chemical constituents of Yinchen–Zhizi herb pair extract

The Yinchen–Zhizi herb pair (YZHP) consists of Herba Artemisiae Scopariae (Yinchen in Chinese) and Fructus Gardeniae (Zhizi in Chinese), and is mainly used to treat icteric hepatitis, itching skin and eczema. However, the bioactive constituents responsible for the pharmacological effects of YZHP are still unclear to date. In this work, a rapid and sensitive method was established to comprehensively study the constituents in YZHP extract by HPLC‐Q‐TOF MS/MS. The analysis was performed on an HPLC system equipped with an Agilent poroshell 120 EC‐C₁₈ column (100 × 2.1 mm, 2.7 mm) working in a gradient elution program coupled to quadrupole‐time‐of‐flight mass spectrometry operating in the negative ion mode. As a result, a total of 46 compounds including 17 from Herba Artemisiae Scopariae and 36 from Fructus Gardeniae were detected and tentatively identified in YZHP extract by comparing the retention time and mass spectrometry and retrieving the reference literature. More importantly, a series of constituents, such as many iridoid glycosides, were reported for the first time in this formula. The HPLC‐Q‐TOF MS/MS method was developed and utilized successfully to identify the major constituents in YZHP extract and would be helpful for further metabolism and pharmacology research on YZHP. Copyright © 2013 John Wiley & Sons, Ltd.     

High‐throughput ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry method for the rapid analysis and characterization of multiple constituents of Radix Polygalae

Radix Polygalae, the dried roots of Polygala tenuifolia and P. sibirica , is one of the most well‐known traditional Chinese medicinal plants. It is an important medicinal plant that has been used as a sedative and to improve memory for a number of years in most of Asia. However, the in vivo constituents of the multiple constituents from Radix Polygalae remain unknown. In the current study, ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry and the MarkerLynx^TM software combined with multiple data processing approach were used to study the constituents in vitro and in vivo. A rapid and efficient method for the characterization of multiple constituents in the herbal medicine Radix Polygalae by ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry is described. In total, 35 compounds in the Radix Polygalae and 13 compounds absorbed into blood were characterized. Of the 35 compounds in vitro, ten were reported for first time. In the 13 compounds in vivo, six were prototype components and seven were metabolites were also elucidated for first time. This work narrowed the range of screening the potentially bioactive components and provided a basis for the quality control and mechanism of action.     

Identification of absorbed constituents and metabolites in rat plasma after oral administration of Shen‐Song‐Yang‐Xin using ultra‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

In this study, a rapid and sensitive method by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry, and Metabolynx^TM software with mass defect filter technique was developed for screening and identification of the metabolites in rat plasma after oral administration of Shen‐Song‐Yang‐Xin capsule (SSYX). A total of 92 SSYX‐related xenobiotics were identified or characterized, including 45 prototypes and 47 metabolites. The results indicated that the absorbed constituents and metabolites mainly came from benzocyclooctadiene lignans, tanshinones, isoquinoline alkaloids and triterpenic acids, while phase I reactions (e.g. hydrogenation, hydroxylation, demethylation) and phase II reaction (glucuronidation) were the main metabolic pathways of these ingredients in SSYX. This is the first study on metabolic profiling of SSYX in rat plasma after oral administration. Furthermore, these findings provide useful information on the potential bioactive compounds, and enhance our understanding of the action mechanism of SSYX. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination and qualitative analysis of six types of components in Naoxintong capsule by miniaturized matrix solid‐phase dispersion extraction coupled with ultra high‐performance liquid chromatography with photodiode array detection and quadrupole time‐of‐flight mass spectrometry

A simple and effective sample preparation process based on miniaturized matrix solid‐phase dispersion was developed for simultaneous determination of phenolic acids (gallic acid, chlorogenic acid, ferulic acid, 3,5‐dicaffeoylqunic acid, 1,5‐dicaffeoylqunic acid, rosmarinic acid, lithospermic acid, and salvianolic acid B), flavonoids (kaempferol‐3‐O‐rutinoside, calycosin, and formononetin), lactones (ligustilide and butyllidephthalide), monoterpenoids (paeoniflorin), phenanthraquinones (cryptotanshinone), and furans (5‐hydroxymethylfurfural) in Naoxintong capsule by ultra high‐performance liquid chromatography. The optimized condition was that 25 mg Naoxintong powder was blended homogeneously with 100 mg Florisil PR for 4 min. One milliliter of methanol/water (75:25, v/v) acidified by 0.05% formic acid was selected to elute all components. It was found that the recoveries of the six types of components ranged from 61.36 to 96.94%. The proposed miniaturized matrix solid‐phase dispersion coupled with ultra high‐performance liquid chromatography was successfully applied to simultaneous determination of the six types of components in Naoxintong capsules. The results demonstrated that the proposed miniaturized matrix solid‐phase dispersion coupled with ultra high‐performance liquid chromatography could be used as an environmentally friendly tool for the extraction and determination of multiple bioactive components in natural products.     

Direct from solid natural products to pure compounds in a single step: Coupling online extraction with high‐speed counter‐current chromatography

Extraction is the most important step in the purification of bioactive compounds from natural products. This study introduces a simple online extraction strategy coupled with high‐speed counter‐current chromatography for efficient extraction and purification of bioactive components from solid natural products. For online extraction strategy, 1.0 g of ground Mangnolia officinalis or Piper nigrum was loaded into a guard column, which was then positioned on the manual injection valve instead of the sample loop. Bioactive components were directly extracted by the mobile phase of high‐speed counter‐current chromatography, and then transferred into high‐speed counter‐current chromatography for purification. In addition, the compatibility of the developed methodology for direct purification of bioactive components from fresh M. officinalis was successfully demonstrated. Obviously, in comparison with traditional offline heat‐reflux extraction, online extraction avoided the instrument, time, solvent, and energy consumption, and purified two phenolic compounds (honokiol and magnolol) from M. officinalis and three alkaloids (piperyline, piperine, and piperanine) from P. nigrum with high extraction efficiency. The superiority of the developed methodology is to establish an easy, rapid, and efficient technique for the purification of a wide variety of bioactive components from solid natural products.     

Screening and isolation of cyclooxygenase‐2 inhibitors from Trifolium pratense L. via ultrafiltration,enzyme‐immobilized magnetic beads,semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography

Nonsteroidal anti‐inflammatory drugs reportedly reduce the risk of developing cancer. One mechanism by which they reduce carcinogenesis involves the inhibition of the activity of cyclooxygenase‐2, an enzyme that is overexpressed in various cancer tissues. Its overexpression increases cell proliferation and inhibits apoptosis. However, selected cyclooxygenase‐2 inhibitors can also act through cyclooxygenase‐independent mechanisms. In this study, using ultrafiltration, enzyme‐immobilized magnetic beads, high‐performance liquid chromatography, and electrospray‐ionization mass spectrometry, several isoflavonoids in Trifolium pratense L. extracts were screened and identified. Semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography were then applied to separate the active constituents. Using these methods, seven major compounds were identified in Trifolium pratense L. As cyclooxygenase‐2 inhibitors: rothindin, ononin, daidzein, trifoside, pseudobaptigenin, formononetin, and biochanin A, which were then isolated with >92% purity. This is the first report of the presence of potent cyclooxygenase‐2 inhibitors in Trifolium pratense L. extracts. The results of this study demonstrate that the systematic isolation of bioactive components from Trifolium pratense L., by using ultrafiltration, enzyme‐immobilized magnetic beads, semi‐preparative high‐performance liquid chromatography, and high‐speed counter‐current chromatography, represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors.