Fragmentation study of iridoid glycosides including epimers by liquid chromatography‐diode array detection/electrospray ionization mass spectrometry and its application in metabolic fingerprint analysis of Gardenia jasminoides Ellis

A high‐performance liquid chromatography‐diode array detection/electrospray ionization mass spectrometry (HPLC‐DAD/ESI‐MS) method was applied to the characterization of ten iridoid glycosides in Gardenia jasminoides Ellis, a traditional Chinese medicine. During the process of structural elucidation, two groups of isomers including two epimers were structurally characterized and differentiated according to their distinctive fragmentation patterns which were closely related to their isomeric differentiations. Subsequently, the major compounds were purified by multi‐dimensional chromatography and semi‐preparative HPLC and the structure identification was confirmed with NMR techniques. The major fragmentation pathways of iridoid glycosides in Gardenia jasminoides Ellis obtained through the MS data were schemed systematically, which provided the best sensitivity and specificity for characterization of the iridoid glycosides especially the isomers so far. Based on the fragmentation patterns of iridoid glycosides concluded, seven major iridoid glycosides were characterized in rat plasma after intravenous administration of Gardenia jasminoides Ellis. Copyright © 2010 John Wiley & Sons, Ltd.     

Iridoid Glycosides from Gardenia jasminoides Ellis

Three new iridoid glycosides, 4″‐O‐[(E)‐p‐coumaroyl]gentiobiosylgenipin ( 1 ), 6′‐O‐[(E)‐caffeoyl]deacetylasperulosidic acid methyl ester ( 2 ), and 6′‐O‐[(E)‐sinapoyl]gardoside ( 3 ), together with seven analogues, 4 – 10 , were isolated from the BuOH extract of the fruits of Gardenia jasminoides Ellis . Their structures were determined by means of spectroscopic analyses, including HR‐ESI‐MS, IR, and ¹H‐ and ¹³C‐NMR, and 2D experiments (COSY, HSQC, and HMBC), and comparison with known related compounds.     

Identification of metabolites of geniposide in rat urine using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Geniposide, an iridoid glycoside, is an important and characteristic compound in the fruits of Gardenia jasminoides Ellis, a commonly used medicinal herb in Chinese traditional and folk medicine for the treatment of inflammation and jaundice. However, few studies have been carried out on the metabolism of geniposide. In this study, we have established a rapid and sensitive method using ultra‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC/ESI‐QTOF‐MS) for analysis of the metabolic profile of geniposide in rat urine after oral administration. A total of ten metabolites were detected and identified by comparing their fragmentation patterns with that of geniposide using Metabolynx? and MassFragment? software tools. The results revealed that the principal metabolism pathways of geniposide in rat occurred after deglycosylation of the irdoid glycoside take place and this is followed by glucuronidation and the pyran‐ring cleavages. The major metabolite, the glucuronic acid conjugate of genipin as observed in vivo, was further confirmed by the in vitro enzymatic study. The results of this work have demonstrated the feasibility of the UPLC/ESI‐QTOF‐MS approach for rapid and reliable characterization of metabolites from iridoid compounds. Copyright © 2011 John Wiley & Sons, Ltd.     

Structural characterization and identification of iridoid glycosides,saponins, phenolic acids and flavonoids in Flos Lonicerae Japonicae by a fast liquid chromatography method with diode‐array detection and time‐of‐flight mass spectrometry

A fast liquid chromatography method with diode‐array detection (DAD) and time‐of‐flight mass spectrometry (TOF‐MS) has been developed for analysis of constituents in Flos Lonicerae Japonicae (FLJ), a traditional Chinese medicine derived from the flower bud of Lonicera japonica. The chromatographic analytical time decreased to 25 min without sacrificing resolution using a column packed with 1.8‐µm porous particles (4.6 × 50 mm), three times faster than the performance of conventional 5.0‐µm columns (4.6 × 150 mm). Four major groups of compounds previously isolated from FLJ were structurally characterized by DAD‐TOF‐MS: iridoid glycosides showed maximum UV absorption at 240 nm; phenolic acids at 217, 242, and 326 nm; flavonoids at 255 and 355 nm; while saponins had no absorption. In electrospray ionization (ESI)‐TOF‐MS experiments, elimination of a glucose unit (162 Da), and successive losses of H₂O, CH₃OH and CO, were generally observed in iridoid glycosides; saponins were characterized by a series of identical aglycone ions; phenolic acids typically generated a base peak at [M–H–caffeoyl]^? by loss of a caffeic acid unit (162 Da) and several marked quinic acid moiety ions; cleavage of the glycosidic bond (loss of 162 or 308 Da), subsequent losses of H₂O, CO, RDA and C‐ring fragmentation were the most possible fragmentation pathways for flavonoids. By accurate mass measurements within 4 ppm error for each molecular ion and subsequent fragment ions, as well as the ‘full mass spectral’ information of TOF‐MS, a total of 41 compounds including 13 iridoid glycosides, 11 phenolic acids, 7 saponins, and 10 flavonoids were identified in a methanolic extract of FLJ. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of chemical constituents in Zhi–Zi–Da–Huang decoction by ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

A sensitive and reliable ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was established to separate and identify the chemical constituents of Zhi–Zi–Da–Huang decoction, a classic traditional Chinese medicine formula. The chromatographic separation was achieved on a Shim‐pack XR‐ODS C₁₈ column (75  × 3.0 mm, 2.2 μm) using a gradient elution program. The detection was performed on a Waters Xevo G2 Q‐TOF mass spectrometer equipped with electrospray ionization source in both positive and negative modes. With the optimized conditions, a total of 82 compounds were identified or tentatively characterized. Of the 82 compounds, 21 compounds were identified by comparing the retention time and MS data with reference standards, the rest were characterized by analyzing MS data and retrieving the reference literature. In addition, 31 compounds were identified from Gardenia jasminoides Ellis, ten compounds were identified from Rheum palmatum L., 33 compounds were identified from Citrus aurantium L., and eight compounds were identified from Sojae Semen Praeparatum. Results indicated that iridoids, anthraquinones, flavonoids, isoflavonoids, coumarins, glycosides of crocetin, monoterpenoids, and organic acids were major constituents in Zhi–Zi–Da–Huang decoction. It is concluded that the developed ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method with high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents of Zhi–Zi–Da–Huang decoction, and the analysis provides a helpful chemical basis for further research on Zhi–Zi–Da–Huang decoction.     

Simultaneous Determination of Five Major Biologically Active Ingredients in Different Parts of Gardenia jasminoides Fruits by HPLC with Diode-Array Detection

A simple, sensitive and specific HPLC method for the simultaneous separation and determination of geniposide, geniposidic acid, chlorogenic acid, crocin 1 and 2 in fruits of Gardenia jasminoides Ellis was developed. Optimum chromatographic performance was obtained with a C₁₈ column and acetonitrile—0.1% aqueous trifluoroacetic acid (v/v) as mobile phase. Isolated peaks were detected at 240 nm for geniposidic acid and geniposide, 330 nm for chlorogenic acid and 440 nm for crocin 1 and 2, respectively. Comparison of spectra recorded with a diode-array detector during elution of peaks enabled determination of method specificity. Quantitative determinations on different parts of gardenia fruit demonstrated that all these compounds were abundant mainly in pericarps and pulps and only iridoid glycosides were also presented in sepals and seeds of the fruits.     

Isolation and purification of six iridoid glycosides from gardenia jasminoides fruit by medium‐pressure liquid chromatography combined with macroporous resin chromatography

Gardeniae fructus is one of the most frequently used herbs in traditional Chinese medicine. In the present study, a process for the enrichment of six iridoid glycosides from Gardeniae fructus was developed using medium‐pressure liquid chromatography combined with macroporous resin and reversed‐phase chromatography. The purities of different fractions from Gardeniae fructus were assessed using quantitative high‐performance liquid chromatography. After fractionation using HPD‐100 column chromatography, a 30% ethanol fraction was selected based on high‐performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis to separate and purify. Based on the orientation analysis results, six compounds—deacetyl asperulosidic acid methyl ester, gardenoside, ixoroside, scandoside methyl ester, genipin‐1‐O‐β‐d‐ gentiobioside, and geniposide—were successfully isolated and purified in three to four combined steps from Gardeniae fructus. The purities of these compounds were found by high‐performance liquid chromatography analysis to be 97.9, 98.1, 95.5, 96.3, 97.1, and 98.7%, respectively. Moreover, their structures were elucidated by NMR spectroscopy and liquid chromatography with tandem mass spectrometry. The separation process was highly efficient, rapid, and accurate, making it a potential approach for the large‐scale production of iridoids in the laboratory and providing several marker compounds for quality control. This procedure may be meaningful for the purification of other natural products used in traditional Chinese medicine.     

Comprehensive separation of iridoid glycosides and triterpenoid saponins from Dipsacus asper with salt‐containing solvent by high‐speed countercurrent chromatography coupled with recycling mode

The roots of Dipsacus asper Wall as a commonly used traditional Chinese medicine are used for tonifying liver and kidney and strengthening bones and muscles. However, an effective separation strategy for comprehensive and rapid separation of the main active compounds from the roots of D. asper is nonexistent. This investigation provided an effective separation method based on AB‐8 macroporous resin column chromatography using different ratios of ethanol in water and two different modes of high‐speed countercurrent chromatography with salt‐containing solvent system for rapid enrichment and separation from the roots of D. asper. The macroporous resin column chromatography was performed on AB‐8 resin using ethanol in water ratios of 10, 30, 40, 50, and 80% as the optimized enrichment conditions for iridoid glycosides and triterpenoid saponins with different polarities. For high‐speed countercurrent chromatography separation, the conventional and recycling modes were combined together to develop a strategy for 12 compounds ( 1 – 12 ) from the enriched parts of 30, 40, and 80% ethanol, including six high‐polarity iridoid glycosides ( 1 – 6 ) using inorganic salt‐containing solvent system and six triterpenoid saponins ( 7 – 12 ). Recycling high‐speed countercurrent chromatography separation was successfully applied to separate two isomers ( 9 and 10 ) after 11 cycles.     

Chemical profiling approach to evaluate the influence of traditional and simplified decoction methods on the holistic quality of Da‐Huang‐Xiao‐Shi decoction using high‐performance liquid chromatography coupled with diode‐array detection and time‐of‐flight mass spectrometry

Da‐Huang‐Xiao‐Shi decoction, consisting of Rheum officinale Baill, Mirabilitum, Phellodendron amurense Rupr. and Gardenia jasminoides Ellis, is a traditional Chinese medicine used for the treatment of jaundice. As described in “Jin Kui Yao Lue”, a traditional multistep decoction of Da‐Huang‐Xiao‐Shi decoction was required while simplified one‐step decoction was used in recent repsorts. To investigate the chemical difference between the decoctions obtained by the traditional and simplified preparations, a sensitive and reliable approach of high‐performance liquid chromatography coupled with diode‐array detection and electrospray ionization time‐of‐flight mass spectrometry was established. As a result, a total of 105 compounds were detected and identified. Analysis of the chromatogram profiles of the two decoctions showed that many compounds in the decoction of simplified preparation had changed obviously compared with those in traditional preparation. The changes of constituents would be bound to cause the differences in the therapeutic effects of the two decoctions. The present study demonstrated that certain preparation methods significantly affect the holistic quality of traditional Chinese medicines and the use of a suitable preparation method is crucial for these medicines to produce special clinical curative effect. This research results elucidated the scientific basis of traditional preparation methods in Chinese medicines.     

Simultaneous determination of eight bioactive constituents of Zhi‐Zi‐Hou‐Po decoction in rat plasma by ultra high performance liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study

Zhi‐Zi‐Hou‐Po Decoction, consisting of Gardenia jasminoides Ellis, Magnolia officinalis Rehd. et Wils., and Citrus aurantium L, is a classical Traditional Chinese Medicine formula for the treatment of depression. In order to make good and rational use of this formula in the future, a sensitive, selective, and reliable ultra high performance liquid chromatography with tandem mass spectrometry method was developed for simultaneous determination of two iridoid glycosides (geniposide and genipin gentiobioside), two lignans (honokiol and magnolol), four flavonoid glycosides (isonaringin, naringin, hesperidin, and neohesperidin), the major bioactive constituents of Zhi‐Zi‐Hou‐Po Decoction, in rat plasma using paeoniflorin as internal standard. Plasma samples were pretreated by a simple protein precipitation with acetonitrile. Chromatographic separation was performed on a shim‐pack XR‐ODS C₁₈ column (75 × 3.0 mm, 2.2 µm) using gradient elution with mobile phase consisting of 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.5 mL/min. Mass spectrometric detection was conducted on a 3200 QTRAP mass spectrometry equipped with electrospray ionization source in negative ionization mode. Quantification was performed using multiple reactions monitoring mode. Calibration curves exhibited good linearity (r > 0.9947) over a wide concentration range for all analytes, and the lower limits of quantification were 10, 5, 1, 5, 1, 5, 1, and 5 ng/mL for geniposide, genipin gentiobioside, honokiol, magnolol, isonaringin, naringin, hesperidin, and neohesperidin, respectively. The intraday and interday precisions at three quality control levels were less than 12.3% and the accuracies ranged from ?11.2 to 10.7%. Extraction recovery, matrix effect, and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of the eight analytes after oral administration of Zhi‐Zi‐Hou‐Po decoction to rats.     

Preparation of five high‐purity iridoid glycosides from Gardenia jasminoides Eills by molecularly imprinted solid‐phase extraction integrated with preparative liquid chromatography

Five iridoid glycosides were prepared using molecularly imprinted solid‐phase extraction combined with preparative high‐performance liquid chromatography. Hydrophilic molecularly imprinted polymers were synthesized using α‐1‐allyl‐2‐N‐acetyl glucosamine, which introduced an abundance of hydrophilic groups into the polymers. Using molecularly imprinted solid‐phase extraction as the sample pretreatment procedure, five iridoid glycosides, gardenoside, geniposide, shanzhiside, geniposidic acid, and genipin‐1‐O‐gentiobioside, were selectively enriched from Gardenia fructus extracts. Preparative high‐performance liquid chromatography then provided iridoid glycosides with a purity >98%. The structures were elucidated by using nuclear magnetic resonance spectroscopy, optical rotation and melting point measurements, and mass spectrometry. The results demonstrate that molecularly imprinted solid‐phase extraction combined with preparative high‐performance liquid chromatography was an efficient, rapid, and economical method for the preparation of bioactive compounds from natural products.     

Efficient purification of high‐purity compounds from the stem of Lonicera japonica Thunb using two‐dimensional preparative chromatography

Purification of high‐purity compounds from traditional Chinese medicines (TCMs) plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of TCMs in chemical composition. In this work, a two‐dimensional preparation method was successfully developed for the preparation of high‐purity compounds from the stem of Lonicera japonica Thunb, based on two novel polar copolymerized RP stationary phases, XAqua C3 and XAqua C18. An XAqua C3 prep column was used to separate the sample in the first‐dimensional preparation, and 14 g of sample was fractionated into eight fractions with a recovery of 82%. An XAqua C18 prep column was selected to prepare high‐purity compounds in the second‐dimensional preparation for its good orthogonality with the XAqua C3 stationary phase. As a result, major compounds in the sample were isolated with more than 99% purity. This method is a potent method to realize the efficient purification of compounds with high purity from the stem of L. japonica Thunb and it shows great potential in the separation of high‐purity compounds from complex samples.     

Targeted characterization of acylated compounds from Scrophulariae Radix using liquid chromatography coupled with Orbitrap mass spectrometry and diagnostic product ion‐based data analysis

Acylated compounds are often present in herbal medicines. In this study, a diagnostic product ion‐based strategy was established to comprehensively characterize acylated compounds in Scrophulariae Radix. After untargeted data acquisition using ultra‐high performance liquid chromatography coupled with Orbitrap mass spectrometry, the data were processed by three‐stage diagnostic product ions. First, diagnostic product ions corresponding to the acyl groups (cinnamoyl, p‐coumaroyl, feruloyl, and caffeoyl) were used to search 90 compounds. Second, these compounds were divided into three categories using diagnostic product ions for phenylethanoid glycosides, iridoid glycosides, and phenylpropanoids, respectively. Last, the linkage position of the acyl group to iridoid glycosides was discriminated via the third‐stage diagnostic product ions. As a result, 90 acylated compounds were characterized, and 37 of them were reported from Scrophulariae Radix for the first time.     

Iridoid-glycoside isolation and purification from Premna fulva leaves

Premna fulva Craib, rich in iridoid glycosides, is widely used to treat periarthritis, osteoproliferation, pain, and other diseases. However, no studies have reported effective purification methods for obtaining iridoid glycosides as active materials. This paper describes an efficient strategy for separating iridoid glycosides from Premna fulva leaves using high-speed counter-current chromatography and preparative high-performance liquid chromatography. A two-phase solvent system, ethyl acetate/n-butanol/water (7.5:2.5:10, v/v), was selected for high-speed counter-current chromatography separation. The proposed method effectively separated and purified four iridoid glycosides and four lignans, including three new iridoid glycosides ( 4–6 ) and five known compounds ( 1–3, 7, 8 ), from Premna fulva leaves, indicating that high-speed counter-current chromatography combined with prep-HPLC can efficiently isolate catalpol derivatives from the genus Premna. Additionally, the in vitro anti-inflammatory activities of all isolated compounds were analyzed using lipopolysaccharide-stimulated RAW 264.7 cells, and the results indicated that six compounds ( 1 and 3–7 ) exhibited potential anti-inflammatory activities.     

LC/MS/MS determination and pharmacokinetic study of iridoid glycosides monotropein and deacetylasperulosidic acid isomers in rat plasma after oral administration of Morinda officinalis extract

Morinda officinalis is a famous traditional Chinese medicine containing iridoid glycoside compounds, such as monotropein and deacetylasperulosidic acid. The aim of the study was to develop a novel and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of the two isomeric iridoid glycosides and then evaluate their pharmacokinetic properties in rats. Selected‐reaction monitoring mode was employed for quantification of two analytes in rat plasma. The calibration curves were linear over their respective concentration range with correlation coefficient >0.995 for both analytes. Precision for monotropein and deacetylasperulosidic acid ranged from 2.5 to 11.9% relative standard deviation, and the accuracy of two analytes was ?2.0–3.7 and ?6.4–10.7% relative error, respectively. This method was successfully applied in pharmacokinetic study after oral administration of M. officinalis extract in rats. The results provided a basis for further research on the bioactivity of M. officinalis. Copyright © 2015 John Wiley & Sons, Ltd.     

Iridoid Glycosides from Hedyotis corymbosa

Three new iridoid glycosides, hedycorysides A–C ( 1 – 3 , resp.), were isolated from the whole plant of Hedyotis corymbosa (Linn .) Lam ., along with four known compounds. Their structures were elucidated by extensive 1D‐ and 2D‐NMR analysis, as well as by HR‐ESI‐MS experiments. The three new compounds are the first benzoylated geniposide derivatives from Hedyotis.     

Iridoid and Phenylethyl Glycosides from Globularia sintenisii

A new iridoid glycoside, sintenoside ( 1 ) and two new phenylethyl glycosides, globusintenoside (=2‐(3,4‐dihydroxyphenyl)ethyl‐O‐6‐O‐feruloyl‐β‐D ‐glucopyranosyl‐(1→4)‐α‐L ‐rhamnopyranosyl‐(1→3)‐4‐O‐caffeoyl‐β‐D ‐glucopyranoside; 2 ) and 3′′′‐O‐methylcrenatoside (=1,2‐O‐[2‐(3,4‐dihydroxyphenyl)ethan‐1,2‐diyl]‐3‐O‐α‐L ‐4‐O‐feruloyl‐rhamnopyranosyl‐β‐D ‐glucopyranose; 3 ) were isolated from the underground parts of Globularia sintenisii, along with three known iridoid glycosides, lytanthosalin, globularin, catalpol, and six known phenylethyl glycosides, verbascoside, isoverbascoside, leucoscepthoside A, plantainoside C, martynoside, and isocrenatoside. The structure elucidation of the isolated compounds was performed by spectroscopic methods (MS and 1D and 2D NMR).     

New Anthraquinone and Iridoid Glycosides from the Stems of Hedyotis hedyotidea

Five new anthraquinone glycosides, hedanthrosides A–E ( 1 – 5 , resp.) and two new iridoid glycosides, hediridosides A and B ( 6 and 7 , resp.), along with two known anthraquinones and four known iridoids, were isolated from the stems of Hedyotis hedyotidea (DC.) Merr . The structures of the new compounds were elucidated on the basis of 1D‐ and 2D‐NMR, and HR‐MS analysis and chemical methods.     

New Iridoid Glycosides from Lamium eriocephalum subsp. eriocephalum

Two new iridoid glycosides, eriobioside ( 1 ) and lamerioside ( 2 ), were isolated from the aerial parts of Lamium eriocephalum subsp. eriocephalum, along with the two known compounds lamiide ( 3 ) and ipolamiide ( 4 ). Their structures were elucidated by spectroscopic methods (UV, 1D‐ and 2D‐NMR) and by mass spectrometry (HR‐ESI‐MS).