Two Novel ent‐Kaurene Diterpenoids from Isodon rubescens

A novel 20‐nor‐ent‐kaurene diterpenoid, rubescensin N ( 1 ), and a new 7,20‐epoxy‐ent‐kaurene diterpenoid, rubescensin O ( 2 ), along with the seven known diterpenoids rabdoternins A–F and xerophilusin N, were isolated from Isodon rubescens collected in Jiyuan prefecture, Henan Province, China. Their structures were established by extensive spectroscopic analysis. Compound 1 is the first example of a naturally occurring 20‐nor‐ent‐kaurene diterpenoid from the Isodon genus plants.     

Simultaneous identification and analysis of cassane diterpenoids in Caesalpinia minax Hance by high‐performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Cassane diterpenoids were successfully and simultaneously identified in Caesalpinia minax Hance by high‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry. A total of 59 peaks were detected, and among them 51 compounds, including 41 furanocassane diterpenoids, 10 furanolactone cassane diterpenoids were simultaneously identified and characterized on the basis of the protonated molecule, retention behavior, and fragments in MS². Ten compounds, including seven novel compounds, were identified or tentatively identified for the first time in C. minax. In a positive ion mode, the fragmentation pathways of cassane diterpenoids were also analyzed for the first time. The relative amounts of the five main diterpenoids (caesalpinin L, caesalpinin F₂, bondcellpin C, caesalpinin E, and ξ‐caesalmin) were simultaneously quantified by high‐performance liquid chromatography. Results showed that the newly discovered and known components of C. minax can be used to determine the material basis of bioactivity; this method can also be applied to analyze cassane diterpenoids in herbal medicines from the genus Caesalpinia belonging to the family Fabaceae.     

Systematic characterization and simultaneous quantification of the multiple components of Rhododendron dauricum based on high‐performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

Rhododendron dauricum L. has been used as a traditional Chinese medicine to treat cough and asthma and relieve phlegm and bronchitis. In this study, a reliable method based on high‐performance liquid chromatography with diode array detection and quadrupole time‐of‐flight tandem mass spectrometry was established to systematically identify and quantify the components in this herb for the first time. A total of 33 compounds were identified, including 24 flavonoids, six phenolic acids, two coumarins and one terpene. Among them, poriolin ( 17 ), farrerol‐7‐O‐β‐d‐ glucopyranoside ( 20 ), and syzalterin ( 30 ) were isolated from this plant for the first time, and quercetin‐3‐β‐d‐ (6‐p‐hydroxy benzoyl) galactoside ( 19 ), quercetin‐3‐β‐d‐ (6‐p‐coumaroyl) galactoside ( 21 ), and myrciacetin ( 23 ) were identified from this genus for the first time. Fragmentation pathways of flavonoids also have been investigated by electrospray ionization mass spectrometry. Moreover, seven bioactive constituents, namely, gallic acid ( 1 ) , scopoletin (6 ), dihydroquercetin ( 7 ), quercetin ( 22 ), kaempferol ( 25 ), 8‐desmethyl farrerol ( 27 ), and farrerol ( 28 ), were simultaneously quantified. The developed method has been validated and applied to analyze ten samples of R. dauricum from Hebei Province successfully. The contents of the seven compounds have been detected and compared.     

LC–DAD–ESI-MS-MS for Characterization and Quantitative Analysis of Diterpenoids from Coleus forskohlii

Qualitative characterization and quantitative analysis of labdane diterpenoids from Coleus forskohlii have been achieved by liquid chromatography hyphenated with photodiode-array detection and tandem electrospray ionization mass spectrometry (LC–DAD–ESI-MSⁿ). By use of this method, thirteen forskolin-type diterpenoids were identified in the crude extract on the basis of their fragmentation mechanisms. Fragmentation rules were deduced from nine forskolin-type standards by ESI-MS in positive-ion mode. It was found that fragmentation behavior varied with the position and number of the substituents on the skeleton; this could be used for convenient identification of this type of compound. Six marker diterpenoids were also quantified, and the quality of both the cultivated and wild plants was evaluated.

     

Rubescensins S and T: Seco‐ent‐Kaurane Diterpenoids from Isodon rubescens var. taihangensis

Two new diterpenoids, rubescensin S (=(1α,6β,14β)‐7α,20‐epoxy‐1,7,14‐trihydroxy‐16‐oxo‐15,16‐seco‐ent‐kauran‐6,15‐olide; 1 ) and rubescensin T (=(1α,6β,11β,20S)‐7α,20‐epoxy‐1,6,7‐trihydroxy‐20‐methoxy‐8,15‐seco‐ent‐kaur‐16‐en‐11,15‐olide; 2 ) were isolated from the Chinese medicinal herb Isodon rubescens var. taihangensis. Compound 1 possesses a unique, unprecedented 15,16‐seco‐ent‐kaurane skeleton. Both compounds exhibited cytotoxic activities against K562 human leukemia cells.     

The in vivo absorbed constituents and metabolites of Danshen decoction in rats identified by HPLC with electrospray ionization tandem ion trap and time‐of‐flight mass spectrometry

Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge, is widely used for the treatment of cardiovascular and cerebrovascular diseases. This research focuses on the in vivo metabolism of Danshen decoction (DSD) in rats. After oral administration of DSD, the absorptive constituents and their metabolites in urine and plasma were analyzed by HPLC coupled with a photodiode array detector and electrospray ionization hybrid ion trap and time‐of‐flight mass spectrometry. Samples were separated on a C₁₈ column by gradient elution using 0.1% (v/v) aqueous formic acid and acetonitrile. As a result, 93 compounds from urine and 38 compounds from plasma were identified. Among them, lipo‐soluble diterpenoids (24 in urine and 15 in plasma) were reported for the first time as in vivo metabolites of DSD. According to the quantities and contents of the identified compounds, tanshinone IIA, cryptotanshinone and tanshinone I were deduced to be the major absorptive diterpenoids of DSD. Moreover, nine water‐soluble phenolics (caffeic acid, ferulic acid, danshensu, etc.) were proved to be the major absorptive constituents as reported. Most of the absorbed constituents underwent sulfation, glucuronidation, hydrogenation and hydroxylation in vivo. This investigation provided scientific evidence to obtain a more comprehensive metabolic profile of DSD. Copyright © 2014 John Wiley & Sons, Ltd.     

Preparative isolation of diterpenoids from Salvia bowleyana Dunn roots by high-speed countercurrent chromatography combined with high-performance liquid chromatography

The root of Salvia bowleyana Dunn (Lamiaceae) is used as a traditional Chinese medicine that has multiple therapeutic effects. In this study, an efficient strategy was developed to separate diterpenoid compounds, which are the main active ingredients in Salvia bowleyana Dunn roots, from complex crude extracts by high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography. A two-phase solvent system comprising n-hexane–ethyl acetate–methanol–water (7:3:7:3, v/v/v/v) was selected for high-speed countercurrent chromatographic separation. Three major diterpenoids, 6α-hydroxysugiol ( 7 ), sugiol ( 8 ), and 6, 12-dihydroxyabieta-5,8,11,13-tetraen-7-one ( 9 ) were obtained at purities of 98.9, 95.4, and 96.2%, respectively, and minor diterpenoids were enriched via one-step separation. The enriched minor diterpenoids were further purified by continuous preparative high-performance liquid chromatography to yield two new norabietanoids ( 1 , 6 ) and four known compounds ( 2 – 5 ). The structures of these new compounds were determined using NMR spectroscopy, high-resolution electrospray ionization mass spectrometry, and electronic circular dichroism spectroscopy. The results suggest that high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography efficiently isolates diterpenoids, including minor components, from complex natural products.     

First report of non‐coloured flavonoids in Echium plantagineum bee pollen: differentiation of isomers by liquid chromatography/ion trap mass spectrometry

Apicultural products have been widely used in diet complements as well as in phytotherapy. Bee pollen from Echium plantagineum was analysed by high‐performance liquid chromatography/photodiode‐array detection coupled to ion trap mass spectrometry (HPLC‐PAD‐MSⁿ) with an electrospray ionisation interface. The structures have been determined by the study of the ion mass fragmentation, which characterises the interglycosidic linkage in glycosylated flavonoids and differentiates positional isomers. Twelve non‐coloured flavonoids were characterised, being kaempferol‐3‐O‐neohesperidoside the major compound, besides others in trace amounts. These include quercetin, kaempferol and isorhamnetin glycosides, with several of them being isomers. Acetylated derivatives are also described. This is the first time that non‐coloured flavonoids are reported from this pollen, with MS fragmentation proving to be most useful in the elucidation of isomeric structures. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid Analysis of 27 Components of Isodon serra by LC–ESI-MS–MS

A new method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry has been developed for simultaneous analysis of 27 components (eighteen diterpenoids, six phenolic acids, and three flavonoids) of Isodon serra, a famous traditional Chinese medicine. Separation on a C₁₈ column was achieved by gradient elution with water and methanol both containing 0.1% formic acid. Identification and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring (MRM) was used for quantification, with switching of electrospray ion source polarity between positive and negative modes in the same chromatographic run. An information-dependent acquisition (IDA) method was used to trigger product ion scans above the MRM signal threshold so that the 27 compounds could be identified by use of enhanced product-ion scans (EPI). The method was fully validated (for linearity, precision, accuracy, and limits of detection and quantification). The results indicated that this simple method was rapid, specific, and reliable. The method was successfully applied to analysis of 45 batches of Isodon serra samples from different sources, and quantification of the compounds in Isodon serra was achieved.     

Characterization and identification of iridoid glucosides,flavonoids and anthraquinones in Hedyotis diffusa by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry

The multiple bioactive constituents in Hedyotis diffusa Willd. (H. diffusa) were extracted and characterized by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MSⁿ). The optimized separation condition was obtained using an Agilent ZorBax SB‐C₁₈ column (4.6×150 mm, 5 μm) and gradient elution with water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid), under which baseline separation for the majority of compounds was achieved. Among the compounds detected, 14 iridoid glucosides, 10 flavonoids, 7 anthraquinones, 1 coumarin and 1 triterpene were unambiguously identified or tentatively characterized based on their retention times and mass spectra in comparison with the data from standards or references. The fragmentation behavior for different types of constituents was also investigated, which could contribute to the elucidation of these constituents in H. diffusa. The present study reveals that even more iridoid glycosides were found in H. diffusa than hitherto assumed. The occurrence of two iridoid glucosides and five flavonoids in particular has not yet been described. This paper marks the first report on the structural characterization of chemical compounds in H. diffusa by a developed HPLC‐ESI‐MSⁿ method.     

Simultaneous identification and quantification of five flavonoids in the seeds of Rheum palmatum L. by using accelerated solvent extraction and HPLC–PDA–ESI/MSn

In this study, flavonoids were extracted using accelerated solvent extraction (ASE) with 80% aqueous methanol. A high-performance liquid chromatography equipped with photodiode array detection and coupled with an electrospray ionization tandem mass spectrometry (HPLC–PDA–ESI/MSⁿ) method has been developed for rapid identification and quantification of flavonoids in the plant extract of Rheum palmatum L. (RPL) seeds from Qinghai. Ten flavonoids from methanolic extract of RPL seeds were screened, of which epicatechin, myricetin, hyperoside, quercitrin and quercetin were identified and quantitatively analyzed for the first time. The absorbance was monitored at 280 nm for epicatechin and 360 nm for other four flavonoids. It was found that the calibration curves for all five analytes showed a good linearity (r² > 0.999) within the test range and the data of the limit of detection (LOD) and limit of quantification (LOQ) for all investigated compounds were less than 2.98 μg mL^?1 based on PDA. The relative standard deviation (R.S.D) values of the intra- and inter-day precisions were 0.98% and 1.65%, respectively. The range of mean recoveries of the five components was 91.3–93.9%, and the R.S.D was 1.3–2.9%. It was also found that the content of quercitrin is the highest in RPL seeds while the contents of epicatechin and myricetin are relatively lower. In addition, the validation procedure confirmed that this method was suitable for providing quality control evaluation of RPL seeds to ensure the therapeutic benefits.     

Simultaneous quantification of 25 active constituents in the total flavonoids extract from Herba Desmodii Styracifolii by high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry

A sensitive and selective high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry method has been developed and validated for the simultaneous determination of 25 active constituents, including 21 flavonoids and four phenolic acids in the total flavonoids extract from Herba Desmodii Styracifolii for the first time. Among the 25 compounds, seven compounds including caffeic acid, acacetin, genistein, genistin, diosmetin, diosmin and hesperidin were identified and quantified for the first time in Herba Desmodii Styracifolii. Chromatographic separation was accomplished on a ZORBAX SB‐C₁₈ (250 mm×4.6 mm, 5.0 μm) column using gradient elution of methanol and 0.1‰ acetic acid v/v at a flow rate of 1.0 mL/min. The identification and quantification of the analytes were achieved using negative electrospray ionization mass spectrometry in multiple‐reaction monitoring mode. The method was fully validated in terms of limits of detection and quantification, linearity, precision and accuracy. The results indicated that the developed method is simple, rapid, specific and reliable. Furthermore, the developed method was successfully applied to quantify the 25 active components in six batches of total flavonoids extract from Herba Desmodii Styracifolii.     

Comprehensive metabolite profiling of Plantaginis Semen using ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry coupled with elevated energy technique

Plantaginis Semen is commonly used in traditional medicine to treat edema, hypertension, and diabetes. The commercially available Plantaginis Semen in China mainly comes from three species. To clarify the chemical composition and distinct different species of Plantaginis Semen, we established a metabolite profiling method based on ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry coupled with elevated energy technique. A total of 108 compounds, including phenylethanoid glycosides, flavonoids, guanidine derivatives, terpenoids, organic acids, and fatty acids, were identified from Plantago asiatica L., P. depressa Willd., and P. major L. Results showed significant differences in chemical components among the three species, particularly flavonoids. This study is the first to provide a comprehensive chemical profile of Plantaginis Semen, which could be involved into the quality control, medication guide, and developing new drug of Plantago seeds.     

Two New Furanoid Diterpenoids from Tinospora sagittata

Two new clerodane‐based furanoid diterpenoids, tinosagittones A and B ( 1 and 2 , resp.), were isolated from the roots of Tinospora sagittata, together with five known diterpenoids, i.e., columbin ( 3 ), its glucoside palmatoside C ( 4 ), isocolumbin ( 5 ), 6‐hydroxycolumbin ( 6 ), and tinophylloloside ( 7 ). Their structures were established by mass spectrometry and spectroscopic methods, especially 2D‐NMR techniques.     

Chemical constituents of Meconopsis horridula and their simultaneous quantification by high‐performance liquid chromatography coupled with tandem mass spectrometry

Meconopsis horridula Hook.f. Thoms has been used as a traditional Tibetan medicine to clear away heat, relieve pain, and mobilize static blood. In this study, a reliable method based on high‐performance liquid chromatography with diode array detection and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry was established for the identification of components in this herb. A total of 40 compounds (including 17 flavonoids, 15 alkaloids, and eight phenylpropanoids) were identified or tentatively identified. Among them, 17 components were identified in the herb for the first time. Compound 39 appears to be a novel compound, which is confirmed as 3‐(kaempferol‐8‐yl)‐2,3‐epoxyflavanone by NMR spectroscopy and mass spectrometry. Moreover, seven major constituents were simultaneously quantified by the developed high‐performance liquid chromatography with tandem triple‐quadrupole mass spectrometry method. The quantitative method was validated and quality parameters were established. The study provides a comprehensive approach for understanding this herbal medicine.     

Chemical Characterization of Phenolic Compounds in Erigeron Injection by Rapid-Resolution LC Coupled with Multi-Stage and Quadrupole-TOF-MS

Rapid-resolution liquid chromatography coupled with electrospray ion trap multistage mass spectrometry (ESI-MS ⁿ ) and electrospray quadrupole-time-of-flight mass spectrometry (ESI-Q-TOF) has been utilized to separate and characterize the phenolic components in Erigeron injections. After extensive separation on the column, each peaks’ potential structures were obtained based on their accurate mass supplied by ESI-Q-TOF and chemical database retrieval, and confirmed by ESI-MS ⁿ . A total of 44 compounds were identified or tentatively characterized and fifteen of them reported for the first time. In addition, the developed method was applied to monitor the elimination of phenolic compounds in rats after intravenous administration of 4 mL kg^?1 Erigeron injection. The caffeoylquinic acids and flavonoids of the Erigeron injection were quickly degraded and most of them reached the low limit of detection (SNR < 3) at 2 h post-injection.     

Development and validation of an ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry method for the simultaneous determination of selected flavonoids in Ginkgo biloba

A rapid and sensitive ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous determination of 13 flavonoids in leaf, stem, and fruit extracts of male and female trees of Ginkgo biloba to investigate gender‐ and age‐related variations of flavonoids content. Chromatographic separation was accomplished on an Acquity UPLC BEH C₁₈ column (50 mm × 2.1 mm id, 1.7 μm) in 5 min. Quantitation was performed using negative electrospray ionization mass spectrometry in multiple reaction monitoring mode. The calibration curves of all analytes showed a good linear relationship (r² ≥ 0.9977) over the concentration range of 1–1000 ng/mL. The precision evaluated by an intra‐ and interday study showed RSD ≤ 1.98% and good accuracy with overall recovery in the range from 97.90 to 101.09% (RSD ≤ 1.67%) for all analytes. The method sensitivity expressed as the limit of quantitation was typically 0.25–3.57 ng/mL. The results showed that the total content of 13 flavonoids was higher in the leaf extract of an old male Ginkgo tree compared to young female Ginkgo trees.     

Identification and quantification of anthocyanins,flavonoids, and phenolic acids in flowers of Rhododendron arboreum and evaluation of their antioxidant potential

The current study aimed to investigate the anthocyanins, non-anthocyanins (flavonoids and phenolic acids), and free radicals scavenging potential in the flowers of Rhododendron arboreum using ultra high performance liquid chromatography with ion mobility quadrupole time of flight tandem mass spectrometry. A total of 25 constituents including nine anthocyanins, six phenolic acids, and ten flavonoids were identified in the flower extract. The major anthocyanins identified were cyanidin-3-O-β-galactoside ( 1 ), cyanidin-3-O-α-arabinoside ( 4 ), and cyanidin-3-O-rhamnoside ( 8 ), while quercetin glycosides were the main identified flavonoids in R. arboreum flowers. Additionally, ultra high performance liquid chromatography methods were developed and validated for the quantification of nine compounds (anthocyanins, flavonoid glycosides, and phenolic acids); five of them were quantified using internal standards. The extracts were analyzed for total phenolics (123.6 mg GAE/g), anthocyanin content (1.76% w/w), and evaluated for antioxidant properties against 2,2-diphenyl-1-picrylhydrazyl radical (IC₅₀: 102.06 and 96.92 μg/mL) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (112.25 and 45.59 μM TE/g) assays. The profiling of R. arboreum for anthocyanins is reported for the first time. The findings suggest that the flowers are a promising source of bioactive constituents and could be used as functional food, antioxidants, and nutraceuticals.     

Liquid Chromatography for Separation and Quantitative Determination of Adrenergic Amines and Flavonoids from Poncirus trifoliatus Raf. Fruits at Different Stages of Growth

The fruits of Poncirus trifoliatus Raf. (Rutaceae) have been used against allergic diseases for generations and still occupy an important place in traditional oriental medicine. They have also been used to treat gastric and duodenal ulcers. Chemical analysis of extracts of Poncirus trifoliatus Raf. fruit at different stages of maturation revealed variations in the concentrations of flavonoids present. Fourteen flavonoids (neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, neoponcirin, poncirin, naringenin, hesperetin, sinensetin, nobiletin, heptamethoxyflavone, 5-O-demethylnobiletin, and tangeretin) and five amines (synephrine, octopamine, N-methyltyramine, hordenine, and tyramine) in extracts from the fruit of Poncirus trifoliatus Raf. were analyzed by HPLC with a C₁₈ reversed-phase column. The concentrations of the four flavonoids naringin, poncirin, narirutin, and neoponcirin was maximum during the first stage of growth and gradually decreased until the fruits were fully developed. The paper also discusses the anatomical variations observed at different stages of development of the fruit.     

New Flavonoids from Lysimachia christinae Hance

A chemical investigation of Lysimachia christinae, a traditional Chinese medicine used as an effective conservative treatment for gall stones, hepatolithiasis, and urinary calculi, resulted in the isolation of two new flavonoids, myricetin 3,3′‐di‐α‐L ‐rhamnopyranoside ( 1 ) and quercetin 3,3′‐di‐α‐L ‐rhamnopyranoside ( 2 ), along with the five known flavonoids quercetin 3‐[O‐α‐L ‐rhamnopyranosyl‐(1→2)‐β‐D ‐galactopyranoside], amentoflavone, hyperin, quercetin 3‐β‐D ‐glucopyranoside, and kaempferol 3‐α‐L ‐rhamnopyranoside. Amentoflavone was reported for the first time from the genus Lysimachia, and quercetin 3‐[O‐α‐L ‐rhamopyranosyl‐(1→2)‐β‐D ‐galactopyranoside] was isolated from this plant for the first time. The structures of the new compounds were elucidated on the basis of their chemical reactions and extensive spectroscopic analyses, including UV, mass, and NMR spectra.