Preparative separation of iridoid glucosides and crocins from Gardeniae Fructus using sequential macroporous resin column chromatography and evaluation of their anti-inflammatory and antioxidant activities.

Iridoid glycosides (geniposide (GP), genipin-1-gentiobioside (GB), etc.) and crocins (crocin Ⅰ (CR1), crocin Ⅱ(CR2), etc.) are two main bioactive components in Gardeniae Fructus (GF), which is a famous traditional Chinese medicine. Iridoid glycosides exhibit many activities and are used to manufacture gardenia blue pigment for the food industry. Crocins are rare natural water-soluble carotenoids that are often used as food colorants. A sequential macroporous resin column chromatography technology composed of HC-500B and HC-900B resins was developed to selectively separate iridoid glucosides and crocins from GF. The adsorption of GP on HC-900B resin was an exothermic process. The adsorption of CR1 on HC-500B resin was an endothermic process. The two kinds of components were completely separated by a sequential resin column. GB and GP were mainly found in product 1 (P1) with purities of 11.38% and 46.83%, respectively, while CR1 and CR2 were mainly found in product 2 (P2) with purities of 12.32% and 1.40%, respectively. The recovery yields of all the compounds were more than 80%. The above results showed that sequential resin column chromatography technology achieved high selectivity and recovery yields. GF extract, P1 and P2 could significantly inhibit the secretion of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-induced RAW264.7 cells, indicating that iridoid glycosides and crocins provide a greater contribution to the anti-inflammatory activity of GF. At the same time, compared to the GF extract and P1, P2 exhibited stronger scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, indicating that crocins may provide a significant contribution to the antioxidant activity of GF.

Hepatoprotective Principles from the Rhizomes of Picrorhiza kurroa.

A methanol extract of rhizomes of Picrorhiza kurroa Royle ex Benth. (Plantaginaceae) showed hepatoprotective effects against D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. We had previously isolated 46 compounds, including several types of iridoid glycosides, phenylethanoid glycosides, and aromatics, etc., from the extract. Among them, picroside II, androsin, and 4-hydroxy-3-methoxyacetophenone exhibited active hepatoprotective effects at doses of 50-100 mg/kg, per os (p.o.) To characterize the mechanisms of action of these isolates and to clarify the structural requirements of phenylethanoid glycosides for their hepatoprotective effects, their effects were assessed in in vitro studies on (i) D-GalN-induced cytotoxicity in mouse primary hepatocytes, (ii) LPS-induced nitric oxide (NO) production in mouse peritoneal macrophages, and (iii) tumor necrosis factor-α (TNF-α)-induced cytotoxicity in L929 cells. These isolates decreased the cytotoxicity caused by D-GalN without inhibiting LPS-induced macrophage activation and also reduced the sensitivity of hepatocytes to TNF-α. In addition, the structural requirements of phenylethanoids for the protective effects of D-GalN-induced cytotoxicity in mouse primary hepatocytes were evaluated.

Quantitative Analysis and Stability Study on Iridoid Glycosides from Seed Meal of Eucommia ulmoides Oliver.

As a traditional Chinese medicine, Eucommia ulmoides Oliver (E. ulmoides Oliv.) is an important medicinal plant, and its barks, male flowers, leaves, and fruits have high value of utilization. The seed meal of E. ulmoides Oliv. is the waste residue produced after oil extraction from seeds of E. ulmoides Oliv. Though the seed meal of E. ulmoides Oliv. is an ideal feed additive, its medicinal value is far from being developed and utilized. We identified six natural iridoid compounds from the seed meal of E. ulmoides Oliv., namely geniposidic acid (GPA), scyphiphin D (SD), ulmoidoside A (UA), ulmoidoside B (UB), ulmoidoside C (UC), and ulmoidoside D (UD). Six natural iridoid compounds were validated to have anti-inflammatory activities. Hence, six compounds were quantified at the optimum extracting conditions in the seed meal of E. ulmoides Oliv. by an established ultra-performance liquid chromatography (UPLC) method. Some interesting conversion phenomena of six tested compounds were uncovered by a systematic study of stability performed under different temperatures and pH levels. GPA was certified to be stable. SD, UA, and UC were only hydrolyzed under strong alkaline solution. UB and UD were affected by high temperature, alkaline, and strong acid conditions. Our findings reveal the active compounds and explore the quantitative analysis of the tested compounds, contributing to rational utilization for the seeds residues of E. ulmoides Oliv.

Targeted discovery and characterization of secoiridoid glycosides from Jasminum pentaneurum Hand.-Mazz by ultra-performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry based on diagnostic ion and neutral loss filtering strategy.

In this study, we proposed an integrated analytical strategy for the rapid and comprehensive discovery of a specific class of secoiridoid glycosides from a Yao medicine, Jasminum pentaneurum Hand.-Mazz. The strategy fully took advantage of the accuracy of ultra-performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry, and the efficiency of diagnostic ion filtering and neutral loss filtering. Twenty-four secoiridoid glycosides, including three known ones and 21 unreported ones, were rapidly discovered and characterized based on the detail analysis of their mass spectrometry data. Particularly, 10-syringicoyl-ligustroside (18) was isolated under the guidance of mass spectrometry analysis. Its chemical structure was elucidated on the basis of extensive spectroscopic data analysis, and absolute configuration was further elucidated by comparison of its experimental and electronic circular dichroism spectra. Furthermore, the mass spectrometry data of 18 was analyzed and the corresponding results indicated that its fragment pathway was fully consistent with the applied diagnostic ion filtering and neutral loss filtering rules, and thus the precision and efficiency of the integrated strategy were validated. The result demonstrated that the proposed integrated strategy could serve as a rapid, accurate, and comprehensive targeted components discovery method to effectively screen out those ingredients of interest from the complex herbal medicines.

A database-guided integrated strategy for comprehensive chemical profiling of traditional Chinese medicine.

A comprehensive chemical profiling of traditional Chinese medicine is the basic issue for further pharmacological research and quality assessment. To facilitate chemical identification and potential components discovery, the present study proposed an integrated identification strategy guided by a self-built component database constructed from literatures to carry out the global profiling of complex matrixes. Lanqin Oral Liquid was applied as example to validate the feasibility of this strategy. Based on LQL Component Database containing 710 compounds, modified MDF windows was established to extract the interested analogues, isoquinoline alkaloids, flavonoids and iridoid glycosides, according to their regular integral masses and mass defect. For compounds with characteristic substructures, such as quinic acids, crocins and some glycoside derivatives, the associated neutral losses and diagnostic fragment ions were collected to assist in profiling. Directly matching the m/z or formulas in database was proposed to components with limited regularity of accurate masses and substructures, like indole alkaloids, sesquiterpenes and some nucleosides. Eventually, 170 ions of 1038 precursor ions were identified or temporarily deduced, including 59 alkaloids, 36 flavonoids, 48 terpenoids, 24 organic acids and their derivatives, 2 oligosaccharides, and 1 lignans. Among them, 52 putative compounds were confirmed by chemical standards. The results indicated that the database-oriented identification strategy could locate potential components quickly and eliminate interfering ions, which have the potential for in-depth analysis of compounds.

[Simultaneous determination of 11 active components in Lonicera japonica flowers and leaves at different development stages by HPLC-DAD].

This study aims to develop an HPLC-DAD method for simultaneous determination of 11 components(6 phenolic acids and 5 iridoids) in Lonicera japonica flowers(LjF) and leaves(LjL), and compare the content differences of LjF at different development stages, LjL at different maturity levels, and between LjF and LjL. One-way ANOVA, principal component analysis(PCA), and orthogonal partial least-squares discriminant analysis(OPLS-DA) were employed to compare the content of the 11 components. The content of total phenolic acids, total iridoid glycosides, and total 11 components in LjF showed an overall downward trend with the development of flowers. The content of total phenolic acids, total iridoid glycosides, and total 11 components in young leaves were higher than those in mature leaves. The results of PCA showed that the samples at different flowering stages had distinguishable differences in component content. The VIP value of OPLS-DA showed that isochlorogenic acid A, chlorogenic acid, and secologanic acid were the main differential components of LjF at different development stages or LjL with different maturity levels. LjF and LjL have certain similarities in chemical composition while significant differences in component content. The content of total phenolic acids in young leaves was significantly higher than that in LjF at various development stages. The content of total iridoid glycosides in young leaves was similar to that in LjF before white flower bud stage. The total content of 11 components in young leaves was significantly higher than that in LjF at green flower bud stage, before and during completely white flower bud stage. LjL have great potential for development. Follow-up research on the pharmacodynamic equivalence of LjF and LjL(especially young leaves) should be carried out to speed up the development and application of LjL.

Rapid identification of chemical constituents in Yinqiao powder using ultra-high-performance liquid chromatography coupled to quadrupole-time-of-flight tandem mass spectrometry using data filtering strategy.

Yinqiao powder is a classic and effective prescription for the treatment of many kinds of pneumonia in China. To date, its chemical constituents have not been determined. A comprehensive identification of its chemical constituents provided a structural basis to discover the potential anti-pneumonia ingredients in Yinqiao powder. This paper developed an ultra-high-performance liquid chromatography (UHPLC) coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) analysis with diagnostic product ions and neutral loss filtering strategy and applied it for the comprehensive chemical profiling of Yinqiao powder, which simplified the structural elucidation of chemical constituents in Yinqiao powder. A total of 124 compounds, comprising 8 C6-C2 glucoside conjugates, 28 iridoid glycosides, 14 lignans, 21 phenylethanol glycosides, 20 triterpenoid saponins, 9 chlorogenic acids, and 24 flavonoids, were rapidly identified in Yinqiao powder, and 32 of these were characterized by comparing their MS/MS data and retention time with reference standards. The results indicated that UHPLC-QTOF-MS/MS method coupled with data filtering strategy was feasible and rational to identify the complex chemical constituents of Yinqiao powder, which would be conducive to discover the active ingredients of Yinqiao powder for the treatment of pneumonia and establish its quality standard.

Secoiridoid glycosides from the fruits of Cornus officinalis.

Five new secoiridoid glycosides, cornusphenosides E-I (1-5), were isolated and characterized from an active fraction of ethanol extract of the fruits of Cornus officinalis. Their structures were determined by extensive spectroscopic data analysis, including 2 D NMR and HRESIMS experiments. In the preliminary assay, compound 5 (when evaluated at 10 µM) showed the neuroprotective effect against H2O2-induced SH-SY5Y cell damage.

Simultaneous determination of 11 active components in Lonicera japonica flowers and leaves at different development stages by HPLC-DAD / 中国中药杂志

This study aims to develop an HPLC-DAD method for simultaneous determination of 11 components(6 phenolic acids and 5 iridoids) in Lonicera japonica flowers(LjF) and leaves(LjL), and compare the content differences of LjF at different development stages, LjL at different maturity levels, and between LjF and LjL. One-way ANOVA, principal component analysis(PCA), and orthogonal partial least-squares discriminant analysis(OPLS-DA) were employed to compare the content of the 11 components. The content of total phenolic acids, total iridoid glycosides, and total 11 components in LjF showed an overall downward trend with the development of flowers. The content of total phenolic acids, total iridoid glycosides, and total 11 components in young leaves were higher than those in mature leaves. The results of PCA showed that the samples at different flowering stages had distinguishable differences in component content. The VIP value of OPLS-DA showed that isochlorogenic acid A, chlorogenic acid, and secologanic acid were the main differential components of LjF at different development stages or LjL with different maturity levels. LjF and LjL have certain similarities in chemical composition while significant differences in component content. The content of total phenolic acids in young leaves was significantly higher than that in LjF at various development stages. The content of total iridoid glycosides in young leaves was similar to that in LjF before white flower bud stage. The total content of 11 components in young leaves was significantly higher than that in LjF at green flower bud stage, before and during completely white flower bud stage. LjL have great potential for development. Follow-up research on the pharmacodynamic equivalence of LjF and LjL(especially young leaves) should be carried out to speed up the development and application of LjL.

Simultaneous determination of three iridoid glycosides of Rehmannia glutinosa in rat biological samples using a validated hydrophilic interaction-UHPLC-MS/MS method in pharmacokinetic and in vitro studies.

The purpose of this study was to develop a method for simultaneous analysis of aucubin, catalpol, and geniposide, which are representative iridoid glycoside constituents of Rehmannia glutinosa, in rat plasma, urine, and feces using hydrophilic interaction ultra high-performance liquid chromatography with tandem mass spectrometry. The three components were separated using 10 mmol/L aqueous ammonium formate containing 0.01% (v/v) formic acid and acetonitrile as a mobile phase by gradient elution at a flow rate of 0.2 mL/min, equipped with a Kinetex® HILIC column (50 × 2.1 mm, 2.6 µm). Quantitation of this analysis was performed on a triple quadrupole mass spectrometer employing electrospray ionization and operated in multiple reaction monitoring mode. The chromatograms showed high resolution, sensitivity, and selectivity with no interference with plasma constituents. In all three iridoid glycosides, both the intra- and interbatch precisions (coefficient of variation %) were less than 4.81%. The accuracy was 96.56-103.55% for aucubin, 95.23-106.21% for catalpol, and 94.50-104.16% for geniposide. The developed analytical method satisfied the criteria of international guidance and was successfully applied to pharmacokinetic studies including oral bioavailability of aucubin, catalpol, and geniposide, and their urinary and fecal excretion ratios after oral or intravenous administration to rats. The new method was also applied to measure plasma protein binding ratios in vitro.

Dynamic analysis of secondary metabolites in various parts of Scrophularia ningpoensis by liquid chromatography tandem mass spectrometry.

The roots of Scrophularia ningpoensis are used as traditional medicines for thousands of years in China, nevertheless the stems and leaves were discarded as non-medicinal parts. Modern research have indicated the chemical constituents in the stems and leaves are similar to the identified in the roots, and the therapeutic effects of stems and leaves are superior to roots for some disease. In the study, the chemical constituents in roots, stems and leaves of S. ningpoensis were analyzed qualitatively by HPLC-Q-TOF-MS/MS. 40 compounds including 17 iridoid glycosides, 15 phenylpropanoids and 8 flavonoids were identified. Meantime, the dynamic accumulations of six index constituents in various parts were measured by HPLC-DAD. The results indicated the S. ningpoensis stems contained high content of aucubin (30.09 mg/g) and harpagide (28.4 mg/g) in August, and the leaves contained high content of harpagoside (12.02 mg/g) in July. The study provides the basis for the full development and utilization of the resource of stems and leaves from S. ningpoensis.

Prediction the contents of fructose, glucose, sucrose, fructo-oligosaccharides and iridoid glycosides in Morinda officinalis radix using near-infrared spectroscopy.

Morindae officinalis radix (MOR) is a famous Chinese herbal medicine which has long history of use in medicine and food. MOR and MOR with steaming process (PMOR) are the most commonly used forms in in clinical and health care. In order to establish a fast and mostly nondestructive quality control method for MOR, 183 beaches of MOR samples and 20 beaches of PMOR samples were collected commercially from major producing areas in Guangdong, Fujian and Guangxi Provinces of China. To predict main components of MOR, a calibration model was established based on near-infrared spectroscopy with partial least square regression. The model was optimized by compared the parameters of root mean square error of prediction (RMSEP), root mean square error of cross validation (RMSECV), coefficient of correlation (R2) and ratio of performance to deviation (RPD). Comparative studies were performed to evaluate the performance of models by different spectra preprocessing methods and different data set. The results showed that the model performance was improved with standard normal variate spectra preprocessing methods and when the data set contained both MOR and PMOR samples. A few PMOR samples were added to MOR samples data set the model predictive performance could be improved. The contents of 14 components were predicted in MOR with lower RMSEP and RMSECV, and higher R2 and RPD, including fructose (12.8 mg/g, 16.3 mg/g, 0.9873, 10.10), glucose (7.28 mg/g, 8.73 mg/g, 0.9611, 6.21 sucrose (9.24 mg/g, 9.10 mg/g, 0.8419, 1.75), GF2(9.42 mg/g, 11.3 mg/g, 0.8526, 2.03), GF3(7.98 mg/g, 9.20 mg/g, 0.8756, 2.74), GF4(6.81 mg/g, 8.93 mg/g, 0.8663, 3.06), GF5(8.13 mg/g, 8.85 mg/g, 0.9001, 3.06), GF6(6.40 mg/g, 6.95 mg/g, 0.9145, 3.27), GF7(5.53 mg/g, 6.15 mg/g, 0.9195, 3.57), GF8(5.40 mg/g, 6.02 mg/g, 0.9179, 3.31), GF9(3.00 mg/g,4.35 mg/g,0.9446, 5.03),GF10(4.08 mg/g, 5.34 mg/g, 0.8983, 3.62), GF11(8.97 mg/g, 7.70 mg/g, 0.8683, 2.01) and iridoid glycosides (4.12 mg/g, 5.51 mg/g, 0.8712, 2.43). The model established in this paper could predict 14 components of MOR. The results would provide a reference method for the quality control of Chinese medical materials and their process products.

Critical Phenological Events Affect Chemical Defense of Plant Tissues: Iridoid Glycosides in a Woody Shrub.

Plants are chemically-complex organisms; each individual contains diverse tissue-types, has the ability to differentially allocate secondary metabolites to these tissues and can change this allocation through time. The interaction of variation in chemical defense of different tissue types and variation in chemical defense through time, however, is rarely examined and has not been studied for iridoid glycoside-producing woody plants. In this study, we quantified allocation of iridoid glycosides (IGs) to the leaves, flowers, fruits, and seeds of 25 individuals of a long-lived shrub (Lonicera x bella Zabel, Caprifoliaceae), at five important phenological timepoints (leaf-out, flowering, fruit appearance, fruit ripening, and fruit dispersal) throughout a growing season. We found that leaves had 2x higher IG concentrations during flowering and fruiting than earlier in the season (after leaf-out), and later in the season (after fruit dispersal). The individual IG driving this increase in leaves during reproduction, secologanin, was also the most abundant IG in semiripe fruits. Flowers and seeds were composed of different proportions of individual IGs than fruits or leaves, but did not change across time and had overall low concentrations of IGs. In L. x bella, phenological events such as flowering and fruiting lead to an increase in leaf chemical defense that is likely to influence interactions with leaf-feeders. Our results stress the importance of considering phenology when sampling plants for the quantification of chemical defenses.

[The study of the polyphenolic complex and iridoid glycosides in various cultivars of edible honeysuckle fruits Lonicera edulis Turcz. ex Freyn].

The high content of minor biologically active substances in the fruits of wild-growing and cultivated varieties of honeysuckle, along with the low knowledge of the chemical composition of domestic varieties, determines the relevance of the study of their qualitative and quantitative composition. The aim of the work was to study the detailed composition of the main groups of biologically active polyphenolic compounds [anthocyanins, flavonoids, hydroxycinnamic acids (HCAs), proanthocyanidins] and iridoids in various cultivars of domestic edible honeysuckle fruits (Lonicera edulis Turcz. Ex Freyn). Material and methods. 15 samples of frozen fruits of edible honeysuckle harvested in Tambov, Voronezh, Moscow regions and Karelia were investigated. The total content of polyphenolic compounds (in terms of gallic acid equivalents) was determined by the modified Folin-Ciocalteu method, the total content of monomeric anthocyanins (in terms of cyanidin-3-glucoside) - by pH-differential spectrophotometry, proanthocyanidins (in terms of procyanidin B2) - by the modified Bate-Smith method. The profiles of individual anthocyanins, iridoids, flavonoids and HCAs was determined by HPLC-DAD/TOF-MS. The carbohydrate profile by capillary electrophoresis and antiradical activity in DPPH test in vitro were investigated as well. Results and discussion. The main groups of polyphenolic compounds were anthocyanins and proanthocyanidins. Cyanidin-3-glucoside was found as predominant among anthocyanins (>85% of their sum). Substantial amounts of iridoids (from 78.0 till 341.8 mg/100 g) were found in the honeysuckle examined. The loganic acid and loganine prevailed among iridoids. The total content of flavonoids varied in the range of 9.2- 46.6 mg/100 g, the main of which was rutin. Among HCAs chlorogenic acid prevailed, which accounted for 85.7-90.4% of the total amount of HCAs (45.9-79.8 mg/100 g). A correlation was found between the amount of polyphenolic compounds and the antiradical properties of honeysuckle fruits in DPPH test. Conclusion. Based on the results of the study the most promising varieties of domestic honeysuckle in terms of the highest content of polyphenolic antioxidants and iridoids with potential anti-inflammatory, hypoglycemic, hypolipidemic, antimicrobial and other types of biological activity were determined for the first time.

Quality Assessment of Medicinal Product and Dietary Supplements Containing Vitex agnus-castus by HPLC Fingerprint and Quantitative Analyses.

In this study, we aimed to evaluate the quality of 11 products sold in Japan (one medicinal product and 10 dietary supplements) containing/claiming to contain chasteberry extract (fruit of Vitex agnus-castus L.) using HPLC fingerprint (15 characteristic peaks), quantitative determination of chemical marker compounds, and a disintegration test. The HPLC profile of the medicinal product was similar to that of the reference standard of V. agnus-castus fruit dry extract obtained from European Directive for the Quality of Medicines (EDQM), whereas the profiles of some dietary supplements showed great variability, such as different proportions of peaks or lack of peaks. Results of the principal component analysis of the fingerprint data were consistent with those of the HPLC profile analysis. The contents of two markers, agnuside and casticin, in dietary supplements showed wide variability; this result was similar to that achieved with the HPLC fingerprint. In particular, agnuside and/or casticin was not detected in two dietary supplements. Furthermore, one dietary supplement was suspected to be contaminated with V. negundo, as evidenced from the results of agnuside to casticin ratio and assay of negundoside, a characteristic marker of V. negundo. Results of the disintegration test showed poor formulation quality of two dietary supplements. These results call attention to the quality problems of many dietary supplements, such as incorrect or poor-quality origin, different contents of the active ingredient, and/or unauthorized manufacturing procedures.

[Comparison of eleven active components in Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and leaves of Lonicero japonica by UPLC].

This present study aims to establish a UPLC method for simultaneously determining eleven components such as new chlorogenic acid,chlorogenic acid,caffeic acid,cryptochlorogenic acid,artichoke,isochlorogenic acid A,isochlorogenic acid B,isochlorogenic acid C,rutin,hibisin and loganin in Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and leaves of Lonicera japonica and comparing the differences in the contents of phenolic acids,flavonoids and iridoid glycosides of Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and leaves of Lonicera japonica.The method was carried out on an ACQUITY UPLC BEH C18column(2.1 mm×100 mm,1.7 µm) by a gradient elution using acetonitrile and 0.1% phosphoric acid.The flow rate was 0.3 mL·min-1.The column temperature was maintained at 30 ℃.The sample room temperature was 8 ℃.The wavelength was set at 326 nm for new chlorogenic acid,chlorogenic acid,caffeic acid,cryptochlorogenic acid,artichoke,isochlorogenic acid A,isochlorogenic acid B and isochlorogenic acid C,352 nm for rutin and lignin,and 238 nm for loganin.The injection volume was 1 µL.The eleven components has good resolution and was separated to baseline.Each component had a wide linear range and a good linear relationship(r≥0.999 6),the average recovery rate(n=9) was 98.96%,100.7%,97.24%,97.06%,99.53%,96.78%,98.12%,95.20%,95.12%,100.2%,98.61%and with RSD was 2.5%,1.4%,1.9%,2.1%,1.7%,1.9%,1.6%,2.0%,1.4%,2.2%,2.0%,respectively.Based on the results of the content determination,the chemometric methods such as cluster analysis and principal component analysis were used to compare the Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and leaves of Lonicera japonica.The results showed that Lonicerae Japonicae Flos and leaves of Lonicera japonica were similar in the chemical constituents,but both showed chemical constituents difference compored to Lonicerae Japonicae Caulis.The established multi-component quantitative analysis method can provide a reference for the quality control of Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and leaves of Lonicera japonica.

Identification of the absorbed components and their metabolites of Tianma-Gouteng granule in rat plasma and bile using ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry.

Tianma-Gouteng granule (TGG), a Chinese herbal formula preparation, is clinically used for the treatment of cardio-cerebrovascular diseases such as hypertension, cerebral ischaemia, acute ischaemic stroke and Parkinson's disease. Although few reports have been published concerning the absorbed prototype components of TGG, the possible metabolic pathways of TGG in vivo remain largely unclear. In this study, a method using UPLC-Q/TOF MS was established for the detection and identification of the absorbed prototype components and related metabolites in rat plasma and bile after oral administration of TGG at high and normal clinical dosages. A total of 68 components were identified or tentatively identified in plasma and bile samples, including absorbed prototypes and their metabolites. The major absorbed components were gastrodin, isorhynchophylline, rhynchophylline, isocorynoxeine, corynoxeine, geissoschizine methyl ether baicalin, baicalein, wogonoside, wogonin, geniposidic acid, leonurine, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucoside and emodin. The main metabolic pathways of these components involved phase I (isomerization, hydrolysis and reduction) and phase II (glucuronidation and sulfation) reaction, and the phase II biotransformation pathway was predominant. The present study provides rich information on the in vivo absorption and metabolism of TGG, and the results will be helpful for further studies on the pharmacokinetics and pharmacodynamics of TGG.

Ultra performance liquid chromatography coupled with principal component and cluster analysis of Swertia chirayita for adulteration check.

This article describes the study to standardize phytochemically and distinguish Swertia chirayita from that of possible substitution/adulteration using ultra performance liquid chromatography (UPLC) with photodiode array detector (PDA) and chemometric tools viz. principal component analysis (PCA) and hierarchical clustering analysis (HCA). Five ecotypes of Swertia chirayita and five possible substitutions, e.g.,Swertia bimaculata (SB), Swertia chordata (SCH), Swertia ciliata (SCL), Swertia paniculata (SP), and Halenia elliptica (HE) collected from different Indian Himalayan region. Samples evaluated for 04 marker compounds- swertiamarin (SM), mangiferin (MF), gentiopicroside (GP), and sweroside (SW). Reverse phase column (Waters Acquity BEH C18, 50 mm × 2.1 mm , 1.7 µm) provided high resolution for all target analytes with binary gradient elution. The detector response was linear (concentration 2.5-125 µg/mL, R2 > 0.999). The limit of detection (LOD) and quantification (LOQ) of targeted compounds was in the range of 1.40-2.06 and 4.57-6.27 µg/mL respectively. The combined relative standard deviation (%RSD) for intra-day and inter-day precision values were less than 2%. The recoveries study comply the method suitability. Chromatogram similarity analysis based on congruence coefficient was higher than 0.925 for the chirayita ecotypes while much lower than 0.629 for possible substitutes. HCA showed that the samples could be clustered (all 5 clusters in two-level) reasonably into different ecotypes and substitutes. HCA together with loading plots has indicated different chemical properties of all five groups. PCA results showed that the discrimination of chirayita ecotypes is because of the presence of SW while SM may have more influence on the targeted substitutes to discriminate from chirayita ecotypes. Therefore, UPLC fingerprint in association with chemometric tools provides a reliable and accurate quality assessment and detection of possible adulteration.

Preparative Separation of Phenylethanoid and Secoiridoid Glycosides from Ligustri Lucidi Fructus by High-Speed Counter-Current Chromatography Coupled with Ultrahigh Pressure Extraction.

Three phenylethanoid glycosides, echinacoside (1), salidroside (3), and acteoside (6), and three secoiridoid glycosides, isonuezhenide (2), nuezhenoside G13 (4), and specnuezhenide (5), have been extracted and separated by a combined method of ultrahigh pressure extraction (UPE) and high-speed counter-current chromatography (HSCCC) from Ligustri Lucidi Fructus. For the UPE, the optimal extraction was developed with conditions including solvent of 90% ethanol, sample to solvent ratio of 1:20 g/mL, pressure of 200 MPa, and time of 2 min, which rendered the yields of compounds 4 and 5 were 15.0 and 78.0 mg/g, respectively. For the HSCCC separation, the strategy of changing flow rates between 1.0 and 2.0 mL/min allowed the acquisition for 2.7 mg of compound 1, 4.5 mg of compound 2, 6.8 mg of compound 3, 5.9 mg of compound 4, 11.2 mg of compound 5, and 2.2 mg of compound 6 in one separation run under the solvent system of ethyl acetate:n-butanol:water (2:1:3, v/v) from 200 mg of the UPE extract. The structures of these phenylethanoid and secoiridoid glycosides were elucidated by extensive spectroscopic methods.

Host Plant Suitability in a Specialist Herbivore, Euphydryas anicia (Nymphalidae): Preference, Performance and Sequestration.

The checkerspot butterfly, Euphydryas anicia (Nymphalidae), specializes on plants containing iridoid glycosides and has the ability to sequester these compounds from its host plants. This study investigated larval preference, performance, and sequestration of iridoid glycosides in a population of E. anicia at Crescent Meadows, Colorado, USA. Although previous studies showed that other populations in Colorado use the host plant, Castilleja integra (Orobanchaceae), we found no evidence for E. anicia ovipositing or feeding on C. integra at Crescent Meadows. Though C. integra and another host plant, Penstemon glaber (Plantaginaceae), occur at Crescent Meadows, the primary host plant used was P. glaber. To determine why C. integra was not being used at the Crescent Meadows site, we first examined the host plant preference of naïve larvae between P. glaber and C. integra. Then we assessed the growth and survivorship of larvae reared on each plant species. Finally, we quantified the iridoid glycoside concentrations of the two plant species and diapausing caterpillars reared on each host plant. Our results showed that E. anicia larvae prefer P. glaber. Also, larvae survive and grow better when reared on P. glaber than on C. integra. Castilleja integra was found to contain two primary iridoid glycosides, macfadienoside and catalpol, and larvae reared on this plant sequestered both compounds; whereas P. glaber contained only catalpol and larvae reared on this species sequestered catalpol. Thus, although larvae are able to use C. integra in the laboratory, the drivers behind the lack of use at the Crescent Meadows site remain unclear.