Screening quality markers (Q-markers) of Xiaoer Chaige Tuire Oral Liquid by in vitro sequential metabolism and in vivo biopharmaceutical analysis.

BACKGROUND: Xiaoer Chaige Tuire Oral Liquid (XCT) is a preparation composed of 7 traditional Chinese medicines including Bupleuri Radix, Puerariae Lobatae Radix, Scutellariae Radix, Gypsum Fibrosum, Artemisiae Annuae Herba, Paeoniae Radix Alba and Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle in proportion. According to traditional Chinese medicine theory, it has the function of dispelling wind evil and relieving exterior syndrome, clearing summer heat and dampness, and reducing internal heat. So, it is indicated for pediatric upper respiratory tract infection caused by exogenous wind-heat. Modern pharmacological studies have indicated that XCT has a variety of activities such as anti-inflammation and antivirus. PURPOSE: To screen potential quality markers (Q-markers) of XCT by tracking in vivo bioactive compounds concomitantly using in vitro sequential metabolism and in vivo biopharmaceutical analysis. METHODS: In vitro metabolic models including artificial gastric juice, intestinal juice, intestinal microbiota, Caco-2 cell monolayer and liver S9 were employed to simulate metabolism of main compounds of XCT in the body. High performance liquid chromatography with diode-array detection (HPLC-DAD) was used to quantitatively determine main components of XCT preparation and its sequential metabolism samples. Ultra performance liquid chromatography with QExactive Orbitrap tandem mass spectrometry (UPLC-QExactive-HF-x-Orbitrap-MS) was used to qualitatively determine in vivo components of XCT preparation in rat plasma and metabolites obtained with liver S9 fraction of rats. RESULTS: Twenty-five compounds were identified from the preparation of XCT. Sequential in vitro metabolism studies indicated that most of these compounds except baicalin and baicalein were stable in artificial gastric juice, albiflorin, glycyrrhizic acid, gallic acid and baicalein were unstable in artificial intestinal juice, daidzin, liquiritin and genistin were hydrolyzed into their aglycones daidzein, liquiritigenin and genistein by intestinal microbiota, and 7 compounds thereout including benzoic acid, puerarin, 3'-methoxypuerarin, paeoniflorin, scopoletin, daidzein and liquiritigenin were shown to be well absorbed with Caco-2 cell monolayer model. These 7 compounds were demonstrated to be metabolized via hydroxylation and glycosylation by liver S9 system. Ten components of XCT preparation including puerarin, baicalin, wogonoside, benzoic acid, daidzein, baicalein, wogonin, oroxylin A, isoscopoletin and isoliquiritigenin were identified from rat plasma by in vivo biopharmaceutical analysis. Most of the compounds screened with both in vitro and in vivo metabolic studies were shown to be active against inflammation and influenza virus. CONCLUSIONS: A screening strategy for potential quality markers (Q-markers) of XCT preparation based on tracking in vivo bioactive compounds using the combination of in vitro sequential metabolism and in vivo biopharmaceutical analysis was established. With this strategy, a total of 12 compounds including puerarin, daidzein, benzoic acid, baicalin, baicalein, wogonoside, wogonin, oroxylin A, 3'-methoxypuerarin, paeoniflorin, scopoletin and liquiritigenin were screened to be potential Q-markers of XCT, which provides a material basis for quality control and development of XCT.

Pharmacological insights into Chukrasia velutina bark: Experimental and computer-aided approaches.

BACKGROUND: Chukrasia velutina is an enthnomedicinally used plant reported to have significant medicinal values. The present study aimed to explore the pharmacological activities of bark methanol extract using in vitro, in vivo and in silico models. METHODS: The study was designed to investigate the pharmacological effects of methanol extract of Chukrasia velutina bark (MECVB) through in vitro, in vivo and in silico assays. Analgesic activity was tested using formalin-induced nociception and acetic acid-induced writhing assays while the antipyretic effect was tested using yeast-induced hyperthermia in mice model. The antioxidant effect was tested using the DPPH and reducing power assay and the cytotoxic screening was tested using the brine shrimp lethality bioassay. In addition, in silico studies were conducted using computer aided methods. RESULTS: In the acetic acid-induced writhing assay, the extract showed 28.36% and 56.16% inhibition of writhing for doses of 200 and 400 mg/kg, respectively. Moreover, a dose-dependent formalin-induced licking response was observed in both early and late phase. In yeast-induced pyrexia, the MECVB exhibited (p < 0.05) antipyretic effect. The extract demonstrated an IC50 value of 78.86 µg/ml compared with ascorbic acid (IC50 23.53 µg/ml) in the DPPH scavenging assay. The compounds sitosterol, 5,7-dimethoxycoumarin and scopoletin were seen be effective in molecular docking scores against COX-I (2OYE), COX-II (6COX) and human peroxiredoxin 5 (1HD2). In ADME/T analysis, 5,7-dimethoxycoumarin and scopoletin satisfied Lipinski's rule of five and thus are potential drug candidates. CONCLUSION: The bark of Chukrasia velutina showed significant analgesic and antipyretic properties and is a potential source of natural anti-oxidative agents.

Comparative Analysis of the Phenolic Profile of Lycium barbarum L. Fruits from Different Regions in China.

Lycium barbarum L. (LB) fruits have high nutritive values and therapeutic effects. The aim of this study was to comprehensively evaluate the differences in phenolic composition of LB fruits from different geographical regions. Different methods of characterization and statistical analysis of data showed that different geographic sources of China could be significantly separated from each other. The highest total phenolic compound (TPC) content was observed in LB fruits from Ningxia (LBN), followed by those from Gansu (LBG) and Qinghai (LBQ). The Fourier transform infrared (FTIR) spectra of LB fruits revealed that LBQ had a peak at 2972 cm-1 whereas there was no similar peak in LBG and LBQ. A new HPLC method was established for the simultaneous determination of 8 phenolic compounds by quantitative analysis of multiple components by a single marker (QAMS), including 4 phenolic acids (chlorogenic acid, caffeic acid, 4-hydroxycinnamic acid, and ferulic acid), 1 coumarin (scopoletin), and 3 flavonoids (kaempferol-3-O-rutinoside, rutin, and narcissoside). It was showed that rutin was the most dominant phenolic compound in LBQ, although the average content of 4 phenolic acids was also high in LBQ, and scopoletin was the richest in LBG. UHPLC-Q-TOF-MS was used to qualitatively analyze the phenolics, which showed LBN was abundant in phenolic acids, LBQ was rich in flavonoids, and coumarins were the most plentiful in LBG. In conclusion, this study can provide references for the quality control and evaluation of phenolics in LB fruits and their by-products.

Joint transcriptomic and metabolomic analysis reveals the mechanism of low-temperature tolerance in Hosta ventricosa.

Hosta ventricosa is a robust ornamental perennial plant that can tolerate low temperatures, and which is widely used in urban landscaping design in Northeast China. However, the mechanism of cold-stress tolerance in this species is unclear. A combination of transcriptomic and metabolomic analysis was used to explore the mechanism of low-temperature tolerance in H. ventricosa. A total of 12 059 differentially expressed genes and 131 differentially expressed metabolites were obtained, which were mainly concentrated in the signal transduction and phenylpropanoid metabolic pathways. In the process of low-temperature signal transduction, possibly by transmitting Ca2+ inside and outside the cell through the ion channels on the three cell membranes of COLD, CNGCs and CRLK, H. ventricosa senses temperature changes and stimulates SCRM to combine with DREB through the MAPK signal pathway and Ca2+ signal sensors such as CBL, thus strengthening its low-temperature resistance. The pathways of phenylpropanoid and flavonoid metabolism represent the main mechanism of low-temperature tolerance in this species. The plant protects itself from low-temperature damage by increasing its content of genistein, scopolentin and scopolin. It is speculated that H. ventricosa can also adjust the content ratio of sinapyl alcohol and coniferyl alcohol and thereby alter the morphological structure of its cell walls and so increase its resistance to low temperatures.When subjected to low-temperature stress, H. ventricosa perceives temperature changes via COLD, CNGCs and CRLK, and protection from low-temperature damage is achieved by an increase in the levels of genistein, scopolentin and scopolin through the pathways of phenylpropanoid biosynthesis and flavonoid biosynthesis.

Quantification of Scopoletin from the Roots of Argyreia Speciosa (Linn. F) Sweet Using HPLC Through the Concept of Design of Experiment.

BACKGROUND: The roots of Argyreia speciosa (Linn. F) Sweet (family: Convolvulaceae) are used in Ayurveda to treat male reproductive and nervous system disorders. OBJECTIVE: Isolation of scopoletin from the roots of Argyreia speciosa, and development and validation of an analytical method using HPLC for the quantification of scopoletin from the root powder of Argyreia speciosa. METHOD: Scopoletin was isolated from chloroform fraction prepared from hydrolyzed methanolic extract and identified using spectral studies. A reverse-phase HPLC-based analytical method was developed and optimized using the Design of Experiment (DoE) approach to estimate scopoletin from the roots of Argyreia speciosa. Scopoletin was separated and quantified using HPLC containing the C18 column and a PDA detector. The optimized mobile phase was methanol: water (pH∼3.2) [25: 75, %v/v]. RESULTS: The Box-Behnken design was used to optimize chromatographic parameters and the extraction procedure. The validation studies showed a linear relationship (r2=0.998) in the range of 1-40 µg/mL. The LOD and LOQ were found to be 0.28 µg/mL and 0.84 µg/mL, respectively, and the recovery values were found to be between 91.94 and 97.86%. The developed analytical method was found to be robust as well. The amount of scopoletin was estimated to be 0.024 ± 0.0016%w/w from dried root powder. CONCLUSION: The recorded chromatogram and amount of scopoletin determined would serve as one of the standardization parameters to access the quality of raw material containing Argyreia speciosa. HIGHLIGHTS: Developed analytical method may be adopted as a part of the standardization procedure for Argyreia speciosa in the quality control laboratory.

Rapid Quantification and Validation of Biomarker Scopoletin in Paederia foetida by qNMR and UV-Vis for Herbal Preparation.

Scopoletin has previously been reported as a biomarker for the standardization of Paederia foetida twigs. This study is the first report on the determination and quantification of scopoletin using quantitative nuclear magnetic resonance (qNMR) in the different extracts of Paederia foetida twigs. The validated qNMR method showed a good linearity (r2 = 0.9999), limit of detection (LOD) (0.009 mg/mL), and quantification (LOQ) (0.029 mg/mL), together with high stability (relative standard deviation (RSD) = 0.022%), high precision (RSD < 1%), and good recovery (94.08-108.45%). The quantification results of scopoletin concentration in chloroform extract using qNMR and microplate ultraviolet-visible (UV-vis) spectrophotometer was almost comparable. Therefore, the qNMR method is deemed accurate and reliable for quality control of Paederia foetida and other medicinal plants without extensive sample preparation.

Development and Validation of a Novel, Rapid Gradient HPLC Method for Simultaneous Estimation of Bioactive Marker Compounds in a Mixture of Convolvulus pluricaulis, Withania somnifera and Bacopa monnieri Extracts.

The use of herbs as medicine is an ancient form of healthcare known to mankind. Standardization of herbal medicines is however a challenging task and is the major bottleneck in their acceptance as the primary therapeutic option. The aim of this study was to develop and validate a simple, rapid HPLC method for standardizing the mixture of extracts of three Medhya Rasayanas (neurotonic), Convolvulus pluricaulis, Withania somnifera and Bacopa monnieri. Simultaneous estimation of the respective bioactive markers of these plants viz., scopoletin, withaferin A, bacoside A 3, bacopaside II, jujubogenin and bacosaponin C has been reported for the first time. The method was developed using Waters Hybrid X-Bridge shield with BEH technology 2.5 µm, 4.6 × 75 mm column and validated according to ICH guidelines. The 20 minutes run time makes the method eco-friendly. The method was linear over a range of 12.5-400 ng/10 µL for scopoletin and 62.5-2,000 ng/10 µL for withaferin A, bacoside A 3, bacopaside II, jujubogenin and bacosaponin C with detection limits of 8.0, 48.3, 30.4, 40.7, 15.6 and 18.9 ng/10 µL and quantification limits of 24.5, 146.5, 92.2, 123.4, 47.4 and 57.4 ng/10 µL, respectively. The correlation coefficient for each analyte was >0.999. The intra-day and inter-day precision was <2%. These results confirmed the precision, accuracy and robustness of the proposed method.

Time-resolved fluorescence and chemometrics-assisted excitation-emission fluorescence for qualitative and quantitative analysis of scopoletin and scopolin in Erycibe obtusifolia Benth.

This paper presents a new strategy for qualitative identification of scopoletin and scopolin in Erycibe obtusifolia Benth using time-resolved (lifetimes) fluorescence and quantitative analysis with chemometrics-assisted excitation-emission matrix (EEM) fluorescence. Due to the significant spectral overlapping among analytes and interference, the use of the more selective time-resolved fluorescence is proposed for qualitative identification in quality control of traditional Chinese medicine (TCM) for the first time. Using the strategy of combining EEM fluorescence with second-order calibration method, i.e. parallel factor analysis (PARAFAC), the simultaneous quantification of scopoletin and scopolin in the complex system of Erycibe obtusifolia Benth was achieved successfully. The predicted concentrations were compared with the values obtained using high performance liquid chromatography-coupled to fluorimetric detector (HPLC-FLD), and no significant differences between them were observed. Therefore, the proposed methods using time-resolved fluorescence for qualitative analysis and EEMs coupled with second-order calibration for quantitative analysis in TCM are comparable and provide a suitable alternative to the chromatography-based method.

Loading of AgNPs onto the surface of boron nitride nanosheets for determination of scopoletin in Atractylodes macrocephala.

In this work, silver nanoparticles prepared by a molten salt method were deposited onto the surface of hexagonal boron nitride nanosheet (NS/AgNP) to from a composite. The synthesized nanocomposite was applied for surface modification of screen-printed electrode (SPE). The modified electrode showed a superior performance for electrochemical detection of scopoletin. The electrochemical behaviour of NS/AgNP/SPE was studied in detail. An electrocatalytic oxidation was observed and used for analytical determination of scopoletin concentration. The response of the proposed electrochemical sensing platform was linear over a wide detection range of 2 µM to 0.45 mM with a low limit of detection (LOD) of 0.89 µM. The NS/AgNP/SPE also showed excellent reproducibility and anti-interference property. In addition, the proposed scopoletin sensor was successfully used for the determination of scopoletin in Atractylodes macrocephala herb samples.

Simultaneous determination of umbelliferone and scopoletin in Tibetan medicine Saussurea laniceps and traditional Chinese medicine Radix angelicae pubescentis using excitation-emission matrix fluorescence coupled with second-order calibration method.

A chemometrics-assisted excitation-emission matrix (EEM) fluorescence method is presented for simultaneous determination of umbelliferone and scopoletin in Tibetan medicine Saussurea laniceps (SL) and traditional Chinese medicine Radix angelicae pubescentis (RAP). Using the strategy of combining EEM fluorescence data with second-order calibration method based on the alternating trilinear decomposition (ATLD) algorithm, the simultaneous quantification of umbelliferone and scopoletin in the two different complex systems was achieved successfully, even in the presence of potential interferents. The pretreatment is simple due to the "second-order advantage" and the use of "mathematical separation" instead of awkward "physical or chemical separation". Satisfactory results have been achieved with the limits of detection (LODs) of umbelliferone and scopoletin being 0.06ngmL(-1) and 0.16ngmL(-1), respectively. The average spike recoveries of umbelliferone and scopoletin are 98.8±4.3% and 102.5±3.3%, respectively. Besides, HPLC-DAD method was used to further validate the presented strategy, and t-test indicates that prediction results of the two methods have no significant differences. Satisfactory experimental results imply that our method is fast, low-cost and sensitive when compared with HPLC-DAD method.

A pre-classification strategy based on UPLC-Triple-TOF/MS for metabolic screening and identification of Radix glehniae in rats.

Traditional Chinese Medicines (TCMs) have gained increasing popularity in modern society. However, the profiles of TCMs in vivo are still unclear owing to their complexity and low level in vivo. In this study, UPLC-Triple-TOF techniques were employed for data acquiring, and a novel pre-classification strategy was developed to rapidly and systematically screen and identify the absorbed constituents and metabolites of TCMs in vivo using Radix glehniae as the research object. In this strategy, pre-classification for absorbed constituents was first performed according to the similarity of their structures. Then representative constituents were elected from every class and analyzed separately to screen non-target absorbed constituents and metabolites in biosamples. This pre-classification strategy is basing on target (known) constituents to screen non-target (unknown) constituents from the massive data acquired by mass spectrometry. Finally, the screened candidate compounds were interpreted and identified based on a predicted metabolic pathway, well - studied fragmentation rules, a predicted metabolic pathway, polarity and retention time of the compounds, and some related literature. With this method, a total of 111 absorbed constituents and metabolites of Radix glehniae in rats' urine, plasma, and bile samples were screened and identified or tentatively characterized successfully. This strategy provides an idea for the screening and identification of the metabolites of other TCMs.

Regeneration-Based Quantification of Coumarins (Scopoletin and Scoparone) in Abutilon indicum In Vitro Cultures.

Abutilon indicum exploited for its immense value has been propagated successfully through multiple shoot induction and somatic embryogenesis. Direct regeneration (8.20 ± 0.83 shoots) was achieved from nodal explants using 0.5 mg/l kinetin (Kn) in MS media. The basal callus from nodal explants turned embryogenic on subsequent introduction of 0.2 mg/l TDZ into the Kn-supplemented media, giving rise to somatic embryos. The embryogenic potential of calli expressed in terms of embryo-forming capacity (EFC) increased from 8.15 EFC to 20.95 EFC after plasmolysis. The phytochemical analysis (HPLC) for the presence of scopoletin and scoparone has revealed a unique accumulation pattern, with higher levels of scopoletin during the earlier stages and scoparone in the later stages of development. The embryogenic calli contained the highest amount of coumarins (99.20 ± 0.97 and 61.03 ± 0.47 µg/gFW, respectively) followed by regenerated plant (9.43 ± 0.20 and 36.36 ± 1.19 µg/gFW, respectively), obtained via somatic embryogenesis. Rapid multiplication of A. indicum equipped with two potent coumarins is important in order to meet the commercial demand for combat against dreadful diseases, thereby providing a new platform for plant-based drugs and their manufacture on a commercial scale.

In vitro propagation and cell cultures of memory tonic herb Evolvulus alsinoides: a best source for elicited production of scopoletin.

Evolvulus alsinoides L. is used for preparation of 'Shankhapushpi', an important popular ayurvedic drug that contributes considerably to the improvement of memory power. The improvement is attributed to the presence of furanocoumarin scopoletin, a metabolite with a wide range of biological activities. This report describes, for the first time, an in vitro culture system for propagation and enhanced production of scopoletin. Different concentrations of auxins and cytokinins individually and in combination were used in Murashige and Skoog (MS) medium to induce shoot regeneration in cotyledonary nodal explants and callus formation in leaf explants. The best response was achieved in MS medium fortified with 5.0 µM 6-benzyladenine (BA) in which 96 % of cultures produced 7.6 ± 0.6 shoots per explant. Regenerated shoots were rooted on MS medium with 5.0 µM indole-3-acetic acid (IAA). Plantlets were successfully acclimatized and established in soil. MS medium fortified with 10 µM BA + 5.0 µM IAA showed maximum growth and accumulation of scopoletin in cell cultures. Cell cultures could be maintained over 24 months. The influences of auxins, cytokinins, organic acids, amino acids, and fungal-derived elicitors on production of scopoletin were studied. Presence of either L-arginine, sodium pyruvate, or yeast extract highly promoted scopoletin production as compared with control and achieved 75.02-, 72.13-, and 57.98-fold higher accumulation, respectively. The results presented herein have laid solid foundation for large-scale production of scopoletin and further investigation of its purification and utilization as a novel pharmaceutical drug.

6-Methoxyflavonoids and Other Constituents from Microliabum polymnioides (Asteraceae).

The flower heads of Microliabum polymnioides afforded scopoletin, 5,4'-dihydroxy-3,6,7-trimethoxyflavone, 3,5,4'-trihydroxy-6,7-dimethoxyflavone and 3,5,7,4'-tetrahydroxy-6-methoxyflavone. The leaves contained hexadecanoic acid, phytol and docosane. This is the first report on the presence of 6-methoxyflavonoids in Microliabum genus.

[Determination of scopolin, chlorogenic acid, scopoletin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C in plants of Erycibe].

OBJECTIVE: An accurate and reliable analytical method for-simultaneous determination of six active components (scopolin, chlorogenic acid, scopoletin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C) in plants of Erycibe was developed. METHOD: Scopolin, chlorogenic acid, scopoletin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C in the samples were well separated in analytical HPLC by gradual elution with methanol-0.1% formic acid solution. The chromatographic condictions: Agilent Poroshell 120 EC-C18 column, flowing rate being 1 mL x min(-1), detecting wavelength at 345 nm. RESULT: Good linearities of scopolin, chlorogenic acid, scopoletin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C were in the range of 0.026 8-2.68, 0.027 0-2.70, 0.008 1-0.81, 0.018 8-1.88, 0.017 6-1.76, 0.019 6-1.96 µg, respectively (r > 0.999 6). The average recoveries of the six components were 98.1%, 98.7%, 100.8%, 100.4%, 99.7%, 101.1%; the relative standard deviations were 2.67%, 2.86%, 2.62%, 1.98%, 2.76%, 2.19%. CONCLUSION: The method is simple, feasible and reproducible and can be used for the quality control of plants of Erycibe.

Optimizing the models for rapid determination of chlorogenic acid, scopoletin and rutin in plant samples by near-infrared diffuse reflectance spectroscopy.

Polyphenols in plant samples have been extensively studied because phenolic compounds are ubiquitous in plants and can be used as antioxidants in promoting human health. A method for rapid determination of three phenolic compounds (chlorogenic acid, scopoletin and rutin) in plant samples using near-infrared diffuse reflectance spectroscopy (NIRDRS) is studied in this work. Partial least squares (PLS) regression was used for building the calibration models, and the effects of spectral preprocessing and variable selection on the models are investigated for optimization of the models. The results show that individual spectral preprocessing and variable selection has no or slight influence on the models, but the combination of the techniques can significantly improve the models. The combination of continuous wavelet transform (CWT) for removing the variant background, multiplicative scatter correction (MSC) for correcting the scattering effect and randomization test (RT) for selecting the informative variables was found to be the best way for building the optimal models. For validation of the models, the polyphenol contents in an independent sample set were predicted. The correlation coefficients between the predicted values and the contents determined by high performance liquid chromatography (HPLC) analysis are as high as 0.964, 0.948 and 0.934 for chlorogenic acid, scopoletin and rutin, respectively.

[Determination of chlorogenic acid, rutin, scopoletin and total polyphenol in tobacco by Fourier transform near infrared spectroscopy].

The objective of the present study was to investigate the feasibility of predicting chlorogenic acid, rutin, scopoletin and total polyphenol in tobacco by Fourier transform near-infrared (FT-NIR) spectroscopy. The partial least squares(PLS) regression method, second derivative and Norris derivative filter were applied in the NIR spectroscopy prediction of chlorogenic acid, rutin, scopoletin and total polyphenol in the range of 7 500 to 4 000 cm(-1). For chlorogenic acid, rutin, scopoletin and total polyphenol, the determination coefficients were 0.976 6, 0.941 9, 0.957 1 and 0.966 6, respectively. The SEP/SEC values for them were < 1.2, and the SD/SEP values for them were > 2. The root mean square error of cross validation (RMSECV) of the four calibration models were 1.938 9, 1.046 2, 0.047 9 and 2.745 2, respectively. NIR spectroscopy was compared with the conventional methods. The results show that the two methods showed no significant difference at the significant level of 0.05. NIR spectroscopy technology can accurately analyze chlorogenic acid, rutin, scopoletin and total polyphenol in tobacco.

[Study on quality standard of Chimonanthus nitens leaf].

OBJECTIVE: To set up the quality standard of Chimonanthus nitens leaf. METHODS: Five compounds were identified by double-development thin layer chromatography, the plate was colorized with 1% vanillin-H2 SO4 solution and warmed up in 110 degrees C; The content of four compounds was determined by HPLC, the chromatography separation was performed on the Elite hypersil ODS2 (250 mm x 4.6 mm, 5 microm) column with gradient elution. The mixture of acetonitrile and water as the mobile phase at a flow rate of 1.0 mL/min was used, the detection wavelength was 344 nm and the column temperature was set at 40 degrees C. RESULTS: The TLC identification was highly specific and spots were clear. In HPLC method, the calibration curve was linear in the range of 5.665 - 56.65 microg/mL for scopoletin (r = 0.9999), the average recovery was 97.06% and RSD was 1.27%; The calibration curve was linear in the range of 2.4312 - 24.312 microg/mL for isofraxidin (r = 0.9999), the average recovery was 102.86% and RSD% was 0.93%; The calibration curve presented a linear range from 2.444 - 24.44 microg/mL for scoparone (r = 0.9999), the average recovery was 102.18% and RSD was 1.81%. The standard curve presented a linear range from 9.012 - 90.12 microg/mL for rutin (r = 0.9992), the average recovery was 104.44% and RSD was 4.2%. CONCLUSION: These methods are reproducible, sensitive and simple and can be used to control the quality of Chimonanthus nitens leaf.

Phenolic constituents of the Chilean herbal tea Fabiana imbricata R. et P.

"Pichi" or "pichi romero" (Fabiana imbricata R. et. P., Solanaceae) is a Chilean plant used as a tea in the Andean regions of Chile and Argentina. A very simple and direct method was developed for the qualitative analysis of polyphenols in the tea by high-performance liquid chromatography (HPLC) with diode array detection and electrospray ionization tandem mass spectrometry. The phenolic constituents identified in the teas were chlorogenic acid (3-O-caffeoylquinic acid), p-hydroxyacetophenone, scopoletin and quercetin derivatives. The glycosides were mainly glucosides from p-hydroxyacetophenone and scopoletin while di- and tri-glycosides from quercetin were the main flavonoids. The content of the main phenolic compounds in the teas (g/100 g lyophilized infusion) was 0.8-1.9 % for scopoletin, 0.4-6.2 % for p-hydroxyacetophenone and 2.1-4.3 % for rutin, respectively. The health-promoting properties reported for this herbal tea can be associated with the presence of several phenolics with known antioxidant, diuretic and antiinflammatory activity.

Rapid and sensitive analysis of three polyphenols in tobacco by CE using homemade C(4)D with a mini detection cell.

A rapid, sensitive, and practical CE with C(4) D detection was developed for the analysis of three polyphenols (rutin, scopoletin, and chlorogenic acid) in tobacco samples. The constructed mini detection cell (12 mm × 10 mm × 10 mm) of C(4) D featured with small inner cell volume (∼2 nL), smaller noise (<0.9 mV), repeatability, high strength and durableness. Three polyphenols were ultrasonically extracted with methanol-water (70:30, v/v) solution following SPE cleanup. The CE method was optimized with the running buffer of 150 mmol L(-1) 2-amino-2-methyl-1-propanol (pH 11.2), and the applied separation voltage of +20 kV over a capillary of 50 µm id × 375 µm od × 50 cm (38 cm to the C(4) D window, 41.5 cm to the UV detector window), which gave a baseline separation of three polyphenols within ca. 6 min. The method provided the limits of quantification (S/N = 10) at about 0.08-0.15 µg g(-1) for three polyphenols, whereas the overall recoveries ranged from 82% to 88%. The proposed method has been successfully applied to measure three polyphenols in the actual tobacco samples, and their contents were calculated and evaluated.