Quantitative analysis of three ingredients in Salvia miltiorrhiza by near infrared spectroscopy combined with hybrid variable selection strategy.

Rosmarinic acid (RA), Tanshinone IIA (Tan IIA), and Salvianolic acid B (Sal B) are crucial compounds found in Salvia miltiorrhiza. Quickly predicting these components can aid in ensuring the quality of S. miltiorrhiza. Spectral preprocessing and variable selection are essential processes in quantitative analysis using near infrared spectroscopy (NIR). A novel hybrid variable selection approach utilizing iVISSA was employed in this study to enhance the quantitative measurement of RA, Tan IIA, and Sal B contents in S. miltiorrhiza. The spectra underwent 108 preprocessing approaches, with the optimal method being determined as orthogonal signal correction (OSC). iVISSA was utilized to identify the intervals (feature bands) that were most pertinent to the target chemical. Various methods such as bootstrapping soft shrinkage (BOSS), competitive adaptive reweighted sampling (CARS), genetic algorithm (GA), variable combination population analysis (VCPA), successive projections algorithm (SPA), iteratively variable subset optimization (IVSO), and iteratively retained informative variables (IRIV) were used to identify significant feature variables. PLSR models were created for comparison using the given variables. The results fully demonstrated that iVISSA-SPA calibration model had the best comprehensive performance for Tan IIA, and iVISSA-BOSS had the best comprehensive performance for RA and Sal B, and correlation coefficients of cross-validation (R2cv), root mean square errors of cross-validation (RMSECV), correlation coefficients of prediction (R2p), and root mean square errors of prediction (RMSEP) were 0.9970, 0.0054, 0.9990 and 0.0033, 0.9992, 0.0016, 0.9961 and 0.0034, 0.9998, 0.0138, 0.9875 and 0.1090, respectively. The results suggest that NIR spectroscopy, along with PLSR and a hybrid variable selection method using iVISSA, can be a valuable tool for quickly quantifying RA, Sal B, and Tan IIA in S. miltiorrhiza.

The key phytochemistry of rosemary (Salvia rosmarinus) contributing to hair protection against UV.

Extracts from rosemary (Salvia Rosmarinus) are analysed for their phytochemistry using LC-MS and the phytochemistry identified. The same extracts were tested for their efficacy to act as antioxidants by both hydrogen-atom transfer (ORAC) and single electron transfer (FRAP). A correlation analysis was performed to identify the key phytochemistry responsible for antioxidant efficacy. The top performing extracts were then tested in a peptide model and in hair with the presence of UV to measure ability to protect against UV-induced peptide and protein damage. Polyphenols (e.g. rosmarinic acid, glycosides of selgin) and abietane diterpenes (e.g. carnosic acid) in rosemary were identified as the principal compounds which enables the extracts to protect hair from UV. OBJECTIVE: The objective of this work was to correlate the phytochemistry of rosemary (Salvia rosmarinus), a botanical with known antioxidant properties, to a UV protection benefit in hair. These data will give insights into mechanisms of UV damage, the ROS formed and their reactivity. METHODS: LC-MS was used to compare the compounds in 10 commercial extracts of rosemary. ORAC (oxygen radical antioxidant capacity) and FRAP (ferric reducing antioxidant power) were used to measure the antioxidant capacity of the rosemary extracts. The ORAC assay measures ability of an antioxidant to react with a peroxyl radical via hydrogen atom extraction and FRAP measures electron transfer through reduction of ferric iron (Fe3+ ) to ferrous iron (Fe2+ ) by antioxidants present in the samples. Correlation of extract composition with antioxidant measures was performed using principal component analysis. Selected extracts were assessed for their ability to protect hair from UV damage in a model peptide system and on hair. In addition, the same methods were used to test rosmarinic acid and carnosic acid, key phytochemistries in the rosemary extracts. The model system was a peptide and its decomposition on exposure to UV was monitored by LC-MS in the absence and presence of the rosemary extracts. Hair degradation in the presence of UV was measured by exposure of UV in an Atlas weatherometer followed by extraction of degraded protein in water. A fragment of the S100A3 protein was used as a marker of UV damage (m/z = 1278) and quantified via LC-MS. RESULTS: Ten rosemary extracts were assessed for antioxidant performance and correlated with their compositions. The phytochemistry in each extract varied widely with a total of 33 individual compounds identified. The differences were most likely driven by the solvent and extraction method used by the supplier with extracts varying in the proportion of polar or non-polar compounds. This did influence their reactivity in the ORAC and FRAP assays and their efficacy in preventing protein damage. Two of the key compounds identified were rosmarinic acid and carnosic acid, with rosmarinic acid dominating in extracts with mainly polar compounds and carnosic acid dominating in extracts with mainly nonpolar compounds. Extracts with higher rosmarinic acid correlated with ORAC and FRAP scores, with UV protection on hair and in the peptide model system. The extracts chosen for hair experiments showed hair protection. UV protection was also measured for rosmarinic and carnosic acid. CONCLUSIONS: Despite the variation in the profile of phytochemistries in the 10 rosemary extracts, likely driven by the chosen extraction method, all rosemary extracts had antioxidant activity measured. This study suggests that the polyphenols (e.g. rosmarinic acid, glycosides of selgin) and abietane diterpenes (e.g. carnosic acid) are the principal compounds which enables the extracts to protect hair from UV.

INTRODUCTION: Les extraits de romarin (Salvia Rosmarinus) sont analysés par LC-MS pour établir et identifier leur profil phytochimique. Les mêmes extraits ont été testés pour leur efficacité à agir comme antioxydants à la fois par transfert d'atome d'hydrogène (ORAC) et par transfert d'électrons uniques (FRAP). Une analyse de corrélation a été réalisée pour identifier les propriétés phytochimiques clés responsables de l'efficacité antioxydante. Les extraits les plus performants ont ensuite été testés dans un modèle peptidique et sur les cheveux en présences d'UV pour mesurer la capacité à protéger contre les dommages induits par les UV su les peptides et protéines. Les polyphénols (par ex. acide rosmarinique, glycosides de selgin) et les diterpènes d'abiétine (par ex. acide carnosique) dans le romarin ont été identifiés comme les principaux composés permettant aux extraits de protéger les cheveux des UV. OBJECTIF: L'objectif de ce travail était de mettre en corrélation la phytochimie du romarin (Salvia rosmarinus), une plante aux propriétés antioxydantes connues, et les bénéfices d'une protection contre les UV dans les cheveux. Ces données fourniront des informations sur les mécanismes des dommages causés par les UV, la formation du ROS et leur réactivité. MÉTHODES: La LC-MS a été utilisée pour comparer les composés de 10 extraits commerciaux de romarin. L'ORAC (Oxygen Radical Antioxidant Capacity/Capacité d'absorption des radicaux d'oxygène) et la FRAP (Ferric Reduction Antioxidant Power/Pouvoir antioxydant de réduction ferrique) ont été utilisés pour mesurer la capacité antioxydante des extraits de romarin. Le dosage ORAC mesure la capacité d'un antioxydant à réagir avec un radical peroxyl par extraction d'atome d'hydrogène et la FRAP mesure le transfert d'électrons par réduction du fer ferrique (Fe3+ ) en fer ferreux (Fe2+ ) par les antioxydants présents dans les échantillons. La corrélation entre la composition de l'extrait et les mesures des antioxydants a été effectuée en analysant les composants principaux. Les extraits sélectionnés ont été évalués pour leur capacité à protéger les cheveux des dommages causés par les UV dans un modèle de système peptidique et sur les cheveux. En outre, les mêmes méthodes ont été utilisées pour tester l'acide rosmarinique et l'acide carnosique, principales caractéristiques phytochimiques dans les extraits de romarin. Le système modèle était un peptide et sa décomposition à l'exposition aux UV a été suivie par LC-MS en l'absence et en présence des extraits de romarin. La dégradation des cheveux en présence d'UV a été mesurée par l'exposition aux UV dans un indicateur de désagrégation Atlas suivi de l'extraction de protéines dégradées dans l'eau. Un fragment de la protéine S100A3 a été utilisé comme marqueur de dommage UV (m/z = 1278) et quantifié par LC-MS. RÉSULTATS: Dix extraits de romarin ont été évalués en termes de performance antioxydante et mis en corrélation avec leurs compositions. La phytochimie de chaque extrait variait considérablement, avec un total de 33 composés individuels identifiés. Les différences étaient très probablement dues à la méthode du solvant et de l'extraction utilisée par le fournisseur avec des extraits variant dans la proportion de composés polaires ou non polaires. Cela a effectivement influencé leur réactivité dans les dosages ORAC et FRAP et leur efficacité dans la prévention des dommages protéiques. Deux des composés clés identifiés étaient l'acide rosmarinique et l'acide carnosique, l'acide rosmarinique dominant dans les extraits contenant principalement des composés polaires et l'acide carnosique dominant dans les extraits contenant principalement des composés non polaires. Les extraits avec un taux d'acide rosmarinique plus élevé étaient mis en corrélation avec les scores ORAC et FRAP, avec une protection UV sur les cheveux et dans le système de modèle peptidique. Les extraits choisis pour les expériences sur les cheveux ont montré une protection des cheveux. La protection contre les UV a également été mesurée pour l'acide rosmarinique et l'acide carnosique. CONCLUSIONS: Malgré la variation des profils phytochimiques dans les dix extraits de romarin, probablement induite par la méthode d'extraction choisie, l'activité antioxydante de tous les extraits de romarin a été mesurée. Les polyphénols (par ex. acide rosmarinique, glycosides de selgin) et les diterpènes d'abiétane (par ex. acide carnosique) dans le romarin ont été identifiés comme les principaux composés permettant aux extraits de protéger les cheveux contre les UV.

Ent-Abietane Diterpenoids from Euphorbia fischeriana and Their Cytotoxic Activities.

The roots of Euphorbia fischeriana have been used as a traditional Chinese medicine for the treatment of tuberculosis and ringworm. In the current study, diterpenoids from the ethyl acetate extract of the roots E. fischeriana and their cytotoxic effects against five cancer lines were investigated. Two new ent-abietane diterpenoids, euphonoids H and I (1-2), as well as their two analogues (3-4) were first isolated from this source. The structures of the two new compounds were elucidated on the basis of spectroscopic data and quantum chemical calculation. Their absolute configurations were assigned via ECD spectrum calculation. The isolated compounds were evaluated for their antiproliferative activities against five cancer cell lines. Compounds 1 and 2 exhibited significant inhibitory effects against human prostate cancers C4-2B and C4-2B/ENZR cell lines with IC50 values ranging from 4.16 ± 0.42 to 5.74 ± 0.45 µM.

Chemical Composition, Anti-Breast Cancer Activity and Extraction Techniques of Ent-Abietane Diterpenoids from Euphorbia fischeriana Steud.

Ent-abietane diterpenoids are the main active constituents of Euphorbia fischeriana. In the continuing search for new anti-breast cancer drugs, 11 ent-abietane diterpenoids (1-11) were isolated from E. fischeriana. The structures of these compounds were clearly elucidated on the basis of 1D and 2D NMR spectra as well as HRESIMS data. Among them, compound 1 was a novel compound, compound 10 was isolated from Euphorbia genus for the first time, compound 11 was firstly discovered from E. fischeriana. These compounds exhibited varying degrees of growth inhibition against the MCF-10A, MCF-7, ZR-75-1 and MDA-MB-231 cell lines in vitro. The experimental data obtained permit us to identify the roles of the epoxy group, hydroxyl group and acetoxyl group on their cytotoxic activities. Extraction is an important means for the isolation, identification, and application of valuable compounds from natural plants. To maximize yields of ent-abietane diterpenoids of E. fischeriana, 17-hydroxyjolkinolide B, jolkinolide B, 17-hydroxyjolkinolide A and jolkinolide A were selected as quality controls to optimize the salting-out-assisted liquid-liquid extraction (SALLE) by response surface methodology (RSM). The optimized conditions for SALLE were 0.47 g sodium dihydrogen phosphate, 5.5 mL acetonitrile and 4.5 mL water at pH 7.5. The experimental values of 17-hydroxyjolkinolide B, jolkinolide B, 17-hydroxyjolkinolide A and jolkinolide A (2.134, 0.529, 0.396, and 0.148 mg/g, respectively) were in agreement with the predicted values, thus demonstrating the appropriateness of the model.

3,4-Seco-Isopimarane Diterpenes from the Twigs and Leaves of Isodon Flavidus.

Three isopimarane diterpenes [fladins B (1), C (2), and D (3)] were isolated from the twigs and leaves of Chinese folk medicine, Isodon flavidus. The chemical structures were determined by the analysis of the comprehensive spectroscopic data, and the absolute configuration was confirmed by X-ray crystallographic analysis. The structures of 1-3 were formed from isopimaranes through the rearrangement of ring A by the bond break at C-3 and C-4 to form a new δ-lactone ring system between C-3 and C-9. This structure type represents the first discovery of a natural isopimarane diterpene with an unusual lactone moiety at C-9 and C-10. In the crystal of 1, molecules are linked to each other by intermolecular O-H···O bonds, forming chains along the b axis. Compounds 1-3 were evaluated for their bioactivities against different diseases. None of these compounds displayed cytotoxic activities against HCT116 and A549 cancer cell lines, antifungal activities against Trichophyton rubrum and T. mentagrophytes, or antiviral activities against HIV entry at 20 µg/mL (62.9-66.7) µM. Compounds 1 and 3 did not show antiviral activities against Ebola entry at 20 µg/mL either; only 2 was found to show an 81% inhibitory effect against Ebola entry activity at 20 µg/mL (66.7 µM). The bioactivity evidence suggested that this type of compound could be a valuable antiviral lead for further structure modification to improve the antiviral potential.

Determination of fingerprints contents of different extracts and parts of six endemic Salvia taxa by GC-MS: Source species for valuable compounds with drug or drug potential.

Public use of Salvia species and their importance in the scientific world is continually increasing. It is known that this use and the importance of Salvia species are mostly due to the terpenoid compounds that they contain. In this context, the terpenoid-steroid-flavonoid contents of extracts of six endemic Salvia (S. kurdica, S. pseudeuphratica, S. rosifolia, S. siirtica, S. cerino-pruinosa var. cerino-pruinosa and S. cerino-pruinosa var. elazigensis) species prepared with different solvents were determined by gas chromatography-mass spectrometry. Within the framework of the ingredient analysis, content analysis of the ethanol extracts of the root, branch, leaf and flower parts of the species collected in the same period between 2015 and 2017 years was performed. In general, extracts prepared with chloroform and ethanol were found to contain a wide variety of compounds while petroleum ether extracts were found to contain much less varied compounds. In addition, in general, root extracts are richer in terpenoid compounds than aerial part extracts. Some species can be used as source species in terms of ferruginol, cryptanol, 6,7-dehydroroyleanone, lup-(20)29-ene-2α-hydroxy-3ß-acetate, salvigenin and ß-sitosterol contents (52,114.28, 75,979.08, 101,247.41, 40,071.29, 33,952.13 and 34,010.90 µg analyte/g extract, respectively).

Differences in diterpenoid diversity reveal new evidence for separating the genus Coleus from Plectranthus.

Covering: up to 2019The large and medicinally important tropical plant genus Plectranthus (Lamiaceae) was recently split into three separate genera on the basis of molecular and morphological evidence; Plectranthus sensu stricto, Coleus and Equilabium. We found striking differences between the diterpenoids which strongly support this taxonomic split. Coleus is characterised by abietanes oxygenated at C-14 such as royleanones, spirocoleons and acylhydroquinones, which could be useful chemotaxonomic markers to distinguish this genus from Plectranthus s.s. In contrast, the abietanes in Plectranthus s.s. lack C-14 oxygenation, but are often acylated with unusual acids. Equilabium species do not seem to produce diterpenoids. The structures of the nearly 240 abietanes so far reported from Coleus and Plectranthus and their distribution are presented. The aim of this Highlight is to provide an overview of the differences in diterpenoid diversity between these newly defined genera, which are relevant to predict which previously understudied species could hold untapped potential for their medicinal and other economic uses, and to underpin future research on how these plants have evolved to synthesise distinct abietane types.

Isolation of diterpenes from Araucaria sp Brazilian brown propolis and development of a validated high-performance liquid chromatography method for its analysis.

Propolis comprises a complex resinous product composed of plant's parts or exudates, pollen, bee wax, and enzymes. Brazilian brown propolis from Araucaria sp displays several biological activities. Considering the lack of validated analytical methods for its analysis, we are reporting the development of a validated high-performance liquid chromatography with photodiode array detector method to analyze Araucaria brown propolis. The crude propolis were extracted and chromatographed, furnishing six main diterpenes. The isolated standards were used to draw the analytical curves, allowing the studies of selectivity, precision, accuracy, recovery, robustness, the determination of limits of detection and limits of quantification. The mobile phase consisted of 0.1% acetic acid in water and acetonitrile, using an octadecylsilane column, 1 mL/min flow rate and detection at 200 or 241 nm. Relative standard deviation values obtained for intra-day and inter-day precision were lower than 4% for all diterpenes. From the five parameters for robustness, wavelength detection and flow rate were the critical ones. Limits of detection and quantification ranged from 0.808 to 10.359 µg/mL and from 2.448 to 31.392 µg/mL, respectively. The recoveries were between 105.03 and 108.13%, with relative standard deviation values around 5.0%. The developed method is precise, sensitive, and reliable for analyzing Araucaria brown propolis.

Application of smartphone in detection of thin-layer chromatography: Case of salvia miltiorrhiza.

In this work, a smartphone-based device was constructed for thin-layer chromatography (TLC) detection and semi-quantitative analysis of the components of Salvia miltiorrhiza. The key construction and shooting parameters were investigated by the relative peak area and signal-to-noise ratio. The best conditions were as follows: shooting height, 17 cm; angle between the UV lamp and TLC plate, 58°; exposure compensation, 0~0.2 EV; and shutter speed under daylight and UV 365 nm, 1/50 s and 1/5 s, respectively. These ideal conditions could be replicated by smartphones from different brands with different versions of software. With good precision, repeatability and stability, the developed device was used for the semi-quantitative analysis of salvianolic acid B, rosmarinic acid, cryptotanshinone, tanshinone I, tanshinone IIA, and miltirone in the TLC analysis of 10 batches of S. miltiorrhiza. The results were compared with those obtained by a TLC densitometric scanner and two common types of image processing software, i.e., Gelanalyzer and ImageJ. Except for salvianolic acid B in the TLC densitometric scanner, all results were not significantly different among these methods, which suggested that smartphones might be a useful tool for the quality control of traditional Chinese medicines.

Sequential extraction of carnosic acid, rosmarinic acid and pigments (carotenoids and chlorophylls) from Rosemary by online supercritical fluid extraction-supercritical fluid chromatography.

A high degree of selectivity is required during the plant extraction process in order to obtain extracts enriched in specific compounds or to avoid the extraction of unwanted ones. Rosemary is well known for its antioxidant compounds (carnosic acid, carnosol and rosmarinic acid). The plant also contains pigments (i.e. carotenoids, chlorophylls) which may cause a colour problem during the use of the extract in cosmetic formulations, for example. Supercritical fluid extraction is considered as a selective technique for plant extraction. Due to the physico-chemical properties of supercritical fluids, related to pressure, temperature and modifier addition, it is possible to carry out sequential extraction with successive conditions to collect different fractions that are rich either in pigments or in bioactive compounds. The aim of this study was to selectively extract bioactive compounds (i.e. carnosic acid and rosmarinic acid) and pigments (carotenoids and chlorophylls) from rosemary using supercritical fluid extraction. The optimisation of the extraction method was carried out using supercritical fluid extraction online coupled with a supercritical fluid chromatography (SFE-SFC) system. Two columns of different polarities were coupled to achieve the separation of the targeted compounds every five minutes during the extraction. Four fractions were obtained: a first one rich in carotenoids obtained with pure CO2 (25°C and 20 MPa), a second rich in carnosic acid obtained with 3% polar modifier (EtOH:water 50/50 v/v), a third fraction rich in rosmarinic acid using 10% of the same modifier and a fourth fraction rich in chlorophylls with 30% of ethanol as modifier. These four samples were then analysed by UHPLC-DAD-ESI-QTOF-HRMS in order to identify other extracted compounds and to study how the selected conditions impacted their extraction.

Anaerobic digestibility of resin acids in primary sludge: Effect of ozone pretreatment.

Resin acids in pulp and paper mills wastewater are potentially partitioned in the solids in post-primary clarification due to higher hydrophobicity with log Kow ∼1.74-5.80. They are known to adversely affect anaerobic digestion (AD) process, although the effect has not been quantified deterministically in control studies. The objective of the present work was to determine the effect of untreated and ozonated spiked resin acids on AD of primary sludge. Batch adsorption tests were conducted to determine the solid-liquid partition coefficient (Kd) of resin acids on the primary sludge. Higher Kd was obtained at pH 4; however, it was decreased by 78-98% at pH 8. Thereafter, batch AD of model resin acids in primary sludge using food to microorganism ratio (S0/X) of 0.5gtCOD/gVSSindicated only 15-20% removal of resin acids in the liquid phase anaerobically. While, ozonation in pure water using 0.74-1.48 mg O3/mg tCOD showed >90% reduction of the test resin acids, an ozone dose of 0.52 mg O3/mg tCOD reduced 50-70% spiked resin acids' load to the digester. However, no further removal of resin acids occurred during AD over 30 days. About 42% reduction in methane production compared to the control digestor occurred in the presence of 150 mg/L of resin acids. When treated with 0.52 mg O3/mg tCOD, methane production improved and was comparable to the control digestor, indicating that resin acids may not be detrimental to AD at a concentration range of 45-75 mg/L.

Supercritical Fluid Chromatography development of a predictive analytical tool to selectively extract bioactive compounds by supercritical fluid extraction and pressurised liquid extraction.

Selective extraction is a great concern in the field of natural products. The interest is to apply specific conditions favouring the solubility of targeted secondary metabolites and avoiding the simultaneous extraction of unwanted ones. Different ways exist to reach selective extractions with suited conditions. These conditions can be determined from experimental studies through experimental design, but a full experimental design takes time, energy, and uses plant samples. Prediction from varied solubility models can also be applied allowing a better understanding of the final selected conditions and eventually less experiments. The aim of this work was to develop and use a chromatographic model to determine optimal extraction conditions without the need for numerous extraction experiments. This model would be applied on the selective extraction of the desired antioxidant compounds in rosemary leaves (rosmarinic and carnosic acids) vs chlorophyll pigments to limit the green colour in extracts. This model was achieved with Supercritical Fluid Chromatography (SFC) and then applied to Supercritical Fluid Extraction (SFE) and Pressurised Liquid Extraction (PLE) assays. SFC models predicted low solubility of chlorophylls for low (5%) and high (100%) percentage of solvent in carbon dioxide. Also, low solubility was predicted with acetonitrile solvent compared to methanol or ethanol. This was confirmed with different extractions performed using SFE with different percentages of solvent (5, 30, and 70%) and with the three solvents used in the SFC models (acetonitrile, methanol and ethanol). Also extractions using PLE were carried out using the same neat solvents in order to confirm the SFC models obtained for 100% of solvent. Globally, extractions validated the SFC models. Only some differences were observed between ethanol and methanol showing the complexity of plant extraction due to matrix effect. For all these extracts, the content of carnosic acid and rosmarinic acid was also monitored and selective extraction conditions of bioactive compounds could be determined.

Differential distribution of characteristic constituents in root, stem and leaf tissues of Salvia miltiorrhiza using MALDI mass spectrometry imaging.

Segmentation-quantification is the most commonly used method for studying the tissue distribution of bioactive constituents in plant, but this method would bring uncontrollable pollution, compound migration and denaturation. Mass spectrometry imaging (MSI), as a new method developed in the past 20 years, has high sensitivity, high spatial resolution, high degree of visualization, and low risk of contamination and degeneration when studying tissue distribution of compounds. For the first time we applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to tissue distribution of characteristic constituents of the medicinal plant Salvia miltiorrhiza. From the collected data, we found the regional differences in root, stem, and leaf tissues, and the ion information with differential distribution characteristics. We also identified 18 bioactive constituents in S. miltiorrhiza with their spatial distribution information. In addition, the plant was divided into five parts, and the identified compounds were analyzed for differences between tissues using LC-MS, which results verified those found from the MSI. It is figured out that MALDI-MSI can be reliably applied to the differential distribution of salvianolic acids and tanshinones.

Quantitative Determination and Validation of Four Ketones in Salvia miltiorrhiza Bunge Using Quantitative Proton Nuclear Magnetic Resonance Spectroscopy.

Salvia mltiorrhiza Bunge (SMB) is native to China, whose dried root has been used as medicine. A few chromatographic- or spectrometric-based methods have already been used to analyze the lipid-soluble components in SMB. However, the methodology of qNMR on the extracts of fresh SMB root has not been verified so far. The purpose of this study was to establish a fast and simple method to quantify the tanshinone I, tanshinone IIA, dihydrotanshinone, and cryptotanshinone in fresh Salvia Miltiorrhiza Bunge root without any pre-purification steps using 1H-NMR spectroscopy. The process is as follows: first, 70% methanol aqueous extracts of fresh Salvia Miltiorrhiza Bunge roots were quantitatively analyzed for tanshinone I, tanshinone IIA, dihydrotanshinone, and cryptotanshinone using 1H-NMR spectroscopy. Different internal standards were tested and the validated method was compared with HPLC. 3,4,5-trichloropyridine was chosen as the internal standard. Twelve samples of Salvia Miltiorrhiza Bunge were quantitatively analyzed by qNMR and HPLC respectively. Then, the results were analyzed by chemometric approaches. This NMR method offers a fast, stable, and accurate analysis of four ketones: tanshinone I, tanshinone IIA, dihydrotanshinone, and cryptotanshinone in fresh roots of Salvia Miltiorrhiza Bunge.

Comparative study of rosemary extracts and several synthetic and natural food antioxidants. Relevance of carnosic acid/carnosol ratio.

The antiradical power, at equal concentrations of active principles, of the following antioxidants were studied using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay: butylated-hydroxyanisole, butylated-hydroxytoluene, tert-butylhydroquinone, ascorbyl palmitate, tocopherol, grape seed extract, olive extract and five rosemary extracts with different concentrations of carnosic acid (CA) and carnosol (COL). The reaction kinetics of DPPH scavenging activity in each studied substance identified significant variations in the time needed to reach the steady state. Rosemary extracts were seen to be more effective than the other compounds. CA had higher antioxidant activity than COL, although COL seemed to react faster with DPPH. The relevance of the CA/COL ratio for the antioxidant activity of rosemary extracts was also analysed. The presence of COL in rosemary extracts increased the antioxidant activity with an optimal CA/COL ratio of 2.5-3.0. Olive extract and grape seed extract seem to be very promising additives for use as technological antioxidants.

Data-Dependent Acquisition and Database-Driven Efficient Peak Annotation for the Comprehensive Profiling and Characterization of the Multicomponents from Compound Xueshuantong Capsule by UHPLC/IM-QTOF-MS.

The state of the art ion mobility quadrupole time of flight (IM-QTOF) mass spectrometer coupled with ultra-high performance liquid chromatography (UHPLC) can offer four-dimensional information supporting the comprehensive multicomponent characterization of traditional Chinese medicine (TCM). Compound Xueshuantong Capsule (CXC) is a four-component Chinese patent medicine prescribed to treat ophthalmic disease and angina. However, research systematically elucidating its chemical composition is not available. An approach was established by integrating reversed-phase UHPLC separation, IM-QTOF-MS operating in both the negative and positive electrospray ionization modes, and a "Component Knockout" strategy. An in-house ginsenoside library and the incorporated TCM library of UNIFITM drove automated peak annotation. With the aid of 85 reference compounds, we could separate and characterize 230 components from CXC, including 155 ginsenosides, six astragalosides, 16 phenolic acids, 16 tanshinones, 13 flavonoids, six iridoids, ten phenylpropanoid, and eight others. Major components of CXC were from the monarch drug, Notoginseng Radix et Rhizoma. This study first clarifies the chemical complexity of CXC and the results obtained can assist to unveil the bioactive components and improve its quality control.

Anti-psoriasis effect of water-processed rosin in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Rosin, an exudate of conifer trees such as Pinus masscnlana (Pinaceae), has been used to treat psoriasis for nearly two thousand years in China despite its so far undefined pharmacology. Unfortunately, the rosin intoxication is noted from time to time, but the water-boiled rosin (WBR) has been documented to be safer. This study was performed to evaluate the in vivo anti-psoriasis efficacy of WBR. MATERIALS AND METHODS: The main phytochemicals in WBR were quantified by high performance liquid chromatography (HPLC). WBR was evaluated in the imiquimod-induced psoriasis-like inflammation mouse model for its anti-psoriasis effect at 130, 260, and 390 mg/kg, which were set according to the dose used for patients. Through a combination of q-PCR, flow cytometry, and histopathological and immunohistochemical (IHC) analysis, the in vivo efficacy was assessed in terms of the psoriasis area severity index (PASI), epidermal keratinocyte proliferation, Th1 and Th17 cell numbers in spleen, and mRNA expressions of inflammatory cytokines. RESULT: Oral administration of WBR ameliorates the psoriasis-like dermatitis in the imiquimod-generated mouse model. In particular, WBR given at 260 or 390 mg/kg significantly restores the normal keratinization of dorsal lesion if compared with the untreated psoriatic mice. Such an effect was addressed to correlate to the Th1/Th17 cell reduction in spleen and the suppressed expression of IL-17A, IL-17F, IL-22, IL-23, TNF-α, K17, and proliferating cell nuclear antigen (PCNA) after the WBR administration. CONCLUSION: WBR is effective in the imiquimod-induced psoriasis-like inflammation mouse model with the efficacy arising from its proliferation inhibition of Th1/Th17 cells and epidermal keratinocytes via the down-regulation of the relevant inflammatory cytokines such as IL-23, IL-17A, and IL-17F. Collectively, WBR harvested and processed in the traditional manner is an efficacious psoriasis-treating agent.

High Yield of Bioactive Abietane Diterpenes in Salvia sclarea Hairy Roots by Overexpressing Cyanobacterial DXS or DXR Genes.

Abietane diterpenoids, containing a quinone moiety, are synthesized in the roots of several Salvia species. Promising cytotoxicity and antiproliferative activities have been reported for these compounds in various cell and animal models. We have recently shown that aethiopinone, an o-naphto-quinone diterpene, produced in the roots of different Salvia species, is selectively cytotoxic against the A375 melanoma cell line. To enhance the synthesis of this abietane diterpenoid, we have engineered the plastidial 2-C-methyl-D-erythritol 4-phosphate-derived isoprenoid pathway in Salvia sclarea hairy roots by ectopic expression and plastid targeting of cyanobacterial genes encoding the 1-deoxy-D-xylulose 5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene, the first two enzymatic steps of the plastidial MEP pathway, from which plant diterpenes primarily derive. Plastid-targeted expression of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase proteins significantly enhanced the yield of aethiopinone by a 3-fold and about 6-fold increase, respectively. The accumulation of other abietane-type diterpenes (ferruginol, salvipisone, and carnosic acid), with interesting antiproliferative activity, was also increased. Compared to our previous data obtained by overexpressing the plant orthologous 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase genes in S. sclarea hairy roots, the results presented here confirm that the bacterial 1-deoxy-D-xylulose-5-phosphate reductoisomerase enzyme plays a major role than the DXS enzyme in the biosynthetic pathway of this class of compounds and that its ectopic expression does not conflict with active hairy root growth, resulting in a balanced trade-off between the transgenic hairy root final biomass and the increased content of o-naphto-quinone diterpenes, with interesting biological activities.

Identification and Quantitative Determination of Resin Acids from Corsican Pinus pinaster Aiton Oleoresin Using 13 C-NMR Spectroscopy.

Twenty-three resin samples have been obtained by tapping from individual Pinus pinaster adult trees grown in Corsica and submitted to acido-basic partition. Identification and quantitative determination of resin acids has been carried out using 13 C-NMR spectroscopy following a method developed by our group. The main components were dehydroabietic acid (up to 37.6 %), levopimaric acid (up to 35.5 %) and abietic acid (up to 24.7 %). A lignan, pinoresinol, has been identified in some samples. Within the 23 compositions, submitted to k-means analysis and Principal Component Analysis, two clusters have been perfectly differentiated, whose compositions were dominated by dehydroabietic acid (Group I, M=23.5 %, SD=6.3) and levopimaric acid (Group II, M=21.2 %; SD=6.2), respectively. Both compositions have been observed in the three locations of harvest.

Relative molar sensitivities of carnosol and carnosic acid with respect to diphenylamine allow accurate quantification of antioxidants in rosemary extract.

We have been developing a high-performance liquid chromatography/photodiode array (HPLC/PDA) employing relative molar sensitivities (RMSs) and adopted it to the accurate quantification of carnosol (CL) and carnosic acid (CA) which are the antioxidants in rosemary extract. The method requires no references of CL or CA and instead uses RMSs with respect to diphenylamine (DPA) whose certified reference material is available from a reagent manufacturer. The molar and response ratios of the analytes to the reference in an artificial mixture of them were determined using 1H-quantitative nuclear magnetic resonance spectroscopy (1H-qNMR) and HPLC/PDA at a wavelength of 284 nm under isocratic condition, respectively, and then RMSs were calculated to be 0.111 for CL/DPA and 0.0809 for CA/DPA as averaged values in three HPLC-PDA instruments. The RMS values varied by up to 1.1% as relative standard deviation. To evaluate the performance of HPLC/PDA with the RMSs, the CL and CA contents in rosemary extracts were determined using DPA as a reference. The CL and CA contents were compared with those determined using calibration curves of CL and CA obtained by HPLC measurement of standard solutions prepared from their reagents whose absolute purities were determined using 1H-qNMR. The differences between the two methods for CL and CA were ≤3% as relative error. This chromatographic method with RMSs allows a simple and reliable quantification when reference of the analyte is unavailable.