[Preparation and effectiveness evaluation of placebo of Jiawei Ermiao Granules].

This study explored the preparation process of the placebo of Jiawei Ermiao Granules and evaluated the placebo effect, aiming to provide qualified placebo samples for clinical trials of Jiawei Ermiao Granules and a reference for the preparation and quality evaluation of placebos of traditional Chinese medicine granules. On the basis of the comprehensive analysis results of Jiawei Ermiao Granules, the orthogonal experiment was conducted to optimize the flavoring agents and colorants. After manual evaluation, the placebo formula was determined as dextrin 10 g, Codonopsis Radix extract 5.0 g, bitter melon extract 1.6 g, Mume Fructus extract 0.3 g, stevioside 0.1 g, sucrose octaacetate 0.004 g, indigo 0.004 g, lemon yellow 0.003 1 g, sunset yellow 0.001 8 g, bitter tea powder 0.001 8 g, caramel 0.001 3 g. Pilot trials were conducted on the placebo formula. The simulation effect of placebo was evaluated independently and comparatively, and the objectively evaluated by electronic nose and electronic tongue. The results showed that the independent manual evaluation of the placebo formula had higher error rate, and the placebo and Jiawei Ermiao Granules showed the similarity of 99.61% in the comparative manual evaluation. The smell similarity between the placebo and Jiawei Ermiao Granules was 99.19%, and the electronic tongue test showed little difference in the taste. In conclusion, the placebo prepared in this study shows a high similarity to Jiawei Ermiao Granules, which is not easy to break the blindness when being applied to clinical trials. This study provides a reference for the preparation and quality evaluation and promotes the large-scale production of placebos of traditional Chinese medicine granules, playing a role in improving the persuasiveness and acceptance of the efficacy of traditional Chinese medicines.

[Determination of absolute configuration of a new triterpenic acid in leaves of Ilex hainanensis].

This study aimed to explore the triterpenic acid components in leaves of Ilex hainanensis. Alkaline water extraction, macroporous resin adsorption, and high performance liquid chromatography were used to separate and purify the triterpenic acid components in leaves of I. hainanensis. The physical and chemical property analysis, MS, NMR spectroscopy, and literature comparison were performed to identify the structures, and a new triterpene acid compound was discovered:(3S, 4R, 5R, 8R, 9R, 10R, 14S, 17S, 18S, 19R)-3,19-dihydroxyursa-12,20(30)-diene-24,28-dioic-acid, and named ilexhainanin F. In addition, according to its structural characteristics, the ~(19)F-NMR Mosher method was further employed to study its absolute configuration. By comparison of the ~(19)F-NMR chemical shifts of Mosher esters, it was determined that the absolute configuration of the 3-position chiral center of the compound was the S configuration.

[Rapid quality evaluation of Glycyrrhizae Radix et Rhizoma and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle based on color digitization and multi-component determination].

To explore the color value changes after processing and further explore the correlations between color values and internal components, we established a rapid evaluation method for the quality of Glycyrrhizae Radix et Rhizoma and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle. In this study, the color values of Glycyrrhizae Radix et Rhizoma and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle were digitized by a spectrophotometer, and the standard ranges of color values of the two herbal medicines were established. Further, a discriminant analysis model was established to quickly and accurately distinguish the two herbal medicines. The content of 9 flavonoids and 1 triterpene in Glycyrrhizae Radix et Rhizoma and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle were determined by HPLC, and Pearson correlation analysis was adopted to analyze the correlations between the color values and the content of 10 components. The standard ranges of L~*, a~*, and b~* values were 65.539 6-68.305 8, 7.296 3-8.467 3, and 29.998 8-32.212 8 for Glycyrrhizae Radix et Rhizoma, and 43.654 3-47.166 4, 14.050 0-15.133 8, and 16.424 6-20.984 8 for Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle, respectively. Glycyrrhizae Radix et Rhizoma had higher L~* and b~* values and lower a~* value than Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle, which indicated that processing with honey decreased the white and yellow values and increased the red value. The original and cross validation of the established discriminant analysis model met the requirements, and the external validation of the model showed the prediction accuracy of 100%. Pearson correlation analysis showed that the a~* value was positively correlated with the content of liquiritin apioside and isoliquiritin apioside(P<0.05), while the L~* and b~* values were negatively correlated with the content of the above two components(P<0.05). After processing with honey, L~* and b~* decreased while a~* increased, and the content of liquiritin apioside and isoliquiritin apioside increased, which was consistent with the content determination results. This study reveals the regularity of the color values of Glycyrrhizae Radix et Rhizoma after processing with honey roasting, as well as the correlations between color values and component content, which provides a basis for the rapid quality evaluation of Glycyrrhizae Radix et Rhizoma and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle.

[Analysis of hydroxylation and O-glycosylation on lysine sites in deer-hide gelatin].

Nano-LC MS/MS was used to analyze trypsin digested deer-hide gelatin(DHG) samples, hydroxylation and O-glycosylation on lysine sites of DHG were comprehensive identified by using PEAKS Studio software. The sites, sorts and amounts of hydroxylation and O-glycosylation on Type Ⅰ collagen α1 chain(COL1 A1) and α2 chain(COL1 A2) of DHG were revealed. As a result, 5 284 peptides were identified from DHG samples, which were mainly from COL1 A1 and COL1 A2. Among these peptides, there were 449 peptides with hydroxylysine, 442 with galactosyl-hydroxylysine, 449 with glucosyl-galactosyl-hydroxylysine. The major modified sites of hydroxylation and O-glycosylation in DHG were shown as follow: α1-9 N and α2-5 N in N-telopeptides, α1-87, α1-174, α1-930, α2-87, α2-174, α2-933 in triple helix domain, and α1-16 C in C-telopeptides. These hydroxylation and O-glycosylation were correlated with the formation and stability of collagen molecules and collagen fibrils. It is feasible for the collagens and peptides dissolving from deer skin collagen fibrils under high temperature and pressure decocting, high temperature and pressure also might destroy inter-molecular covalent cross-linking and help those glycol-peptides formations. The present study provided ideas and strategies for the in-depth investigation on DHG chemical constituents, and showed good theoretical significance and application value.

Collagen derived species-specific peptides for distinguishing donkey-hide gelatin (Asini Corii Colla).

Objective: As an important food therapy product with traditional Chinese medicine (TCM) applications, donkey-hide gelatin (Asini Corii Colla, ACC) has been used for thousands of years. However, till now few effective strategy had been proposed to distinguish ACC from other animal hide gelatins, especially closely related horse- and mule-hide gelatins, which was an embarrassment of ACC quality control. Methods: Combined mass spectrometry and bioinformatic methods have been applied to identify and verify two ACC-specific peptides (Pep-1 and Pep-2) capable of distinguishing ACC from other closely related animal gelatins with high selectivity. Results: It confirmed that these two peptides could be not only used for distinguishing ACC from highly homologous horse-hide and mule-hide gelatins as well as other animal hide gelatins. Conclusion: The present study provides a simple method for species-specific peptides discovery, which can be used for assessing the quality of animal gelatin products, and ensure they are authenticable and traceable.

Comprehensive Comparison of Two Color Varieties of Perillae Folium Using Rapid Resolution Liquid Chromatography Coupled with Quadruple-Time-of-Flight Mass Spectrometry (RRLC-Q/TOF-MS)-Based Metabolic Profile and in Vivo/in Vitro Anti-Oxidative Activity.

Perillae Folium (PF), which is extensively used as a dietary vegetable and medicinal herb, contains two varietal forms corresponding to purple perilla leaf (Perilla frutescens var. crispa) and green perilla leaf (Perilla frutescens var. frutescens). However, the components and efficacy of different PF varieties remain underexplored so far. In the present work, a nontargeted rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (RRLC-Q/TOF-MS)-based metabolomics approach was developed to investigate the difference in the chemical compositions between green PF and purple PF. A total of 71 compounds were identified or tentatively identified, among which 7 phenolic acids, 10 flavonoids, and 9 anthocyanins were characterized as differential metabolites. In addition, heatmap visualization and ultraperformance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-TQ-MS/MS)-based quantitative analysis revealed that flavonoids and anthocyanins especially had higher contents in purple PF. Furthermore, the anti-oxidative activities of two varietal PFs were evaluated in vivo zebrafish and in vitro human umbilical vein endothelial cells (HUVECs). The results showed that the purple PF had more pronounced anti-oxidative activities than did the green PF, which may be due to the presence of anthocyanins and a higher concentration of flavonoids in its phytochemical profile. The outcome of the present study is expected to provide useful insight on the comprehensive utilization of a PF resource.

[Analysis of quality difference based on Astragalus membranaceus var. mongholicus in genuine region].

This work is to establish the fingerprint of Astragalus membranaceus var. mongholicus by HPLC-ELSD method, and to analyze the simulated wildness degree of A. membranaceus var. mongholicus in the genuine region of Inner Mongolia, Ningxia and Gansu. Compared with wild A. membranaceus var. mongholicus, the quality differences of A. membranaceus var. mongholicus in the genuine region were analyzed by identification of chromatographic peaks and similarity evaluation, cluster analysis(CA), principal components analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA). HPLC fingerprints of A. membranaceus var. mongholicus in different genuine regions are established. The qualitative analysis of mass spectrometry identified 18 components. The similarity evaluation shows that the similarity of 32 batches of A. membranaceus var. mongholicus samples was 0.688-0.993. Among them, the similarity of samples in Shanxi, Inner Mongolia, Ningxia is 0.688-0.993, 0.835-0.989, 0.934-0.988, respectively and the similarity of samples in Gansu is 0.729-0.876 except No. 25 sample. The results of CA show that the samples of A. membranaceus var. mongholicus can be grouped into four categories according to the production area except the No. 11 and No. 25 samples. The results of PCA indicate that 32 batches of A. membranaceus var. mongholicus samples can be clustered according to quality and origin, and the quality of A. membranaceus var. mongholicus in Inner Mongolia is the closest to the wild breed. The results of OPLS-DA indicate that there are six components that can distinguish the wild and domestic A. membranaceus var. mongholicus, which are malonylastragaloside Ⅰ, astragaloside Ⅰ, calycosin-7-O-ß-D-glycoside-6″-O-malonate, calycosin-7-O-ß-D-glycoside, formononetin-7-O-ß-D-glycoside-6″-O-malonate, and astrapterocarpan-3-O-ß-D-glycoside-6″-O-malonate. The established method can be used to analyze differences between A. membranaceus var. mongholicus origin and planting environment, and can provide references for the protection and replacement of wild A. membranaceus var. mongholicus resources, and the cultivation, processing and production of A. membranaceus var. mongholicus.

[Comparative study of water characteristic components of Glycyrrhiza uralensis from three geographical regions by chemical pattern recognition combined with muti-component qualitative and quantitative analysis].

Thirty-two batches of cultivated and wild Glycyrrhiza uralensis were obtained from three geographical regions. Comparative study of water characteristic components of G. uralensis from three geographical origins was conducted by PCA,OPLS-DA chemical pattern recognition combined with LC-TOF/MS and muti-component analysis. The similarity of fingerprints of 32 batches of medicinal materials ranged from 0. 903 to 0. 999. Patterns recognition could be used to distinguish cultivated G. uralensis in Gansu and Xinjiang areas from cultivated and wild plants in Inner Mongolia. Then a total of thirty-one common constituents were identified by LC-TOF/MS analysis coupled with standard compounds information. The contents of four flavonoid glycosides and five saponins were determinated by HPLC and compared using One-way ANOVA. The results showed that there was no significant difference in the contents of 5 triterpenoid saponins among the three regions,but the contents of 4 flavonoid saponins showed the trend of Inner Mongolia >Gansu≈Xinjiang( P<0. 05). In the same Inner Mongolia region,the contents of 4 flavonoid glycosides and 5 triterpenoid saponins in wild plant was significantly higher than that in cultivated plants( P<0. 01). In addition,the contents of liquiritin,isoliquiritin,licorice-saponin A_3,22ß-acetoxyl-glycyrrhizic acid and uralsaponin B in Gansu and Xinjiang were obviously lower than those in Inner Mongolia,but the contents of glycyrrhizic acid,the main component of G. uralensis,were not different in the three geographical regions. In Inner Mongolia,the contents of liquiritin,isoliquiritin,licorice-saponin A_3,licorice-saponin G_2 and glycyrrhizic acid in wild plants were significantly higher than those in cultivated plants. In conclusion,qualitative/quantitative analysis of multi-index components combined with pattern recognition could effectively evaluate the quality of cultivated and wild licorice in different regions. It was helpful for us to understand the reality of licorice in different regions,and provided scientific basis for the development and comprehensive utilization of licorice resources.

[HPLC characteristic fingerprints of Xinshenghua Keli].

Xinshenghua Keli is known as the "preferred prescription of postpartum", with large demand in the field of gynecologic medicine. However, the quality of the preparation is uneven in the market, so its clinical efficacy cannot be guaranteed. In order to improve and establish its quality control standard, high performance liquid chromatography (HPLC) was used to establish the fingerprint of Xinshenghua Keli. The detection was performed on Agilent 5 HC-C18 (2) column(4.6 mm×250 mm, 5 microns) with methanol-0.1% formic acid solution as mobile phase for gradient elution, at a flow rate of 1 mL·min⁻¹ with column temperature of 25 °C. The injection volume was 10 µL and detection wavelength was set at the maximum value between 210.0 nm and 400.0 nm by Photo-Diode Array (PDA) detector. The fingerprint of 12 batches of high-quality Xinshenghua Keli was established and 43 common peaks were identified. The similarities of crowned products, 10 batches of ordinary ones made by Jiangsu Rongyu Pharmaceutical and 10 batches produced by different manufacturers were evaluated. The composition identification and source analysis for the common peaks were performed by comparing the retention time of herbal medicines and ultraviolet absorption spectrum, along with high performance liquid chromatography-mass spectrometry (HPLC-MS) technology. The established fingerprint of Xinshenghua Keli, has proven to have good precision, stability and repeatability through the methodology validation, so it can be used to comprehensively evaluate the quality of Xinshenghua Keli.

[Design and implementation of Xinshenghua granule traceability system based on data authenticity].

The quality of traditional Chinese medicine affects the clinical efficacy and the market research at home or abroad, which is closely related to the standardization of production. At present, Chinese market still exist in drug quality and nonstandard production phenomenon. In addition to the existing GMP production standards, it is still necessary to ensure the authenticity and reliability of traditional Chinese medicine by means of retrospect. Based on the Xinshenghua granule, starting from the authenticity of the whole process of production, this study designs and constructs the traditional Chinese medicine traceability system which is equipped with the Internet platform by using two-dimensional code as a trace tool. By making authentic record of the production process and quality transfer, it is convenient for consumers to know the drug information. The production traceability system provides guarantee for the standardization of traditional Chinese medicine development, in order to obtain the production source，the testing quality, whereabouts and responsibility.

Potential hypoglycaemic activity phenolic glycosides from Moringa oleifera seeds.

Moringa oleifera seed has remarkable curative effects on reducing blood pressure, blood sugar and enhancing human immunity. In this study, one novel phenolic glycoside (1) together with four known compounds 2-5 were isolated from the macroporous resin adsorption extract of M. oleifera seeds, and the compound 3 was reported for the first time from this plant. The structure of the new crystalline compound was determined on the basis of spectroscopic analyses including mass spectrometry, 1D and 2D NMR experiments. The hypoglycaemic activity of isolated compounds was investigated with HepG2 cell and STZ-induced mice. It was found that compound 1, 4 and 5 could promote the glucose consumption of insulin resistance cells and reduce blood glucose levels of STZ-induced mice. This study concludes that compound 1, 4 and 5 may be developed as new and safe hypoglycaemic drugs.

Identification of the Allergenic Ingredients in Reduning Injection by Ultrafiltration and High-Performance Liquid Chromatography.

Reduning injection is a traditional Chinese medicine injection which has multiple functions such as clearing heat, dispelling wind, and detoxification. Although Reduning injection was widely utilized, reports of its allergenicity emerged one after another. However, there is little research on its allergenic substances. The aim of this study is to evaluate the sensitization of Reduning injection and explore the underlying cause of the anaphylactic reaction. The main ingredients in Reduning injection were analyzed before and after ultrafiltration. Ultrafiltrate Reduning injection, unfiltered Reduning injection, egg albumin, Tween-80, and nine effective components in Reduning injection were utilized to sensitize guinea pigs. The serum 5-hydroxytryptamine level was used to assess the sensitization effect of Reduning injection. We found a significant decrease in Tween-80 content comparing to other components in the injection after ultrafiltration. Unfiltered Reduning injection, Tween-80, chlorogenic acid, and cryptochlorogenin acid caused remarkable anaphylactoid reaction on guinea pigs while ultrafiltration Reduning resulted in a significantly lower degree of sensitization. Our results suggest that ultrafiltration could significantly reduce the sensitization of Reduning injection, which is likely due to the decrease of Tween-80. We also conjectured that the form of chlorogenic acid and cryptochlorogenin acid within the complex solution mixture may also affect the sensitizing effect.

Hepatoprotective triterpene saponins from the roots of Glycyrrhiza inflata.

Two novel oleanane-type triterpene saponins, licorice-saponin P2 (1) and licorice-saponin Q2 (3), together with nine known compounds 2, 4-11, have been isolated from the water extract of the roots of Glycyrrhiza inflata. The structures of these compounds were elucidated on the basis of spectroscopic analysis, including 2D-NMR experiments (1H-1H COSY, HSQC, HMBC and ROESY). In in vitro assays, compounds 2-4, 6 and 11 showed significant hepatoprotective activities by lowering the ALT and AST levels in primary rat hepatocytes injured by D-galactosamine (D-GalN). In addition, compounds 2-4, 6, 7 and 11 were found to inhibit the activity of PLA2 with IC50 values of 6.9 µM, 3.6 µM, 16.9 µM, 27.1 µM, 32.2 µM and 9.3 µM, respectively, which might be involved in the regulation of the hepatoprotective activities observed.

Antioxidant and anti-inflammatory activities of six flavonoids separated from licorice.

Licorice, the roots and rhizomes of several Glycyrrhiza species (Leguminosae), is an important natural sweetening agent and a widely used herbal medicine. In this work, six flavonoids, 5-(1,1-dimethylallyl)-3,4,4'-trihydroxy-2-methoxychalcone (1), licochalcone B (2), licochalcone A (3), echinatin (4), glycycoumarin (5) and glyurallin B (6), were isolated from the extracts of licorice (Glycyrrhiza inflata and Glycyrrhiza uralensis). Their structures were elucidated using various spectroscopic methods. To our knowledge, compound 1 was isolated from natural plants for the first time. All the isolates were tested by antioxidant and anti-inflammatory assays. Compounds 2, 4 and 5 showed strong scavenging activity toward the ABTS(+) radical, and compounds 1, 2, 3, 5 and 6 exhibited potent inhibition of lipid peroxidation in rat liver microsomes compared with the reference controls. Compounds 1-4 dose-dependently inhibited LPS induced reactive oxygen species (ROS) production in RAW 264.7 cells. Furthermore, compounds 1-5 were demonstrated to inhibit the production of nitric oxide (NO), interleukin-6 (IL-6) and prostaglandin E2 (PGE2) in LPS-induced macrophage cells. Moreover, the contents of the six compounds, in different Glycyrrhiza species, were quantified by HPLC-MS.

A green and efficient protocol for large-scale production of glycyrrhizic acid from licorice roots by combination of polyamide and macroporous resin adsorbent chromatography.

A green and efficient method for large-scale preparation of glycyrrhizic acid from licorice roots was developed by combination of polyamide and macroporous resin. The entire preparation procedure consisted of two simple separation steps. The first step is to use polyamide resin to remove licorice flavoniods from the licorice crude extract. Subsequently, various macroporous resins were tried to purify glycyrrhizic acid, and HPD-400 showed the most suitable adsorption and desorption properties. Under the optimized conditions, a large-scale preparation of glycyrrhizic acid from licorice roots was carried out. A 20 kg raw material produced 0.43 kg of glycyrrhizic acid using green aqueous ethanol as the solvent. The purity of glycyrrhizic acid was increased from 11.40 to 88.95% with a recovery of 76.53%. The proposed method may be also extended to produce large-scale other triterpenoid saponins from herbal materials.

[Multi-index determination and optimization of liquirtin separated from polyamide resin].

To optimize the separation process of liquirtin from glycyrrhiz by static, dynamic adsorption and desorption experiments on polyamide resin, with liquirtin, isoliquiritin and glycyrrhizic acid as the study index. The optimum process conditions were that the pH of solution was regulated to be 7.0, the concentration of liquirtin was 1.296 g x L(-1), the volume of loading buffer was 3 BV. After absorption, efforts shall be made to elute resin with water, 10%, 20%, 30% ethanol (3 BV for each), collect 20% ethanol eluted fraction, and recover solvents. The results showed lower contents of such impurities as isoliquiritin and isoliquiritin in extracts sepaprated under this process conditions, as well as an increase in purity of liquirtin from 4.86% to 88.5%. The method was simple and feasible, it could obtain a higher purity in extracts from liquirtin and provide basis for industrialized separation and preparation of liquirtin.

[Comparison of activated carbon and ultrafiltration technique in the production process of huoxue tongluo injection].

OBJECTIVE: To research the applicability of activated carbon and ultrafiltration technique in the production process of Huoxue Tongluo Injection. METHODS: The kinetic-turbidimetric method was used to determine the content of bacterial endotoxins in Huoxue Tongluo solution. Particle size change in Huoxue Tongluo solution was determined by nanometer particle size instrument before and after the use of different concentration activated carbon and different molecular weight ultrafiltration membrane. RESULTS: The removal efficacy of bacterial endotoxins was 65.2%, 77.5%, 80.4% by using three concentrations of active carbon at 0.05%, 0.10%, 0.30% in Huoxue Tongluo Injection, respectively. It was above 95% by using cutoff molecular weight both 5 kDa and 10 kDa ultrafiltration membrane. Measure results by nanometer particle size instrument showed that particle size of filter liquor by 10 kDa cutoff molecular weight ultrafiltration membrane was much smaller than that of by use of different concentration activated carbon. CONCLUSION: Ultrafiltration method is more suitable to the removal of bacterial endotoxins. The solution is more clear after using ultrafiltration method, and large particles of solution is removed. The ultrafiltration method provides the basis for injection production.

Screening and characterization of natural antioxidants in four Glycyrrhiza species by liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Licorice, derived from the dried roots and rhizomes of several species of genus Glycyrrhiza L. (Leguminosae family), has been traditionally used in herbal medicine for over 4000 years. In recent years, the interest in antioxidative constituents in licorice has greatly increased. In this work, a new method based on 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) spiking test combined with HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis was developed to screen and identify the antioxidants in licorice. The results of the method validation indicated that the developed method was reliable and repeatable. Compared with DPPH on-line method, the HPLC-Q-TOF MS/MS method combined with DPPH spiking test offered much higher sensitivity and resolution. Using this method, 35 radical scavengers were screened from four Glycyrrhiza species (G. inflata, G. glabra, G. pallidiflora and G. uralensis), and 21 of them were unambiguously or tentatively identified by HPLC-Q-TOF MS/MS. Among the 21 identified flavonoids, 10 compounds had been reported to possess antioxidative activities in the previous studies, and the radical scavenging activities of the other 11 compounds were reported for the first time. The effects of six purified flavonoids on DPPH radical and lipid peroxidation were evaluated for validation of the developed method. The results indicated that HPLC-Q-TOF MS/MS coupled with DPPH treatment is an efficient and powerful method to discover the potential antioxidative compounds from the complex natural product mixtures. In this study, the identified components with free radical scavenging activity, would help to explain the therapeutic benefit of licorice in the treatment of human disease associated with oxidative stress.

Two new ginkgolides from the leaves of Ginkgo biloba.

Two new diterpenoid compounds, ginkgolide P(1) and ginkgolide Q(2), were isolated from the leaves of Ginkgo biloba L. Their structures were elucidated by various spectroscopic methods, and the structure of 1 was further confirmed by X-ray crystallographic analysis. The activities of the compounds were evaluated against platelet aggregation induced by platelet activating factor (PAF), and the preliminary structure-activity relationship was also discussed.

Structural characterization and identification of oleanane-type triterpene saponins in Glycyrrhiza uralensis Fischer by rapid-resolution liquid chromatography coupled with time-of-flight mass spectrometry.

Oleanane-type triterpene saponins (OTS) are major active ingredients in Glycyrrhiza uralensis. In this work, a rapid-resolution liquid chromatography with time-of-flight mass spectrometry (RRLC/TOF-MS) method has been developed to characterize and identify OTS from G. uralensis. The major diagnostic ions and fragmentation pathways from thirteen OTS have been characterized for the first time. At a low fragmentor voltage of 120 V in positive ion mode, the precursor ion [M + H](+) or/and [M + Na](+) was obtained for accurate determination of molecular formula. When the fragmentor voltage was increased to 425 V, abundant characteristic fragment ions were observed for structural characterization. Neutral losses of sugar moieties, such as glucuronic acid (GlcA, 176 Da), glucose (Glc, 162 Da) and rhamnose (Rha, 146 Da), were commonly observed in the MS spectra for prediction of the sugar number and sequences. Other typical losses included AcOH (60 Da), CH(2)O (30 Da), 2 × H(2)O (2 × 18 Da) and HCOOH (46 Da) from [Aglycone + H-H(2)O](+) (named [B](+)), corresponding to the presence of a C(22)-acetyl group, C(24)-hydroxyl group, C(22)-hydroxyl group or C(30)-carboxyl group on the aglycone moiety, respectively. In particular, characteristic ring cleavages of the aglycone moieties on A- and B-rings were observed. Based on the fragmentation patterns of reference compounds, nineteen OTS have been identified in an extract of G. uralensis, thirteen of which were unambiguously identified and the other six were tentatively assigned.