Integration of Zebrafish Model and Network Pharmacology to Explore Possible Action Mechanisms of Morinda officinalis for Treating Osteoporosis.

Osteoporosis (OP) is a metabolic bone disease affecting nearly 200 million individuals globally. Morinda officinalis F.C.How (MOH) has long been used as a traditional herbal medicine for the treatment of bone fractures and joint diseases in China. However, it still remains unclear how the compounds in MOH work synergistically for treating OP. In this study, we used prednisolone (PNSL)-induced zebrafish OP model to screen the antiosteoporosis components in MOH. A network pharmacology approach was further proposed to explore the underlying mechanism of MOH on OP. The PNSL-induced zebrafish model validated that two anthraquinones, one iridoid glycoside, and two saccharides exerted antiosteoporotic effect. We constructed the components-targets network and obtained the enriched Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. A total of 26 candidate compounds of MOH and 257 related targets could probably treat OP through regulating osteoclast differentiation and MAPK signaling pathway. Our work developed a strategy to screen the antiosteoporosis components and explore the underlying mechanism of MOH for treating OP at a network pharmacology level.

Development and validation of a supercritical fluid chromatography method for fast analysis of six flavonoids in Citri Reticulatae Pericarpium.

The study is aimed to develop a rapid and efficient supercritical fluid chromatography coupled with diode-array detection (SFC-DAD) method for simultaneous quantification of six flavonoids in Citri Reticulatae Pericarpium (CRP). The chromatographic parameters including stationary phase, mobile phase composition, back pressure, temperature and flow rate, were systematically optimized with single-factor test. The results indicated that the six compounds were baseline separated on an Agilent Zorbax RX-SIL column using gradient elution within 10 min. The optimized mobile phase was constituted of carbon dioxide (CO2) and methanol. And the parameters were set as follows: backpressure, 95 bar; temperature, 45 °C; flow rate, 0.8 mL/min. The optimized method showed satisfactory retention and resolution for the analytes. The method was validated to demonstrate its selectivity, linearity, precision, accuracy and robustness. Thus, the verified SFC method was successfully applied to quantification of flavonoids present in CRP samples. The results indicated that SFC has the potential to become an excellent alternative for the analysis of flavonoids in herbal medicines, owning to its intrinsic features like high efficiency, separation speed and eco-friendly characteristics.

Identification of anti-inflammatory components in Sinomenii Caulis based on spectrum-effect relationship and chemometric methods.

The present study aimed to identify the anti-inflammatory components in Sinomenii Caulis (SC) based on spectrum-effect relationship and chemometric methods. A phytochemical investigation of SC extract was performed firstly and afforded eleven potential bioactive compounds. The HPLC fingerprints of 19 batches of SC samples were evaluated by the chemometric methods such as similarity analysis (SA) and hierarchical clustering analysis (HCA). The anti-inflammatory effects of these samples were determined by inhibition of Nitric Oxide (NO) production. Partial least squares regression (PLSR) and artificial neural network (ANN) were used to explore the spectrum-effect relationship of SC. The results indicated that there was a close correlation between chemical fingerprint and anti-inflammatory activity of SC, and peaks 8, 9, 12, 13, 14, 16, 19 and 22 might be potential anti-inflammatory compounds in SC. The verification experiments by testing individual compounds and a combination of them indicated that sinomenine (P8), magnoflorine (P13), menisperine (P16) and stepharanine (P19) were the major anti-inflammatory compounds in SC. Collectively, the present study established the spectrum-effect relationship mode of SC and discovered the anti-inflammatory compounds in SC, which could be used for exploration of bioactive components and quality control of herbal medicines.

Simultaneous Quantification of Five Flavanone Glycosides in Rat Plasma by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry: Application to a Comparative Pharmacokinetic Study of Aurantii Fructus Immaturus and Aurantii Fructus Extracts.

Background: Aurantii Fructus Immaturus (AFI) and Aurantii Fructus (AF) are two traditional citrus herbs with health-promoting and nutritive properties. Objective: This paper presents the first attempt to simultaneously investigate the absorption of five major flavanone glycosides, namely narirutin, naringin, hesperidin, neohesperidin, and poncirin, in rat plasma following a single oral administration of AFI and AF extracts to rats. Methods: The plasma concentrations were determined by liquid-liquid extraction followed by a rapid and sensitive ultra-performance LC-tandem mass spectrometry method. Pharmacokinetic parameters were analyzed by noncompartmental modeling using DAS software. Results: The developed method was validated and successfully applied to the pharmacokinetic study of these five flavanone glycosides. Conclusions: The comparison of the pharmacokinetic parameters of flavanone glycosides showed that the absorption of AF extract was lower, while the elimination was relatively rapid, compared with those of AFI extract. Highlights: This study may be useful for further utilization of these two citrus herbs.

Chemical profiling and quantitation of bioactive compounds in Platycladi Cacumen by UPLC-Q-TOF-MS/MS and UPLC-DAD.

Platycladi Cacumen (PC) is a traditional Chinese medicine used for the treatment of hemorrhages, cough, asthma and hair loss. To get a better understanding of the chemical constituents in PC, ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) and diagnostic ion filtering strategy were firstly employed for chemical profiling of PC. A total of 43 compounds including organic acids and derivatives, flavonoids as well as phenylpropanolds were unambiguously or reasonably identified. Coumarin and lignan were reported for the first time in PC. Chemical variation of 39 batches of PC from different geographical origins and 10 batches of processed product of PC was subsequently investigated by quantitation of nine major flavonoids. The results determined by UPLC coupled with diode array detection (UPLC-DAD) and hierarchical cluster analysis (HCA) indicated that the contents of flavonoids in PC samples differ greatly. This work provides an efficient approach to comprehensively evaluate the quality of PC.

Comparison of Aurantii Fructus Immaturus and Aurantii Fructus based on multiple chromatographic analysis and chemometrics methods.

To get a better understanding of the bioactive constituents in Aurantii Fructus Immaturus (AFI) and Aurantii Fructus (AF), in the present study, a comprehensive strategy integrating multiple chromatographic analysis and chemometrics methods was firstly proposed. Based on segmental monitoring, a high-performance liquid chromatography (HPLC)-variable wavelength detection method was established for simultaneous quantification of ten major flavonoids, and the quantitative data were further analyzed by hierarchical cluster analysis (HCA) and principal component analysis (PCA). A strong cation exchange-high performance liquid chromatography (SCX-HPLC) method combined with t-test and one-way analysis of variance (ANOVA) was developed to determine synephrine, the major alkaloid in AFI and AF. The essential oils were analyzed by gas chromatography-mass spectrometry (GC-MS) and further processed by partial least squares discrimination analysis (PLS-DA). The results indicated that the contents of ten flavonoids and synephrine in AFI were significantly higher than those in AF, and significant difference existed in samples from different geographical origins. Also, 9 differential volatile constituents detected could be used as chemical markers for discrimination of AFI and AF. Collectively, the proposed comprehensive analysis might be a well-acceptable strategy to evaluate the quality of traditional citrus herbs.

Comprehensive analysis of chemical constituents in Xingxiong injection by high performance liquid chromatography coupled with mass spectrometry.

Xingxiong injection (XXI) is a widely used Chinese herbal formula prepared by the folium ginkgo extract and ligustrazine for the treatment of cardiovascular and cerebrovascular diseases. Compared with the pharmacological studies, chemical analysis and quality control studies on this formula are relatively limited. In the present study, a high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) method was applied to comprehensive analysis of constituents in XXI. According to the fragmentation rules and previous reports, thirty ginkgo flavonoids, four ginkgo terpene lactones, and one alkaloid were identified. A high performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC-QQQ MS) method was then applied to quantify ten major constituents in XXI. The method validation results indicated that the developed method had desirable specificity, linearity, precision and accuracy. The total contents of ginkgo flavonoids were about 22.05-25.51 µg·mL(-1) and the ginkgo terpene lactones amounts were about 4.41-8.70 µg·mL(-1) in six batches of XXI samples, respectively. Furthermore, cosine ratio algorithm and distance measurements were employed to evaluate the similarity of XXI samples, and the results demonstrated a high-quality consistency. This work could provide comprehensive information on the quality control of Xingxiong injection, which be helpful in the establishment of a rational quality control standard.

Fragmentation study of iridoid glycosides and phenylpropanoid glycosides in Radix Scrophulariae by rapid resolution liquid chromatography with diode-array detection and electrospray ionization time-of-flight mass spectrometry.

Rapid resolution liquid chromatography (RRLC) coupled with diode array detection (DAD) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) method was applied to the mass spectral study of a series of naturally occurring iridoid glycosides and phenylpropanoid glycosides in Radix Scrophulariae, which provides higher speed and increased sensitivity without loss of resolution. With dynamic adjustment as the key role of the fragmentor voltage and confirmed with authentic standards, valuable structural information regarding the nature of both the glycoside skeletons was thus obtained. Most compositions were found to possess organic acid moiety such as cinnamoyl, caffeoyl and ferulyol. Besides extensive fragmentation of the carbohydrate moiety, losses of the hydroxyl and glucose residue units showed in the spectra, permitting the exploration of the skeleton and the identity of substituents in the molecule. Ten major iridoid glycosides and 10 phenylpropanoid glycosides were identified or tentatively characterized based on their retention times, UV and TOF MS data. The major fragmentation pathways of PGs in Radix Scrophulariae obtained through the MS data was schemed systematically for the first time, which provides a reference for other PGs derivatives.

An in vivo microdialysis measurement of harpagoside in rat blood and bile for predicting hepatobiliary excretion and its interaction with cyclosporin A and verapamil.

Harpagoside, a major bioactive iridoid glucoside in genus Scrophularia, has been widely used in clinical practice for the treatment of pain in the joints and lower back for its neuroprotective and anti-inflammation activities. To investigate the pharmacokinetics and hepatobiliary excretion, an in vivo microdialysis method coupled with high performance liquid chromatography was developed to monitor the concentration of harpagoside in blood and bile. The harpagoside bile-to-blood distribution ratio (AUC(bile)/AUC(blood)) up to 986.28+/-78.46 significantly decreased to 6.41+/-0.56 or 221.20+/-18.92 after co-administration of cyclosporin A or verapamil. The results indicated that harpagoside went through concentrative elimination from the bile which was probably regulated by P-glucoprotein, providing possible clinical trials of co-administration of transporter inhibitors to decrease drug efflux, thus to enhance the curative effects.

Application of liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry for screening and quantitative analysis of C21 steroids in the roots and rhizomes of Cynanchum paniculatum.

A liquid chromatography-mass spectrometry method is presented for the quantification of C21 steroids in the roots and rhizomes of Cynanchum paniculatum. Eight C21 steroids, including five steroidal aglycones and three steroidal glycosides, were simultaneously analyzed by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry. The extracted ion current chromatograms were extracted from the total ion current chromatogram using characteristic ions produced by target compounds for peak determination. Chromatographic separation was achieved on a C18 reversed-phase column within 60 min, using an acetonitrile/water gradient. For comparision, six C. paniculatum samples from different locations were investigated by the established method, and the results indicated that the different geographical origin significantly influenced the C21 steroid composition. The method was observed to have the necessary sensitivity, selectivity, precision, and accuracy, and to be suitable for quality control of herbal medicines and their preparations.

Simultaneous qualification and quantification of eight triterpenoids in radix achyranthis bidentatae by high-performance liquid chromatography with evaporative light scattering detection and mass spectrometric detection.

An HPLC with evaporative light scattering detection (ELSD) and ESI-MS was established for the simultaneous determination of eight triterpenoids in Radix Achyranthis Bidentatae. The optimal chromatographic conditions were achieved on a Zorbax C18 column by linear gradient elution with 0.08% v/v aqueous formic acid and ACN as the mobile phase at the flow rate of 0.8 mL/min. Temperature for the detector drift tube was set at 101 degrees C and the nitrogen flow rate was 2.8 L/min. The identities of the analytes were accomplished by comparing retention times and mass data with those of reference compounds. The validation of the method included tests of linearity, sensitivity, repeatability, recovery, and stability. All the calibration curves of the eight triterpenoids showed good linear regression (R2 >0.997) within the test ranges. The method provides desirable repeatability with overall intra- and interday variations of less than 4.9%. The obtained recoveries varied between 93.6 and 98.1% while the RSDs were below 3.9% (n = 3). The analysis results indicate that the content of investigated triterpenoids in Radix Achyranthis Bidentatae from different locations was greatly diverse, and the triterpenoids could be used as chemical markers for the discrimination of genuine and ungenuine crude drugs.

[Study on fingerprints of astragalosides injection by HPLC-ELSD].

OBJECTIVE: To develop an HPLC method for fingerprint determination of the astragalosides injection. METHOD: Analyses were carried out at 25 degrees C on a Zorbax SB-C18 column (4.6 mm x 250 mm,5 microm). Mobile phase A was water, and B was acetonitrile. The analysis followed a linear gradient program. Initial conditions were 15% B; 0 to approximately 65 min, changed to 60% B. The flow rate was 0.8 mL x min(-1). The drift tube temparature of the ELSD was 105 degrees C, and gas flowrate was 2.7 L x min(-1). RESULT: The RSD of relative retention time of the common peaks in the plant material, the extract and the injection fingerprints was less than 0.1%, and there was good similarity among 10 batches of samples. CONCLUSION: The method was reliable and simple which could be used for quality control of the injection.

Simultaneous determination of the major isosteroidal alkaloids and their glucosides in the bulbs of Fritillaria by high performance liquid chromatography coupled with evaporative light scattering detection.

AIM: To establish an HPLC-ELSD method for the simultaneous determination of five major bioactive isosteroidal alkaloids and gluco-alkaloids in the bulbs of Fritillaria namely peimissine, imperialine, sinpeinine A, imperialine-3 beta-glucoside and yibeinoside A. METHODS: A Nova-Pak C18 column (150 mm x 3.9 mm ID) was used. The chromatography was carried out with a linear gradient programming. The mobile phase was acetonitrile-water (containing 0.1% diethylamine) and the flow rate was 1.0 mL.min-1. RESULTS: The linear range of peimissine was 13.1-288.2 mg.L-1 (r2 = 0.9975), imperialine-3 beta-glucoside 7.7-169.4 mg.L-1 (r2 = 0.9993), yibeinoside A 7.3-160.6 mg.L-1 (r2 = 0.9997), imperialine 16.5-363.0 mg.L-1 (r2 = 0.9992), sinpeinine A 8.7-191.4 mg.L-1 (r2 = 0.9942). CONCLUSION: The method is accurate with overall intra- and inter-day variation less than 5% and recovery more than 95%. The method was successfully applied to analyze five major bioactive alkaloids and gluco-alkaloids in three Fritillaria bulbs.

[Studies on chemical constituents of Pholidota yunnanensis].

OBJECTIVE: To investigate the chemical constituents from Pholidota yunnanensis. METHOD: Various chromatographic techniques were employed for isolation and purification of the constituents. The structures were elucidated by chemical and spectral analyses. RESULT: Seven compounds were obtained and they were identified by spectroscopic analysis as n-nonacosane, cyclopholidone, n-dotriacontanoic acid, n-octacostyl ferulate, cyclopholidonol, cycloneolitsol and beta-sitosterol, respectively. CONCLUSION: n-octacostyl ferulate and cycloneolitsol were isolated from genus Pholidota for the first time.

[Studies on the chemical constituents of Dendrobium fimbriatum].

AIM: To investigate the chemical constituents of Dendrobium fimbriatum Hook. METHODS: Various chromatographic techniques were employed for isolation and purification of the constituents. The structures were elucidated by IR, MS, 1HNMR, 13CNMR and 2D-NMR. RESULTS: Eight compounds were obtained and identified by spectral analysis as fimbriatone, confusarin, crepidatin, physcion, rhein, ayapin, scopolin methyl ether and n-octacostyl ferulate. CONCLUSION: Fimbriatone is a new compound, physcion and rhein are firstly isolated from Genus Dendrobium. The others are found from this species for the first time. Fimbriatone showed potential inhibitory effects on BGC cell line.

HPLC-ELSD determination of triterpenoids and triterpenoid saponins in Ilex pupurea leaves.

AIM: To establish an HPLC-ELSD method for the quantification of pedunculoside, ziyuglycoside I and rutundic acid, in the leaves of Ilex purpurea Hassk. METHODS: By optimizing the chromatographic conditions of HPLC and the parameters of ELSD to study the methodology. Column: Allsphere ODS-2 (250 mm x 4.6 mm ID, 5 microns), mobile phase: methanol-water (59:41), flow rate: 1.0 mL.min-1, drift tube temperature: 59 degrees C, gas flow rate: 2.38 L.min-1. RESULTS: The calibration curves were linear in the range of 2.56-25.60 micrograms for pedunculoside, 1.64-16.40 micrograms for ziyuglycoside I and 3.74-37.40 micrograms for rutundic acid. The average recovery of pedunculoside was 96.3%, RSD 1.59% (n = 5), ziyuglycoside I 97.3%, RSD 3.82% (n = 5), rutundic acid 97.7%, RSD 2.11% (n = 5). All of RSDs of the precision were less than 4% (n = 6), and the reproduciblities less than 5% (n = 6). CONCLUSION: The method is simple, accurate, effective and feasible. It can be used for the determination of the contents of pedunculoside, ziyuglycoside I and rutundic acid in leaf of Ilex purpurea Hassk.

[Determination of epimedin C and icariin in Herba Epimedii by HPLC].

OBJECTIVE: To establish a RP-HPLC method for the determination of epimedin C and icariin in Herba Epimedii. METHOD: Chromatographic conditions: Hypersil BDS-C18 column, acetonitrice and water containing 0.05% phosphoric acid as gradient eluents, G1315A photodiode array detector at 272 nm. RESULT: The average recovery rate of epimedin C was 99.7%, RSD 1.5% (n = 9), icariin 102.5%, RSD 1.1% (n=9). CONCLUSION: The method is reliable, stable and well repeatable. It provides the useful information for quality evaluation.