Analysis of the marker compounds of Rhodiola rosea L. (golden root) by reversed phase high performance liquid chromatography

An HPLC method permitting the first simultaneous detection of 5 marker compounds (salidroside, rosarin, rosavin, rosin, rosiridin) of R. rosea was developed. A separation was achieved within 27 min by using C-18 column material, a phosphate buffer/acetonitrile gradient system and at a separation temperature of 60 degrees C. All five compounds could be detected at concentrations as low as 0.62 microg/ml and were clearly assignable in R. rosea plant material and commercial products. Therefore, this quantitative and qualitative applicability of the method offers efficient and reliable means for the evaluation of R. rosea and products thereof.     

Use of SPE—TLC for Quality Control of Rhodiola rosea Extracts

SPE and TLC have been used for qualitative and quantitative analysis of salidroside, rosavin, rosarin, and rosin in commercially available dry extracts from Rhodiola rosea roots. The best separation of all the compounds was achieved on silica gel TLC plates with ethyl acetate—methanol—water, 77 + 13 + 10 (v/v), as mobile phase. UV detection was performed at λ = 215 nm for salidroside and at λ= 245 nm for the rosavins (rosavin, rosarin, and rosin). Detection limits for salidroside and the rosavins were 90 ng and 60 ng per spot, respectively. Results from quantitative analysis confirmed the manufacturer’s declaration of the amounts of salidroside and the rosavins in the extracts.

     

Phenylpropanoid glycosides from Rhodiola rosea

Rhodiola rosea L. (Golden Root) has been used for a long time as an adaptogen in Chinese traditional medicine and is reported to have many pharmacological properties. Along its known secondary metabolites tyrosol (1), salidroside (rhodioloside) (2), rosin (3), rosarin (4), rosavin (5), sachaliside 1 (6) and 4-methoxy-cinnamyl-O-beta-D-glucopyranoside (7), four compounds were isolated from aqueous methanol extract of the plant and identified as cinnamyl-(6'-O-beta-xylopyranosyl)-O-beta-glucopyranoside (8), 4-methoxy-cinnamyl-(6'-O-alpha-arabinopyranosyl)-O-beta-glucopyranoside (9), picein (10) and benzyl-O-beta-glucopyranoside (11) by UV, MS and NMR methods. Compounds 8 and 9 are new natural compounds whereas compounds 10 and 11 were isolated first time from R. rosea. Also the compounds 6 and 7 are isolated earlier only from the callus cultures of the plant but not from the differentiated plant.     

Comparison of electrospray ionization and atmospheric pressure chemical ionization techniques in the analysis of the main constituents from Rhodiola rosea extracts by liquid chromatography/mass spectrometry

Rhodiola rosea L. (Golden Root) has been used for a long time as an adaptogen in Chinese traditional medicine and is reported to have many pharmacological properties. A liquid chromatographic (LC) method with mass spectrometric (MS) detection based on selected ion monitoring (SIM) was developed for determining salidroside, sachaliside 1, rosin, 4-methoxycinnamyl-O-beta-glucopyranoside, rosarin, rosavin, cinnamyl-(6'-O-beta-xylopyranosyl)-O-beta-glucopyranoside, 4-methoxy-cinnamyl-(6'-O-alpha-arabinopyranosyl)-O-beta-glucopyranoside, rosiridin and benzyl-O-beta-glucopyranoside from the callus and plant extracts in one chromatographic run. Good linearity over the range 0.5-500 ng ml(-1) for salidroside, 2-2000 ng ml(-1) for rosavin and 2-500 ng ml(-1) for benzyl-O-beta-glucopyranoside was observed. The intra-assay accuracy and precision within quantitation ranges varied between -10.0 and +13.2% and between 0.7 and 9.0%, respectively. Optimization of the ionization process was performed with electrospray and atmospheric pressure chemical ionization techniques using four different additive compositions for eluents in the LC/MS scan mode, using both positive and negative ion modes. The best ionization sensitivity for the compounds studied was obtained with electrospray ionization when using pure water without any additives as the aqueous phase.     

Simultaneous Determination of Six Active Compounds in Rhodiola L. by RP-LC

Rhodiola L. has a long history as traditional Chinese medicine (TCM) with a medicinal efficacy similar to Ginseng and Manyprickle Acathopanax roots. There exist three classes of important active constituents, i.e., phenylethanoids (salidroside, p-tyrosol), phenylpropanoides (rosarin, rosavin, rosin), and monoterpene (rosiridin) in this TCM. In this study, by optimizing the extraction, separation and analytical conditions, a sensitive and accurate high performance liquid chromatographic method has been developed for the simultaneous determination of the six active compounds in the different species of Rhodioa L. for the first time. The analysis was performed on a Purospher STAR C18 column at 30 °C using 20 mmol L⁻¹ aqueous ammonia acetate/methanol gradient system at a flow rate of 1.0 mL min⁻¹ and photodiode array detection (DAD ) at wavelengths 276, 250 and 205 nm, respectively. The optimized method provided good linear relation (r ² > 0.9993 for all the target compounds), satisfactory precision (RSD values less than 1.53%) and good recovery (from 96.3–104.8%). The limits of detection ranged between 0.012 and 0.047 μg mL⁻¹ for the different analytes. The developed method has been successfully applied to analysis and quality control of Rhodiola L.     

LC/MS/MS identification of glycosides produced by biotransformation of cinnamyl alcohol in Rhodiola rosea compact callus aggregates

Cinnamyl alcohol was added to the media of compact callus aggregates (CCA) of Rhodiola rosea for stimulating the production of cinnamyl glycosides. The biotransformation reaction produced high amounts of rosin, while only a very low amount of rosavin was produced. As the consumption rate of cinnamyl alcohol was much higher than production of rosin, the aqueous methanol extracts of compact callus aggregates were studied by liquid chromatography-mass spectrometric methods and four new unexpected biotransformation products of cinnamyl alcohol were identified.     

Simultaneous quantification of Panax and Epimedium species using Rapid Resolution Liquid Chromatography (RRLC)

Two Rapid Resolution Liquid Chromatography (RRLC) methods have been developed and validated for simultaneous quantification of eight major ginsenosides from Panax species, namely, R1, Rg1, Re, Rf, Rb1, Rb2, Rc, and Rd, and flavonoids from Epimedium species, namely, epimedins A, B, and C and icariin. The analyses were performed using an Agilent 1200 series RRLC system with Phenomenex Luna C18-HST and Zorbax Eclipse XDB columns. The separation was performed with a gradient mobile phase of A (pure water) and B (acetonitrile) at a flow rate of 1.0 mL/min and 2.5 mL/min, respectively. Both columns were kept at 40 degrees C with the detection wavelength set at 203 nm. Specific eluted compounds were identified by using reference samples of ginsenosides R1, Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd, and epimedins A, B, C and icariin. Baseline separation was achieved in less than 15 minutes for the Phenomenex Luna column and 4 minutes for the Zorbax Eclipse column. Characteristic RRLC profiles were established for complex mixtures of ginsenosides from Panax species and flavonoids from Epimedium species. Both methods developed here are effective for the quality control of formulated products containing both Panax and Epimedium varieties.     

Analysis of selected stilbenes in Polygonum cuspidatum by HPLC coupled with CoulArray detection

The roots of three varieties of Polygonum cuspidatum were analyzed for resveratrol and its analogs. The powder of the dried roots was extracted with aqueous ethanol (60% v/v) and the extracts obtained were analyzed using RP HPLC with coulometric detection. A simple HPLC method with a multichannel CoulArray detector was developed for the determination of four stilbenes: resveratrol, its glucoside piceid, piceatannol, and its glucoside astringin. Analyses were carried out on a LiChrospher C18 (125 x 4.6 mm id, particle size 5 microm) column with a mobile phase of ammonium acetate (pH 3) and ACN in gradient mode. Four compounds were monitored by a CoulArray electrochemical detector. Potentials of eight electrochemical cells in series were set in the range of 200-900 mV. Optimization of the mobile phase pH was performed. Calibration curves showed good linearity with correlation coefficients (r(2))--more than 0.9975.     

Determination of pharmacologically active compounds in root extracts of Cassia alata L. by use of high performance liquid chromatography

A simple high performance liquid chromatography (HPLC) method was developed and validated for the determination of six phenolic compounds, five anthraquinones (rhein, aloe-emodin, emodin, chrysophanol and physcion) and a flavonoid (kaempferol), in root extracts from Cassia alata L. Solid-phase extraction, using C(18) cartridges, was used to remove interfering substances from the root extracts. The extracts were analyzed on a C(18) column using an isocratic mobile phase which consisted of acetonitrile, methanol, and 10mM aqueous ammonium acetate (25:55:20, v/v). Identification of the analytes was performed by use of standards and on-line mass spectrometric detection using atmospheric pressure chemical ionization. The concentration of the phenolic compounds in the root extracts was determined using HPLC with ultraviolet detection at 260nm. The limits of detection obtained for the anlytes were in the range of 0.23-4.61ppm. The overall R.S.D. precision values (intra- and inter-day) for the retention times and peak-areas were lower than 0.16 and 2.10%, respectively. In addition, the recovery of the developed method for the analysis of these phenolic compounds was determined, and ranged from 81.2+/-4.3 to 106+/-2%.     

马来松香酸基酰胺化合物的合成及除草活性

以马来松香酸为原料,经酰氯化和与苯胺类化合物的N-酰化反应后,得到9个新颖的马来松香酸基酰胺类化合物,其结构均经1H NMR、13C NMR、IR和元素分析确证。初步的除草活性测试表明,在浓度为100μg/mL时,目标产物对油菜的胚根生长有一定抑制作用,其中化合物3c(R=4-ClC6H4)的活性最好,抑制率为66.1%。     

Separation of the insecticidal pyrethrin esters by capillary electrochromatography

A simple, rapid technique for the direct separation and quantification of the six insecticidally active pyrethrin esters in typical extracts and commercial formulations by capillary electrochromatography (CEC) has been described. The separation of the pyrethrin esters was achieved by optimizing several parameters including the length of stationary phase, the mobile phase composition and column temperature. The mobile phase composition had the most pronounced effect toward resolving these structurally similar compounds. A ternary mobile phase composed of acetonitrile-aqueous buffer-tetrahydrofuran (55:35:10) provided the elutropic solvent strength needed to resolve the six esters from an extract mixture in under 16 min. A 25 cm packed bed of Hypersil 3 microm C18 stationary phase was used with the ternary mobile phase at 25 degrees C and 30 kV voltage. These conditions also yielded excellent separation of the pyrethrin esters in two different commercially available insecticidal formulations. In addition, the developed CEC method was shown to be a fast and easy way of quantifying the amount of these esters in typical pyrethrin formulations.     

Comparative analysis of radical scavenging and antioxidant activity of phenolic compounds present in everyday use spice plants by means of spectrophotometric and chromatographic methods

Comparative analysis of radical scavenging and antioxidant activities of phenolic compounds present in everyday use spice plants was carried out by means of spectrophotometric and chromatographic methods. Six spice plant samples, namely onion (Allium cepa), parsley (Petroselinum crispum) roots and leaves, celery (Apium graveolens) roots and leaves and leaves of dill (Anethum graveolens) were analyzed. Total amount of phenolic compounds and radical scavenging activity (RSA) was the highest in celery leaves and dill extracts and was the lowest in celery roots. Comparing commonly used spectrophotometric analysis of 2,2-diphenyl-1-picrylhydrazyl (DPPH) RSA of extracts with the results obtained using reversed-phase chromatographic separation with on-line post-column radical scavenging reaction detection, good correlation was obtained (R(2)=0.848). Studies using HPLC system with electrochemical detector showed that bioactive phytochemicals can be separated and antioxidant activities of individual compounds evaluated without the need of a complex HPLC system with reaction detector. The results obtained using electrochemical detection correlate with the RSA assayed using spectrophotometric method (R(2)=0.893).     

Biological fingerprinting analysis by liquid chromatography/mass spectrometry for evaluation of DNA structural selectivity of multiple compounds in natural products

A method by combination of centrifugal ultrafiltration (CUF) sampling with liquid chromatography-mass spectrometry (LC-MS) analysis was established to evaluate the DNA structure and sequence selectivity of the multiple compounds in a small molecule library. The developed method was applied to analyze the extracts of natural products Coptis chinensis Franch and Rheum palmatum (L.). From the obtained biological fingerprinting chromatograms, 7 compounds in C. chinensis Franch and 11 in R. palmatum (L.) were screened out as DNA binding agents. Most of these compounds were identified by standards and LC-MS analysis after the sample pretreatment with the DNA immobilized cartridge. DNA structural binding preference of the multiple active compounds in these two extracts was then evaluated simultaneously without purification.     

Liquid chromatographic method for analysis of saponins in Maesa balansae extract active against leishmaniasis

A liquid chromatographic method was developed for the separation of six related triterpenoid saponins in Maesa balansae extracts with different purity, active against leishmaniasis. As stationary phase a Hypersil BDS C18 column (3 microm), 100 x 4.6 mm was used. The mobile phase was a mixture of methanol, acetonitrile, 5% (m/v) ammonium acetate, pH 6.5 and water. A linear gradient was developed for the analysis of crude extracts. An isocratic method was developed to analyze purified samples that mainly contained saponins 3 and 4, the most active saponins. The isocratic LC method was optimized and the robustness was evaluated with an experimental design. The method showed good selectivity, repeatability, linearity and sensitivity.     

Antimicrobial activities of the CH2Cl2-CH3OH (1:1) extracts and compounds from the roots and fruits of Pycnanthus angolensis (Myristicaceae)

This study was designed at evaluating the antimycobacterial, antibacterial and antifungal activities of the CH2Cl2-CH3OH (1:1) extracts and isolated compounds, namely 3,4-dimethoxy-3',4'-methylenedioxy-7,7'-epoxylignan (1), genkwainin (2), pycnanthulignene C (3), 4,5-dimethoxy-3',4'-methylenedioxy-2,7'-cycloligna-7,7'-diene (4), pycnanthulignene A (5) from the roots, and calycosin (6), biochanin A (7) and prunetin (8), from the fruits of Pycnanthus angolensis. The microplate alamar blue assay and the broth microdilution method were used to determine the minimal inhibitory concentration (MIC) and minimal microbicidal concentration of the samples. The H+-ATPase-mediated proton pumping assay was used to evaluate one of the possible mechanisms of action of the extracts and isolated compounds. The results of MIC determinations showed that the extract from roots was able to prevent the growth of all the studied organisms, including mycobacteria, fungi, and Gram-positive and Gram-negative bacteria. All tested compounds showed antimicrobial activities to different extents, compound 1 and 8 exhibiting the best antimicrobial spectrum, with 92.3% of the tested organisms being sensitive. The results obtained in this study also showed that the extracts as well as most of the compounds were able to inhibit the H(+)-ATPase activity. The overall results provided evidence that P. angolensis and some of its components might be potential sources of antimicrobial drugs against tuberculosis, bacterial and fungal diseases.     

High-speed analyses using rapid resolution liquid chromatography on 1.8-microm porous particles

The potential of high-speed analyses by rapid resolution liquid chromatography (RRLC) and RRLC/MS on 1.8-microm porous particles packed into short columns operated at high flow-rate was investigated and compared with the performance of 5-microm porous particles packed into conventional columns. Using similar chemistries, the ease of conversion from conventional HPLC to an RRLC method was demonstrated. In order to display the practicality of RRLC separations, the analysis of pesticides in crops and catechins in Japanese green tea was selected. Using the Japanese Food Hygiene Law method, which employs a conventional 5-microm RP column (250 mm x 4.6 mm) for quantification of pesticides in crops, the analysis time was 25 min under isocratic conditions. Using the RRLC method on the short (50 mm x 4.6 mm) column packed with 1.8-microm porous particles, the same separation could be performed in 0.8 min with the RRLC/MS method without a loss in resolution. At the highest flow rate, compared to the conventional method, the time could be reduced by a factor of 31. In gradient elution, the fastest separation of catechins in Japanese green tea was achieved by RRLC on 50-mm x 4.6-mm id or 50-mm x 2.1-mm id RRLC columns packed with 1.8-microm particles. The analysis time at 5 mL/min was less than 1 min. Compared to the conventional HPLC method on a 150-mm column packed with 5-microm particles, time was reduced by a factor of 15. The effect of other experimental parameters such as the column temperature, acquisition rate of the detector and the influence of cell volume on chromatographic performance was also investigated. After the optimization, the analysis precision under the fastest RRLC conditions was examined. RSDs of retention time and peak area were 0.2% and 0.47%, respectively.     

Simultaneous quantification of Echinacea species, Flos Lonicerae, Radix Scutellaria and Fructus Forsythiae combinations by rapid resolution liquid chromatography

Echinacea angustifolia and E. purpurea are commonly used in North America for their anti-bacterial effects. Flos Lonicerae, Radix Scutellaria and Fructus Forsythiae are traditional Chinese medicinal herbs commonly used for the treatment of complaints such as pneumonia, acute upper respiratory tract infection, and acute bronchitis. A reproducible, simple, and reliable rapid resolution liquid chromatographic (RRLC) method has been developed to analyze extracts of products formulated containing E. angustifolia, E. purpurea, Flos Lonicerae, Radix Scutellariae and Fructus Forsythiae simultaneously in one run in less than 6 minutes. The method uses a C18-HST column, a mobile phase consisting of 0.1% aqueous phosphoric acid solution and acetonitrile, and UV detection at 327 nm and 229 nm. A stability test was performed that revealed that chlorogenic acid is more stable in acidic pH, and hence it is best to keep the extract of E. augustifolia, E. purpurea, Flos Lonicerae, Radix Scutellariae and Fructus Forsythiae in mild acidic conditions at approximately pH 5.     

Chemical profiling and separation of bioactive secondary metabolites in Maca (Lepidium peruvianum) by normal and reverse phase thin layer chromatography coupled to desorption electrospray ionization‐mass spectrometry

Maca is a Peruvian tuberous root of the Brassicaceae family grown in the central Andes between altitudes of 4000 and 4500 m. The medicinal plant is a nutraceutical with important biological activities and health effects. In this study, we report a rapid high‐performance thin layer chromatography (HPTLC)‐(?)desorption electrospray ionization (DESI)‐mass spectrometry (MS) method to profile and separate intact glucosinolates without prior biochemical modifications from the hydromethanolic extracts of two phenotypes, red and black Maca (Lepidium peruvianum) seeds. In the first stage of the plant's life cycle, aromatic glucosinolates were the main chemical constituents whereby six aromatic, three indole, and one aliphatic glucosinolate were tentatively identified. At the seedling stage, glucolepigramin/Glucosinalbin was the most predominant precursor, rather than Glucotropaeolin, which is mainly found in hypocotyls and roots. These findings lead us to suggest that glucolepigramin/glucosinalbin play a major role as active precursors in the biosynthetic pathways of other secondary metabolites in the early stages of plant development. Between red and black Maca seeds, only minor differences in the relative abundances of glucosinolates were observed rather than different plant metabolites. For the first time, we report six potential plant antibiotics, phytoanticipins: glycosylated ascorbigens and dihydroascorbigens from Maca seeds. We also investigated a targeted reverse phase C₁₈ functionalized TLC‐DESI‐MS method with high sensitivity and specificity for Brassicaceae fatty acids in Maca seeds and health supplements such as black Maca root lyophilized powder and tinctures. The investigation of secondary metabolites by normal and reverse phase TLC‐DESI‐MS methods, described in this study, can aid in their identification as they begin to emerge in later stages of development in plant tissues such as leaves, hypocotyls, and roots.     

Camptothecinoids from the seeds of Taiwanese Nothapodytes foetida

Two new alkaloids, 9-methoxy-18,19-dehydrocamptothecin (1) and 5- hydroxymappicine-20-O-beta-glucopyranoside (2a/2b as a racemic mixture), together with nine known compounds: camptothecin (3), 9-methoxy-camptothecin (4), 5-hydroxycamptothecin (5a/5b racemic mixture), 5-hydroxy-9-methoxycamptothecin (6a/6b racemic mixture), diosmetin (7), apigenin (8), apigenin-7-O-glucopyranoside (9), rosin (cinnamyl-O-beta-D-glucopyranoside) (10) and amarantholidoside IV (11) were isolated from the immature seeds of Nothapodytes foetida (Wight) Sleumer. The structures were elucidated by spectroscopic analyses. In the present research, compounds 1, 3, 4, 5a/5b and 6a/6b, also showed in vitro cytotoxicity against six cancer cell lines (HepG2, Hep3B, MDA-MB- 231, MCF-7, A549, and Ca9-22). Among them, compound 1 exhibited significant cytotoxicity against these cancer cell lines, with IC(50) of 0.24-6.57 microM. Furthermore, HPLC profiles were developed for qualitative and quantitative analysis of these active constituents in different parts of this plant, including mature and immature seeds, leaves, stems and roots. The results revealed that compounds 3 and 4 have the highest concentrations, which are found in the roots part of the plant.     

RP-HPLC method for the quantitative analysis of naturally occurring flavonoids in leaves of Blumea balsamifera DC

A selective and sensitive reversed-phase (RP) high-performance liquid chromatographic method is developed for the quantitative analysis of five naturally occurring flavonoids of Blumea balsamifera DC, namely dihydroquercetin-7,4'-dimethyl ether (DQDE), blumeatin (BL), quercetin (QN), 5,7,3',5'-tetrahydroxyflavanone (THFE), and dihydroquercetin-4'-methyl ether (DQME). These compounds have been isolated using various chromatographic methods. The five compounds are completely separated within 35 min using an RP C18, Nucleosil column and with an isocratic methanol-0.5% phosphoric acid (50:50, v/v) mobile phase at the flow rate of 0.9 mL/min. The separation of the compounds is monitored at 285 nm using UV detection. Identifications of specific flavonoids are made by comparing their retention times with those of the standards. Reproducibility of the method is good, with coefficients of variation of 1.48% for DQME, 2.25% for THFE, 2.31% for QN, 2.23% for DQDE, and 1.51% for BL. The average recoveries of pure flavonoids upon addition to lyophilized powder and subsequent extraction are 99.8% for DQME, 99.9% for THFE, 100.0% for BL, 100.6% for DQDE, and 97.4% for QN.