Highly selective monitoring of in-source fragmentation sapogenin product ions in positive mode enabling group-target ginsenosides profiling and simultaneous identification of seven Panax herbal medicines.

In-source fragmentation of ginsenosides in the positive ESI mode (pISF-G) frequently occurs, which results in little fragment information useful for the structural elucidation. We are aimed to unveil the genesic mechanism and explore its potential significance in quality control of Ginseng and the related compound formulae. By applying six high-resolution mass spectrometers from Agilent, Waters, and Thermo Fisher, we could primarily demonstrate the susceptibility of pISF-G. The ion clusters in the positive full-scan MS1 spectra were generated from the protonated sapogenins by successive elimination of H2O, and showed specificity for ginsenoside classification. Selective ion monitoring (SIM) of the sapogenin product ions could delineate group-target ginsenoside profiles from Ginseng. A high-selectivity characteristic chromatogram (CC) was elaborated for Ginseng, on the Vion™ IMS-QTOF mass spectrometer by IM (ion mobility) separation and quadrupole filtering of four sapogenin fragments (m/z 407.37/CCS 206.24 Å2; m/z 423.36/CCS 211.26 Å2; m/z 439.36/CCS 209.60 Å2; m/z 457.37/CCS 217.81 Å2). Chemometric analysis, based on the CC data of seven Ginseng drugs (P. ginseng, P. quinquefolius, P. notoginseng, Red ginseng, leaf of P. ginseng, P. japonicus, and P. japonicus var. major), disclosed 35 marker compounds. We could readily discriminate among P. ginseng, P. quinquefolius, and P. notoginseng, in 15 different compound formulae by identifying these marker compounds on both the Vion IMS-QTOF and QTrap 4500 mass spectrometers. Conclusively, SIM of the pISF-G sapogenin product ions renders a new concept of CC enabling the group-target profiling of ginsenosides and authentication of Ginseng and the related compound formulae.

[Determination of five protopanaxadiol ginsenosides in ginseng by solid-phase extraction-ultra performance liquid chromatography].

A new method based on solid-phase extraction-ultraperformance liquid chromatography (SPE-UPLC) was developed for the determination of five protopanaxadiol ginsenosides in ginseng. The ginsenosides were extracted from ground ginseng samples using water-saturated n-butanol and purified on a hydrophilic solid-phase extraction column. Chromatographic separation was achieved on an ACQUITY UPLC BEH Shield RP18 column (100 mm×2.1 mm, 1.7 µm) by linear gradient elution using an acetonitrile/water mobile phase. Five protopanaxadiol ginsenosides were detected by a photodiode array detector, and they showed a strong positive linear correlation (r2>0.999) in the range of 5-500 µg/mL. In addition, the instrument precision ranged between 0.95% and 2.62% (n=6), with the sample stability between 0.90% and 2.15% (n=8) within 22 h. Intra- and inter-day repeatabilities were 5.35%-6.47% (n=6) and 5.56%-6.34% (n=8), respectively. Sample recoveries and the corresponding relative standard deviations (RSDs) were 87.16%-101.92% and 1.54%-4.01% (n=6), respectively. Hydrophilic chromatography materials were used in SPE, and the extract was directly loaded and purified without pretreatment. Besides, with the use of UPLC, the analysis time was greatly shortened. The developed method is simple and rapid, with high throughput, thus being suitable for the quantitative analysis of the five protopanaxadiol ginsenosides in ginsengs.

[Study on transformation rules of terrestrosin D in course of Tribuli Fructus stir-frying based on simulated processing technology].

The contents of terrestrosin D and hecogenin from Tribuli Fructus were determined before and after stir-frying. The results showed that the content of terrestrosin D was decreased significantly,and the content of hecogenin was increased significantly after such processing. In order to verify the inference that terrestrosin D was converted to hecogenin by stir-frying,the quantitative variation rules of terrestrosin D and hecogenin were studied by simulated processing technology,and the simulated processing product of terrestrosin D was qualitatively characterized by ultra performance liquid chromatography/time of flight mass spectrometry( UPLC-TOF/MS) to clarify its transformation process during stir-frying. The results showed that the content of terrestrosin D was decreased significantly at first and then a platform stage appeared with the prolongation of processing time at a certain temperature. Raising the stir-frying temperature could further decrease the content of terrestrosin D and delay the time that the platform stage appeared. When the processing was simulated at higher temperatures( 220 ℃ and 240 ℃),the content of hecogenin was increased gradually with the increase of processing temperature and the prolongation of processing time. In the process of stir-frying,the deglycosylation reaction of terrestrosin D to hecogenin was not completed in one step. The deglycosylation reaction occurred first at the end of the sugar chain,and then other glycosyl units in the sugar chain were sequentially removed from the outside to the inside to finally form the hecogenin. This study provides a basis for further revealing the detoxification mechanism of stir-fried Tribuli Fructus.

A Metabolomic Approach for the Discrimination of Red Ginseng Root Parts and Targeted Validation.

Ginsenosides are used as existing markers of red ginseng (RG) quality, and ginsenoside ratios are also indicative of the different components of red ginseng. For the analysis and classification of ginsenoside content, red ginseng was separated into three parts, namely, main roots, lateral roots, and fine roots, and each extract was subjected to ultra-performance liquid chromatography quadruple time-of-flight mass spectrometry (UPLC-QToF-MS) with multivariate statistical analysis. Principal component analysis (PCA) showed a clear discrimination between the extracts of main roots and fine roots and suggested discrimination markers (four for the main roots and five for the fine roots). The fine root markers were identified as ginsenoside. We identified two markers for the main roots of red ginseng in this study. Moreover, the contents of 22 ginsenosides were analyzed in all three components of red ginseng. Fine roots have the highest protopanaxadiol (PPD)/protopanaxatriol (PPT) ratio. The PPD group of ginsenosides, which is quantitatively dominant in fine roots, clearly distinguishes the main roots from the other parts.

Combination of HPLC-MS and QAMS as a new analytical approach for determination of saponins in ginseng containing products.

Conventional liquid chromatographic methods coupled with ultraviolet detection with low-wavelength range are lacking selectivity and sensitivity to determine both polar and less polar ginsenosides. Also the lack of standard substances for such quality control methods is leading to development of the approaches using single standard for quantitative analysis of multi-component system (QAMS). The objective of present study was to establish and compare for the first time liquid chromatography-ultraviolet detection and liquid chromatography-mass spectrometry QAMS methods for the simultaneous determination of protopanaxatriol-type and protopanaxadiol-type ginsenosides in a variety of ginseng products. Sixteen polar and less polar ginsenosides were separated on a reversed-phase C18-column (150mm×2.0mm, 2.2µm) with a mobile phase consisting of 0.1% formic acid in water and acetonitrile. Components were then detected by means of ultraviolet and mass spectrometry detection. Characteristic sapogenin fragmentation signals with m/z 423 and 425 for two major groups of ginseng saponins allowed their simultaneous determination in a single chromatographic run, while the use of ultraviolet detection tends to give overvalued results. Structural correlation between the relative response factors and saponin structure was demonstrated. The method was linear (R2 >0.999) and sensitive (LODs, 0.01-0.03mg/mL) within the concentration range tested. Concentrations of individual ginsenosides and several quality control parameters were determined in ginseng root extracts and commercial ginseng products of different types (root slices, tablets and tea samples), and results showed that ginsenoside content can be successfully measured by means of QAMS approach.

Two new dammarane-type triterpene sapogenins from Chinese red ginseng.

Two new dammarane-type triterpene sapogenins were isolated from the Chinese red ginseng. The new sapogenins were named as 24,26-dihydroxy-panaxdiol (1) and 24-hydroxy-panaxdiol (2). Their structures were elucidated by the combined analysis of NMR and mass spectrometry as 20(S),25(R)-epoxydammarane-3ß,12ß,24ß,26-tetraol (1) and 20(S),25-epoxydammarane-3ß,12ß,24α-triol (2). The complete signal assignments of the two compounds were carried out by 2D NMR spectral and NOE differential spectroscopy analysis.

A new triterpenoid saponin from Clinopodium chinense (Benth.) O. Kuntze.

A new triterpene saponin, 3ß,16ß,23α,28ß,30ß-pentahydroxyl-olean-11,13(18)-dien-3ß-yl-[ß-D-glucopyranosyl-(1→2)]-[ß-D-glucopyranosyl-(1→3)]-ß-D-fucopyranoside, was named Clinoposaponin D (1), together with six known triterpene saponins, buddlejasaponin IVb (2), buddlejasaponin IVa (3), buddlejasaponin IV (4), clinopodisides D (5), 11α,16ß,23,28-Tetrahydroxyolean-12-en-3ß-yl-[ß-D-glucopyranosyl-(1→2)]-[ß-D-glucopyranosyl-(1→3)]-ß-D-fucopyranoside (6) and prosaikogenin A (7), and two known triterpenes, saikogenin A (8) and saikogenin F (9) were isolated from Clinopodium chinense (Benth.) O. Kuntze. Their structures were elucidated on the basis of 1D, 2D NMR and MS analysis. Meanwhile, the effects of all compounds on rabbit platelet aggregation and thrombin time (TT) were investigated in vitro. Compounds 4 and 7 had significant promoting effects on platelet aggregation with EC50 value at 53.4 and 12.2 µM, respectively. In addition, the highest concentration (200 µM) of compounds 2 and 9 shortened TT by 20.6 and 25.1%, respectively.

Fast Centrifugal Partition Chromatography Fractionation of Concentrated Agave (Agave salmiana) Sap to Obtain Saponins with Apoptotic Effect on Colon Cancer Cells.

Separation of potentially bioactive components from foods and plant extracts is one of the main challenges for their study. Centrifugal partition chromatography has been a successful technique for the screening and identification of molecules with bioactive potential, such as steroidal saponins. Agave is a source of steroidal saponins with anticancer potential, though the activity of these compounds in concentrated agave sap has not been yet explored. In this study, fast centrifugal partition chromatography (FCPC) was used coupled with in vitro tests on HT-29 cells as a screening procedure to identify apoptotic saponins from an acetonic extract of concentrated agave sap. The three most bioactive fractions obtained by FCPC at partition coefficients between 0.23 and 0.4 contained steroidal saponins, predominantly magueyoside b. Flow cytometry analysis determined that the fraction rich in kammogenin and manogenin glycosides induced apoptosis, but when gentrogenin and hecogenin glycosides were also found in the fraction, a necrotic effect was observed. In conclusion, this study provides the evidence that steroidal saponins in concentrated agave sap were potential inductors of apoptosis and that it was possible to separate them using fast centrifugal partition chromatography.

Ginseng Saponins in Different Parts of Panax vietnamensis.

Chemical and pharmacological studies of Panax vietnamensis (Vietnamese ginseng; VG) have been reported since its discovery in 1973. However, the content of each saponin in different parts of VG has not been reported. In this study, 17 ginsenosides in the different underground parts of P. vietnamensis were analyzed by HPLC/evaporative light scattering detector (ELSD). Their contents in the dried rhizome, radix, and fine roots were 195, 156, and 139 mg/g, respectively, which were extremely high compared to other Panax species. The content of protopanaxatriol (PPT)-type saponins were not much different among underground parts; however, the content of protopanaxadiol (PPD)- and ocotillol (OCT)-type saponins were greatly different. It is noteworthy that the ginsenoside pattern in the fine roots is different from other underground parts. In particular, despite the content of PPD-type saponins being the highest in the fine roots, which is similar to other Panax species, the total content of saponins was the lowest in the fine roots, which is different from other Panax species. The ratios of PPT : PPD : OCT-type saponins were 1 : 1.7 : 7.8, 1 : 1.6 : 5.5, and 1 : 4.8 : 3.3 for the rhizome, radix, and fine roots, respectively. OCT-type saponins accounted for 36-75% of total saponins and contributed mostly to the difference in the total saponin content of each part.

Characterization of metabolites of 20(S)-protopanaxadiol in rats using ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

In this study, ultra-performance liquid chromatography (UPLC)/quadrupole-time-of-flight mass spectrometry (QTOF-MS) was applied to the rapid analysis of 20(S)-protopanaxadiol (PPD) metabolites in rats after oral administration, enabling the structural characterization of 23 metabolites in plasma, bile, urine, and feces. 16 of these, including M1-M5, M9, and M11-M15, have not been previously reported. The results also indicated that demethylation, dehydration, dehydrogenation, oxidation, deoxidation, and glucuronidation were the major metabolic reactions of PPD in vivo. This study provides important information about the metabolism of PPD which will be helpful for fully understanding its mechanism of action. Furthermore, structural modification of PPD in vivo may aid in obtaining new chemical derivatives for pharmacological screening.

Glaucogenin E, a new C21 steroid from Cynanchum stauntonii.

Glaucogenin E (1), a new C(21) steroid sapogenin, along with three known ones (2-4) were isolated from the rhizomes of Cynanchum stauntonii (Decne.) Schltr. ex Levl. Their structures were established mainly by the spectroscopic analysis, including 2D NMR. All the isolated compounds were evaluated for their cytotoxicity against human cancer cell lines HeLa, Bel-7402, SGC-7901 and BGC-823.

Antimicrobial activity and phytochemical study of Vernonia glabra (Steetz) Oliv. & Hiern. in Kenya.

Infectious diseases are prevalent and life threatening in Kenya. Majority of the sick are seeking herbal remedies in search of effective, safe, and affordable cure. This project aims to investigate the antimicrobial activity and presence of active phytochemical compounds in different parts of Vernonia glabra; a plant used by herbalists in various regions of Kenya, for the treatment of gastrointestinal problems. The plant sample was collected in January 2010 in Machakos, and different parts dried at room temperature under shade, ground into powder and extracted in Dichloromethane: Methanol in the ratio 1:1, and water. These crude extracts were tested against Staphylococcus aureus, Escherichia coli, Candida albicans, and Aspergillus niger for antimicrobial activity using disc diffusion technique. Minimum inhibitory concentrations (MICs) for active crude extracts were done using disc diffusion technique after the failure of agar and broth dilution methods. It was observed that the organic crude extracts of flower, leaf, stem, root, and/or entire plant, showed activity against at least one of the four micro-organisms screened, and at concentrations lower than the aqueous crude extracts. Organic crude extract of the leaf showed the highest activity against Staphylococcus aureus (mean inhibition zone of 1.85), recording higher activity than the commercially used standard antibiotic (Streptomycin mean inhibition zone of 1.30). The organic crude extract of flower showed significant activity only against S. aureus, with the lowest MIC of 1.5625 mg/100µl, compared to streptomycin with M.I.C of 6.25 mg/100µl. Thin Layer Chromatography-Bioautography Agar-Overlay showed that, flower alkaloids (50% active), root sapogenins (43.8% active), and root terpenoids (38.5% active) were identified as the potential antibacterial compounds against S. aureus. These results suggest that, V. glabra contains phytochemicals of medicinal properties and justify the use of V. glabra in traditional herbal medicine for the treatment of microbial based diseases. However, research on toxicity which is missing in this study is recommended for V. glabra in order to verify, validate and document the safety of this medicinal plant to the society.

A modified spectrophotometric assay to estimate deglycosylation of steroidal saponin to sapogenin by mixed ruminal microbes.

BACKGROUND: The lack of a method for measuring deglycosylation of saponins in ruminal fluid has limited the ability to investigate the impact of these compounds on rumen microorganisms. A simple spectrophotometric assay was adapted and a protocol developed to enable measurement of steroidal saponin and sapogenin in ruminal fluid. The procedure was used for in vitro determination of deglycosylation activity of rumen bacteria obtained from cattle fed or not fed Yucca schidigera saponin, and to determine the relative deglycosylase activities of extracellular and cell-associated enzymes from ruminal content. RESULTS: Modifications to the spectrophotometric assay (i.e. heating time shortened to 10 min and 0.5 mL dH(2)O added to the reaction mixture) improved the stability of the optical density (425 nm) of the chromophore for up to 24 h post-reaction. Centrifugation (12 000 x g, 20 min) enabled differential estimations of steroidal saponin and sapogenin in ruminal fluid. Steroidal saponin added to defaunated ruminal fluid (dRF) or clarified ruminal fluid (cRF) was recovered completely from the mixture as saponin + sapogenin (99.1% and 100.6%, respectively), whereas saponin recovery from the supernatant of dRF was greatly reduced (P < 0.001) compared to that from supernatant of cRF (58.5 vs. 98.7%). Saponin recoveries from the supernatants of dRF and cRF did not differ between donor cattle fed or not fed Yucca schidigera saponin (59.2 vs. 57.3% and 98.4 vs. 99.3%, respectively). The majority (89-90%) of saponin added to a ruminal extracellular enzyme preparation was recoverable in supernatant after 24 h, compared with only 26-32% remaining in supernatant from incubation with a cell-associated enzymes fraction. CONCLUSION: Mixed rumen bacteria deglycosylate steroidal saponin to sapogenin, at activity levels unaffected by prior exposure to saponin, but they were unable to degrade the sapogenin core structure. Deglycosylation activity occurred primarily in the cell-associated enzyme fraction of ruminal content.

An improved facile method for extraction and determination of steroidal saponins in Tribulus terrestris by focused microwave-assisted extraction coupled with GC-MS.

An improved fast method for extraction of steroidal saponins in Tribulus terrestris based on the use of focus microwave-assisted extraction (FMAE) is proposed. Under optimized conditions, four steroidal saponins were extracted from Tribulus terrestris and identified by GC-MS, which are Tigogenin (TG), Gitogenin (GG), Hecogenin (HG) and Neohecogenin (NG). One of the most important steroidal saponins, namely TG was quantified finally. The recovery of TG was in the range of 86.7-91.9% with RSD<5.2%. The convention heating reflux extraction was also conducted in order to validate the reliability of this new FMAE method. The yield of total steroidal saponins was 90.3% in a one-step FMAE, while the yield of 65.0% was achieved during heating reflux extraction, and the extraction time was reduced from 3 h to 5 min by using less solvent. The method was successfully applied to analyze the steroidal saponins of Tribulus terrestris from different areas of occurrence. The difference in chromatographic characteristics of steroidal saponins was proved to be related to the different areas of occurrence. The results showed that FMAE-GC-MS is a simple, rapid, solvent-saving method for the extraction and determination of steroidal saponins in Tribulus terrestris.

High-performance liquid chromatographic fingerprint for quality control of Terminalia arjuna containing Ayurvedic churna formulation.

Because Ayurvedic herbal preparations contain a myriad of compounds in complex matrixes, it is difficult to establish quality control standards for raw materials and to standardize finished Ayurvedic drugs. A novel, accurate, and valid fingerprint method was developed using HPLC for quality control of a traditional Ayurvedic Arjuna churna formulation, which is used as a cardiotonic drug. Comprehensive comparison of chromatograms of standardized formulation of Arjuna churna and marketed formulations revealed eight characteristic peaks in chromatograms, which unambiguously confirmed the presence of authentic raw material used in the formulation on the basis of their retention time values and UV data. An HPLC fingerprint was also developed for total sapogenins present in Terminalia arjuna. The six common peaks observed in chromatograms of isolated sapogenins, standardized formulations, and marketed formulations can serve as a quality control tool for qualitative estimation of total saponin glycosides present in an Arjuna churna formulation.

[Determination of content and entrapment efficiency of 20 (S)-protopanaxadiol in pharmacosomes by RP-HPLC method].

OBJECTIVE: To establish a RP-HPLC method for content and entrapment efficiency of 20 (S)-protopanaxadiol in pharmacosomes. METHOD: The separation was performed with a COSMOSIL 5 C18-MS-II column (4.6 mm x 250 mm, 5 mmicrom) using methanol-water (95:5) as the mobile phase and detected at 203 nm. The flow rate was 1.0 mL x min(-1) and 50 microL sample solution was injected for each time. RESULT: The calibration curve was linear within the range 0.1-0.5 mg x mL(-1) (r = 0. 9999) , the intra-day RSD and inter-day RSD were less than 2% and the average recovery was between 101.44%-103.11% (n = 3). CONCLUSION: The method is simple, accurate, sensitive and applicable for determination of content and entrapment efficiency of 20 (S)-protopanaxadiol pharmacosomes.

[Research on the quality changes in pre-and-post-processed pieces of Radix Polygalae].

OBJECTIVE: To study the quality changes in pre-and-post-processed pieces of Radix Polygalae. METHODS: The changes of the content of senegenin and polygalacic acid were studied. RESULTS: The content of polygalacic acid in post-processed piece was lower than that in pre-processed piece. The content of senegenin had no obvious difference. CONCLUSION: The processing can make the quality change.

Jamaican bitter yam sapogenin: potential mechanisms of action in diabetes.

Sapogenin has been proposed to be the active component responsible for the beneficial effects of Jamaican bitter yam (Dioscorea polygonoides) in the management of diabetes. Most of the research activities on bitter yam have focused on the role sapogenin play in the management of diabetes. Changes in weight, activities of carbohydrate digestive and transport enzymes, alterations in the intestinal morphology, changes in blood lipids, reduction in lipid peroxidation and the prevention of liver damage associated with diabetes have all been attributed to bitter yam sapogenin supplementation. Also, the possible exploitation of bitter yam for nutraceutical/pharmaceutical purposes is based on the high saponin content. There are however, concerns about the beneficial claims of the findings especially with regard to the possible adverse effects that may accrue in the clinical applications. This review therefore provides an overview of the findings in this research area with a view to proposing the potential mechanisms whereby the supplement of bitter yam sapogenin extract exert its hypoglycemic and hypolipidemic properties and the probable adverse effects in diabetes mellitus.

Physical, morphological and chemical characteristics, oil recovery and fatty acid composition of Balanites aegyptiaca Del. kernels.

Balanites aegyptiaca Del. kernels were chemically, physically and morphologically characterized. Crude oil (49.0%) and crude protein (32.4%) were the two major constituents of the kernels. Phytic acid content was relatively high compared to other legumes. In contrast, antitryptic activities of the kernel flours were very low. Sapogenin contents of the full fat, defatted and testa flours were 1.5, 2.7 and 3.0%, respectively. The hardness of the kernel was found to be about 10.4 x 10(5) N/m2, which was somewhat high. The morphological structure of the kernel using a scanning electron microscope revealed that the protein matrix was embedded in a lake of oil droplets. Oil recovery, as a function of pressing time, pressure, temperature and particle size was investigated. With increasing temperature up to 70 degrees C at 400 bar, for 120 min, an oil recovery of 79.4% was obtained. Using an expeller at 115 degrees C, about 85% of the kernel oil was recovered. The reduction of particle size had a negative effect on oil recovery under the same conditions. The fatty acid composition was not affected by the pressing temperature up to 115 degrees C. The total amount of the unsaturated fatty acids was found to be up to 74.8% (50 degrees C) and 75.1% (115 degrees C) of the total fatty acids content.

How to distinguish garlic from the other Allium vegetables.

The establishment of international monographs for herbs is in progress. Here, we propose both a marker compound and a method for its analysis for the identification of garlic bulbs and their products. The constituents in 26 kinds of fresh edible parts of Allium vegetables and three types of garlic preparations were analyzed. Sulfur compounds are the most characteristic constituents in garlic, but manufacturing processes of garlic products dramatically affect these constituents. Thus, no sulfur compound could be specified as a universal marker of identification applicable for any type of garlic. On the other hand, garlic contains other characteristic compounds, namely, saponins. After analyzing Allium vegetables and garlic preparations, we concluded that sapogenins, especially beta-chlorogenin, may be a viable candidate for identifying and distinguishing garlic from other Allium vegetables.