Improved Extraction Method and Chromatographic Separation of Ginsenosides from Ginseng Roots,Leaves and Ginseng Drug Preparations

A new extraction method for ginsenosides from ginseng roots, ginseng leaves and ginseng drug preparations by Sep-Pak C₁₈ cartridges has been studied. Ginsenoside extraction by Sep-Pak cartridges is a rapid, efficient, reproducible method. In addition, the extracts were analyzed by high performance thin layer chromatography (HPTLC) and reverse phase high performance liquid chromatography (HPLC). The major components of ginseng saponins were effectively separated using an ODS-120T column.     

177 Saponins,Including 11 New Compounds in Wild Ginseng Tentatively Identified via HPLC-IT-TOF-MSn,and Differences among Wild Ginseng,Ginseng under Forest,and Cultivated Ginseng

Wild ginseng (W-GS), ginseng under forest (F-GS, planted in mountain forest and growing in natural environment), and cultivated ginseng (C-GS) were compared via HPLC-DAD and HPLC-IT-TOF-MSⁿ. A total of 199 saponins, including 16 potential new compounds, were tentatively identified from 100 mg W-GS (177 saponins in W-GS with 11 new compounds), F-GS (56 saponins with 1 new compound), and C-GS (60 saponins with 6 new compounds). There were 21 saponins detected from all the W-GS, F-GS, and C-GS. Fifty saponins were only detected from W-GS, including 23 saponins found in ginseng for the first time. Contents of ginsenosides Re (12.36–13.91 mg/g), Rh₁ (7.46–7.65 mg/g), Rd (12.94–12.98 mg/g), and the total contents (50.52–55.51 mg/g) of Rg₁, Re, Rf, Rb₁, Rg₂, Rh₁, and Rd in W-GS were remarkably higher than those in F-GS (Re 1.22–3.50 mg/g, Rh₁ 0.15–1.49 mg/g, Rd 0.19–1.49 mg/g, total 5.69–18.74 mg/g), and C-GS (Re 0.30–3.45 mg/g, Rh₁ 0.05–3.42 mg/g, Rd 0.17–1.68 mg/g, total 2.99–19.55 mg/g). Contents of Re and Rf were significantly higher in F-GS than those in C-GS (p < 0.05). Using the contents of Re, Rf, or Rb₁, approximately a half number of cultivated ginseng samples could be identified from ginseng under forest. Contents of Rg₁, Re, Rg₂, Rh₁, as well as the total contents of the seven ginsenosides were highest in ginseng older than 15 years, middle–high in ginseng between 10 to 15 years old, and lowest in ginseng younger than 10 years. Contents of Rg₁, Re, Rf, Rb₁, Rg₂, and the total of seven ginsenosides were significantly related to the growing ages of ginseng (p < 0.10). Similarities of chromatographic fingerprints to W-GS were significantly higher (p < 0.05) for F-GS (median: 0.824) than C-GS (median: 0.745). A characteristic peak pattern in fingerprint was also discovered for distinguishing three types of ginseng. Conclusively, wild ginseng was remarkably superior to ginseng under forest and cultivated ginseng, with ginseng under forest slightly closer to wild ginseng than cultivated ginseng. The differences among wild ginseng, ginseng under forest, and cultivated ginseng in saponin compositions and contents of ginsenosides were mainly attributed to their growing ages.     

Simultaneous LC Determination of Ginsenosides Using a Modified Extraction Procedure and an Improved Step Gradient Program

A novel, accurate and precise high performance liquid chromatographic method has been developed for simultaneous determination of seven important ginsenosides (Rg1, Re, Rf, Rb1, Rc, Rb2 and Rd) in ginseng products. The separation was performed on a Shim-pack VP-ODS column (5 μm, 150 ×2 mm i.d) with ultraviolet detection at 200 nm by using the improved step gradient elution program. The LODs (S/N = 3) were in the range 0.29 to 1.33 ng μL⁻¹. All calibration curves showed a good linearity (R² > 0.998) over the ranges tested. The recoveries obtained from spiked sample were between 95.1% and 98.7%. The proposed method was successfully applied to several ginseng pharmaceutical samples. For the sample preparation, a modified extraction method was made to improve the extraction efficiency by evaluation of five solvent systems. The results demonstrated that the extraction with methanol-water (80:20, v/v) is suitable method preferably for the extraction of the ginsenosides. On leave from Department of Pharmacy and Applied Chemistry, Jilin Institute of Chemical Technology, Jilin 132022, P. R. China     

Comprehensive Investigation on Ginsenosides in Different Parts of a Garden-Cultivated Ginseng Root and Rhizome

Background: Ginseng is widely used as herb or food. Different parts of ginseng have diverse usages. However, the comprehensive analysis on the ginsenosides in different parts of ginseng root is scarce. Methods: An ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) combined with UNIFI informatics platform and ultra-high-performance liquid chromatography-charged aerosol detection (UHPLC-CAD) were employed to evaluate the different parts of cultivated ginseng root. Results: 105 ginsenosides including 16 new compounds were identified or tentatively characterized. 22 potential chemical markers were identified, 20, 17, and 19 for main root (MR) and fibrous root (FR), main root (MR) and branch root (BR), and main root (MR) and rhizome (RH), respectively. The relative contents of Re, Rb₁, 20(R)-Rh₁, Rd, and Rf were highest in FR. The relative content of Rg₁ was highest in RH. The total relative content of pharmacopoeia indicators Rg₁, Re, and Rb₁ was highest in FR. Conclusion: The differences among these parts were the compositions and relative contents of ginsenosides. Under our research conditions, the peak area ratio of Rg₁ and Re could distinguish the MR and FR samples. Fibrous roots showed rich ingredients and high ginsenosides contents which should be further utilized.     

Determination of Seven Major Ginsenosides in Different Parts of Panax quinquefolius L.（American Ginseng） with Different Ages

Ginsenosides Rgl, Re, Rb1, Rc, Rb2, Rb3, and Rd in different parts of the American ginseng plant were investigated. The extraction process was a pressurized microwave-assisted extraction（PMAE）. The seven ginsenosides were separated and determined by high-performance liquid chromatography（HPLC） with a ultraviolet（UV） detector, at 203 nm. The experiment results showed significant variations in the individual ginsenoside contents of the American ginseng in different parts and ages of the plant. The results demonstrated that the leaves, root hairs, and rhizomes of Panax quinquefolius L. contained higher ginsenoside contents, followed by the main roots and stems. The leaves contained dramatically higher levels of ginsenoside Rg1 Rb3, and Rd than the other four parts. Higher contents of Rb1 and Re were present in the main roots, root hairs, and rhizomes. The amount of ginsenoside content in the stems was the lowest. The total content of the seven ginsenosides in main roots, root hairs and rhizomes increased with the age of the plant. In contrast, the ginsenoside contents in the leaves and stems decreased with a year of growth.     

Microwave-assisted extraction of ginsenosides from ginseng root

The extractions of ginsenosides Rg₁ and Rb₁ from ginseng root under atmospheric pressure by focused microwave-assisted technique have been investigated. The parameters used for the optimization were solvent composition, extraction time, and applied microwave power. The ginsenosides were quantified by high-performance liquid chromatography equipped with UV/Vis detector. The results of the 15-min microwave-assisted extraction (0.28% of Rg₁ obtained in 70% water-ethanol and 1.31% of Rb₁ obtained in 30% water-ethanol under 150 W of microwave power) were better than that from 10-h conventional solvent extraction (0.22% of Rg₁ and 0.87% of Rb₁ obtained in 70% water-ethanol).     

State-of-the-art separation of ginsenosides from Korean white and red ginseng by countercurrent chromatography

Ginseng (Panax ginseng C. A. Meyer) has been one of the most popular herbs used for nutritional and medicinal purposes by the people of eastern Asia for thousands of years. Ginsenosides, the mostly widely studied chemical components of ginseng, are quite different depending on the processing method used. A number of studies demonstrate the countercurrent chromatography (CCC) separation of ginsenosides from several sources; however, there is no single report demonstrating a one-step separation of all of these ginsenosides from different sources. In the present study, we have successfully developed an efficient CCC separation methodology in which the flow-rate gradient technique was coupled with a new solvent gradient dilution strategy for the isolation of ginsenosides from Korean white (peeled off dried P. ginseng) and red ginseng (steam-treated P. ginseng). The crude samples were initially prepared by extraction with butanol and were further purified with CCC using solvent gradients composed of methylene chloride–methanol–isopropanol–water (different ratios, v/v). Gas chromatography coupled with flame ionization detector was used to analyze the components of the two-phase solvent mixture. Each phase solvent mixture was prepared without presaturation, which saves time and reduces the solvent consumption. Finally, 13 ginsenosides have been purified from red ginseng with the new technique, including Rg₁, Re, Rf, Rg_2, Rb₁, Rb₂, Rc, Rd, Rg₃, Rk₁, Rg₅, Rg₆, and F₄. Meanwhile, eight ginsenosides have been purified from white ginseng, including Rg₁, Re, Rf, Rh_1, Rb₁, Rb₂, Rc, and Rd by using a single-solvent system. Thus, the present technique could be used for the purification of ginsenosides from all types’ ginseng sources. To our knowledge, this is the first report involving the separation of ginsenoside Rg₂ and Rg₆ and the one-step separation of thirteen ginsenosides from red ginseng by CCC.     

Structure elucidation of sweet-tasting cycloartane-type saponins from ginseng oolong tea and Abrus precatorius L. leaves

The composition of ginseng oolong a widely used tea product was investigated. A new sweet-tasting cycloartane-type saponin 3β-O-[β-D-glucuronopyranosyl(1→2)]-β-D-6-acetylglucopyranosyl)-(20S,22S)-3β,22-dihydroxy-9,19-cyclolanost-24-en-26,29-oic acid (9), along with five new compounds, was identified in the methanolic extract of the ginseng oolong tea. Structural elucidation was conducted by spectroscopic methods, including 1D- and 2D-NMR, HR-MS, HPLC-LITMS/MS. It was shown that these compounds have different sugar chains connected to the abrusogenin molecule. Then saponin profiles of four commercially available ginseng oolong samples and Abrus precatorius L. leaves were compared and the presence of three known abrusosides and six new acetyl and malonyl abrusogenin derivatives was proved. The original ginsenosides were not detected in the extract, therefore, abrusosides could serve as a replacement responsible for the sweet taste of the tea.     

Detection and Confirmation of Ginsenosides in Horse Urine by GC–MS and LC–MS

Ginseng has been used by the Chinese as a traditional herbal medicine for thousands of years. In view of the growing popularity in the use of ginseng preparations as natural remedies and food supplements worldwide, there is an increasing concern for their abuse in both human and animal sports. Ginsenosides are considered the major constituents of ginseng responsible for its pharmacological properties. In this study, a method was developed for the detection and confirmation of a number of ginsenosides in horse urine. The intact ginsenosides were detected and confirmed at 5–100 ng mL^?1 by LC–MS², and two deglycosylation metabolites, namely protopanaxadiol and protopanaxatriol, could both be detected and confirmed at 2 ng mL^?1 by GC–MS² after trimethylsilylation. The above GC–MS and LC–MS methods were then applied to study the in vitro metabolism of ginsenosides Rg1 and Rb1 and the in vivo urinary metabolites after oral administration of Rg1 to horses. Results obtained reveal the very first evidence for the existence of the metabolites, Rg1 and protopanaxatriol, as glucuronides in urine.     

Solid-Phase Extraction and High-Performance Liquid Chromatography for Simultaneous Determination of Important Bioactive Ginsenosides in Pharmaceutical Preparations

A robust method based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection has been developed for simultaneous determination of six important ginsenosides (Rg1, Re, Rb1, Rc, Rb2, and Rd) in pharmaceutical preparations. For sample preparation, simple and efficient extraction by ultrasonication, combined with solid-phase extraction (SPE) for clean-up, was effective without consuming large amounts of solvent. Chromatographic separation was performed on an ODS column with optimized gradient elution by means of a dual-solvent-pumping system. The validated method results in excellent separation, and quantitative determination is highly precise and accurate. The problem of co-elution of ginsenosides Rg1 and Re is also solved, with good resolution (R_S approx. 1.5). Intraday variation was between 0.2 and 4.4% and interday variation was between 0.4 and 6.5% (n=5 for both). The accuracy was satisfactory—in the range 93.9 to 103.4% from replicate evaluation at three different spiking concentrations. Overall limits of detection based on a typical injection volume of 5 μL were from 1.16 to 1.58 ng μL⁻¹. The validated method enabled complete assessment for quality control of ginseng samples. The technique may be performed with less sample preparation and, consequently, reduced possibility of sample loss.     

Green and Efficient Extraction of Polysaccharide and Ginsenoside from American Ginseng (Panax quinquefolius L.) by Deep Eutectic Solvent Extraction and Aqueous Two-Phase System

In this study, a green and effective extraction method was proposed to extract two main compounds, ginsenosides and polysaccharides, from American ginseng by combining deep eutectic solvents (DESs) with aqueous two-phase systems. The factors of type of DESs, water content in DESs, the solid–liquid ratio, extraction temperature, and extraction time were studied in the solid–liquid extraction. Then, the aqueous two-phase system (DESs-ethylene oxide–propylene oxide (EOPO)) and salty solution exchange (EOPO-salty solution) was applied for the purification of polysaccharides. The content of the polysaccharides and ginsenosides were analyzed by the anthrone–sulfuric acid method and HPLC method, which showed that the extraction efficiency of deep eutectic solvents (DESs) was better than conventional methods. Moreover, the antioxidant activities of ginseng polysaccharides and their cytotoxicity were further assayed. The advantages of the current study are that, throughout the whole extraction process, we avoided the usage of an organic reagent. Furthermore, the separated green solvent DESs and EOPO could be recovered and reused for a next cycle. Thus, this study proposed a new, green and recyclable extraction method for extracting ginsenosides and polysaccharides from American ginseng.     

Determination of Pentachloronitrobenzene and Its Metabolites in Ginseng by Matrix Solid-Phase Dispersion and GC–MS–MS

A rapid method was developed for the determination of pentachloronitrobenzene (PCNB) and its metabolites pentachloroaniline, pentachlorothioanisole residues in ginseng. Extraction and clean-up were carried out in a single step and analysis was accomplished by gas chromatography–mass spectrometry with multiple reaction monitoring. The main parameters affecting extraction yield and selectivity, such as type and amount of dispersant material, clean-up co-sorbent and extraction solvent were evaluated. The best results were obtained using 1 g ginseng, 2 g florisil as dispersant sorbent, 0.5 g neutral alumina as clean-up co-sorbent, and subsequent extraction with 10 mL acetone–n-hexane (5:5, v/v) with assisted sonication and repeated with another 5 mL of the same solvent mixture. The method was validated by analysis of ginseng samples fortified at different concentration levels (0.01–0.10 mg kg^?1). Average recoveries (n = 5) ranged from 85 to 95% with relative standard deviation between 2.5 and 11.2%. Spiked blank samples were used as standards to counteract the matrix effect observed in the chromatographic determination. The detection limits ranged from 0.2 to 0.9 µg kg^?1 in ginseng. The method was applied to the analysis of PCNB and its metabolite residues in commercial ginseng samples.     

Ultra-high performance liquid chromatography/ion mobility time-of-flight mass spectrometry-based untargeted metabolomics combined with quantitative assay unveiled the metabolic difference among the root,leaf, and flower bud of Panax notoginseng

Despite Panax notoginseng (Sanchi: the root and rhizome) is globally popular serving as the source of food additives, health-care products, and traditional Chinese medicines (TCMs), the saponin difference between the root (PNR) and two aerial parts (leaf, PNL; flower bud, PNF) that can be vicariously used, remains unclear. Authentication of Sanchi, particular from the Chinese patent medicines (CPMs), poses great challenges. Ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS)-based untargeted metabolomics and quantitative assay by UHPLC-UV were utilized to establish the “Identification Markers” for Sanchi. Targeted monitoring of multiple identification markers was performed for authenticating Sanchi simultaneously from 15 different CPMs. Dimension-enhanced profiling by UHPLC/IM-QTOF-MS in the negative high-definition MS^E (HDMS^E) mode and in-house library-driven peak annotation could characterize totally 328 ginsenosides (133 from PNR, 125 from PNL, and 161 from PNF). Multivariate statistical analysis of the PNR/PNL/PNF samples (45 batches) identified 27 potential markers. Five major markers (notoginsenoside R1, ginsenosides Rg1, -Rb1, -Rb2, and -Rb3) thereof were quantitatively assayed by a fully validated UHPLC-UV (detected at 203 nm) approach. The application of selective ion monitoring (SIM) of 12 differential saponins coupled with UHPLC separation could precisely identify Sanchi from 15 different CPMs (45 batches). Holistic difference in ginsenosides among three parts of P. notoginseng was unveiled, and the markers deduced may assist to identify the illicit substitution of leaf or flower as the root in the TCM compound formulae. Conclusively, the integration of untargeted metabolomics and quantitative analysis can provide reliable information enabling the precise authentication of TCM.     

Rapid characterization of triterpene saponins from Conyza blinii by liquid chromatography coupled with mass spectrometry

Conyza blinii Le'vl is a medicinal herb used for the treatment of inflammation in Chinese folk medicine. Its major bioactive constituents are triterpene saponins, most of which contain 6–8 sugar residues. In this report, electrospray ionization tandem mass spectrometry fragmentation behaviors of bisdesmosidic triterpene saponins (conyzasaponin A, B, and C) were studied in both positive and negative ion modes with an ion‐trap mass spectrometer. In full scan mass spectrometry, these saponins gave predominant [M–H]^? and [M+Na]⁺ ions, which determined the molecular weights. In tandem mass spectrometry (MSⁿ, n = 2–4), the [M–H]^? and [M+Na]⁺ ions yielded fragments [Y_0α–H]^? and [B_α+Na]⁺, which were diagnostic for the structures of the triterpene skeleton and sugar chains. The structural elucidation was approved by accurate mass data using IT‐TOF‐MS. An interpretation guideline based on MSⁿ (n = 2–4) diagnostic ions was proposed in order to elucidate the chemical structures of unknown triterpene saponins in C. blinii extract. The saponins in C. blinii were separated by liquid chromatography with a methanol/acetonitrile/water solvent system, and then analyzed by ion‐trap and IT‐TOF mass spectrometers. Based on the interpretation guideline, a total of 35 triterpenoid saponins were tentatively identified. Among them, 15 saponins had been previously reported, and the other 20 saponins were reported from Conyza species for the first time. This study indicates that LC/MS is a powerful technology for the rapid characterization of complicated saponins in herbal extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of Different Extraction Methods in Pharmacopoeia on the Content and Structure Transformation of Ginsenosides

Ginseng (Panax ginseng C. A. Meyer), a perennial herb, possesses immunostimulatory, anticarcinogenic, antiemetic, and antioxidative biological activities. In recent years, more and more people have paid attention to the extraction methods and quality evaluation of ginseng. China, the United States, Europe, Japan, and Korea have all had the quality standards and content determination methods of ginseng. The different treatment methods are adopted before the determination of ginseng samples and the content limits of the index components, such as ginsenoside Rb₁, ginsenoside Rg₁, and ginsenoside Re exist differences. The similarities and differences of ginseng content detection methods in pharmacopoeias of different countries have been analyzed by a research group, but the comparison of the effects of different methods on the ginsenoside content and structural transformation has not been reported. In this paper, ginsenosides in ginseng were extracted according to four national pharmacopoeias and analyzed quantitatively and qualitatively by UPLC-Q-Exactive-MS and HPLC-UV. It was illustrated that the pretreatment method has a significant influence on the content determination of ginseng. The yield of rare saponins was increased by heating concluded from both the qualitative and quantitative comparison. Finally, a simple and feasible extraction method was optimized by response surface method at room temperature. The analysis of the preparation method and process optimization of the four pharmacopoeias can provide important reference information for the revision of ginseng standards.     

Identification of new trace triterpenoid saponins from the roots of Panax notoginseng by high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Triterpenoid saponins are the major bioactive constituents of Panax notoginseng. In the study reported here, the fragmentation behavior of triterpenoid saponins from P. notoginseng was investigated by electrospray ionization tandem mass spectrometry (ESI‐MSⁿ)and high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC/ESI‐MSⁿ). Analyses revealed that product ions from glycosidic and cross‐ring cleavages can give a wealth of structural information regarding the nature of the aglycone, sugar types, the sequence and linkage information of sugar units. It is noted that different glycosylation positions remarkably influenced the fragmentation behaviors, which could assist in the differentiation of saponin analogues. To rationalize this characteristic, the collision energy required for various glycosidic cleavages was investigated. According to the summarized fragmentation rules, identification of triterpenoid saponins from the roots of P. notoginseng could be fulfilled, even when reference standards were unavailable. Furthermore, minor and trace constituents were enriched and detected by eliminating the major constituents in one of the saponin fractions. As a result, a total of 151 saponins, including 56 new trace ones, were identified or tentatively characterized from saponin fractions based on their retention times, HPLC/HRMS, HPLC/ESI‐MSⁿ fragmentation behaviors and comparison with literature data. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparative study on chemical components and anti‐inflammatory effects of Panax notoginseng flower extracted by water and methanol

Methanol and water are commonly used solvents for chemical analysis and traditional decoction, respectively. In the present study, a high‐performance liquid chromatography with ultraviolet detection method was developed to quantify 11 saponins in Panax notoginseng flower extracted by aqueous solution and methanol, and chemical components and anti‐inflammatory effects of these two extracts were compared. The separation of 11 saponins, including notoginsenoside Fc and ginsenoside Rc, was well achieved on a Zorbax SB C18 column. This developed method provides an adequate linearity (r ² > 0.999), repeatability (RSD < 4.26%), inter‐ and intraday variations (RSD < 3.20%) with recovery (94.7–104.1%) of 11 saponins concerned. Our data indicated that ginsenoside biotransformation in PNF was found, when water was used as the extraction solvent, but not methanol. Specifically, the major components of Panax notoginseng flower, ginsenosides Rb1, Rc, Rb2, Rb3, and Rd, can be near completely transformed to the minor components, gypenoside XVII, notoginsenoside Fe, ginsenoside Rd2, notoginsenoside Fd, and ginsenoside F2, respectively. Total protein isolated from Panax notoginseng flower is responsible for this ginsenoside biotransformation. Additionally, methanol extract exerted the stronger anti‐inflammatory effects than water extract in lipopolysaccharide‐induced RAW264.7 cells. This difference in anti‐inflammatory action might be attributed to their chemical difference of saponins.     

人参中人参皂苷的直接高压微波辅助降解

采用高效液相色谱-电喷雾质谱联用法测定了人参提取液中的人参皂苷. 考察了天然人参皂苷发生降解的条件, 同时研究了单体人参皂苷Rg1, Re, Rb1, Rc, Rb2和Rd的降解, 并对降解产物进行了分析. 结果表明, 随着提取压力的升高, 提取液中天然人参皂苷的含量逐渐减少, 同时产生多种次级人参皂苷. 当微波提取压力达到600 kPa, 提取时间为10 min时, 提取液中的主要天然人参皂苷达到完全降解, 次级人参皂苷Rg3含量达到最高. 在单体人参皂苷Rb1, Rc, Rb2和Rd的降解产物中均得到人参皂苷Rg3.     

Determination of Seven Major Ginsenosides in Different Parts of Panax quinquefolius L.(American Ginseng) with Different Ages

Ginsenosides Rg1,Re,Rb1,Rc,Rb2,Rb3,and Rd in different parts of the American ginseng plant were investigated.The extraction process was a pressurized microwave-assisted extraction(PMAE).The seven ginsenosides were separated and determined by high-performance liquid chromatography(HPLC) with a ultraviolet(UV) detector,at 203 nm.The experiment results showed significant variations in the individual ginsenoside contents of the American ginseng in different parts and ages of the plant.The results demonstrated that the leaves,root hairs,and rhizomes of Panax quinquefolius L.contained higher ginsenoside contents,followed by the main roots and stems.The leaves contained dramatically higher levels of ginsenoside Rg1,Rb3,and Rd than the other four parts.Higher contents of Rb1 and Re were present in the main roots,root hairs,and rhizomes.The amount of ginsenoside content in the stems was the lowest.The total content of the seven ginsenosides in main roots,root hairs and rhizomes increased with the age of the plant.In contrast,the ginsenoside contents in the leaves and stems decreased with a year of growth.     

Characterization of steroidal saponins in saponin extract from Paris polyphylla by liquid chromatography tandem multi-stage mass spectrometry

Rhizoma Paridis saponins are bioactive steroidal saponins derived from Paris polyphylla. Optimization of the ionization process was performed with electrospray ionization tandem mass spectrometry in both positive and negative-ion modes. Negative-ion ESI was adopted for generation of the precursor deprotonated molecules to achieve the best ionization sensitivity for the analytes. Positive ionization was used to choose the most abundant fragment ion. Furthermore, according to the characteristic fragmentation behavior of known steroidal saponins isolated from this plant (polyphyllin D, formosanin C, gracillin, Paris H, Paris VII, and dioscin), 23 constituents were structurally characterized on the basis of their retention time and ESI analyses, including four pairs of naturally occurring isomers. Five of these 23 constituents were new compounds. The analytical method of LC–MS ⁿ in positive and negative-ion modes has been developed for the direct structural elucidation of steroid saponins of this kind in plant extracts. Yanjun Zhang and Wenyuan Gao contributed equally to this paper.