The localization of the alkaloids in Coptis chinensis rhizome by time-of-flight secondary ion mass spectrometry.

Background: Understanding the spatial distribution of active compounds can effectively evaluate the quality of decoction pieces of traditional Chinese medicine (TCM). Traditional methods are economical and practical but lack chemical information on the original distribution. Time-of-flight secondary ion mass spectrometry (TOF-SIMS), with the advantage of non-destructive detection of samples, can directly analyze the distribution of chemical compounds on the surface of various samples. Methods: In this study, TOF-SIMS image analysis technology was used to detect TCM for the first time. Taking Coptis rhizome (CR) as an example, a commonly used TCM, the distribution of the compounds in the cross-section of CR was studied. Meanwhile, ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLCQQQ-MS/MS) was used to verify the results of TOF-SIMS. Results: The distribution of nine active compounds: berberine, epiberberine, coptisine, palmatine, columbamine, jatrorrhizine, tetrahydricheilanthifolinium, and oxyberberine, was well imaged in the cross-section of CR by TOF-SIMS. The content of berberine and epiberberine was the highest; Palmatine distribution in the pith was more than that in other parts; Oxyberberine was mainly concentrated in the cork and xylem rays. Normalization analysis showed contents of these compounds increased along with the growth years. The result was consistent with UPLC-QQQ-MS/MS. Conclusion: The TOF-SIMS method can display the spatial distribution status of the active compounds of herbs, providing a basis for selecting the medicine site with non-destructive and fast detection.

Novel 2,5-Diarylcyclopentenone Derivatives from the Wild Edible Mushroom Paxillus involutus and Their Antioxidant Activities.

Wild edible mushrooms are important as a source of nutraceuticals and for the discovery of bioactive metabolites as pharmaceuticals. In this work, 10 rare 2,5-diarylcyclopentenone derivatives were isolated from the wild edible mushroom Paxillus involutus (Batsch) Fr., including eight novel compounds termed involutenone A-H (1-8) and two previously identified compounds (9-10). Their structures were established using high-resolution electrospray ionization mass spectroscopy and 1D and 2D nuclear magnetic resonance data. The absolute configurations of compounds 1-3 and 6-8 were assigned based on the comparison of the experimental and calculated electronic circular dichroism data. The antioxidant activities of 1-8 were tested through DPPH free radical scavenging, hydroxyl radical scavenging, and superoxide anion radical scavenging assays. Compounds 3, 5, 6, and 7 demonstrated significant antioxidant activity compared to the positive control (tert-butylhydroquinone). These compounds could be effective natural antioxidants with considerable pharmaceutical value.

Discovery of chemical markers for improving the quality and safety control of Sinomenium acutum stem by the simultaneous determination of multiple alkaloids using UHPLC-QQQ-MS/MS.

Sinomenium acutum stem is a popular traditional Chinese medicine used to treat bone and joint diseases. Sinomenine is considered the only chemical marker for the quality control of S. acutum stem in mainstream pharmacopeias. However, higenamine in S. acutum stem is a novel stimulant that was banned by the World Anti-Doping Agency in 2017. Therefore, enhancing the quality and safety control of S. acutum stem to avoid potential safety risks is of utmost importance. In this study, a fast, sensitive, precise, and accurate method for the simultaneous determination of 11 alkaloids in S. acutum stem by ultrahigh-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) was established. This method successfully analyzed thirty-five batches of S. acutum stem samples. The average contents of sinomenine, magnoflorine, coclaurine, acutumine, higenamine, sinoacutine, palmatine, magnocurarine, columbamine, 8-oxypalmatine, and jatrorrhizine were 24.9 mg/g, 6.35 mg/g, 435 µg/g, 435 µg/g, 288 µg/g, 44.4 µg/g, 22.5 µg/g, 21.1 µg/g, 15.8 µg/g, 9.30 µg/g, and 8.75 µg/g, respectively. Multivariate analysis, including principal component analysis (PCA), orthogonal partial least square method-discriminant analysis (OPLS-DA), and hierarchical cluster analysis (HCA), were performed to characterize the importance and differences among these alkaloids in S. acutum stem samples. As a result, sinomenine, magnoflorine, coclaurine, acutumine, and higenamine are proposed as chemical markers for quality control. Higenamine and coclaurine are also recommended as chemical markers for safety control. This report provides five alkaloids that can be used as chemical markers for improving the quality and safety control of S. acutum stem. It also alerts athletes to avoid the risks associated with consuming S. acutum stem.

Discovery of chemical markers for identifying species, growth mode and production area of Astragali Radix by using ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry.

BACKGROUND: Astragali Radix (AR) is a well-known Chinese herbal medicine. The quality of AR can be affected by many factors such as species, growth mode and production area, but there are still no chemical markers to distinguish it. PURPOSE: To explore chemical markers for improving the quality assessment of AR and discover chemical markers for identifying species, growth mode and production area of AR. METHODS: A highly sensitive, efficient and accurate method based on ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) for simultaneous quantitative determination of 14 major chemical components (five flavonoids and nine triterpene saponins) in 94 batches of AR from China, Republic of Korea and Germany was developed for the first time. To explore chemical markers and assess changes in the contents of 14 compounds in the 94 batches of AR samples from different regions, hierarchical clustering analysis (HCA) and principal component analysis (PCA) were performed. RESULTS: Astragaloside III was not only an important chemical marker for distinguishing two species of AR, i.e.: Astragalus mongholicus and A. membranaceus, but also a potential chemical marker for the classification of cultivated and semi-wild AR. In addition, in the batches of cultivated AR, the content of isoastragaloside II and cyclocephaloside II were greater in batches from the region of Shaanxi Province than that of other Provinces in China, but the content of calycosin-7-O-ß-D-glucoside and astragaloside IV, which are the quality control markers of AR required by the Chinese Pharmacopoeia, were higher than that of other Provinces in China. In addition, the content of calycosin-7-O-ß-D-glucoside, ononin, calycosin and astragaloside I could be used to identify samples of AR collected from China, Republic of Korea and Germany. CONCLUSION: This UHPLC-QQQ-MS/MS method could be applied to the quantitative evaluation of AR and could be an important and meaningful reference to develop chemical markers for quality control of AR.

Inhibitory Effects of Ginsenoside Ro on the Growth of B16F10 Melanoma via Its Metabolites.

Ginsenoside Ro (Ro), a major saponin derived and isolated from Panax ginseng C.A. Meyer, exerts multiple biological activities. However, the anti-tumour efficacy of Ro remains unclear because of its poor in vitro effects. In this study, we confirmed that Ro has no anti-tumour activity in vitro. We explored the anti-tumour activity of Ro in vivo in B16F10 tumour-bearing mice. The results revealed that Ro considerably suppressed tumour growth with no significant side effects on immune organs and body weight. Zingibroside R1, chikusetsusaponin IVa, and calenduloside E, three metabolites of Ro, were detected in the plasma of Ro-treated tumour-bearing mice and showed excellent anti-tumour effects as well as anti-angiogenic activity. The results suggest that the metabolites play important roles in the anti-tumour efficacy of Ro in vivo. Additionally, the haemolysis test demonstrated that Ro has good biocompatibility. Taken together, the findings of this study demonstrate that Ro markedly suppresses the tumour growth of B16F10-transplanted tumours in vivo, and its anti-tumour effects are based on the biological activity of its metabolites. The anti-tumour efficacy of these metabolites is due, at least in part, to its anti-angiogenic activity.

Discovery of markers for discriminating the age of cultivated ginseng by using UHPLC-QTOF/MS coupled with OPLS-DA.

BACKGROUND: Ginseng (Ginseng Radix et Rhizoma, Panax ginseng C.A. Meyer) is gaining more publicity in modern society due to its health benefit and huge value in market. In the practice of grading and pricing of ginseng, the age is one of the major factor influencing the price and grade of ginseng. Therefore, the age discrimination is an important task for the quality control of ginseng. However, the traditional morphological methods are too subjective to be reproductive in discrimination. PURPOSE: To establish a method that can discriminate the ginseng samples with different cultivation years. STUDY DESIGN: To analyze the correlation between chemical compositions and cultivation years of cultivated ginseng samples of different age and thus discover potential quality marker (Q-marker) for discriminating the age of cultivated ginseng. METHODS: In the present study, the ultra-high performance liquid chromatography coupled with the quadrupole-time of flight mass spectrometry (UHPLC-QTOF/MS) were utilized for the age discrimination and marker discovery. A statistical data processing procedure was established to screen markers and reduce the false positive rate. RESULTS: The results showed that the ginseng samples from 2- to 6-year-old could be well separated in the orthogonal projections on the latent structure - discrimination analysis (OPLS-DA) using the markers screened by the established statistical procedure, which could reduce approximately 20% of the insignificant markers and false positive discoveries. Ultimately, more than 50 compounds contributing to the age discrimination were identified including one new compound (malonylginsenoside). One negative marker (1038.4825@8.98) was discovered for the 2-year-old ginseng, and an equation was established to effectively predict the age of 3- to 6-year-old of ginseng. CONCLUSION: The constructed method can discriminate the ginseng samples with different cultivation years and is a complement to the traditional discrimination method of ginseng age.

Supplementation of American ginseng berry extract mitigated cisplatin-evoked nephrotoxicity by suppressing ROS-mediated activation of MAPK and NF-κB signaling pathways.

Nephrotoxicity induced by cisplatin in 30% of all cisplatin treated patients seriously limits its clinical implication as a widely used anticancer agent, and may even cause patients to alter or give up cisplatin therapy. The purpose of this study is to test a protective effect of American ginseng berry extract (AGBE) on cisplatin-induced nephrotoxicity in mice. In this study, the histopathological changes and elevated levels of serum creatinine (CRE) and urea nitrogen (BUN) caused by cisplatin were significantly diminished by AGBE treatment. Oxidative stress caused by cisplatin, evidenced by increases in kidney tissues malondialdehyde (MDA) content, cytochrome P450 E1 (CYP2E1), renal 4-hydroxynonenal (4-HNE) levels and decreases of glutathione (GSH) and superoxide dismutase (SOD) contents, was significantly ameliorated by AGBE pretreatment. The expression levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were inhibited by AGBE treatment, suggesting a suppression of inflammatory response. Additionally, AGBE clearly inhibited cisplatin-induced activations of nuclear factor-kappa B (NF-κB) and mitogen activated protein kinase (MAPK) signal pathways. Supplementation of cisplatin-intoxicated mice with AGBE also significantly reduced apoptotic protein levels of Bax, cleaved caspase-3, cytochrome c and increased anti-apoptotic protein Bcl-2. These findings highlight nephroprotective effect of AGBE against cisplatin-evoked nephrotoxicity through ROS-mediated MAPK and NF-κB signaling pathways.

Effect of B-complex vitamins on the antifatigue activity and bioavailability of ginsenoside Re after oral administration.

BACKGROUND: Both ginsenoside Re and B-complex vitamins are widely used as nutritional supplements. They are often taken together so as to fully utilize their antifatigue and refreshing effects, respectively. Whether actually a drug-nutrient interaction exists between ginsenoside Re and B-complex vitamins is still unknown. The objective of this study was to simultaneously investigate the effect of B-complex vitamins on the antifatigue activity and bioavailability of ginsenoside Re after their oral administration. The study results will provide valuable theoretical guidance for the combined utilization of ginseng and B-complex vitamins. METHODS: Ginsenoside Re with or without B-complex vitamins was orally administered to mice to evaluate its antifatigue effects and to rats to evaluate its bioavailability. The antifatigue activity was evaluated by the weight-loaded swimming test and biochemical parameters, including hepatic glycogen, plasma urea nitrogen, and blood lactic acid. The concentration of ginsenoside Re in plasma was determined by liquid chromatography-tandem mass spectrometry. RESULTS: No antifatigue effect of ginsenoside Re was noted when ginsenoside Re in combination with B-complex vitamins was orally administered to mice. B-complex vitamins caused to a reduction in the bioavailability of ginsenoside Re with the area under the concentration-time curve from zero to infinity markedly decreasing from 11,830.85 ± 2,366.47 h·ng/mL to 890.55 ± 372.94 h·ng/mL. CONCLUSION: The results suggested that there were pharmacokinetic and pharmacodynamic drug-nutrient interactions between ginsenoside Re and B-complex vitamins. B-complex vitamins can significantly weaken the antifatigue effect and decrease the bioavailability of ginsenoside Re when simultaneously administered orally.

Purity assessment of ginsenoside Rg1 using quantitative 1H nuclear magnetic resonance.

Ginseng herbs comprise a group of the most popular herbs, including Panax ginseng, P. notoginseng and P. quinquefolius (Family Araliaceae), which are used as traditional Chinese medicine (TCM) and are some of the best-selling natural products in the world. The accurate quantification of ginsenoside Rg1 is one of the major aspects of its quality control. However, the purity of the commercial Rg1 chemical reference substance (CRS) is often measured with high-performance chromatography coupled with an ultraviolet detector (HPLC-UV), which is a selective detector with unequal responses to different compounds; thus, this detector introduces probable error to purity assessments. In the present study, quantitative nuclear magnetic resonance (qNMR), due to its absolute quantification ability, was applied to accurately assess the purity of Rg1 CRS. Phenylmethyl phthalate was used as the internal standard (IS) to calibrate the purity of Rg1 CRS. The proton signal of Rg1 CRS in methanol-d4 at 4.37ppm was selected to avoid interfering signals, enabling accurate quantitative analysis. The relaxation delay, number of scans, and NMR windowing were optimized for data acquisition. For post-processing, the Lorentz/Gauss deconvolution method was employed to increase the signal accuracy by separating the impurities and noise in the integrated region of the quantitative proton. The method validation showed that the developed method has acceptable sensitivity, linearity, precision, and accuracy. The purity of the commercial Rg1 CRS examined with the method developed in this research was 90.34±0.21%, which was obviously lower than that reported by the manufacturer (>98.0%, HPLC-UV). The cross-method validation shows that the commonly used HPLC-UV, HPLC-ELSD (evaporative light scattering detector) and even LC-MS (mass spectrometry) methods provide significantly higher purity values of Rg1 CRS compared with the qNMR method, and the accuracy of these LC-based methods largely depend on the amount of the sample that was loaded and the properties of the impurities.

Complete (1)H-NMR and (13)C-NMR spectral assignment of five malonyl ginsenosides from the fresh flower buds of Panax ginseng.

BACKGROUND: Ginsenosides are the major effective ingredients responsible for the pharmacological effects of ginseng. Malonyl ginsenosides are natural ginsenosides that contain a malonyl group attached to a glucose unit of the corresponding neutral ginsenosides. METHODS: Medium-pressure liquid chromatography and semipreparative high-performance liquid chromatography were used to isolate purified compounds and their structures determined by extensive one-dimensional- and two-dimensional nuclear magnetic resonance (NMR) experiments. RESULTS: A new saponin, namely malonyl-ginsenoside Re, was isolated from the fresh flower buds of Panax ginseng, along with malonyl-ginsenosides Rb1, Rb2, Rc, Rd. Some assignments for previously published (1)H- and (13)C-NMR spectra were found to be inaccurate. CONCLUSION: This study reports the complete NMR assignment of malonyl-ginsenoside Re, Rb1, Rb2, Rc, and Rd for the first time.

Characterization of active phenolic components in the ethanolic extract of Ananas comosus L. leaves using high-performance liquid chromatography with diode array detection and tandem mass spectrometry.

HPLC-DAD-MS was utilized to investigate the phytochemical constituents in ethanolic extract of Ananas comosus L. leaves (EEACL) responsible for antidiabetic, antihyperlipidemic and antioxidative effects. Eight phenylpropane diglycerides, together with two hydroxycinnamic acids, three hydroxycinnamoyl quinic acids, four phenylpropane monoglycerides, three flavones and six phenylpropanoid glycosides were detected, and their proposed structures were elucidated based on HPLC retention time, UV and MS profiles. Meanwhile, a new HPLC-DAD-MS method was established for the identification and characterization of phenylpropane diglycerides in natural plants.

[Comparison of chemical compositions between Dendrobium candidum and Dendrobium nobile].

OBJECTIVE: To compare the chemical compositions between Dendrobium candidum (D. candidum) and Dendrobium nobile (D. nobile). METHOD: Relative area and content of every chromatographic peak in the ammonic chloroform extracts of D. candidum were compared with those of D. nobile with high performance liquid chromatography-mass spectrometry. RESULTS: The relative area of alkaloids accounted for 2.34% and 41.87% in D. candidum and D. nobile, respectively. The relative area of 25 identical compositions took up 97.12% in the total area of D. candidum and 50.09% in that of D. nobile, and contents of 23 composition were higher in D. candidum than those in D. nobile. CONCLUSIONS: The quantity and contents of alkaloids are remarkably different between D. candidum and D. nobile. However, D. candidum has a higher quality than D. nobile in terms of the same chemical compositions.

A new minor triterpene saponin from kaixin-san prescription.

A new minor oleanane-type saponin, ginsenoside-R(OA) (1), has been isolated from the extract of Kaixin-San, a prescription composed of Panax ginseng and other medicinal herbs, together with 15 known ginsenosides. The structure of 1 was established as 3-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucuronopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside by chemical and spectroscopic evidence. In addition, the presence of ginsenoside-R(OA) in ginseng was confirmed by HPLC-ESI-MS.

[Identification of Panax notoginseng and its preparations by LC/MS].

AIM: To develop a method of identifying the existing of Panax notoginseng in products of traditional Chinese medicine compound Danshen. METHODS: Total ion chromatograms (TIC) of Panax notoginseng, P. ginseng, and P. quinquefolius were obtained by means of LC/MS. Extracted ion chromatograms (EIC) posses m/z of 770, 800, 932, 946 and 1,108 of above-mentioned three herbs was compared between species. EIC 800 and 946 were selected as differentiation marks to distinguish P. notoginseng from the other two species. The EIC 800 and 946 of P. notoginseng were also compared to the EICs obtained by the same method from Chinese patent medicines of compound Danshen pellet, compound Danshen tablet, and compound Danshen injection. EIC 800 and 946 of P. notoginseng and its products possess similar peaks, relative retention time, and relative integral areas. Main chemical constitutes of P. notoginseng were also identified by using LC/MS/MS. RESULTS: EIC 800 and 946 were obviously different between P. notoginseng, P. ginseng, and P. quinquefolius. Patent medicines of compound Danshen pellet, compound Danshen tablet, which consist of extractions from P. notoginseng, possess the characteristic EICs. The selected EICs were stable and reproductive. CONCLUSION: EIC 800 and 946, which correspond to ginsenoside Rg1, Re, and their isomers, can be used as identifying mark of P. notoginseng to differentiate it from other herbs, and also can be used to tell apart P. notoginseng from other herb extractions in Chinese patent medicines of compound Danshen.

[Studies on second metabolites of an endophytic fungus (II)].

OBJECTIVE: To study the chemical constituents from the cultured mycelia of a fungus Cephalosporium which accelerate the growth of plant. METHOD: The constituents were isolated by column chromatography and identified by advanced physical and spectral analysis. RESULT: Eleven compounds including 3-isopropyl-6-(1-methylpropyl) piperazine-2,5-dione(I), choline sulfate(II), 2-[(2-hydroxy tetracosanoyl) amino]-1,3,4-octadecatriol(III) were isolated and identified. CONCLUSION: Compound I, II were isolated from Cephalosporium genus for the first time.