Qin Meng

Interaction of L-arginine with κ-casein and its effect on amyloid fibril formation by the protein: multi-spectroscopic approaches.

Herein, the interaction of l-arginine (ARG) with κ-casein, and its effect on amyloid fibril formation of the protein, have been investigated in vitro by resonance light scattering (RLS), fluorescence, UV-Vis absorption spectroscopy and transmission electron microscopy (TEM) under simulated physiological conditions. The results indicated that ARG inhibited fibril formation by reduced and carboxymethylated κ-casein (RCMκ-CN), and there was interaction between ARG and RCMκ-CN, proved by the observation of enhancement in RLS intensity attributed to the formation of RCMκ-CN-ARG complex. It was also demonstrated that ARG strongly quenched the intrinsic fluorescence of RCMκ-CN through a static quenching mechanism. The corresponding thermodynamic parameters (ΔH, ΔS and ΔG) were tested to show that the binding process was spontaneous and mainly enthalpy driven with an unfavorable entropy, and both hydrogen bond and van der Waals forces played a key role in the binding of ARG and RCMκ-CN. The determined value of the distance r between ARG and RCMκ-CN Trp97 residue evaluated by fluorescence resonance energy transfer (FRET) was 2.94nm. Furthermore, the conformational investigation from synchronous fluorescence showed that the RCMκ-CN Trp97 residue was placed in a less polar environment and more difficultly exposed to the solvent after addition of ARG.

Anti-colon carcinoma cell activity of ginsenosides from the acid hydrolysate of Panax ginseng

Six dammarane ginsenosides, 20(R)-dammar-25-ethoxy-3β,12β,20-triol (1), 3β,6α,12β-triol22,23,24,25,26,27-hexanordammaran-20-one (2), dammar-(E)-20(22)-ene-3β,12β,25-triol (3), 20(S)-dammar-3β,12β,20,25-tetrol (4), and 20(R)-protopanaxadiol (5), 20(R)-protopanaxatriol (6), were isolated from the acid hydrolysate of Panax ginseng. Compound 1 was only mentioned in a patent literature, and there is no spectroscopic data elsewhere. This paper is the first to elucidate the structure of compound 1 on the basis of spectroscopic evidence. The biological activities of compounds 1–6, crude ginsenosides, and the ginsenoside-hydrolysate were determined in human colon carcinoma cell. Compounds 1–3 and the ginsenoside-hydrolysate exhibited anti-human colon carcinoma cell activity, with IC50 of 66.1, 72.4, 50.1, and 25.7 μg/mL, respectively.

Interaction of the ginsenosides with κ-casein and their effects on amyloid fibril formation by the protein: Multi-spectroscopic approaches.

The interaction of the ginsenosides (GS) including ginsenoside Rg1, Rb1 and Re with κ-casein and the effects of GS inhibiting amyloid fibril formation by κ-casein have been investigated in vitro by fluorescence and ultraviolet spectra. Results showed that Rg1 and Rb1 had dose-dependent inhibitory effects on reduced and carboxymethylated κ-casein (RCMκ-CN) fibril formation, while Re resulted in an increase in the rate of fibril formation. The enhancement in RLS intensity was attributed to the formation of new complex between GS and RCMκ-CN, and the corresponding thermodynamic parameters (ΔH, ΔS and ΔG) were assayed. The steady-state ultraviolet-visible absorption spectra had also been tested to observe if the ground-state complex formed, and it showed the same result as RLS spectra. The binding constants and the number of binding sites between GS and RCMκ-CN at different temperatures had been evaluated from relevant fluorescence data. According to the Förster non-radiation energy transfer theory, the binding distance between RCMκ-CN and GS was calculated. The fluorescence lifetime of RCMκ-CN was longer in the presence of GS than in absence of GS, which was evident that the hydrophobic interaction plays a major role in the binding of GS to RCMκ-CN. From the results of synchronous fluorescence, it could be deduced that the polarity around RCMκ-CN Trp97 residue decreased and the hydrophobicity increased after addition of Rg1 or Rb1. Based on all the above results, it is explained that Rg1 and Rb1 inhibited amyloid fibril formation by κ-casein because the molecular spatial conformation and physical property of κ-casein changed causing by the complex formation between GS and κ-casein.

Structural identification of neopanaxadiol metabolites in rats by ultraperformance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry

RATIONALE Neopanaxadiol (NPD) is one of the major ginsenosides in Panax ginseng C. A. Meyer (Araliaceae) that has been suggested to be a drug candidate against Alzheimers disease. However, few data are available regarding its metabolism in rats. METHODS In this study, a method of ultraperformance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/QTOFMS) was developed to identify major metabolites of NPD in the stomach, intestine, urine and feces of rats, with the aim of determining the main metabolic pathways of NPD in rats after oral administration. RESULTS UPLC/QTOFMS revealed two metabolites in the stomach of rats, one metabolite in the intestine and two metabolites in feces. One metabolite, named M2, was isolated and purified from rats feces, which was identified as (20S,22S)-dammar-22,25-epoxy-3β,12β,20-triol based on extensive NMR spectroscopy and mass spectrometry data. The main metabolites of NPD in rats were the products of epoxidation, dehydrogenation and hydroxylation. NPD was predominantly metabolized by 20,22-double-bond epoxidation and rearrangement to yield an expoxidation product (M2). CONCLUSIONS Based on the profiles of the metabolites, possible metabolic pathways of NPD in rats were proposed for the first time. This study provides new and available information on the metabolism of NPD, which is indispensable for further research on metabolic pathways of dammarane ginsengenins in vivo.

The effects of ginsenosides to amyloid fibril formation by RCMκ-casein

When not incorporated into the casein micelle, isolated κ-casein spontaneously forms amyloid fibrils under physiological conditions, and is a convenient model for researching generic aspects of fibril formation. Ginsenosides have recently attracted much research interest because of the effects on aging diseases, which are always associated with amyloid fibril formation, for example, Alzheimers, Parkinsons, and Huntingtons diseases. In addition, the mechanism remains unclear that ginsenosides exert the effects against aging diseases. To address these aspects, we have investigated the ability of ginsenoside Rb1, Rc, Rg1, and Re influencing fibril formation by RCMκ-casein (reduced and carboxymethylated κ-casein), with the methods of Thioflavin T fluorescence assay, transmission electron microscopy (TEM), and intrinsic fluorescence spectroscopy. The results showed that ginsenoside Rb1 and Rg1 inhibited obviously RCMκ-CN fibrillation in both the initial rate and final level of ThT fluorescence. On the contrary, ginsenoside Re had a few effect on promoting RCMκ-CN fibril formation, proved by thick and larger fibrils observed frequently in TEM. While Rc did not influence RCMκ-CN fibrillation. It is demonstrated that Rg1 prevent RCMκ-CN fibril formation by stabilising RCMκ-CN in its native like state. Additional chemical structure difference of ginsenosides and the effects on fibril formation are also implicated.

Tissue distribution and excretion study of neopanaxadiol in rats by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry

Neopanaxadiol (NPD), a major ginsenoside in Panax ginseng C. A. Meyer (Araliaceae), was reported to have neuroprotective effect. In this study, a method of ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/QTOF-MS) was developed and validated for quantitative analysis of NPD in tissues, urine and feces, using liquid-liquid extraction (LLE) to isolate NPD from different biological samples, and chromatographic separation was performed on an Agilent Zorbax Stable Bond C18 (2.1 × 50 mm, 1.8 µm) column with 0.1% formic acid in water and acetonitrile. All standard calibration curves were linear (all r(2) > 0.995) within the test range. After oral administration, NPD was extensively distributed to most of the tissues without long-term accumulation. The higher levels were observed in stomach and intestine, followed by kidney and liver. Approximately 64.56 ± 20.32% of administered dose in feces and 0.0233 ± 0.0356% in urine were found within 96 h, which indicated that the major elimination route was fecal excretion. This analytical method was applied to the study of NPD distribution and excretion in rats after oral intake for the first time. The results we found here are helpful for us to understand the pharmacological effects of NPD, as well as its toxicity.

Structure of Acid Hydrolysate of Total Ginsenosides and Their Cytotoxic Activity

Bioassay-guided fractionation of the cytotoxic acid hydrolysate of the total ginsenosides of Panax ginseng C. A. Meyer (Araliaceae) afforded a new ginsenoside, (20S,22S)-dammar-22,25-epoxy-3β,12β,20-triol (1), along with five known ginsenosides, 20R,24R-dammar-20(24)-epoxy-3β,12β,25-triol (2), 20R-panaxdiol (3), dammar-(E)-20(22)-ene-3β,12β,25-triol (4), 20R-protopanaxadiol (5), and 20R-dammar-25-ethoxy-3β,12β,20-triol (6). Their structures were elucidated on the basis of spectroscopic data. Among the compounds isolated, compound 1 showed strong cytotoxic activity against human cancer cell lines SW1116, HCT116, and A549.