The Fate and Intermediary Metabolism of Soyasapogenol in the Rat.

Research suggests that soyasaponins are poorly absorbed in the GI tract and that soyasaponin aglycones or soyasapogenols are absorbed faster and in greater amounts than the corresponding soyasaponins. Therefore, it is important to understand the bioavailability of these compounds for the potential development of functional foods containing their components. In this paper, to investigate the metabolic characteristics of soyasapogenols A and B, the pharmacokinetic parameters in rats were determined via oral and intravenous administration. The liver metabolites of soyasapogenols were identified using UPLC-/Q-TOF-MS/MS, and their metabolic pathways were also speculated. The results show that, after oral administration, there was a bimodal phenomenon in the absorption process. Tmax was about 2 h, and soyasapogenol was completely metabolized 24 h later. The bioavailability of soyasapogenol was superior, reaching more than 60%. There were sixteen metabolites of soyasapogenol A and fifteen metabolites of soyasapogenol B detected in rat bile. Both phase I and II metabolic transformation types of soyasapogenols, including oxidation, dehydrogenation, hydrolysis, dehydration, deoxidization, phosphorylation, sulfation, glucoaldehyde acidification, and conjugation with cysteine, were identified. In addition, soyasapogenol A could be converted into soyasapogenols B and E in the metabolic process. These results suggest that it is feasible to use soyasapogenols as functional ingredients in nutraceuticals or food formulations.

Computer-Aided Design of Molecularly Imprinted Polymers for Simultaneous Detection of Clenbuterol and Its Metabolites.

Molecular imprinting technology (MIT) offers an effective technique for efficient separation and enrichment of specific analytes from complicated matrices and has been used for illicit veterinary drug detectionin recent years due to its high selectivity, good chemical stability, and simple preparation. The development of in silico-based approaches has enabled the simulation of molecularly imprinted polymers (MIPs) to facilitate the selection of imprinting conditions such as template, functional monomer, and the best suitable solvent. In this work, using density functional theory (DFT), the molecularly imprinted polymers of clenbuterol and its metabolites were designed by computer-aided at B3LYP/6-31 + G (d, p) level. Screening molecular imprinting components such as functional monomers, cross-linkers, and solvents has been achieved in the computational simulation considerations. The simulation results showed that methacrylic acid (MAA) is the best functional monomer; the optimal imprinting ratio for both clenbuterol (CLB) and its dummy template molecule of phenylephrine (PE) to functional monomer is 1:3, while the optimal imprinting ratio for the two dummy template molecules of CLB's metabolites is 1:5. Choosin gethyleneglycol dimethacrylate (EDGMA) as a crosslinker and aprotic solvents could increase the selectivity of the molecularly imprinted system. Atoms in Molecules (AIM) topology analysis was applied to investigate the template-monomer complexes bonding situation and helped to explain the nature of the reaction in the imprinting process. These theoretical predictions were also verified by the experimental results and found to be in good agreement with the computational results. The computer-simulated imprinting process compensates for the lack of clarity in the mechanism of the molecular imprinting process, and provides an important reference and direction for developing better recognition pattern towards CLB and its metabolite analytes in swine urine samples at the same time.

A novel quartz crystal microbalance sensor array based on molecular imprinted polymers for simultaneous detection of clenbuterol and its metabolites.

For the rapid and robust detection of both parent clenbuterol (CLB) and its metabolites in swine urine samples, a novel quartz crystal microbalance (QCM) sensor array for CLB detection based on molecularly imprinted polymers (MIPs) was developed in this investigation. At first, clenbuterol and the structural analogs of its metabolites, 4-Aminohippuric acid (AHA) and 4-hydroxymandelic acid (HMA), were chosen as molecular templates. Through computational molecular modeling, the optimum ratio between the functional monomer and molecular template was selected. The surface imprinting method was applied to modify QCM electrode surface to graft a thin MIP film. The grafting polymer was characterized by Fourier-transformed infrared spectrometry (FTIR) and atomic force microscopy (AFM), respectively. After then, an array system composed of three sensors was employed to test the responses with different solutions and the principal component analysis (PCA) was adopted to analyze the corresponding data. As a result, for the designed sensor to clenbuterol, a linear equation y=100.07x-722.96 (R2=0.9928) was found between the sensor frequency shift ΔF and negative logarithm of clenbuterol concentration (-lgC). The limitation of detection (LOD) was 3.0ng/mL, which is lower than the Codex Alimentarius Commission regulations residue limit 10µg/L. The corresponding data of the three template solutions were analyzed by PCA, obtaining 100% recognition. The result demonstrated the feasibility that the developed method could be applied to detect whether the livestock was feed with CLB nutrient redistribution agent by checking the urine samples.

Evaluation of cytotoxicity and immune modulatory activities of soyasaponin Ab: an in vitro and in vivo study.

To improve the immune efficacy of protein subunit vaccines, novel adjuvants are needed to elicit a suitable protective immune response and to promote long term immunologic memory. In this work, soyasaponin Ab, a major constituent among group A soyasaponins in soybeans was purified and prepared from soy hypocotyls. The immunomodulatory effects of soyasaponin Ab both in vitro and in vivo were investigated, and its pro-immunomodulatory molecular mechanism was also studied. For in vitro assays, with mouse macrophage cell line RAW264.7 as the studying model, both cytotoxicity and immune stimulatory activity were investigated to evaluate the potential of soyasaponin Ab as the vaccine adjuvant. The results indicated that soyasaponin Ab could be significantly safer than Quillaja saponins (QS). Soyasaponin Ab showed no toxicities over the tested concentration ranges compared to QS. Soyasaponin Ab was proved able to promote releases of inflammatory cytokines like TNFα and IL-1ß in a dose-dependent manner. Furthermore, NF-κB signalling was also activated by soyasaponin Ab effectively. In addition, with TLR4 gene expression of RAW264.7 cell inhibited by RNA interference, immune stimulatory effects by soyasaponin Ab dropped down significantly. On the other hand, the in vivo experiment results showed that anti-ovalbumin (OVA) IgG, IgG1, IgG2a, IgG2b were significantly enhanced by the soyasaponin Ab and QS groups (p<0.05 or p<0.01). The results suggested that compared to QS, soyasaponin Ab may represent a viable candidate for effective vaccine adjuvant. TLR4 receptor dependent pathway may be involved in immune stimulatory effects of soyasaponin Ab.

Haemolytic activity and adjuvant effect of soyasaponins and some of their derivatives on the immune responses to ovalbumin in mice.

Immunological adjuvants are agents that enhance specific immune responses to vaccines. At present more studies are needed to identify a suitable adjuvant for a particular vaccine with maximum safety and efficacy. In this study, six soyasaponins (Aa, Ab, Af, Ba, Bb, and Bb') and three soyasaponin Ab-derivatives (AbDs) were selected to evaluate their toxicities and adjuvant activities. The haemolytic activity assay was performed to evaluate the toxicity of the tested soyasaponins and AbDs. Immunoadjuvant activity was investigated in vivo and in vitro using a splenocyte proliferation assay and sera indirect ELISA. Our results demonstrated that soyasaponins and AbDs showed a slight haemolytic effect to 0.5% red blood cell. Except for the Af and Ba groups, other soyasaponins and AbDs groups stimulated by concanavalin A (ConA) and lipopolysaccharide (LPS) showed a greater proliferative response at appropriate doses (0.01-10 µg/ml) compared with the control and Alum groups. Anti-OVA IgG, IgG1, IgG2a, and IgG2b were significantly enhanced by the soyasaponins (Ab, Ba, Bb, and Bb'), QS and AbDs groups (p < 0.05 or p < 0.01). In addition, three AbDs indicated a tendency of immunoadjuvant potential improvement after structural modification. Moreover, Ab-D2 showed adjuvant activity at the lowest injection dose among the three AbDs. In conclusion, these results suggested that soyasaponins together with their derivatives may represent viable candidates for effective vaccine adjuvants due to their higher immune response and lower or non-haemolytic effects.

Efficient protocol for isolation and purification of different soyasaponins from soy hypocotyls.

Soyasaponins are naturally occurring triterpenoid glycosides associated with many biological activities. The aim of the present study was to develop an effective method for isolation and purification of differently glycosylated, acetylated, and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soyasaponins from soy hypocotyls. Both gel filtration using Sephadex LH-20 chromatography (Amersham Pharmacia Biotech AB; elution phase: methanol, flow rate: 3.0 mL/min, sample loading: 60 mg) and high-speed countercurrent chromatography (stationary phase: n-butanol-acetic acid (5.0%, v/v), mobile phase: water flow rate: 3.0 mL/min, sample loading: 100 mg) could effectively fractionate isoflavones and soyasaponins from the crude extract with yield of soyasaponin complexes 20.5 mg and 22.3 mg, respectively. After fractionation, the soyasaponin complexes could be purified further using preparative HPLC to separate individuals. A total of nine soyasaponins, triacetyl soyasaponin Ab (yield 1.55%, HPLC purity >98%), Aa (2.68%, >99%), Ab (18.53%, >98%), Ae (0.85%, >98%), Ba (0.63%, >91%), Af (1.12%, >85%), Bb (3.45%, >98%) and Be (0.59%, >76.8%) were obtained. DDMP-conjugated groups, αg (2.06%, >85%), ßg (7.59%, >85%), and γg (0.29%, >85%) that were very labile even in mild conditions, were also collected. The method described here can be used as an effective protocol to separate different soyasaponins occurring in the original sample.

Preparation and characterization of a molecularly imprinted polymer by grafting on silica supports: a selective sorbent for patulin toxin.

A new molecularly imprinted polymer (MIP) has been prepared on silica beads using the radical "grafting from" polymerization method for selective extraction of minor contaminant mycotoxin of patulin (PTL). After the introduction of amino groups onto the silica surface with 3-aminopropyltriethoxysilane, azo initiator onto the silica surface was achieved by the reaction of surface amino groups with 4,4'-azobis(4-cyanopentanoic acid). The scale-up synthesis of MIP was then carried out in the presence of 6-hydroxynicotinic acid as template substitute, functional, and cross-linking monomers. The prepared sorbent was characterized using FT-IR spectroscopy, scanning electron microscopy, elemental analysis, and the adsorption-desorption selectivity, and the capacity characteristic of the polymer was investigated by a conventional batch adsorption test and Scatchard plot analysis. The results indicated that coated polymers had specific adsorption to PTL as compared with its co-occurring 5-hydroxymethyl-2-furaldehyde (hydroxymethylfurfural (HMF)), at the same bulk concentration for solution of PTL and HMF, the maximum absorbance in the solid-phase extraction (SPE) method to PTL were 93.97% or 0.654 µg/mg while to HMF they were 76.89% or 0.496 µg/mg. Scatchard analysis revealed that two classes of binding sites were formed in PTL-MIP with dissociation constants of 3.2 × 10(-2) and 5.0 × 10(-3) mg/mL and the affinity binding sites of 8.029 and 1.364 mg/g, respectively. The recoveries of PTL were more than 90% for the developed MISPE and around 75% for the traditional liquid-liquid extraction in spiked apple juice samples. It was concluded that the method is suitable for the scale-up synthesis of PTL-MIP grafted on silica, and the polymer can be effectively applied as SPE coupled with high-performance liquid chromatography (HPLC) for the determination of PTL in apple juice or other related products.