1,Study On Two New Monascus Metabolites With Fluorescence Isolated From Red Yeast Rice 2,Development Of Gold Immunochromatography Assay For Rapid Detection Of Deoxynivalenol (DON)

Red yeast rice is produced by growing Monascus sp.on rice to produce a red-colored product.Although for years it has been known that there are six pigments and other metabolic products from Monascus sp.,such as alcohols,organic acids,and substances with a wide range of biological and therapeutic benefits,including anticarcinogenic,antioxidative,and hypolipidemic activities,which have been described,in the past decade,some new pigments and metabolites have been isolated and their chemical structures have been characterized. However,some fluorescent compound products from Monascus sp.have received less attention.Therefore,the first part in this study,two new Monascus metabolites from red yeast rice were studied.1 Two new Monascus metabolites with similar fluorescence spectra(λex=396 nm,λem=460 nm) and UV absorption spectra were detected.They were isolated by rechromatography on a silica gel column and semipreparative HPLC,and two strong blue fluorescent compounds were obtained.Their structures were elucidated by electrospray ionization mass spectrometry(ESI-MS),electrospray ionization tandem mass spectrometry(ESI-MS/MS), intensive ESI-MS,and nuclear magnetic resonance spectroscopy(1H NMR,13C NMR,COSY, and HMBC) studies.High-resolution mass spectrometry indicated the molecular formulas C₂₁H₂₄O₅ and C₂₃H₂₈O₅.The two new compounds,named monasfluore A(MFA) and monasfluore B(MFB),respectively,contain a alkyl side chain,γ-lactone,and propenyl group, whereas themore lipophilic compound,MFB,is a higher homologue of MFA,with the more lipophilic octanoyl instead of the hexanoyl side chain.2 A specific HPLC method has been developed and validated for the simultaneous determination of MFA and MFB in red yeast rice.Chromatographic separation was achieved at room temperature using a 250×4.6 mm I.D.,5μm Symmetry C₁₈ column(Waters),with isocratic elution of water/acetonitrile(23:77,v/v) at a flow rate of 0.8 mL/min.The average recoveries of the method for MFA and MFB were 85.2%and 82.3%,respectively,and the RSD were 4.2%and 5.3%,respectively.The linear equations of MFA and MFB were Y=772.5X-289.2 and Y=2376.6X+1346.4 respectively,and the correlation coefficients were 0.9994 and 0.9972,respectively.The linear ranges of MFA and MFB were from 0.5 to 10 ng/mL and from 0.25 to 5 ng/mL,respectively.20 samples of red yeast rice were analyzed directly by this method.The results showed that the maximum production of MFA and MFB by NO.13 Monascus sp.were 81.4 and 26.3 g/kg,respectively,and the minimum production of MFA and MFB by NO.14 Monascus sp.were 0.01 and 0.003 g/kg,respectively.3 The stabilties of MFA and MFB for strong acid,strong alkali,heat and light were studied, and the interaction between BSA and MFA and MFB were also studied.The results showed that the stabilties of MFA and MFB for alkali,heat,light were poor,but the stabilties of MFA and MFB for strong acid were better than for strong alkali.MFA is a lower homologue of MFB,with the less lipophilic hexanoyl instead of octanoyl side chain,and this results in a less interaction with BSA.4 The inhibition proliferation of cancer cell lines,Hep G2 and A549,and human normal cell lines,WI-38,were determined by methyl thiazolyl tetrazolium(MTT) assay method.The results indicate that the notable inhibition proliferation on A549 and Hep G2 are found in MFA and MFB,but without too significant inhibition toward normal fibroblasts WI-38 at the same concentrations of 5-20μg/mL.The concentrations of 50%percent inhibition(IC₅₀) of MFA in the two tumor cell lines(HepG2 and A549),were found to be around 12.4 and 16.3μg/mL, respectively,and IC₅₀ of MFB in HepG2 and A549 were about 7.1 and 10.6μg/mL These results indicate that the length of the saturated side chain on the ketonic carbonyl group of MFB is an important factor toward the inhibition proliferation. Deoxynivalenol(DON) is produced largely by Fusarium fungi,which are widespread in nature and commonly contaminate many cereal grains such as wheat,corn,barley,oats,and rye intended for human and animal consumption.At present,the determination of DON is normally carried out using gas chromatography(GC) with either electron capture or mass spectrometric(MS) detection,or high-performance liquid chromatography(HPLC) with ultraviolet(UV) detection,fluorescence detection after postcolumn derivatization or MS detection.The merit of chromatographic methods is that they allow highly sensitive measurements of individual toxins simultaneously.But they are thought to be unsuitable for measuring many samples within a short time,because lengthy sample preparation such as column cleanup and derivation is required to achieve high sensitivity.Furthermore, high-priced measurement machines and highly qualified personnel are required in order to apply these methods,and the running costs are generally high.In addition,some researchers have established enzyme-linked immunosorbent assay(ELISA) for DON,and it is also unsuitable for on-site detection because it requires labor-intensive operations including incubation,washing,and enzymatic reactions during signal generation.There is a need for rapid and cheap but reliable methods that can be conducted and interpreted by users who are close to the site of contamination.Therefore,the second part in this study,a colloidal gold immunochromatographic strip(ICS) test for rapid detection of DON in wheat and maize samples was developed.1 For the development of a sensitive ICS test,it is necessary for the optimal pH and amount of antibody for the labeled gold colloid.After centrifugation,the titer of supernatant and original antibody was compared by ELISA to determine the optimal pH value.The minimum amount of anti-DON Mab was determined by adding NaCl to colloidal gold particles containing different amounts of Mab.After reaction with NaCl,a colloidal gold solution containing minimum Mab could be kept reddish or red-orange color.It was found that the optimal pH value for the labeled colloidal gold was 7.0 and the minimum amount of Mab was 30μg for 1 mL colloidal gold. 2 A total of 0.74μL per 1 cm of DON-cationic bovine serum albumin(CBSA)(0.8 mg/mL) conjugate and goat anti-mouse IgG antibody(1.0 mg/mL) were sprayed onto NC membrane(P 40) as the T and C line,and a total of 5μL/cm of colloidal gold-labelled anti-DON Mab diluted three times,was jetted on the treated conjugate pad.In addition,the nonspecific reaction was removed by adding 0.005%anion surfactant to the solution,and it appears that anion surfactant may neutralize the positive charge of the CBSA.3 The cross-reacted rates between the DON rapid test strip and aflatoxin B1 nivalenol,T-2, HT-2,ZEN,and citrinin were poorly.The ICS test had a visual detection limit of 50 ng/mL for DON.The validity of DON rapid test strip was at least 5-6 months at 25℃.4 The sample preparation methods for the ICS were investigated.It was found that the test results of ICS were not significantly affected by size of the sample,different water,mixing time and standing time in this experminement.To perform the test,5 g of sample was extracted in a ratio of 1:20 with water by shaking for 2 min,standing 2 min,and then the extract directly test without further cleanup steps.After 10 min,the test results were obtained, and the whole process was about 15 min.5 Analysis of DON in wheat and maize samples revealed that data obtained from ICS test were in a good agreement with those obtained from ELISA.The results demonstrate that the ICS test can be used as a reliable,rapid,cost-effective and convenient qualitative tool for on-site screening technique of DON in wheat and maize samples.