Risk Assessment Of Phthalates Exposure In Chinese Rice Wine And Study On Antioxidant Activities Of Chinese Rice Wine

Objects:To get information of Chinese Rice Wines(CRW) consumption of adult males in Shanghai, to detect the pollution levels of phthalates (phthalic acid esters, PAEs) in commercial CRW and to assess the health risk of PAEs cumulative exposure in adult males in Shanghai.To establish and modify the antioxidant activity evaluation system and methods in vitro, to observe the antioxidant ability in vitro and in vivo and to determine the phenolics content and distribution of CRW.Methods:1. Risk Assessment of PAEs Cumulative Exposure in Commercial CRW to Adult Males in ShanghaiThe investigated population were the adult male residents in Shanghai in 18～80 years old,720 objects were selected using multi-stage stratified random sampling method. Through household survey, to get the information of the consumption of CRW in different packaging types through drinking and cooking of adult males in Shanghai and then obtained the average daily intake(mL) as total.164 samples were obtained from randomly sampling in commercial CRW in local Supermarkets, which were in different brands and different packaging types,16 were in Urn or altar,42 were in Glass bottle,60 were in Plastic Bucket and 46were in Plastic Bag. The distribution and concentration of the 16 PAEs were determined by GC-MS according to the method in GB/T21911-2008 Determination of Phthalates in Food.Dietary intake of PAEs from CRW in adult males in Shanghai was simulated by Monte Carlo simulation, based on the PAEs content in commercial CRW and the CRW consumption in the population, and health risk associated with PAEs intake was evaluated by applying Cumulative Exposure risk assessment method.2. Study On Antioxidant Activities of CRW in vitroBased on the literature and preliminary experiments, antioxidant activity evaluation method system in vitro of CRW was established, which covered different detected mechanisms methods selected according to the laboratory conditions and the situation of CRW. Then the method which can simultaneously determine the main phenolics in CRW by high performance liquid chromatograph (HPLC) was established. Using these established methods, the antioxidant activity in vitro and phenolics content of 51 commercial CRW from 4 different places of origin were determined, and the data were compared and analyzed among different ingredients, types and ages. Finally, a modified determination method of oxygen radical absorbance capacity(ORAC) in CRW by fluorescence spectrophotometer was tried. Commercial CRW were used as determined material, ORAC were determined by a fluorescence spectrophotometer using half fluorescence decay time as the observation index, Calibration curve of Trolox as the quantitative standard. The detection limit, repeatability precision, parallel precision and the recovery rates of spiked samples of the method were determined.3. Study On Antioxidant Activities of CRW in vivoThe effect of CRW on the antioxidant ability in aged mice induced by D-galactose was observe. CRW were concentrated to 1/3 of initial volume, and the amount of total phenolic compounds and total flavones were determined. Sixty ICR mice were randomized into 5 groups, the D-galactose were injected neck back subcutaneously at doses of 125 mg/kg·d to the mice in model group and CRW groups, the mice in CRW groups were oral administrated concentrated CRW at three different doses of 1.5,3.0,9.0 mg/kg-d respectively, the effect of CRW on the content of malondialdehyde (MDA), protein carbonyl group (PCO), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum, brain and liver were observed after 45 d.Results:1. The effective objects was 634, considering the consumption both drinking and cooking, the frequency of average daily intake of CRW in 0.1-49.9 mL was 575 (90.69%), that in 50.0-99.9 mL was 40(6.31%), and that in 100.0-300.0 mL was 19 (3.00%), the minimum value of average daily intake of CRW was 6.25 mL, the median was 13.72 mL and the maximum was 300 mL.6 of the 16 PAEs in CRW samples were detected in at least one sample, but the detection rate were not high, the order of detected frequency from high to low was 25 (15.24%) of DIBP,24 (14.63%) of DMP,22 (13.41%) of DBP,22 (13.41%) of DEHP, 11 (6.71%) of DEP and 10 (6.10%) of BBP, and the detected content was 0.05106-0.17432 mg/L.rice wine, the PAEs detection rate was different among the CRW in different packing types, all PAEs were not detected among the samples in urns or altars,4 PAEs(DEP, DIBP, DBP and DEHP)were detected among the samples in glass bottle, and 6 PAEs were all detected among the samples in plastic buckets and bags.The average exposure level of the 6 PAEs (DMP, DEP, DIBP, DBP, BBP and DEHP) to adult males in Shanghai were 0.0066～0.0071 μg/(d·kg), and the highest exposure level were 0.1374～0.5405 μg/(d·kg). The cumulative exposure health risk index(HI) based on the average exposure level of the 5 PAEs (DEP, DIBP, DBP, BBP and DEHP) was 0.001141 (1/876), and the HI based on the highest exposure level of those was 0.062969 (1/16), both are far less than 1. Whether in the condition based on the average or highest exposure level, the main source of health risk are from DBP and DEHP. The carcinogenic risk index Rs of DEHP based on the median and maximum cumulated DEHP exposure level was 1/22556 and 1/291 of the present liver cancer incidence in adult males in Shanghai.2. Antioxidant activity evaluation detection system in vitro of CRW including 6 indicators was established, including Superoxide Anion Radical Scavenging Rate(SARSR), Hydroxyl Radical Scavenging Rate(HRSR),Oxygen Radical Absorbance Capacity(ORAC), Trolox Equivalence Antioxidant Capacity(TEAC), DPPH(1,1 diphenyl-2-trinitrobenzene hydrazine) Radical Scavenging Rate, Ferric Reducing Antioxidant Power (FRAP) and Total Phenolics(TP).A methodology employing reversed-phase high-performance liquid chromatography (RP-HPLC) combined with UV-vis detector had been developed and validated for simultaneous determination of thirteen phenolic compounds, including gallic acid(GA), protocatechuic acid(PRCA), vanillic acid(VA), syringic acid(SRA), caffeic acid(CA), ferulic acid(FA), p-coumaric acid(pCA), sinapic acid(SA), chlorogenic acid(CHA), (+)-catechin(CAT), (-)-epicatechin(EPI), quercetin(QUE) and rutin(RUT), in rice wine. Phenolic compounds were separated with a C18 reversed-phase column by gradient elution using 3% acetic acid aqueous solutionin (solvent A) and acetonitrile (solvent B) (0 min,5% B; 5 min,8% B; 10 min,15% B; 20 min,25% B and 25 min,5% B) as the mobile phase at a flow rate of 1.0 ml/min, the column temperature was at 38℃, UV-vis detector was set at 280 nm. Rice wine sample(20 μL) filtrated through a 0.45 am polyvinylidene fluoride membrane was injected into the HPLC directly. Results showed that the chromatograms of all analytes were observed within 20 min, all calibration curves were linear (R2> 0.995) within the range, limits of detection (LOD) ranged from 0.02 to 0.06 ug/mL, recoveries of 88.07-106.80% and relative standard deviations of 0.05-5.36%were obtained for simultaneous determination of these phenolic compounds.SARSR of commercial CRW was (59.85+9.35)%, HRSR was (59.42+9.63)%, ORAC was (3.97±0.72) μmol TE/ml、TEAC was (3.09±0.44) μmol TE/mL、FRAP was (2.67±0.33) μmol TE/ml、TP was (271.18±49.92) μg PE/mL. The total phenolics content of the 13 constituents measured by HPLC was (61.34±11.85) μg/mL, the highest content among the individual constituents was (29.78±8.17) μg/mL of CAT and the lowest one was (0.65±0.18) μg/mL of CA. The antioxidant activity in vitro and a variety of phenolics content of the millet rice wines were lower than those of the glutinous rice wine and rice wine. Among the CRW original from Jiangsu, Zhejiang and Shanghai, there were no significant difference in the antioxidant activity in vitro and phenolics content in different winerys, types and ages. Good correlations were observed among the selected 6 indexes presented the antioxidant activity in vitro. Phenolics content was closely related with antioxidant activity in vitro of CRW. Ten of the 13 detected phenolics were positively correlated with one or more antioxidant activity indicators, some of which the content was very low while the regression coefficient was relatively larger.In the Method established of ORAC determination in CRW by Fluorescence Spectrophotometry, excitation wavelength was 490nm, Emission wavelength was 517nm. The linear regression equation between relative half fluorescence decay time and Trolox concentration was Y= 4.3897X+9.0303, the R-squared was 0.9978, detection range was 0.28-10.00μmol Trolox/mL. The repeatability precision and parallel precision of this established method were 3.02% and 2.69% respectively. The recovery rates of spiked samples were in the range of 93.00%～102.00%. The ORAC value of determined Chinese rice wines were form 4.52 to 5.36μmol Trolox/mL3. The total phenol content in rice wine concentrate used in the experiment were 719.2 μgPE/mL, total flavonoid content were 499.0 μgCE/mL, Which retained more than 95% phenolic active ingredients of the original CRW. In serum, brain and liver of the mice in model group, the content of MDA and PCO were higher and the content of SOD and GSH-Px were lower than those in the normal group (P<0.01). Compared with the index in serum, brain and liver of the mice in model group, the content of MDA and PCO decreased and the content of SOD and GSH-Px increased significantly in the Middle and High dose CRW groups.Conclusions:1. CRW was generally consumed by the adult males in Shanghai, although multiple PAEs are detected in commercial CRW, but such exposure levels raised a very low level of health risk and carcinogenic risk in the target adult males in Shanghai.2. The 6 indexes including SARSR, HRSR, ORAC, TEAC, FRAP and TP can be used to overall evaluation of antioxidant activity in vitro of CRW. The established HPLC method was a simple, accurate and rapid method, and it is suitable for determining the phenolic compounds in various types of rice wines. CRW had a strong antioxidant activity in vitro and phenolics content was positively correlated with it, the role of a variety of trace amounts of phenolic substances in CRW to the antioxidant activity in vitro should not to ignore. Among the CRW original from Jiangsu, Zhejiang and Shanghai, there were no significant difference in the antioxidant activity in vitro and phenolics content in different winerys, types and ages. With good accuracy, precision and stability, ORAC fluorescence spectrophotometry simplifies the operation procedure, shorten the experiment time, it can be used to determine the ORAC in Chinese rice wine of different brands and different ages.3. CRW can lighten the antioxidant ability decreasing of aged mice induced by D-galactose in a certain degree.