Studying On The Detection And Control Of Aflatoxin Contamination In Fivetypical Chinese Medicinal Materials,Preparation And Simple Application Of Ochratoxin Immunoassay Strips

The mycotoxins in Traditional Chinese medicines（TCMs）mainly included aflatoxins（AFs）,ochratoxins（OTA）,fumonisins（FBs）,T-2 toxins,etc.Studies have shown that these toxins are toxicity.Among them,AFs and OTA are easy to pollute TCMs,which is a serious threat to the use of drug.OTA is second only to AFs,both of which are human carcinogens.Therefore,it is essential to study the rapid detection of mycotoxins and research on pollution prevention and control technology is crucial in common herbal medicines.This paper studies the following contents:1.Determination of aflatoxins in five typical traditional Chinese medicine decoction pieces by IAC-HPLC-FLD methodFour aflatoxins（AFB₁,AFB₂,AFG₁ and AFG₂）in TCM decoction pieces ware detection by utilizationing IAC-HPLC-FLD method,which extracted with 70%methanol,purified by immunoaffinity column,and separated by liquid chromatography-fluorescence detection.The pretreatment method was optimized,so that this method can simultaneously satisfy five kinds of TCM decoction,which included Polygalaceae,Platycladi Seed,Cassia,Codonopsis pilosula,and Coix seed.114 batches of 5 Chinese herbal medicines was collected and tested,26 batches of Platycladi Seed(AFs 1.22⁴6.67μg·kg^-1,AFB₁ 1.22³1.40μg·kg^-1),4 batches of Chinese wolfberry(AFs1.97⁴1.13μg·kg^-1,AFB₁ 1.97³6.40μg·kg^-1),1 batch of Cassia(AFs 13.65μg·kg^-1,AFB₁ 12.60μg·kg^-1)was detected positive,Codonopsis was not detected,13 batches of Polygalaceae(AFs 2.40¹94.46μg·kg^-1,AFB₁ 2.03⁹0.04μg·kg^-1),the positive rate was 41%,the over-standard rate is 15%.2.Discussion on the key points of aflatoxin to occur during the initial processing of Polygalaceae,and the method of detoxificationAflatoxin was seriously polluted in Polygala,but there ware few studies on the key points of its toxin production and the control of mold resistance.This chapter investigates and collects the initial processing of Polygala from Shanxi origin through field visit,and simultaneously simulates the initial processing of Polygala in the laboratory to test the production of aflatoxins B₁,B₂,G₁ and G₂ under various initial conditions,including burying,sweating,core pulling,etc.the decoction pieces obtained from the initial processing were storage at 28°C and the humidity ware 82%⁹0%;96%respectively.After storaged,the occurrence of toxins was determined,to investigate the occurrence of aflatoxins in the processing-storage process.Firstly,the samples obtained in each of the initial processing periods ware all negative.Secondly,the culture results under the two kinds of humidity conditions showed that when the humidity was 96%,the mold was severely spoiled within 7 days,and detected the contents of AFB₁ and AFs were 4.01μg·kg^-1,5.36μg·kg^-1,respectively.Therefore,the harvested fresh products ware treated in a timely manner and dried,which was essential for the anti-mild storage of the Polygala.For further experiments,the two batches of toxins with higher and lower than the limit of toxin content ware collected,No.14(AFs,AFB₁,159.44,68.66μg·kg^-1)and No.39(AFs,AFB₁,2.08,2.08μg·kg^-1).Firstly,the content of aflatoxin ware measured under water and 75%ethanol washing.The removal rate of AFB₁ in No.14 ware 92.47%,64.01%after washed with water and 75%ethanol;No.39 with no toxin after washed with water and 75%ethanol,and the removal rate of AFB₁ was≤100%.Secondly,the change of toxin content in the process from decoction to granules was determined,and AFs,AFB₁ became 60.61,11.97μg·kg^-11 in the No.14,the transfer rate ware 38.01%,17.43%,respectively.Toxin was not detected and its transfer rate was ND in the No.39.3.Discussion on the key points of aflatoxin to occur during the initial processing of Platycladi Seed,and the method of detoxificationThe detoxification of mycotoxins in Platycladi Seed which mainly refers to the physical,chemical,biological and other methods of removing,destroying and reducing the action of toxins.Monitoring of medicines in the processing and storage of production areas will prevent mycotoxins becoming a source of pollution to human health.The production conditions of AFB₁,AFB₂,AFG₁ and AFG₂ were detected by simply simulating the initial process.The results showed that no AFs ware detected during the simulation in the unfinished products.In addition,AFs detected in the unfinished and the finished products which were collected in the last year exceeded the limit and the pollution was serious.In the process of initial processing of the medicinal material base,there ware two situations where that have been contaminated by toxins before being obtained from cypress,or contaminated by toxins during the process of storing cypress,which further illustrates the importance of monitoring the initial link of traditional Chinese medicine processing.Two batches in Platycladi Seeds ware selected that with toxin content higher and below than the limit.The initial contents of AFs and AFB₁ toxin were 23.95,23.17μg·kg^-1;3.94,3.94μg·kg^-1;Detoxification toxin experiments were carried out to determine the changes of toxin content with AFs under water and 75%ethanol washing.The removal rate of AFB₁ after washing with water and 75%ethanol ware 77.69%,≤100%,and the removal rate of AFs ware 78.41%,≤100%;,No.2 sample with no toxins was detected in the two removal methods,and the removal rates of AFB₁ and AFs were all≤100%after washing.4.Preparation and simple application of the OTA immunoassay stripThe self-assembly colloidal gold immunoassay strip of OTA was preparated that based on the competition principle and the colloidal gold-labeled antibody probe by optimizing the coupling ratio of the gold-labeled antibody,the resuspension system,the C-line and the T-line concentration.Added the antibody of 5μg,10 mM PBS（1%sucrose,0.02%MgSO₄,0.1%Tween 20）in a resuspension solution,used NC140 nitrocellulose membrane,with a visual detection limit of 2.5μg·mL^-1 and 10²0 detection time.The samples collected by 70%methanol-water extraction,to investigate the influence of sample matrix on the detection of colloidal gold test strips.The method was to meet the requirements of the EU limit standard of15μg·kg^-1.At the same time,the established LC-MS/MS method for multitoxin detection confirmed the sample detection results of the immunochromatographic analysis method.