| Exp.1Optimized HPLC Method for Determining Free Gossypol inCottonseed MealA method of high-performance liquid chromatographic (HPLC) wasdeveloped for the determination of free gossypol in cottonseed meal. Thegossypol in cottonseed meal was extracted with acetone by ultrasound,centrifugation, suction filter, rotating evaporation, and then dissolved withmixture of acetonitrile and0.2%phosphoric acid (85/15, v/v). The optimalconditions of HPLC were as follows: the column was Agilent TC-C18(250mmx4.6mm,5μm), mobile phase was mixture of acetonitrile and0.2%phosphoricacid (85:15, v/v), with a flow rate of1.0mL/min. UV detected wavelength wasset at235nm, with injected sample volume of20μL and column temperature of25℃. The equation of the standard curve was y=189347x+24969(R=0.99999),which had a highly linear relationship when gossypol concentration rangedfrom0.1224ug/mLto78.336ug/mL, and the minimum detection limit ofgossypol was0.52mg/kg. The relative standard deviation of gossypol extractedfrom cottonseed stability at4℃within36hours was1.51%～3.43%.The average recoveries of the method were94.72%～99.49%, with relativestandard deviation of1.72%～3.13%. In conclusion, the results showed that theproposed HPLC method was an efficient means to measure free gossypol incottonseed meal.Exp.2Qualitative analysis of gossypol and its derivatives in cottonseedmeal by high performance liquid chromatography ionization-iontrap-time-of-flight mass spectrometryThe content of free gossypol determined by spectrophotometer method andHPLC in cottonseed meal was700.05,60.04mg/kg, respectively. To explainthe significant difference between the two content determined the two methods,LCMS-IT-TOF method was conducted for qualitative analysis of free gossypoland its derivatives. Samples were separated by a Shimadzu Shim-pack XR-ODScolumn(2.0mm×75mm,2.2μm) using gradient elution with a mobile phase of0.1%formic acid-acetonitrile, and analyzed by HPLC-ESI-MS. Objectiveswith similar molecular weight to gossypol were scanned and analyzed withmultistage mass spectrometry in negative ion mode. On the basis offragmentation pathways of gossypol, molecular structures of gossypolderivatives were specultated with Shimadzu Formula Predictor softwareaccording to rules of organic compounds degradation, isotope, unsaturation,nitrogen, and MSninformation, Result showed that cottonseed meal containedtwelve kinds of gossypol derivatives. Among them, molecular structures fromseven kinds of derivatives were speculated and analyzed. These derivatives originated from gossypol with the loss of hydrone or isopropyl methyl group, ormethyl substituting hydrogen in hydroxyl, or nitrogen substituting oxygen inaldehyde and prodcing C=N Structure, or aldehyde substituting methyl inisopropyl. Results above showed that content of so called free gossypol incottonseed meal determined by spectrophotometer method include real feeegossypol and twelve kinds of gossypol derivatives, these derivatives may havesimilar biological functions as gossypol.Exp.3Effects of gossypol-acetic acid on growth performance and serumbiochemical indicators of tilapia, Oreochromis niloticus×O.aureusAn8-week feeding experiment was conducted to evaluate the effects ofgossypol–acetic acid on growth performance and hematological index of tilapia,Oreochromis niloticus×O.aureus. Fish with initial body weight of1.27±0.06gwere fed with basal diet supplemented with0(control diet),300,600,900,1200mg/kg gossypol-acetic acid. Results showed that weight gains (WG) weregradually decreased and feed conversion ratios (FCR) were gradually increasedwith the increasing adding amouts of gossypol-acetic acid. WG of600,900,1200mg/kg gossypol-acetic acid groups decreased13.08%,20.13%,29.76%,FCR increased8.16%,14.96%,26.51%, compared with control grouprespectively. WG of900,1200mg/kg gossypol–acetic acid groups weresignificantly lower than that of control (P <0.05), and FCR of1200mg/kggossypol–acetic acid groups were significantly higher than that of control (P <0.05). Serum ALT of1200mg/kg group was significantly higher than the control group (P<0.05); There was no significant difference in serum TP, ALB, UREAamong treatments (P>0.05). Results above showed that, when dietarygossypol-acetic acid supplementation reached900mg/kg， the growthperformance of Oreochromis niloticus×O.aureus was decreased；when dietarygossypol-acetic acid supplementation reached1200mg/kg，the liver functionof Oreochromis niloticus×O.aureus was damaged. |
PDF Full Text Request