The Profiles,Structural Changes during Oxidation and in Vitro Antitumor and Anti-Inflammatory Activities of Linusorbs in Flaxseed(Linum Usitatissimum L.)
Author:Zou Xian Guo
Supervisor:deng ze yuan
Flax（Linum usitatissimum L.）orbitides are a series of minor hydrophobic cyclic peptides with special structure existing in flax seed and rhizome,and mainly dissolved in oil during flaxseed processing.To date,more than 20 types of flax orbitides have been found,and their difference in structure was only one or two amino acid residues.Studies have shown that flax orbitides played an important role in the oxidation process of flaxseed oil and affect oils’quality.Other studies have shown that flax orbitides exhibited various biological activities,such as binding with biomacromolecules and metal ions,immunosuppressive,antioxidant,anti-inflammatory and anticancer activities.Therefore,natural existing flax orbitides have the potential value of being used as drugs or drug substitutes.However,so far,studies reporting the biological characteristics of flax orbitides were limited,and reported researches were not deeply enough.To deeply understand the physical and chemical characteristics and biological function of flax orbitides as well as increasing its exploitation and application in functional foods,this study evaluated the difference in the distribution and content of orbitides from 13 Chinese cultivated oil and fiber flax samples using silica gel column separation and HPLC/ESI-QTOF-MS/MS detection technology.Besides,this study explored the change pattern of flax orbitides during the oxidation of flaxseed oil,and the relationship between flax orbitides and common oxidation indicators.In addition,this study investigated the in vitro antitumor and anti-imflammatory activites as well as underlying molecular mechanism of crude flax orbitides and 2different structural flax orbitides.The main research results were listed as follows:1.The differences in proximate compositions,fatty acids profile and micronutrients including orbitides as well as tocopherol in flaxseeds or their oils from13 Chinese cultivated oil and fiber flax samples were compared and evaluated.Totally 14 types of orbitides in 13 oils were identified and quantified by HPLC-ESI-QTOF-MS/MS,which varied from 272.90 to 434.43μg/g of oil,and the highest content of total orbitides was found in fiber flax Zhongya4.Only oil flax Dingya23 showed significantly higher oil content（41.36%）withα-linolenic acid（ALA）level（55.4%of fatty acids）over fiber flax,thus it is a qualified cultivar.And the mean proportions of ALA andγ-tocopherol in oils from oil flax were similar to those in oils from fiber flax（51.37 vs 52.20%,and 34.16 vs 33.79 mg/100 g of oil respectively）.In addition,the fiber flax showed higher mean levels of crude protein,total orbitides and orbitide[1–9-NαC]-linusorb B2 than those of oil flax.2.[1–9-NαC]-linusorb B2 and[1–9-NαC]-linusorb B3 with different structures were prepared using silica gel column chromatography and AKTA purification technology.The purity of 2 flax orbitides were 99.99%（[1–9-NαC]-linusorb B2）and97.45%（[1–9-NαC]-linusorb B3）respectively detected by HPLC,and their structures were confirmed by NMR.In addition,orbitide[1–9-NαC]-linusorb B3 crystal was obtained using solvent crystallization method.3.The change pattern of flax orbitides during the oxidation of flaxseed oil and the possibility of using methionine sulfone（Msn）-containing orbitides as oxidation indicators were explored.After 4 days’heating,oxidation values slightly increased（p>0.05）with significant decrease in methionine（Met）-containing peptides（p<0.05）instead ofγ-tocopherol（p>0.05）.During the whole oxidation process,methionine sulfoxide（Mso）-containing orbitides increased and then decreased,Msn orbitides were increasing.Results showed that Met orbitides was oxidized to Mso orbitides and further to Msn ones.However,as oxidation time continues increasing（4-24 days）,oxidation values significantly increased（p<0.05）with significant reduction ofγ-tocopherol（p<0.05）.It demonstrated that Met-containing peptides were more readily oxidized compared withγ-tocopherol and showed certain antioxidant activity.Besides,high logarithmic correlations were found between oxidation values and Msn-containing orbitides（0.94-1.00）,such as between total carbonyl compounds and orbitide[1–8-NαC],[1-MetO2]-linusorb B1（64.95 lnx–52.14,R2=0.99,Dingya23oil）.Therefore,Msn-containing orbitides may be good indicators to evaluate the oxidation process of flaxseed oil.4.The influnce of crude orbitides isolated from flaxseed on the growth of SGC-7901 cancer cells were investigated.Results showed that 120 and 200μM crude flaxseed orbitides significantly killed off SGC-7901 cancer cells by inducing apoptosis in a dose-dependent manner,which were confirmed by the appearance of nuclear shrinkage and DNA fragmentation.The molecule mechanism was that the mitochondrial apoptosis pathway-related protein cytochrome C（Cyt C）was released from mitochondria to cytosol,associated with the activation of caspase 9 and 3,the increase of Bax/Bcl-2 ratio and the cleavage of PARP.5.Cellular uptake of methionine containing orbitide[1–9-NαC]-linusorb B2 and isoleucine containing[1–9-NαC]-linusorb B3 in SGC-7901 cells were detected,and their antitumor activities in the aspect of inducing apoptosis as well as its underlying molecule mechanism were investigated.Results showed that the accumulation of 2orbitides in cells increased with the increase of incubation time,and the absorption efficiency of[1–9-NαC]-linusorb B3（19.46%,36 h）was higher than that of[1–9-NαC]-linusorb B2（15.15%,36 h）.Both orbitides can decrease cell viabilities in a dose-and time-dependent manner and the IC50 value in 36 h was 195.55 and 223.07μM,respectively.Besides,through the evaluation of mitochondrial membrane potential（Δφm）,Cyt C release,caspase,death receptor activation and cleavage of PARP,[1–9-NαC]-linusorb B3 exhibited more obvious apoptosis induction on SGC-7901 cells than[1–9-NαC]-linusorb B2.The mechanisms were:[1–9-NαC]-linusorb B3-induced cell apoptosis might involve a mitochondrial-and death receptor-mediated intrinsic and extrinsic pathway;however,[1–9-NαC]-linusorb B2induced cell apoptosis by the involvement of only extrinsic pathway.6.The anti-proliferative effects of[1–9-NαC]-linusorb B2 and[1–9-NαC]-linusorb B3 on SGC-7901 cells as well as the specific mechanisms were investigated.Results showed that the 2 flax orbitides could dose-dependently induce cell cycle arrest of SGC-7901 cells in the G1 phase,accompanied with the downregulation of CDK2,CDK4,cyclin D3,cyclin E as well as upregulation of p21WAF1/CIP1 and p27KIP1IP1 analyzed by Western blot,thus inhibit cells proliferation.However,[1–9-NαC]-linusorb B2 was more sensitive in suppressing AKT activation than[1–9-NαC]-linusorb B3.In addition,although two orbitides treatment activated p38 MAPK,JNK,and ERK protein expressions,inhibitor of JNK（SP600125）,neither p38（SB203580）nor ERK（PB98095）,promoted[1–9-NαC]-linusorb B2-or[1–9-NαC]-linusorb B3-induced G1 phase arrest.7.The anti-inflammatory effects of[1–9-NαC]-linusorb B2 and[1–9-NαC]-linusorb B3 as well as the specific mechanisms were investigated.Results showed that both two flax orbitides at low not high concentrations（1-4μM）exhibited in vitro anti-inflammatory activities in LPS-induced THP-1 macrophages through the suppression of pro-inflammatory cytokines（TNF-α,IL-1βand IL-6）as well as the deactivation of NF-κB pathway（p-IKKα/β,p-IκBαand p-p65-NF-κB）.Besides,their inhibitory concentrations towards the release of NO and protein expression of COX-2were limited to 1-4μM.It is interesting to note,their concentrations in deactivating NF-κB pathway were different:for[1–9-NαC]-linusorb B3,1 and 2μM were enough,and for[1–9-NαC]-linusorb B2,4μM was needed.