| Walnut(Juglans regia L.)is a kind of nut with higher nutritional value.It is abundant in phenolic substances,which have a lot of functions including cardiovascular protection,anti-oxidation and immune regulation.Fenitrothion(FNT)is a widely used organphosphorus pesticide which is toxic to the nervous system,inmune system,and reproductive system.Previous studies have found that walnut polyphenols have reducing effects on the immune toxicity induced by FNT.However,the active components with the immune protective effects in Walnut Polyphenol Extract(WPE)still remain unclear.In this paper,total polyphenols content and immune protection of walnut and other three nuts were compared firstly.The study researched the immune protective effects of walnut polyphenols on the spleen lymphocytes induced by FNT.WPE was separated into several different components by silica gel chromatography,and the immune protective effects of different components were measured.High performance liquid chromatography electrospray ionization well/time of flight tandem mass spectrometry(HPLC-ESI-TOF-MS)was used to identify the active components of walnut polyphenols.The main results were as follows:(1)The total phenolic content of the unprocessed walnut,apricot kernel,peanut,and pine nut polyphenol extracts were determined by Folin-Phenol method.The content of total polyphenols in WPE was higher,which was mainly concentrated in the walnut seed coat.MTT cell proliferation assay was used to compare effects of walnut,apricot kernel,peanut,and pine nut polyphenol extracts on the immune toxicity of splenic lymphocytes induced by FNT.The results showed that WPE had a significant effect on mitigating the immunity toxicity of spleen lymphocytes,while the polyphenols extracted from walnut seed coat had better effect of relieving immune toxicity among all the parts of walnut being studied.(2)MTT cell proliferation assay and ELISA technique were used to further study the immune protective effects of WPE.The effects of WPE were studied on the T/B lymphocyte toxicity and the production of cytokines interleukin-4,interferon-?,interleukin-6,and granzyme B which were induced by FNT.The results showed that the immune toxicity of FNT on spleen lymphocytes was significantly alleviated.The proliferation of T lymphocytes and the secretion of IL-4,IFN-?,and granzyme B were significantly increased by WPE.(3)In order to further study the active components of walnut polyphenols with immune protection,11 fractions of FO-F10 were separated from WPE by different concentrations of the eluent with silica gel column chromatography.F8 showed the optimal immune protection in 11 components at the ratio of chloroform to methanol was 2:8 in the eluent.(4)The components of walnut polyphenols and active fraction F8 were analyzed by HPLC-ESI-TOF-MS The results showed that there were 19 kinds of phenolic compounds in walnut polyphenols,mainly belonging to ellagitannins(13 kinds),gallic tannins(3 kinds),phenolic acids(1 kind),and flavonoids(2 kinds).The relative contents of ellagitannins,gallic tarnins,phenolic acid and flavonoids were 82.80%,9.75%,3.74%,and 1.65%,respectively.F8 was consisted of ellagic acid,ellagic acid pentose isomer,ellagic acid hexose isomer and quercetin,which belongs to phenolic acid(62.05%),ellagitannins(21.93%),and flavonoids(16.02%),respectively.The content of ellagic acid and ellagitannins in F8 were83.98%,and the ratio of them to quercetin were 5.24:1.The above results showed that the active components of walnut polyphenols,mainly consisted of ellagic acid and ellagitannins,recovered the proliferation rate of spleen lymphocyte,the cell viability of T-lymphocyteand,and the levels of cell cytokines IL-4,IFN-?,as well as granzyme B,indicating that the FNT-induced splenocyte toxicity was significantly reduced.The main active components of walnut polyphenols were ellagic acid and ellagitannins,which played an important role in the immune protection of walnut polyphenols. |
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