| Cryptotaenia japonica Hassk.is a perennial herbaceous plants belonging to umbrelliferae,and mainly distributes in northeast region of China,Shaanxi province,Yunnan province and Hunan province.Cryptotaenia japonica Hassk.is a kind of medicinal and edible vegetable with special aromatic smell.The tender stem and leaf often are used as vegetables,and also can be used as Chinese medicine treatment of pneumonia,skin diseases such as carbuncle.Studies showed that Cryptotaenia japonica Hassk.contain a variety of active ingredients,and the flavonoids is one of the main bioactive components.At present,the investigations of Cryptotaenia japonica mainly focuses on cultivation and the reports about its active ingredients are also concentrated on essential oils.Research on the optimization of the extraction method and identification of its flavonoids,and the its biological activity and mechanism are relatively scarce.Therefore,this paper optimized the extracting,separating technology conditions of flavonoids from Cryptotaenia japonica Hassk.,and main types and content of flavonoids and phenolic acid compounds in Cryptotaenia japonica Hassk.were also analyzed.The antioxidant activity,antibacterial activity,anti-inflammatory activity of Cryptotaenia japonica Hassk.and its mechanism of flavonoids from Cryptotaenia japonica Hassk.were investigated.These results provide scientific basis for the development and utilization of Cryptotaenia japonica Hassk.in the future.The main results were as follows:(1)The experimental results of optimization of the extraction technology of flavonoids from Cryptotaenia japonica Hassk.showed that the flavonoid yields obtained by microwave-assisted,ultrasound-assisted and enzymatic-assisted extraction methods were 27.01 ±0.66,33.11 ±4.54 and 47.62±5.86 mg/g,respectively.Comprehensive comparison suggests that the yield of enzyme-assisted extraction is much higher than the yield of ultrasound-assisted extraction and microwave-assisted extraction,but its extraction time is longer,and the process is relatively complex and cumbersome.The study of biological activity showed that ultrasound-assisted extraction was more beneficial to the release of active ingredients from Cryptotaenia japonica Hassk.than enzyme-assisted extraction.The optimum macroporous resin LS-46D was selected from six kinds of macroporous resins by using the index of adsorption capacity,adsorption rate,desorption capacity and desorption rate,and its optimal separation condition was investigated by dynamic adsorption-desorption test.The results showed that the optimal separation conditions were as follows:the concentration of the sample solution was between 5.07 and 6.02 mg/mL,the adsorption flow rate was 3.0 mL/min,and the ratio of diameter to height was 1:6.The sample was first washed with distilled water until colorless,and then eluted with 70%ethonal under a flow rate of 2.0 mL/min.Finally,150 mL eluent was collected.Under this condition,the recovery rate of flavonoids was 98.84%and the purity increased 3.5 times than crude extract.(2)The results of UPLC-MS/MS detection of different extracts from Cryptotaenia japonica Hassk.showed that the Cryptotaenia japonica Hassk.contain three kinds of flavonoids(luteolin,apigenin,and diosmite)and six kinds of phenolic acids(gallic acid,chlorogenic acid,vanillic acid,and caffeic acid,p-coumaric acid,ferulic acid).Among them,luteolin,apigenin,p-coumaric acid,gallic acid,and chlorogenic acid are the main components.The ethyl acetate fraction,ultrasonic-assisted extracts,and n-butanol fration contain relatively high concentration and types of active ingredients in all samples.In particular,the content of luteolin was the highest and its content in the three samples reached 10 6806.1,20267.9 and 5072.7 μg/g,respectively.The apigenin content ranks the second highest,and the contents of these three samples are 13780.8,1346.0 and 1157.2 μg/g,respectively.(3)The results of in vitro antioxidant activity assay indicated that the antioxidant capacity of different extracts was extremely significantly related to the content of flavonoids and polyphenols.Among the nine samples,EAF had the best antioxidant capacity,and its IC50 values for DPPH,ABTS,and reducing power were 0.08±0.05 mg/mL,3.62±1.36 μg/mL,and 0.44±0.11 mg/mL,respectively.The antibacterial test results showed that all nine samples had certain antibacterial activity,and the EAF had the best antibacterial effect.At the concentration of 800 mg/g,the inhibition zone of EAF against four strain baterial(Escherichia coli,Staphylococcus aureus,Bacillus subtilis andβ-hemolytic streptococcus)was 21.13 ± 0.16,16.11 ± 0.75,17.15 ± 2.05 and 20.78 ± 0.03 mm,respectively.Its minimum inhibitory concentration(MIC)were all less than 12.5 mg/mL,except to β-hemolytic streptococcus(50 mg/mL).The combined compositional analysis showed that luteolin,apigenin and p-coumaric acid were the main active ingredients of Cryptotaenia japonica Hassk.contribute to the antioxidant and antibacterial activities.(4)Enzyme-linked immunosorbent assay(ELISA)experiments showed that the ethyl acetate extract(EAF),petroleum ether extract(PEF)and n-butanol extract(NBF)displayed well inhibition activity of the release of NO,IL-1β,IL-6 and TNF-α in LPS-induced mouse RAW264.7 cells when they at a concentration of 20-100 μg/mL,and the water-soluble extract(AQF)was at a concentration of 200～600 μg/mL.RT-qPCR,Western blot and luciferase reporter gene technology were used to further prove that Cryptotaenia japonica Hassk can inhibit the transcriptional activation of NF-κB in RA264.7 cells induced by LPS,and down-regulate the production of IL-1β,IL-6,TNF-αinRNA,and their protein expression produces to exert the anti-inflammatory activity.Combined the analysis results of bioactive compounds,we can deduce that the main anti-inflammatory ingredients in EAF are luteolin,apigenin and p-coumaric acid. |
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