Chemical Composition Of Fubaiju And The Protective Effects And Mechanism Against Oxidative Stress In ARPE-19 Cells

Chrysanthemum morifolium cv.Fubaiju,which was cultivated in Hubei Province,had been registered as one of the National Geographic Product Marking for agricultural products.Fubaiju is a traditional medicinal and edible plant with excellent quality and large production,and one of the three most important chrysanthemum cultivars in Traditional Chinese Medicine.However,the chemical components of flavonoids and phenolic acids and bioactivates of Fubaiju have not been reported,not even the comparable study between different cultivars of chrysanthemums.Chrysanthemum has been primarily consumed with hot-H2O as a tea product,however,few studies have investigated the health effects of the hot-H2O extracts of chrysanthemum,and the research results from their organic solvent extracts may not truly reflect the beneficial effects of drinking chrysanthemum tea.Chrysanthemum were referred to have the effect of "brightening eyes" in traditional Chinese medicine books,however,there were no study of how chrysanthemum may protect the retinal pigment epithelium cells.The main results of this study were summarized as follows:(1)The chemical composition of Fubaiju extracted with hot-water was investigated using ultra-performance liquid chromatography-ion mobility spectrometry/quadrupole time-of-flight mass spectrometry(UPLC-IMS-QTOF-MS).The ethanol extract was included so that the results of the hot-water extract can be related to the alcohol extracts from previous studies and the possible use in nutraceutical products development.As shown in the results:a total of 28 compounds were detected and identified in the hot-water extract and the ethanol extract.Tuberonic acid glucoside and tasumatrol B were detected in the Fubaiju hot-water extract and ethanol extract for the first time.Then the total phenolic content(TPC),anti-inflammatory and free radical scavenging activities of the Fubaiju hot-water and ethanol extract were also investigated.As shown in the results:The TPC value,relative ABTS·+(ABTS),DPPH·(RDSC)and oxygen radical(ORAC)scavenging activities of Fubaiju hot-water extract were 33.48 mg gallic acid equivalents(GAE)/g,121.57,110.11 and 1638.99?mol Trolox equivalents(TE)/g,respectively.The TPC,ABTS,RDSC and ORAC values of Fubaiju ethanol extract were 2.89 mg GAE/g,10.29,11.28 and 260.56?mol TE/g,respectively.The hot-water extract had greater total phenolic content and free radical scavenging activities than its counterpart ethanol extract under the same experimental conditions.The hot-water extract of chrysanthemum was more effective in suppressing the lipopolysaccharide(LPS)-induced tumor necrosis factor-a(TNF-?),interleukin-6(IL-6)and cyclooxygenase-2(COX-2)mRNA expressions at lower concentrations of 1,10 and 100?g/mL.(2)The chemical components of Fubaiju,Hangbaiju,Hangtaiju,Gongju,Huaiju and Kunlun Xueju and Kunlun Miju extracted with hot-water were detected by UPLC/Q-TOF-MS analysis.75%methanol extracts were included,so that the chemical composition results from the present study may be related and compared with previous studies.As shown in the results:total of 28 and 34 compounds were detected and identified using UPLC/Q-TOF-MS analysis from the hot-water and 75%methanol extracts,respectively.For the first time,6,8-C,C-diglucosylapigenin and eriodictyol-7-O-glucoside were detected in the Snow chrysanthemum(Kunlun Xueju and Kunlun Miju),and acetylmarein was detected in HangJu,GongJu and HuaiJu.The extracts were also examined for their radical scavenging and anti-inflammatory activities in vitro and the results from the present study showed that hot-water extracts had greater or comparable anti-inflammatory,free radical scavenging,and cellular antioxidative activities as their counterpart 75%methanol extracts,along with greater or comparable level of TPC values on a per chrysanthemum flower weight basis.Among all the C.morifolium samples,Fubaiju 2(FBJ2)hot-water extract had the greatest ability to decrease the LPS-induced IL-6,IL-1? and COX-2 mRNA expressions and their expressions were decreased by 92%,88%and 61%,respectively,compared to the LPS treated group.Among all the hot-water extracts of C.morifolium samples,FBJ2 had the greatest level of TPC and ORAC values,which were 9.53 mg GAE/g and 1349.13 ?mol TE/g,respectively.In addition,both extracts of Fubaiju significantly inhibited the H2O2-induced intracellular reactive oxygen species(ROS)production in cultured adult retinal pigment epithelium cell line-19(ARPE-19)cells.(3)Fubaiju hot-water extract was used to examine the protective effects and mechanism against the H2O2-induced oxidative stress in ARPE-19 cells.The hot-water extract of Fubaiju significantly restored the H2O2-induced decrease of cell viability and suppressed the excessive production of ROS.Compared to the H2O2 treated group,100?g/mL Fubaiju hot-water extract also remarkably decreased the expression of cleaved caspase-3,cleaved poly(ADP-ribose)polymerase(PARP)and the ratio of Bax/Bcl2 by 70.4%,68.4%and 35%,respectively,suggesting that Fubaiju could protect ARPE-19 cells from apoptosis under oxidative stress induced by H2O2.In addition,pretreatment with 100?g/mL Fubaiju hot-water extract significantly increased the expression of phase II enzymes including catalase,glutamate-cysteine ligase catalytic subunit(GCLc),superoxide dismutase(SOD),heme oxygenase-1(HO-1)and NAD(P)H:quinone oxidoreductase 1(NQO-1)by 155.2%,113.4%,267.6%,203.0%and 347.8%,respectively.Furthermore,pre-incubation with 100?g/mL Fubaiju hot-water extract remarkably increased the phosphorylation of Akt and the translocation of nuclear factor(erythroid-derived-2)-like 2(Nrf2).The HO-1 expressions were significantly decreased when the Nrf2 was silenced by siRNA or the Akt was blocked by LY294002,and the nuclear translocation of Nrf2 was also inhibited by the inhibition of Akt.The observations indicated that the Fubaiju hot-water extract may activate the PI3K/Akt dependent Nrf2/HO-1 signaling pathway to protect the ARPE-19 cells against oxidative stress caused by H2O2.In summary,the chemical components of flavonoids and phenolic acids of C.morifolium cv.Fubaiju were identified.Comparatively studied Fubaiju and chrysanthemums from other different locations.The protective effects against oxidative stress and the underlying mechanism of Fubaiju in ARPE-19 cells were revealed in this study.These results may be used to promote the consumption of Fubaiju as functional tea,which have potential value in preventing oxidative stress induced RPE cell damage.