Assessment Of Antioxidant And Anti-Inflammatory Activities Of Phenolic-Rich Extracts From Salvadora Persica L Leaves

Background Inflammation is the first reaction of the innate immune system in response to the various exogenous and endogenous aggressors.However,excess production of intracellular reactive oxygen species(ROS),free radicals(Hydroxyl)and microbial infections lead chronic inflammation and pyroptosis via hypersecretion of IL-1? active and GSDMD cleavage activated by NLRP3 assembly to caspase1 pathway.Many factors lead to ROS hypersecretion such as oxidative stress,excessive mitochondrial function,microbial infection and LPS stimulus.It's known that synthetic antioxidants are used to host the immune system,but they are cytotoxic.Therefore,it's important to explore safe and efficient antioxidant agents from natural compounds.Salvadora persica L(Sp)plant has been widely used in traditional African and Asian medical practices due its various bioactive phenolic compounds such as rutin and quercetin.However,the phenolic compounds in the leaves of Sp have not been thoroughly explored.Also,Sp leaves extract and rutin effects on inflammation and pyroptosis models induced by IL-1 ? active and GSDMD cleavage have not been explored.In the present study,we hypothesized that the Sp leaves could contain a higher concentration of the bioactive phenolic compound.Also,Sp leaves could inhibit inflammation and pyroptosis induced by the NLRP3 inflammasome complex.In the present study,we opted first to optimize the identification and yield of total phenolic compounds in the Sp leaves extract and access their antioxidant activities.We then quantified the rutin and quercetin compound in the flavonoid rich fraction(S1TF7).And we investigated the mechanisms inhibitory effects of rutin and S1TF7 on inflammation and pyroptosis induced by IL-1? active,and GSDMD cleavage in LPS stimulated THP-1 monocyte cell.PART-1: BIOACTIVE PHENOLIC COMPONENTS AND ANTIOXIDANT ACTIVITIES OF WATER-BASED EXTRACTS AND FLAVONOID-RICH FRACTIONS FROM SALVADORA PERSICA L.LEAVESAims: i)Identify the bioactive phenolic compounds present in the Sp leaves extracts;ii)Quantify total phenol phenolic content(TPC)and total flavonoid content(TFC)of different Sp leaves extract;iii)Evaluate the antioxidant capacity of the different Sp leaves extract in DPPH,ABTS,FRAP and DCFH assays;iv)Evaluate the cell viability and cellular antioxidant capacity of flavonoid-rich from young leaves of Sp in Raw-cell 264.7.Material and Methods: i)Both the young leaves(S1)and the old leaves(S2)of Sp were extracted and purified to optimize the yield of total phenolic compounds.The fraction from S1 and S2 were used to identify the bioactive phenolic compounds using LC-ESI-QTOF MS Analysis.ii)Total phenol phenolic content(TPC)and Total flavonoid content(TFC)of different Sp leaves extract were determined by Folin-Ciocalteu and colorimetric methods respectively;iii)We applied the DPPH,ABTS,and FRAP assays to determine the free-radical scavenging activities of different Sp leaves extract;iv)Flavonoid-rich fraction from young leaves of Sp(S1TF7)were selected to determine the cell viability in Raw-cell 264.7 using MTS assay;v)Cellular antioxidant capacity of S1TF7 was evaluated on LPS-induced intracellular ROS production in Raw-cell 264.7 by using DCFH assay.Results: i)Six flavonoids;isoquercitrin,kaempferol-3-neohesperidoside,myricetin-3-galactoside,apigenin-O-hexoside,isorhamnetin and isorhamnetin-3-neohesperidoside were identified for the first time in Sp leaves,except that isorhamnetin-3-neohesperidoside was not detected in S2;ii)S1TF7 has the highest TFC(358.88±0.12 mg rutin equivalent/g)and TPC(180.82±0.82 mg gallic acid equivalent/g),and it exhibited significant(p < 0.05)free-radical scavenging activity in DPPH(IC50% = 14.64 ± 0.29?g/m L)and ABTS(IC50% = 200.41 ± 1.85?g/m L)assays which was similar to Butylated hydroxytoluene(BHT)as a positive control;iii)S1TF7 inhibited LPS-stimulated ROS production in raw-cell-264.7 with no significant toxicity at 200?g/m L(p > 0.05).Conclusion The study concluded that Sp young leaves,specifically flavonoid-rich fraction(S1TF7)appears to contain high content of phenolics correlated to strong antioxidant activity,which could support these young leaves as to prevent oxidative disease for the rural peoples,and it merits further pharmacological investigations.PART-2: FLAVONOID-RICH FRACTION FROM SALVADORA PERSICA LEAVES INHIBITS INFLAMMATION AND PYROPTOSIS INDUCED VIA HYPERSECRETION OF IL-1? AND GSDMD IN LPS STIMULATED THP-1 CELLAims: i)Quantify the rutin compound in the S1TF7 and evaluate their cell viability in THP-1 cell using CCK-8 assay;ii)Determine the effects of S1TF7 and rutin on IL-1? active,GSDMD cleavage,NLRP3 activation,and caspase1 in LPS stimulated THP-1 cell;iii)Determine the effects of S1TF7 on ROS generation and AMPK in LPS stimulated THP-1;iv)Determine the direct effects of S1TF7 on pyroptosis model in LPS stimulated THP-1 cell.Material and Methods: i)HPLC analysis was performed to quantify the rutin and quercetin in the S1TF7.CCK-8 assay was used to determine the not cytotoxic concentration of rutin and S1TF7 in THP-1 cell.ii)RT-PCR analysis was used to quantify the effects of S1TF7 on m RNA expression of IL-1?,NLPR3 and caspase1 in LPS-1 stimulated THP-1 cell.quantified using RT-PCR analysis.iii)Wb assay was performed to measure the effects of S1TF7 and rutin on the protein expressions of IL-1 ? active,GSDMD cleavage,NLRP3,caspase1 active and AMPK in LPS stimulated THP-1 cell.iv)Flow cytometry assay was performed to determine the inhibitor effect of S1TF7 on the NLRP3 activation through inhibition the ROS/AMPK pathway in THP-1 cell.v)The TME image was used to observe the effects of S1TF7 on pyroptosis induces by LPS in THP1 cell.Results: i)S1TF7 contained a high level of rutin and quercetin with no cytotoxicity effects in THP1 cells(p > 0.05).ii)S1TF7 and rutin significantly inhibited the hypersecretions of IL-1? active and GSDMD cleavage induced in LPS-stimulated THP-1 cells(p < 0.001).iii)S1TF7 and rutin showed a significant inhibition effects on m RNA and protein level of NLRP3 expression and on m RNA level of caspase1(p < 0.01)in LPS stimulated THP-1 cell.iv)S1TF7 inhibited the NLRP3 activation via ROS/AMPK pathway with potent effect at 200 ?g/m L in THP-1 stimulated cell(p < 0.001).v)S1TF7 inhibited pyroptosis which was induced by LPS(2?g/m L),as shown in the TEM image.Conclusion Sp leaves as in flavonoid-rich fraction(S1TF7)and rutin single natural flavonoid inhibited the inflammation and pyroptosis by directly inhibiting production of IL-1? active GSDMD cleavage,NLRP3/caspase1.The S1TF7 attenuated NLRP3 activation through inhibition of the ROS intracellular production and AMPK activation.The Sp young leaves can be to prevent the chronic inflammation and pyroptosis in pulmonary diseases such as neutrophilic asthma.