Study On Immunomodulatory And Antioxidant Activity Of GouQi Chewable Tablets

ObjectiveTo study the immunomodulatory function and antioxidant effect of Gou Qi Chewable Tablets(GQCT),and try to explain the mechanism of immunomodulatory function of GQCT from the antioxidant pathway.BackgroundLycium Chinese is a kind of deciduous perennial shrub belonging to the family Lycium,Solanaceae,and Tubiflorae.And mostly distributed in the Loess Plateau with low temperature and arid environment.The fruit of the plant is a traditional medicinal and food plant resource,known as "Gou Ti Zi","Gou Qi Dou",and "Hong Er Zhui".The fresh fruit of Lycium Chinese is dark red,oblong,or ovoid.It has few seeds and the flesh is juicy and sweet.The Lycii Fructus is mostly made of sun-dried,air-dried,or roasted.Lycium barbarum is usually made by sun exposure,air drying,or baking.Lycium barbarum L.is a general name for Chinese wolfberry varieties distributed in northwest China which is included in the Pharmacopoeia of the People’s Republic of China.The nutrient content of fresh Lycium barbarum L.fruits is higher than that of dried fruits,but there are many problems in the harvesting and transportation process such as they’re vulnerable to damage in transportation due to the thin skin,and the preservation period is short in the early stage.Based on the processing and development characteristics of fresh Chinese wolfberry fruit,the research group improved the processing technology,and prepared GQCT from fresh fruit homogenate.Now we are studying the modulation of immune function and antioxidant activity of the organism.Research contentMice were orally administered with GQCT for 4 weeks to observe the regulatory effect of GQCT on their immune function.The antioxidant activities of GQCT in vivo were observed by detecting the contents of antioxidant enzymes and malondialdehyde in serum.And in vitro antioxidant capacity was achieved by evaluating its free radical clearing ability.Thus observing the antioxidant activity of GQCT in vivo and in vitro.A hydrogen peroxide-induced oxidative damage model of macrophages was established.The effects of GQCT on phagocytosis function,secretion function,and the heme oxygenase(HO-1)mRNA expression level of macrophages were observed to elucidate the mechanism of immune function regulation from the perspective of antioxidant.Regulation of GQCT on the immune function of normal mice160 SPF Balb/C male mice(body weight:20±2g)were used to conduct experiments.40 were used for serum hemolysin determination and organ determination,40 were used for mouse carbon clearance test,and 32 were used for NK cell activity determination.48 were used for the spleen lymphocyte transformation experiment and spleen lymphocyte proliferation experiment after obtaining serum.After the mice were adaptively reared for 3 days,the experimental mice were randomly divided into 4 groups according to their body weight,including the blank control group,low,medium,and high dose groups of GQCT(0.25,0.5,and 1.5 g·kg-1.BW).The experimental animals were administrated for 28 days according to the dosing volume of 10 mL·kg-1-BW and weighed regularly.Study on Antioxidant Activity of GQCTTo investigate the in vivo antioxidant capacity of GQCT after administrated,the content of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and malondialdehyde(MDA)in the serum of the mice were determined.The experiments on the scavenging ability of GQCT on DPPH+,ABTS+,and OH-free radicals were performed to determine the in vitro antioxidant activity.Protective Effect of GQCT on Raw264.7 macrophage injury induced by H2O2The Raw264.7 cell was damaged by 600 μM H2O2 to establish the oxidatively damaged model.0.03125,0.0625,0.125 g·L-1 GQCT was added to study the protective effect on oxidative damage of macrophages,and to verify whether antioxidation can improve the immune function of the body by increasing macrophageactivity.ResultsRegulation of GQCT on the immune function of normal miceThe carbon clearance index method was used as the phagocytic index.In the blank control group,the phagocytic index a was 2.587±0.940.Compared with the blank control group,the phagocytic index was increased in the GQCT-M and GQCT-H group,and there was a significant difference in GQCT-H group.Compared with the blank control group,the serum hemolysin level of mice in the administration group was significantly increased after oral administration for 28 d of the GQCT-L,GQCT-M,and GQCT-H group(P<0.01),among them the GQCT-L group is the most obvious.The results showed that the spleen index and thymus index were highly significantly increased in the GQCT-H group(P<0.01).Compared with the blank control group,the NK cell activity of GQCT in GQCT-L,GQCT-M,and GQCT-H groups was significantly increased(P<0.05,P<0.01).In the conA-induced splenic lymphocyte transformation experiment of mice,compared with the blank group,the GQCT-L,GQCT-M,and GQCT-H group could significantly promote the conA-induced splenic lymphocyte transformation of mice(P<0.05,P<0.01),among them the GQCT-L group is the most obvious,up to 47.8%.Study on Antioxidant Activity of GQCTCompared with the blank group,the serum GSH-Px activity in the GQCT-M group was significantly increased(P<0.05).The serum MDA content in the GQCT-H group was significantly decreased(P<0.05).There was no difference of the serum SOD activity in GQCT-L,GQCT-M,and GQCT-H group.In vitro antioxidant activity evaluation experiment,it was found that the GQCT-H group cleared DPPH+ in a concentration-dependent manner ABTS+ and hydroxyl radical,ABTS+ and hydroxyl radical scavenging rate reached 100%when the mass concentration was 44.9g·L-1,DPPH+ radical scavenging rate reached 95.24%±0.02%,when the mass concentration was 8.7g·L-1,GQCT cleared 50%DPPH+.The IC50 of ABTS+ and hydroxyl radical were 1.637±0.198 2.041±0.025 and 10.273±0.035 g·L-1,respectively.These results indicated that GQCT had good scavenging ability on three kinds of free radicals,and the scavenging ability was in the order of DPPH+>ABTS+>OH-.Protective effects of Gou Qi Chewable tablets on Raw264.7 macrophage injury induced by hydrogen peroxideThe phagocytic function of Raw264.7 cells was decreased and the mRNA expression of Heme oxygenase-1(HO-1)was increased under the induced of 600 μM H2O2(P<0.01).GQCT at 0.0625 g·L-1 and 0.125 g·L-1 could significantly improve the phagocytic function of Raw264.7 macrophages(P<0.05).The concentration of GQCT at 0.0625 g·L-1 and 0.125 g·L-1 could significantly increase the TNF-α content of Raw264.7 macrophages(P<0.01).The concentration of 0.0625 and 0.125 g·L-1 of GQCT could significantly reduce the increase of HO-1 mRNA expression caused by oxidative stress(P<0.05).Conclusion1.This study confirmed that GQCT can significantly improve the activity of NK cells,spleen lymphocyte proliferation,thymus index,spleen index,macrophage phagocytosis index and mouse serum hemolysin antibody level.It is suggested that GQCT has the effect of improving immune function.2.This study confirmed that GQCT has antioxidant capacity in vivo and in vitro through free radical scavenging experiments in vitro and serum antioxidant experiments in vivo.3.GQCT can enhance Raw264.7 macrophage activity through antioxidant,and improve immune function by promoting TNF-α secretion and decreasing ho-1 mRNA expression induced by oxidative stress.