| ObjectiveSkin photoaging is a premature aging process caused by long-term exposure to ultraviolet(UVA and UVB).Clinically,severely photo-aged skin is characterized by a rough texture,dryness,wrinkles,lack of elasticity and irregular pigmentation,etc.Not only does skin photoaging induce skin dysfunction,but it also related to the cutaneous carcinogenesis.It seems that the most effective way to prevent skin photoaging is wearing protective clothes or application of sunscreen to reduce the amount of skin exposure to sun light,but sometimes also limited.After an investigation of domestic and foreign literature,I found that skin photoaging is related to the excessive generation of ROS and inflammatory induced by UV radiation,which suggest that application of anti-inflammatory drugs or antioxidants may be a method to prevent skin photoaging.House fly(Musca Demestica)larvae commonly known as maggot or grain worm and its desiccate is a traditional Chinese medicine named Luo Xianzi,of which extraction display several bioactivity such as anti-bacteria,anti-virus,and immunomodulation.Previous study of our group indicated that maggot extraction possess anti-inflammatory,anti-atherosclerotic,anti-oxidation and anti-aging activity.As oxidative stress and inflammatory response play an important role in the process of skin photoaging,Luo Xianzi extraction may have the effect of interfering or preventing the occurrence of skin photoaging.In this paper,we used Luo Xianzi as raw material and explored an appropriate and convenient method to extract the antioxidant peptide.Then,whether the antioxidant peptide has protective effect to UV-induced skin photoaging mouse model was observed and its possible protective mechanism was studied on histopathology,oxidative stress and inflammatory response.Methods?.Study on preparation technology of Luo Xianzi antioxidant peptides(i)Preparation of antioxidant peptides by "protein extraction and enzymatic hydrolysis method" : The protein of Luo Xianzi was extracted by "alkali dissolved acid precipitation method" and then hydrolyzed by alkaline protease Alcalase.The DPPH free radical scavenging rate was used as an evaluation index to optimize the enzymatic hydrolysis conditions of Luo Xianzi protein by single factor test and orthogonal test.The enzymatic Hydrolysate of Luo Xianzi was separated by stepwise ultrafiltration(10kD and 5kD filters).The activity of DPPH free radicals scavenging and yield of each fraction was determined.(ii)Preparation of antioxidant peptides by “water extraction and alcohol precipitation method”: The DPPH radical scavenging activity of supernatant was determined,and the yield was calculated.(iii)Molecular weight distribution of supernatant and precipitate were analyzed by TSKgel G2000 SW gel column.A suitable antioxidant peptide preparation method was selected by comparison of molecular weight distribution,DPPH radical scavenging activity and yield.(iv)Evaluation of antioxidant activity of Luo Xianzi antioxidant peptides : The scavenging activity of DPPH free radical,ABTS radical and hydroxyl radicals were determined and the potent antioxidant vitamin C was used as control.The antioxidant activity was evaluated by comparing the median elimination concentration(EC50)of each radical with vitamin C.?.Study on the protective effect of Luo Xianzi antioxidant peptide on UV-induced skin photo aging model.Fifty-six female Balb/c mice were randomly divided into 7 groups: Normal control group(NC),Model control group(MC),Ointment base group(OC),Luo Xianzi antioxidant peptide Low dose group-2%(L),Middle dose group-4%(M),High dose group-8%(H)and Vitamin E group(VE).The mice were shaved at the back each day and were exposed to UV irradiation after drug treatment for 1 h.The experiment was conducted 6 days a week for 6 weeks.The cumulative dose of UVA and UVB irradiation was 9.12 and 91.2 J/cm2,respectively(to simulate the ratio of UVA to UVB of sunlight).Finally,skin appearance of each group was observed and photographed and thickness of the mice back skin was measured to evaluate the protective effect of Luo Xianzi antioxidant peptide on UV-induced mouse skin photoaging model.Mice skin tissue morphology was observed by H&E staining slices,aldehyde fuchsin staining slices and transmission electron microscopy slices.The activity of SOD and GSH-Px and the contents of MDA,MMP-1,MMP-3,TNF-??IL-1? and IL-6 in the skin tissue of mice were determined to explore the possible mechanism of protective effect of Luo Xianzi antioxidant peptide on mouse skin photoaging model.Results?.Study on preparation technology of Luo Xianzi antioxidant peptides(i)Preparation of antioxidant peptides by “protein extraction and enzymatic hydrolysis method”:The optimal enzymolysis conditions were as follows: the amount of enzyme was 7000 U/g,the substrate concentration was 1%,the enzymolysis reaction time was 15 min,the enzymolysis temperature was 50 ? and the pH value was 7.5.The enzymatic hydrolysate of Luo Xianzi protein was separated by stepwise ultrafiltration with 10 kD and 5kD filters and three molecular segments were collected,of which the molecular weight ranges were MW> 10 kD,5kD <MW <10kD and MW <5kD and yield of each fraction were 9.06 ± 0.14%,2.13 ± 0.04% and 1.30 ± 0.02%,respectively.The DPPH free radical scavenging activity of the three molecular segments increased with the increment of the concentration and showed a dose-effect relationship.The DPPH free radical scavenging activity of the fraction(MW > 10kDa)was significantly lower than the fraction(MW <10 kDa)and the strongest is the fraction(MW < 5kDa).(ii)Preparation of antioxidant peptides by “water extraction and alcohol precipitation method”: The yield of antioxidant peptides extracted by water extraction and alcohol precipitation method is 13.33 ± 0.31%.(iii)A certain proportion of large molecule weight protein was contained in the supernatant extracted by alkali-soluble acid precipitation method,while no large molecule weight protein was detected in the supernatant extracted by water extraction and alcohol precipitation method and the molecular weight of peptides in the supernatant was about 4 kDa.Besides,with a simple process,small pollution and high yield of antioxidant peptides,water extraction and alcohol precipitation method is more suitable for the preparation of antioxidant peptides.(iv).Evaluation of antioxidant activity of Luo Xianzi antioxidant peptides: The median elimination concentration(EC50)of DPPH free radical,hydroxyl radical and ABTS radicals were 0.117,2.232 and 2.546 mmol/L.The EC50 ratio of vitamin C and Luo Xianzi antioxidant peptide were 1 : 6.5(DPPH·),1 : 3.97(ABTS·+)and 0.84 : 1(OH·),respectively.The result compared with the potent antioxidant Vitamin C suggested that Luo Xianzi antioxidant peptide is a good antioxidant.?.Study on the protective effect of Luo Xianzi antioxidant peptide on UV-induced skin photoaging model.Compared with the NC group,the skin thickness of the MC was significantly increased and erythema,peeling and rough hardened skin were observed.The skin thickness of the middle dose group and the high dose group were significantly decreased,erythema and peeling were decreased.The result of HE staining and aldian magenta staining showed that the medium and high dose groups suppressed UV-induced stratum corneum,excessive proliferation of keratinocytes,irregular distribution of keratinocyte,loss of dermal papilla,disorder of elastic fibers.Also,middle dose and high dose of Luo Xianzi antioxidant peptide suppressed UV-induced increase of MDA,MMP-1,MMP-3,TNF-?,IL-1? and IL-6 content and decrease of SOD and GSH-PX activity.ConclusionThe optimal enzymolysis conditions of Luo Xianzi protein extracted by “alkali dissolved acid precipitation method” were as follows: the amount of enzyme was 7000 U/g,the substrate concentration was 1%,the enzymolysis reaction time was 15 min,the enzymolysis temperature was 50 ? and the pH value was 7.5.The fraction(MW < 10 kDa)had potent DPPH free radical scavenging activity while its yield was low.The molecular weight of the peptide in the supernatant that was extracted by “water extraction and alcohol precipitation method” was about 4 kDa and no high molecular weight protein was detected.The yield of these peptide was 13.33 ± 0.31%,which showed potent scavenging activites of DPPH free radical,ABTS radical and hydroxyl free radical.Therefore,it is more suitable for the preparation of antioxidants by extraction of small molecular peptides using water extraction and alcohol precipitation method.Topical use of Luo Xianzi antioxidant peptides has a protective effect on the mice skin photoaging model and it may exert its effectiveness through the inhibition of UV-induced skin lipid peroxidation,increase of antioxidant enzymes activity(SOD and GSH-Px)and decrease of the expression of inflammatory factors and content of MMPs. |
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