Antioxidant And Antitumor Activity Of Potato Protease Inhibitors(PPIs)

The waste liquid produced from the preparation of potato starch contained a large amount of protein,which is usually recovered by acid heat treatment to form an irreversible precipitate,thereby losing its functionality and being used in low-value applications such as feed,which is not conducive to the rational utilization of protein resources.Potato contain a variety of protease inhibitors?PPIs?,accounting for more than 50% of the total protein.The extraction of PPIs from the waste liquid has the advantages of easy extraction,low cost,high safety and wide application.Some research reviewed that PPIs might regulate the activity of proteolysis in vivo,which had the functions of lowering blood glucose levels,inhibiting the growth of some tumors,reducing toxic and side effects,protecting the liver and maintaining the healthy immune system,and was of great significance for maintaining normal life activities.Therefore,the efficient and convenient extraction of PPIs from starch industrial waste liquid and the full development of its functionality,as well as more potential applications in the field of food played an important role in promoting the high-value and industrial use of potato waste liquid and the development of new functional food factors.In this paper,a simple and mass production method for the preparation of PPIs had been established.The antioxidant activity in vitro and its changes under different processing conditions of PPIs had been studied.Meanwhile,the antioxidant and antitumor effects had been further investigated at the cell level,which provided a theoretical basis for the high-value utilization of potato protein resources.The main results were as follows:1.PPIs were prepared by salting-out precipitation method?SOP?and isoelectric-point precipitation?IPP?method with different combination sequences.The optimal combination sequence,?NH₄?₂SO₄ saturation,pH value of isoelectric point separation,heating temperature and time were selected with the extraction rate as the index,and their physicochemical properties were determined.According to the results,the extraction method was SOP-IPP.The optimal process conditions were as follows: salting out the whole protein at 40% saturation?NH₄?₂SO₄,separating soluble PPIs at pH 3.0,heating in water bath at80? for 10 min to remove other proteins.The PPIs obtained by this optimal preparation process had stable solubility,suitable emulsification and foaming properties in the pH range of 2-10,and its amino acid composition ratios were relatively balanced and rich and had the potential to interact with free radicals.2.The antioxidant activity in vitro of PPIs and the effect of processing on its stability were evaluated by five methods.The results showed that the scavenging ability of PPIs to ABTS was the best,followed by the inhibition of lipid peroxidation,scavenging ability to DPPH radical,reducing power and scavenging ability to hydroxyl radical.Specially,the IC₅₀ value of PPIs on the inhibition of lipid peroxidation was lower than that of Ve,showing obvious advantages.The effect of temperature on the antioxidant activity of PPIs was different in different systems.The antioxidant activity of PPIs decreased rapidly at high temperature for a long time.In the concentration conditions of this study,the reducing power,scavenging ability to DPPH and hydroxyl radical of PPIs were low,and the trend of change affected by temperature was gentle.However,the scavenging ability to ABTS and inhibition of lipid peroxidation of PPIs could maintain better stability when the temperature was lower than 50? and 25?,respectively.The stable range of the antioxidant activity of PPIs treated with different pH values was different under different evaluation systems.The scavenging ability of PPIs to hydroxyl radical was not significantly affected by pH;the inhibition of lipid peroxide was stable under pH 2-8;the scavenging ability to ABTS radical was stable at a high level in the range of pH 6-11;the reducing power first decreased slowly and then increased gradually with the increase of pH value;the scavenging ability to DPPH radical decreased slowly with the increase of pH value.In general,the antioxidant activity of PPIs was stable in a wide range of pH.However,the effect of ultrahigh high-pressure treatment on the antioxidant activity of PPIs was not significant in a certain period of time.3.The antioxidant activity of PPIs was investigated at the cell level.Firstly,it was found that PPIs had significant?p<0.05?antioxidant effect in cells by PSC and CAA.The cells oxidative damage model of HepG2 and HUVEC induced by H₂O₂ were used for further study.The results of cell viability and ROS showed that PPIs had a significant?p<0.05?inhibitory effect on cell oxidative damage.The morphological changes of injured cells were examined by inverted phase contrast microscopy,which found that PPIs could significantly reduce the number of damaged cells,maintain the morphological of the cells and prevent cells from shrinking.Moreover,the level of SOD and NO of injured cells were significantly increased,and the level of MDA and LDH were significantly reduced after treatment with PPIs.4.The anti-tumor of PPIs was investigated at the cell level.PPIs could significantly inhibit the proliferation of chronic myeloid leukemia K562 cell line(IC₅₀=178.21 mg/m L),human gastric cancer MKN45 cell line(IC₅₀=186.60 mg/m L)and human gastrointestinal stromal tumor GIST882 cell line(IC₅₀=10.53 mg/mL)in vitro,among them the most targeted was GIST882 cell line.The dose-response curve showed that the inhibition rate of GIST882 cell proliferation was dependent on the concentration of PPIs within 48 h.In addition,flow cytometry was used to detect cell cycle and apoptosis.The results showed that PPIs could block GIST882 cells in G₂/M phase,and improved the apoptosis rate,and inhibit the proliferation of tumor cells in vitro.GIST882 cells stained with AO-EB double staining were observed with a fluorescent inverted microscope.It was found that the cell density and number decreased with the increase of PPIs concentration,and apoptotic bodies appeared.In addition,the results of wound healing experiments in vitro showed that PPIs could also inhibit the migration ability of GIST882 cells and prevent the metastasis and invasion of tumor cells.In conclusion,PPIs had satisfactory antioxidant activity in vitro and had better tolerance under different processing conditions.Meanwhile,the cell level experiments showed that PPIs was not toxic and could effectively protect cells from the oxidative damage of H₂O₂ and inhibit the growth of tumor cells.Thus,it had the potential to be used as a functional food factor.