Study On Preparation And Anti-Osteoporosis Activity Of Phospholipids From Tilapia Head

Tilapia is one of the key freshwater fishes in Chinese aquatic industry.Its processed products are mainly frozen tilapia fillets,which produce a large amount of fish head,skin,bones and other scraps in the processing process.Tilapia head is rich in phospholipid,which can be used as raw materials for aquatic health food.Phospholipids are the basic components of cell membranes,and have many important physiological functions such as inhibiting inflammation,regulating blood lipids,improving memory,and delaying aging.These physiological activities are closely related to their fatty acid composition and substituent groups(containing amino bases or alcohols)linked by phosphoric acid.In recent years,it has been found that phospholipids have a biological role in the treatment of bone diseases.In this paper,tilapia head phospholipid(TH-PL)was obtained by silica gel column chromatography using tilapia head as a raw material.And the effect of TH-PL on bone formation and bone resorption were studied at the cellular level.The details are as follows:1.The preparation,properties and structure identification of tilapia head phospholipidsFirstly,the tilapia head was used as raw material to extract the total lipid by solvent method.The total lipid was separated by silica gel coluln chromatography to obtain TH-PL.TH-PL accounted for 1.52%of the dry weight of tilapia head.Secondly,it was found by Fourier transform infrared spectroscopy that TH-PL has a characteristic functional group of a phospholipid.Gas chromatographic analysis showed that TH-PL contains 17 kinds of fatty acids,and the number of carbon atoms is between 14 and 22.Saturated fatty acids accounted for 34.84%of total fatty acids and C16:0 is the main part.Monounsaturated fatty acids accounted for 22.05%,of which C18:1 is the most abundant.In addition,TH-PL was also rich in polyunsaturated fatty acids,accounting for 38%,of which C18:2 n-6 was the highest.The contents of DHA and EPA were 7.98%and 0.51%,respectively.Finally,it was confirmed by thin layer chromatography and liquid chromatography-mass spectrometry that TH-PL mainly contains four phospholipid components,which are phosphatidyl choline(PC,65.17%),sphingomyelin(SM,17.60%),lysophosphatidylcholine(LPC,11.86%)and phosphatidyl ethanolmines(PE,2.62%).PC was mainly composed of molecular species such as 18:0/24:1,16:0/18:1,12:0/24:2,18:0/18:2,18:0/18:1,18:0/22:1,18:0/24:2 and 22:6/22:6.SM was mainly composed of molecular species such as 16:1/16:0,18:1/24:1,16:0/26:2,16:0/24:1,16:0/18:1 and 16:0/26:1.LPC was mainly composed of molecular species such as 16:0,18:1 and 18:2.PE was mainly composed of molecular species such as 22:6/22:6,18:0p/18:1 and 18:0/20:4.In addition,phospholipids containing DHA and EPA chains were detected in PC,and their relative contents were 5.1 1%and 0.62%,respectively.2.The effect of tilapia head phospholipids on proliferation and differentiation of MC3T3-E1 cellsIn order to investigate the effect of tilapia head phospholipids on the proliferation and differentiation of MC3T3-E1 cells,the osteogenic model was established by MC3T3-E1 cells in vitro,and the effect of tilapia head phospholipids was evaluated.In the process of index determination,the cell morphology of MC3T3-E1 was observed by inverted phase contrast microscope,the proliferation of MC3T3-E1 cells was determined by MTT assay,the alkaline phosphatase(ALP)activity of cells was detected by p-nitrophenyl phosphate(p-NPP)method,the expression of type ? collagen(COL I)and osteocalcin(OCN)was determined by enzyme-linked immunosorbent assay,and the alizarin red staining method was used for the determination of calcium nodules.The results showed that after adding TH-PL,the cell morphology and growth state were not affected compared with the control,and there was no significant promotion of cell proliferation.However,TH-PL significantly increased cellular ALP activity,promoted the expression of COL ? and OCN,and the formation of cellular calcium nodules.Compared with the control,when the final concentration of TH-PL added in the medium was 100 ?g/mL,the expression levels of ALP,COL I and OCN increased by 56.97%,49.15%and76.73%,respectively.The mineralized nodule rate increased by 65.44%.The above results showed that TH-PL did not significantly promote the proliferation of MC3T3-E1 cells,but significantly promoted the differentiation and mineralization of MC3T3-El cells.3.The effect of tilapia head phospholipids on osteoclast formation and apoptosisIn order to investigate the effect of tilapia head phospholipids on osteoclast formation and apoptosis,RANKL was used to induce RAW264.7 cell differentiation to establish osteoclast model,and MTT assay was used to detect the effect of TH-PL on the viability of RAW264.7 cells.The effect of TH-PL on osteoclast formation was evaluated by anti-tartaric acid phosphatase(TRAP)staining.Flow cytometry was used to detect the apoptosis of osteoclasts.The mitochondrial membrane potential of osteoclasts was determined by fluorescent staining with rhodamine 123 reagent.The expression of caspase-3 in osteoclasts was detected by Western blotting.The results showed that TH-PL had no toxic side effects on RAW264.7 cells.TH-PL with a concentration of 50 and 100?g/mL significantly inhibited the differentiation of RAW264.7 cells into osteoclasts,promoted osteoclast apoptosis,decreased osteoblast mitochondrial membrane potential,and increased caspase-3 expression in cells.Compared with the model group,TH-PL with a concentration of 100 ?g/mL increased the total apoptotic rate of osteoclasts to 24.17%,and the cell mitochondrial membrane potential decreased to 76.10%.Based on the above results,we judged that TH-PL has a significant inhibitory effect on osteoclast formation,and it is speculated that TH-PL induces osteoclast apoptosis through the mitochondrial pathway.