Study On The Authenticity Identification Of Sika Deer Skin Glue Based On Proteomics And Its Prescription Technology And Immunomodulatory Activity

Deerskin glue comes from the Manchu royal court and is made from the skins of sika deer（Cervus nippon）or red deer（Cervus elaphus）using a variety of special processing techniques.Ancient books have recorded that deerskin glue is non-toxic and has a good effect of nourishing qi and blood.So far,a large number of studies have shown that deerskin glue has the effects of nourishing blood,tonifying Yang,improving immunity and anti-fatigue.In this study,the sample was digested by sequence-level trypsin,and the quadrupole time-of-flight high-resolution mass spectrometry（QTOF）IDA mode was used,combined with database identification,and a unique peptide of sika deer skin glue was screened out,which can distinguish sika deer.The gelatin is steamed from the skin of other species of deer and common pigs,horses,cattle and sheep.The identification sequence is FSLADAINTEFK and the characteristic ion m/z is 678.3457.The screened sika deer skin glue peptide T1 and the deer skin glue characteristic peptide T2 that have been researched were synthesized,and the triple quadrupole mass spectrometer MRM mode was used for multi-reaction monitoring,and the characteristic molecular ion pair of T1 was selected m/z678.7→235.1,the characteristic molecular ion pair of T2 is selected m/z 725.0→274.1 for detection.The results show that T1 is a specific peptide of sika deer skin glue,which can distinguish sika deer from similar deer species and glue made from the skins of pigs,horses,cattle and sheep,and when mixed with different proportions of sika deer skin glue,miscellaneous skin glue there is no interference,and the peak area is directly proportional to the amount of incorporation,indicating that the method has strong specificity.T2 is unique to deerskin glue,and it can be detected in the animal samples of the deer genus.There is no signal in such species as pigs,horses,cattle and sheep,indicating that the existing T2 peptides are characteristic peptides of deerskin glue.A stable,sensitive and highly specific method for authenticity and quality control of sika deer skin glue has been established.Orthogonal experiment was conducted on the technological parameters of the formula"Shenqilujiao"containing deer skin glue.The optimal extraction technological parameters obtained by screening were:8 times the amount of water to decoct the medicinal materials3 times,and the time of each decoction one hour,three parallel verification experiments were carried out on the optimal process obtained from the screening,and all the indicators in the results maintained a high level.To study the iconic components of ginseng in Shenqilujiao,the content of ginsenoside Rg₁and Rb₁was determined by using HPLC phase,and the established exclusive detection method was investigated methodologically,and the stability and reproducibility of the method were investigated.It is proved that the detection method of ginsenoside Rg₁and Rb₁in the extract of Shenqilujiao is reasonable and feasible.The immunomodulatory effects of deer skin glue and the extract of Sengqilujiao containing deer skin glue formula were studied.Normally developed mice were used as the research object,the immune organ index after administration was calculated,and the serum cytokines（interleukin-2,interleukin-4,interferon-γ,tumor necrosis factor-αof each group of mice were detected by ELISA method）,The content changes were detected by flow cytometry for the changes in the distribution of T lymphocyte subgroups（CD4⁺,CD8⁺）in each group of mice.The results showed that the different doses of deer skin glue and Shenqilujiao extract can significantly increase the thymus index and spleen index of normal mice（P<0.05 or P<0.01）;the high dose group of deer skin glue and the medium,high doses group of Shenqilujiao extract promoted the secretion of serum cytokines（IL-2,IL-4,IFN-γ,TNF-α）in normal mice,and was concentration-dependent（P<0.01）;The high-dose group of deer skin glue and Shenqilujiao extract can increase the CD4⁺ratio of peripheral blood lymphocyte subsets in normal mice（P<0.01）,and each dose group of deer skin glue and Shenqilujiao extract can increase the peripheral blood of mice.Blood CD4⁺/CD8⁺value（P<0.05 or P<0.01）;Deer skin glue and Shenqilujiao extract did not cause obvious abnormal pathological stimulation or damage to the spleen tissue morphology of normal mice.The different doses of deer skin glue and Shenqilujiao extract have a certain immunoregulatory effect on the immune function of mice.They are safe,non-toxic and effective,and have strong development and utilization value.