| Almond oil of small white apricot has been a common food and medicine resource inXinjiang for a long time. Traditionally, the Uygur residents in Xinjiang believe almond oilhave functions of nerves relief, anti-inflammatory, anti-mutagen and anti-bacteria. The currentissues, such as aging almond trees, endangering wild species and low degree of deepprocessing and comprehensive utilization, have limited development of almond oil industry inXinjiang. Although biological functions of special regional resources have drawn wideattention today, study on fingerprint of Xinjiang small white apricot almond oil and itsfunctions of antioxidant and immunomodulatory regulation is blank. The followinginvestigations were conducted based on small white apricot resources inventory in Xinjiang:Small white apricot almond oil from different areas in Xinjiang was extracted based onprevious researches to analyze the major biological active substances using GC-MStechnology and determine its fingerprint information; then, clustering and similarity analysiswere used to build the fingerprint library of bioactive substances. The varieties and quality ofsmall white apricot almond oil were identified and monitored with principal componentanalysis (PCA) and partial least squares discriminant analysis (PLS-DA); Small white apricotalmond from Kuche was taken as sample to undertake chromatographic studies for almond oilsamples prepared by different ways. The scavenging effects of different almond oil samples toDPPH, hydroxyl, superoxide anion, ABTS and other free radicals were investigated. Smallwhite apricot almond oil was composed by complex biological active substances. Dose-effectof antioxidant activity of almond oil was studied based on PLSR analysis;Immunocompromised rat model was established. Samples from supercritical carbon dioxideextraction during the study of antioxidant performance were prepared as base materials.Immunomodulatory effects of small white apricot almond oil were studied for blank feedinggroup (BS), model group (MC), model almond oil sample group (SF). Its immunomodulatoryeffects to immunocompromised rats were examined according to assessment of immune organindex, immune factors in serum (liver) and slices of immune tissues; Serum samples obtainedfrom rats of control group parallel to the immunomodulatory experiments were investigated.Differentially expressed proteins of serum were quantitatively identified through iTRAQlabeling and high performance liquid chromatography-electrospray ionization tandem massspectrometry (LC-ESI-MS/MS). Functional annotation to the differentially expressed proteinsidentified was conducted with expression pattern clustering (EPC) and metabolic pathway(Pathway) analysis to determine their most important biochemical metabolic pathways andsignal transduction pathways. The results and conclusions are as follows:(1)13model samples were screened from15basic almond oil samples using GC-MS and cluster analysis to develop fingerprint of characteristic compositions of small whiteapricot almond oil. Similarities and principle component analysis for fingerprint wereconducted for10randomly selected adulteration samples and other oil products. The resultsfully demonstrated that the proposed fingerprint was effective to assess quality of small whiteapricot almond oil samples (products). A qualitative discriminant model was developed fordifferent varieties of grease and almond oil based on PLS-DA. All correlation coefficients ofpredicted values and observed values were0.95or above. The samples were100%identified.This indicated that comprehensive use of fingerprint information and PLS-DA discriminantcan accurately detect and distinguish small white apricot almond oil and other grease.(2) Kuche small white apricot almond oil samples prepared by supercritical CO2extraction (SCDE), mechanical crushing extraction (MCE), ultrasonic assisted extraction(UAE) and Soxhlet extraction (SE) were analyzed using GC and GC-MS technologies. Totalof33kinds of common active substances screened, including fatty acid (19species), sterolsand terpenes (14species) showed significant differences (P﹤0.05, exclude α-tocopherol).The samples prepared by different methods presented different scavenging capacity to DPPH,hydroxyl, superoxide anion and ABTS. Samples prepared by SCDE and MCE showed thescavenging capacity to free radicals presented a trend to be stronger than VEcontrol group,especially to superoxide anion.(3) The correlation between samples prepared by different methods and antioxidantindicators were analyzed using PLS2model. Samples prepared by UAE presented relativelypoorer scavenging capacity to all of four kinds of free radicals. SCDE samples were stronglypositively correlated to oxidative stress indicators (DPPH radicals and hydroxyl radicals);compared with almond oil samples prepared by SCDE and MCE, SE samples showed positivecorrelations to all four antioxidant indicators, however, the correlations were relatively weak.(4) The correlations between specific active substances and antioxidant indicators wereanalyzed through PLS1model. Scavenging capacity to DPPH free radicals showedsignificantly positive correlations with cis-oleic acid△9, linoleic acid△9,12, linoleic acid△8,11,linolenic acid△6,9,12, eicosatrienoic acid△8,11,14, α(γ)-tocopherol, spinach sterol△7,22,β-sitosterol△5and squalene; scavenging capacity to hydroxyl free radicals had significantlypositive correlations with linoleic acid△9,12,△5-stigmasterol△5,22, β-sitosterol△5, γ-tocopheroland squalene; scavenging capacity to free radicals of superoxide anion showed significantlypositive correlations with palmitoleic acid, arachidonic acid△5,8,11,14, α-tocopherol,△5-avenasterol△5,24(28)and△7-avenasterol△7,24(28); scavenging capacity to ABTS freeradicals showed significantly positive correlations with trans-oleic acid△9, linoleic acid△9,12,linoleic acid△6,9,12, α-tocopherol,△5-avenasterol△5,24(28)and squalene. Squalene, linoleic acid△9,12and α-tocopherol presented significant correlations with three or more antioxidantindicators, which may be the important base materials. Squalene△2,6,10,14,18,22, arachidonic acid△5,8,11,14,△5-avenasterol△5,24(28)and linolenic acid△6,9,12may play the key synergism infree radical scavenging system.(5) It was found from immunocompromised rat model and Enzyme-linkedimmunosorbent assay that Xinjiang small white apricot almond oil can significantlystrengthen spleen index, thymus index and liver index of immunocompromised rat (P﹤0.05)and improve tissue structures of immune organs. Its improvements to IgA, IgM, IgG, IL-12and SOD of rat serum and liver were highly significant (P﹤0.01). For IL-2, TNF-α, GSH-Pxand MDA, the improvements were relatively significant (P﹤0.05).(6) Serum differential proteomics study results showed that Xinjiang small white apricotalmond oil can significantly change protein expression profile of immunocompromised rats.Total of14common differential proteins were screened from251varieties of differentiallyexpressed proteins through cluster analysis. Enrichment analysis was conducted for14varieties of common differential proteins based on KEGG Pathway database. Total of35ofpathways for10kinds of common differential proteins were obtained, of which V-IIII sectionof immunoglobulin heavy chain (VH26) were related with20pathways;5innate immunepathways, including interactions with staphylococcus aureus infection, phagosome,pathogenic E. coli infection, primary immunodeficiency, complement and blood coagulationwere the most frequently. The relevant common differential proteins included haptoglobinprotein (HP), keratin (KRT10, KRT42), tubulin α-8chain (TUBA8), V-III section ofimmunoglobulin k chain (MOPC63), α-1-antiprotease (SERPINA1) and T-prokinin (KNG1).(7) Through STRING interactive analysis to unknown pathways of four commondifferential proteins, including serum deprivation-reactive protein (SDPR), T-syntheticproteinⅠsubunit (CCT3), N-Acetyl-L-Alanine amidase (PGLYRP2) and chromogranin-A(CHGA), suppressor pathways associated with tumors and microbial infections werepredicted.(8) The antioxidant and immunomodulatory effects of small white apricot almond oilwere demonstrated again by the analysis of pathways about differential proteins andbiological effects about related active substances. There were some foreseeable multiplecorrelations among active substances, such as unsaturated fatty acids, β-sitosterol△5,squalene△2,6,10,14,18,22, α-tocopherols, and14common differential proteins.This study aims to the fingerprint of major bioactive substances of Xinjiang almond andits application to look for a species and quality identification and monitoring model. Thepotential values of this food and medicine resource were further investigated throughevaluation of its biological function and molecular mechanisms. The results provided a goodtheoretical basis for development of functional products and quality certification, which had important significance for accelerating process from resource advantages to economyadvantage, protection of local high quality resources and quality upgrade. |
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