Immunomodulatory Effects Of N-butanol Extract And The Compound Isolated From Ligustri Lucidi Fructus In Immunosuppressed Mice

Immunosuppressive diseases are common in husbandry,which caused huge economic loss.To prevent the farm animals immunosuppressive,the exploitation of immune enhancers has been attracted much attention besides adjuvants and vaccines.Ligustri Lucidi Fructus(LLF)is the dry and ripe fruit of Ligustrum lucidum Ait.It is sweet and bitter in flavor,cool in nature and attributes to the liver and kidney meridians.Its main effects are invigorating liver and kidney,strengthening muscles and bones,and improving the eyesight.It can be used to enrich Yin due to the Yin deficiency,the deficiency of kidney manifested as spontaneous seminal emission,weakness of the waist and knees,failure of eyesight,etc.Current research showed that LLF contains terpenoids,phenylethanol,flavonoids and other components,which are able to improve the immunomodulatory,and with the effect of anti-inflammatory,antioxidant as well.Our preview study demonstrated that the LLF extracted by n-butanol(LLF-Bu)could enhance the immunity in vitro,and hydroxytyrosol could be its one of the most major elements in the LLF-Bu.However,the immunomodulatory effect of LLF-Bu and hydroxytyrosol in vivo is unknown.In this study,LLF-Bu was prepared by liquid-liquid extraction.Thin layer chromatography(TLC)and high-performance liquid chromatography(HPLC)were used to identify and determine the content of hydroxytyrosol in the LLF-Bu.Meanwhile,the immunomodulatory effects of LLF-Bu and hydroxytyrosol in immunosuppressed mice were investigated.The following results have been achieved so far:(1)The identification and content determination of hydroxytyrosol in LLF-Bu.LLF-Bu was prepared by liquid-liquid extraction,the identification and content of hydroxytyrosol was determined by TLC and HPLC.The LLF-Bu extract was analyzed by silica gel thin layer plate(chloroform: methanol: water: formic acid = 8:2:0.1:0.1 as developing solvent).After drying,the plate was developed with iodine vapor.The results showed that,the LLF-Bu and hydroxytyrosol appeared the similar spots in the same distance on the plate.The HPLC condition of content determination of hydroxytyrosol:5 μL of LLF-Bu were injected onto a Utimate-XBC18 HPLC column(250 mm×4.60 mm×5 μm).The mobile phase consisted of water: methanol: formic acid was 85:15:0.12 in volume.The column was operated at 28°C with a flow rate of 0.8 m L/min.UV detection was performed at 280 nm.The standard curve was y=6592.5x-43832(r=0.9999)and the content of hydroxytyrosol in the range of 33.33~200 μg/m L showed a good linear relationship with the peak area.The separation of the main peak was 2.318,the theoretical plate number was 11476,and the precision,repeatability and recovery met the requirements of Chinese Veterinary Pharmacopoeia.The above methods were suitable for the identification and content determination of hydroxytyrosol in LLF-Bu,and the content of hydroxytyrosol was 1.5964 mg/g.(2)The immunomodulatory effect of LLF-Bu in immunosuppressed mice.Immunosuppressive mice model: 12 KM mice were randomly divided into 4 groups.The 4 groups were high,middle,low-dose of cyclophosphamide(200,150 or 100 mg/kg,via intraperitoneal injection,once a day for 3 days)and healthy group,respectively.The dosage of cyclophosphamide was selected to evaluate the morphological changes of spleen tissue.The results showed that,the immunosuppressive mice model was successfully established by evaluating the cellularity of white pulp when the dose of cyclophosphamide was 100 mg/kg.In this dosage,the the cellularity of white pulp decreased moderate.While 150,200 mg/kg cyclophosphamide induced the reticuloendothelial cells and macrophage proliferation,besides the decrease of cellularity of white pulp.Therefore,100 mg/kg cyclophosphamide was injected intraperitoneally for 3 days to establish the immunosuppressive mice model.Immunomodulatory effect of LLF-Bu in immunosuppressed mice: 150 KM mice were randomly divided into 6 groups,which were high,middle,low-dose of LLF-Bu groups(800,400 or 200 mg/kg),positive control group(levamisole 40 mg/kg),model group and healthy group,respectively.The experimental groups were treated with cyclophosphamide at a dosage of 100 mg/kg via intraperitoneal injection,once a day for 3days.Then the mice were intragastrically administered with different doses of LLF-Bu,once a day for 7 days.The results showed that,compared with the model group,the cellularity of white pulp and myeloid cells in red pulp of mice spleen tissue were increased in different dose groups.The thymus index was significantly increased in different dose groups(P<0.05),the spleen index was markedly increased in 800 mg/kg dosage group(P<0.05).The proliferation ability of T and B lymphocytes and phagocytic function of macrophage were significantly enhanced in 400,800 mg/kg dosage group(P<0.05).The serum level of Ig G was significantly upregulated in 400,800 mg/kg dosage group(P<0.05).The level of MDA was markedly downregulated in 800 mg/kg dosage group(P<0.05).The results indicated that LLF-Bu could enhance immunity of immunosuppressed mice.(3)The immunomodulatory effect of hydroxytyrosol in immunosuppressed mice.150 KM mice were randomly divided into 6 groups,which were high,middle,low-dose of hydroxytyrosol groups(50,10 or 2 mg/kg),positive control group(levamisole40 mg/kg),model group and healthy group,respectively.The experimental groups were treated with cyclophosphamide at a dosage of 100 mg/kg via intraperitoneal injection,once a day for 3 days.Then the mice were intragastrically administered with different doses of hydroxytyrosol,once a day for 7 days.The results showed that,the cellularity of white pulp of mice spleen tissue was increased in different dose groups.The splenic myeloid cells were increased in 2 mg/kg dosage treatment group.In contrast,in 10,50 mg/kg dosage treatment groups were decreased(P<0.05).The thymus index of mice was significantly increased in different dose groups compared with the model group(P<0.05),and there was significant difference between the three dose groups(P<0.05).The spleen index was significantly increased in 2,10 mg/kg dosage groups(P<0.05),but in 50 mg/kg dosage group was markedly decreased(P<0.05).The proliferation ability of T and B lymphocytes were significantly activated in 10,50 mg/kg dosage groups(P<0.05).The phagocytic function of macrophages was significantly enhanced in 50 mg/kg dosage group(P<0.05).The serum level of Ig G was significantly upregulated in 10 and 50 mg/kg dosage groups(P<0.05).The level of MDA was markedly downregulated in 50 mg/kg dosage group(P<0.05).The results indicated that hydroxytyrosol could enhance immunity of immunosuppressed mice.In conclusion,the content of hydroxytyrosol in LLF-Bu was 1.5964 mg/g.LLF-Bu and hydroxytyrosol have immune-enhancing effect on immunosuppressed mice.