Haiqing FU,Xi LIN,Huaying FU,Wei CAI,Tuanwei CHEN

1.Jinshan College of Fujian Agriculture and Forestry University,Fuzhou 350002,China;

2.College of Crop Science of Fujian Agriculture and Forestry University,Fuzhou 350002,China;

3.College of Food Science of Fujian Agriculture and Forestry University,Fuzhou 350002,China

Effects of Compound Phellinus igniarius(L.ex Fr.) Quel.Oral Liquid’s Polysaccharide on the Immunologic Function of Mice

Haiqing FU1*,Xi LIN1,Huaying FU2,Wei CAI1,Tuanwei CHEN3

1.Jinshan College of Fujian Agriculture and Forestry University,Fuzhou 350002,China;

2.College of Crop Science of Fujian Agriculture and Forestry University,Fuzhou 350002,China;

3.College of Food Science of Fujian Agriculture and Forestry University,Fuzhou 350002,China

In this paper,the effects of compoundP.igniariusoral liquid’s crude polysaccharide on the immunologic function of mice were studied from four aspects, namely,carbon clearance test of mice,macrophage phagocytosis of chicken red blood cells in the enterocoelia of mice(semi-in vivo method),the ratio of organ to body weight,natural killer(NK)cell activity in mice(the determination of lactate dehydrogenase assay).The results showed that high-dosage group(40 mL/kg)of compoundP.igniariusoral liquid can obviously enhance the ability of carbon clearance of mice;middle-(20 mL/kg)and high-dosage groups can significantly enhance the phagocytic rate and phagocytic index of chicken erythrocyte of mouse macrophage; low-(10 mL/kg),middle-and high-dosage groups can significantly enhance NK cell activity of mice.These showed that compoundP.igniariusoral liquid can enhance mononuclear-macrophage and NK cell activity.In conclusion,compoundP.igniariusoral liquid’s polysaccharide can enhance immunologic function and significantly improve the specific and nonspecific immunologic function.

Phellinus igniarius(L.ex Fr.)Quel.;CompoundPhellinus igniariusoral liquid;Crude polysaccharide;Mice;Immunologic function

P hellinus igniarius(L.ex Fr.) Quel.is aPhellinusfungus belonging to family Polyporaceae of Polyporales in Basidiomycetes, commonly known asPhellinus igniarius(L.ex Fr.)Quél.,Jew’s Earon mulberry tree,Sangchen,Husunyan,etc. It is a kind of precious medical fun gus[1],it is also the most effective medicinal fungus in the field of biological cancer therapy[2].Modern researches have showed that the polysaccharide ofP.igniariushas an antitumous effect[3-4],which can improve immunity by cellular regulation and humoral immunity,thereby inhibiting the gr-owth and metastasis of tumor cells[5-8],what’s more,it has not toxic side effect[9-10].Currently,the materials for pharmacological research ofP.igniariusare mainly the extractives of fruit bodies ofP.igniarius[11-14],but resour-ces of fruit bodies ofP.igniariuswill run out,and artificial cultivation is hard to be realized,so it is of great significance to replace fruit bodies with fermentation mycelia.Some researches have indicated that the nutritional ingredients between the two have not significant difference,and fermentation mycelia ofP.igniariuseven have more constituents[15-16]. Now,the researches on immunologic function of oral liquid made from the polysaccharide ofP.igniariusand other extractives from edible and medicinal materials are few[17-18].In the paper, to know the immunologic function of

Materials and Methods

Experimental samples

P.igniarius oral liquid(positive control)was made from the fermentation broth of P.igniarius liquid and the extracting solution of P.igniarius mycelia polysaccharides[19-21],its ingredient was crude polysaccharide of P.igniarius(the content was 10.0 ml/kg);compound P.igniarius oral liquid was composed of the above P.igniarius oral liquid(the content accounted for 60%,V/V),Gynostemma pentaphyllum(Thunb.)Makino,Aloe,Codonopsis pilosula and Lycium chincnse Mill.(each content accounted for 10%, V/V,the content of crude polysaccharide was 10.0 mg/ml).P.igniarius strain was provided by Fungus Research Center of Fujian Agriculture and Forestry University and the code was Ph.l0001.

Experimental animals

The experimental animals were ICR mice,they were clean grade and male,the weights were among 18-20 g,and purchased from Shanghai Slac Laboratory Animal Co.,Ltd.The number of animal production license was SCXK(Hu)2012-0002,and the number of animal occupancy license was SYXK(Min)2013-0003.The temperature of animal room was 22-25℃and its relative humidity was 55%-70%.

The experimental animals were randomly divided into 10 groups according to weight,each group had 10 mice,and each experimental group contained 5 groups,thus there were such two experimental groups as immunity group 1 and immunity group 2. Group 1 carried out carbon clearance experiment;group 2 carried out macrophage phagocytosis of chicken red blood cells in the enterocoelia of mice,the ratio of organ to body weight and the determination of NK cell activity.Each experimental group contained three dosage groups and two control groups,namely low-(the content of compound P.igniarius oral liquid was 10 mL/kg),middle-(the content of compound P.igniarius oral liquid was 20 mL/kg)and high-(the content of compound P.igniarius oral liquid was 40 ml/kg)dosage groups, positive control group(the content of P.igniarius oral liquid was 40 ml/kg) and negative control group(the content of normal saline was 20 ml/kg), mice of each group were continuously lavaged for 15 d.

Instruments and equipment

Key instruments were Thermo YL. C.4-PY.04 low-temperature highspeed centrifuge,Biorad550 YL.A.1-MB.01 microplate reader,Thermo YL. C.4-EY.01 CO2 incubator,Shimadzu YL.A.1-ZY.07 UV2450 ultraviolet spectrophotometer,ESCO YL.C.1-GZ. 12 biosafety cabinet,YL.C.3-XW.31 Lycra inverted microscope,Mettler-AE200 YL.A.2-TP.08 electronic scales,YL.C.4-PY.04 constant temperature incubator,96-hole culture plate(flat base),etc.

Main reagents

Main reagents were Indian ink for injection,20%chicken red cell suspension(fresh chicken blood was got to place in conical flask with glass beads, then the flask was shaken fully along one direction to defiber;and then it was washed with normal saline for three times with the centrifugal speed of 2000 r/min for 10 min,followed by clearing supernate,finally 20%V/V chicken red cell suspension was made),Giemsa dye liquor,YAC-1 cell, Hanks liquid(pH 7.2-7.4),PBS buffer solution(pH7.2-7.4),RPMI1640 cell culture fluid,calf serum,lithium lactate, INT,PMS,NAD,Tris-HCL buffer solution(pH 8.2)1%NP40 and trypan blue, etc.

Experimental methods[22]

The determination of mouse carbon clearance testOne hour after the last administration,mice of each group were weighed,and diluent Indian ink (1:8)was injected into caudal vein of mice according to the mouse weight of 10 ml/kg,and timing immediately;2 and 10 min after injection,20 μl blood from inner canthus veinplex was respectively got,then they were immediately put into 2 ml 0.1%Na2CO3solution.Taking Na2CO3solution as negative control,the optical density(OD) was measured at the wave length of 600 nm using ultraviolet spectrophotometer.Finally,mice were sacrificed, their livers and splenic organs were taken out,and all superficial bloodiness was soaked up,then weighing respectively and calculating the phagocytic index a.

Macrophage phagocytosis of chicken red blood cells in the enterocoelia of mice(semi-in vivomethod)and the ratio of organ to body weightOne hour after the last administration,1 ml 20%chicken red cell suspension was injected into the enterocoelia of every mouse.After 30 min,these mice were killed by cervical vertebra dislocation and fixed in rat boards with the supine position,then the abdominal skin was cut open and 2 ml normal saline was injected into the enterocoelia,followed by rotating rat boards for 1 min.After that,1 ml peritoneal lavage fluid was extracted and averagely dropped onto two glass slides,followed by putting into enamel box lined with wet gauze,and then they were moved to 37℃incubator for 30 min.After incubating,normal saline was used for rinsing to get rid of unposted cells.Drying in the air,1:1 acetone methanol solution was used for fixing,4%(V/V)Giemsa-phosphate buffer was used for dyeing 3 min,and distilled water was used for rinsing and airing.Afterwards,the slices were read under oil immersion lens,and phagocytic percentage and phagocytic index were calculated.And splenic organs and thymuses of mice were taken out, the blood was soaked up,then weighing respectively,thymus index and spleen index were counted.

The determination of NK cell activity of mice(the determination of lactate dehydrogenase assay)The mice were killed by cervical vertebra dislocation,their spleens were taken out under aseptic condition and put on small flat dishes with sterile Hank’s solution,and riven gently with tweezers,then after filtrating with 200-mesh sieve,single-cell suspension for standby application was made.Cell suspension was washed by Hank’s solution twice with the centrifugalspeed of 1 000 r/min for 10 min.The supernatant was discarded and the cytoplasm was bounced,0.5 ml sterile water was put into for 20 s,after the splitting decomposition of red blood cells,0.5 ml twofold Hank’s solution and 8 ml Hank’s solution were put into again with the centrifugal speed of 1 000 r/min for 10 min,then 1 ml RPMI1640 complete culture solution containing 10%calf serum was used for resuspending,and living cell population was counted by trypan blue dyeometer(the number should be above 95%),finally,cell concentration was adjusted to 2×107/ml using RPMI1640 complete culture solution.100 uL target cell and 100 ul effector cell (the ratio of effector cell to target cell was 50:1)were got to put onto U-type 96-hole culture plate;100 μl target cell and 100 μl nutrient solution were put in natural releasing holes,and 100 μl target cell and 100 μl 1%NP40 were put in the biggest releasing holes of target cell.There were three parallel holes for all above items,and they were all cultured in 37℃and 5%CO2incubator for 4 h,then 96-hole culture plate was centrifuged for 5 min with the centrifugal speed of 1 500 r/min,and each hole absorbed 100μl supernate, meantime,100 μl LDH matrix solution was added for keeping 3 min,each hole was added 1 mol/L HCl 30 μl,the optical density(OD)was determined at 490 nm of microplate reader,thereby NK cell viability was calculated.

Statistical treatment of data

Results and Analysis

Effects of samples on mouse weight

The mice were lavaged for two weeks using different dosages of compound P.igniarius oral liquid, P.igniarius oral liquid and normal saline,and its effects on mouse weight were studied,the results were in Table 1 and Table 2.And there was no significant difference between the effects of the samples on the growth of mouse weight of each group and that of negative control group(P＞0.05),that was, the samples had no effect on the growth of mouse weight.

Carbon clearance test of mice

Phagocytosis test is an important indicator determining the effects of drugs on specific and nonspecific immunologic functions,the measuring methods were generally divided into in vivo and in vitro(including semi-in vivo).In the experiment,the method of in vivo was adopted firstly to carry out carbon clearance test of mice,and phagocytic index a of each group was determined and counted(in Table 3). The greater the index,the higher the phagocytic activity that had on granular foreign body[23].The results showed that compared with negative control group,high-dosage group and positive control group of samples can obviously improve phagocytic index a,moreover,there was no obvious difference (P＜0.05),thus it can be seen that the samples can significantly improve the carbon clearance ability of mice.

Macrophage phagocytosis of chicken red blood cells in the enterocoelia of mice

In the experiment,the method of semi-in vivo was used to study the macrophage phagocytosis of chicken red blood cells in the enterocoelia of mice,the results were shown in Table 4.The greater the phagocytic percentage,the stronger the phagocytic function of peritoneal macrophage.In the samples,compared with negative control group,middle-and highdosage groups as well as positive control group can obviously enhance the phagocytic rate and phagocytic index of macrophage phagocytosis of chicken red blood cells in the enterocoelia of mice,and the differences were obvious(P＜0.05),indicating that the samples had enhancement function on phagocytic ability of macrophages.

The determination of thymus index and spleen index

The experiment studied the effects of the samples on immune organs like spleen and thymus,etc.,the results were in Table 5.Compared with negative control group,thymus index and spleen index of other dosage groups had not significant difference(P＞0.05).

The determination of NK cell viability

NK cells are immune cells with heterogeneous multifunction and natural killer cells having nothing to do with specific immune response.They had regulating effects to multiple cells in vivo,especially for T and B lymphocytes.The cell lysis mediated by thecells was not limited by major histocompatibility complex(MHC).They had strong scavenging action not only for cancer cell,but also for virus,intracellular bacterium and aging differentiated cell.Their lethal effect didn’t need pre-existing immunity or sensitization,and the lethal effect was earlier than their effector cells,which was the first line of defense against tumour. They also played a certain role in the pathogenesis of anti-virus and autoimmune disease,thus they had important significance in anti-tumor and immune adjustment.Therefore,finding the substances enhancing NK cell viability was of important significance to immune adjustment and anti-tumor[24]. The experiment studied the effects of the samples on NK cell viability,the results were in Table 6.In the samples,compared with negative control group,low-,middle-and high-dosage groups as well as positive control group can significantly improve NK cell viability,and the differences had obvious(P＞0.05)or extremely obvious significance(P＞0.01),showing that the samples can significantly improve NK cell viability.

Table 1Effects of samples(immunity group 1)on mouse weight

Table 1Effects of samples(immunity group 1)on mouse weight

Group The number of animals Initial weight weight P Weight increment P Final Negative control 10 19.2±0.6 33.0±1.4-13.8±1.5-Low-dosage group 10 19.3±1.1 33.4±1.0 0.375 14.1±1.0 0.573 Middle-dosage group 10 19.2±0.8 32.7±0.8 0.505 13.5±1.1 0.573 High-dosagegroup 10 18.9±0.9 33.0±0.8 1.000 14.1±1.2 0.573 Positive control 10 19.5±0.7 32.4±0.8 0.186 12.9±1.1 0.095

Table 2Effects of samples(immunity group 2)on mouse weight

Table 2Effects of samples(immunity group 2)on mouse weight

Group The number of animals Initial weight weight P Weight increment P Final Negative control 10 19.0±0.7 33.0±1.4-14.0±1.6-Low-dosage group 10 19.1±0.7 33.5±1.2 0.303 14.4±1.4 0.523 Middle-dosage group 10 19.2±0.8 33.0±1.1 1.000 13.8±1.6 0.749 High-dosagegroup 10 19.5±0.7 33.1±0.9 0.836 13.6±1.3 0.523 Positive control 10 19.2±0.6 32.6±0.7 0.409 13.4±1.0 0.339

Table 3Carbon clearance test of mice

Table 3Carbon clearance test of mice

Compared with negative control,*P＜0.05,**P＜0.01.

Table 4Macrophage phagocytosis of chicken red blood cells in the enterocoelia of mice (X±S)

Table 5The determination of thymus index and spleen index

Table 5The determination of thymus index and spleen index

Group The number of animals index∥%P Spleen index∥%P Thymus Negative control 10 0.21±0.02-0.35±0.03-Low-dosage group 10 0.20±0.04 0.913 0.33±0.04 0.203 Middle-dosage group 10 0.21±0.06 1.000 0.33±0.03 0.263 High-dosagegroup 10 0.22±0.06 1.000 0.36±0.07 0.666 Positive control 10 0.20±0.04 0.983 0.34±0.05 0.420

Table 6The determination of NK cell viability

Table 6The determination of NK cell viability

Compared with negative control,*P＜0.05,**P＜0.01.

Group The number of animals NK cell viability∥% Conversion value of NK cell viability P Negative control 10 24.96±9.14 0.52±0.11-Low-dosage group 10 31.71±4.48 0.60±0.05*0.043 Middle-dosage group 10 32.29±5.51 0.60±0.06*0.031 High-dosagegroup 10 46.60±9.22 0.75±0.10**0.001 Positive control 10 47.41±9.99 0.76±0.10**0.001

Conclusions

The results showed that highdosage group of compound P.igniarius oral liquid can obviously improve the carbon clearance ability of mice; middle-and high-dosage groups can obviously enhance the phagocytic rate and phagocytic index of macrophage phagocytosis of chicken red blood cells in the enterocoelia of mice;low-, middle-and high-dosage groups can significantly improve NK cell viability; however,each dosage group had not effect on the weight,spleen index and thymus index of mice.These showed that compound P.igniarius oral liquid can enhance mononuclearmacrophage and NK cell activity.In conclusion,compound P.igniarius oral liquid’s polysaccharide can enhance immune function and significantly improve the specific and nonspecific immunologic functions.

Discussions

The experiment was based on the preliminary study,P.igniarius oral liquid was taken as positive control,and it has already showed good effect of enhancing immunologic function in preliminary study[23].And compound P.igniarius oral liquid generally had the same function.From specific test items,compared with positive control group,high-dosage group of compound P.igniarius oral liquid almost had same value or slightly lower value in improving carbon clearance ability of mice,enhancing phagocytic rate and phagocytic index of macrophage phagocytosis of chicken red blood cells in the enterocoelia of mice,and improving NK cell viability,these revealed that P.igniarius polysaccharide played an important role in enhancing immunization,after compounding,the whole immunologic function weakened in some ways because of the reduction of dosage.However,in terms of economics,low-cost raw materials made the production cost of compound P.igniarius oral liquid lower, showing the better market application prospect.

Compound research of counterparts have mainly focused on fungi polysaccharides[17-18],the experiment was a compound try on P.igniarius polysaccharide and nonfungal polysaccharide,and the results showed a better immune effect.How to make compound better or which materials should be adopted for enhancing im-mune effect should be studied further.

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Responsible editor:Nanling WANG

Responsible proofreader:Xiaoyan WU

復(fù)方桑黃口服液粗多糖對小鼠免疫功能的影響

傅海慶1*，林希1，傅華英2，蔡瑋1，陳團偉3
（1.福建農(nóng)林大學(xué)金山學(xué)院，福建福州350002；2.福建農(nóng)林大學(xué)作物科學(xué)學(xué)院，福建福州350002；3.福建農(nóng)林大學(xué)食品科學(xué)學(xué)院，福建福州350002）

研究復(fù)方桑黃口服液粗多糖對小鼠免疫功能的影響。從小鼠碳廓清實驗、小鼠腹腔巨噬細胞吞噬雞紅細胞實驗（半體內(nèi)法）、臟器與體重比值、小鼠自然殺傷（NK）細胞活性測定（乳酸脫氫酶測定法）等四個方面進行研究。結(jié)果表明：復(fù)方桑黃口服液的高劑量組（40 ml/kg）能明顯增強小鼠碳廓清能力，中（20 ml/kg）、高劑量組能明顯增強小鼠巨噬細胞吞噬雞紅細胞的吞噬百分率和吞噬指數(shù)，低（10 ml/kg）、中、高劑量組能明顯增強小鼠NK細胞活力功能。這表明復(fù)方桑黃口服液具有增強單核-巨噬細胞功能、增強NK細胞活力功能，復(fù)方桑黃口服液具有增強免疫功能的作用，可顯著提高機體特異性和非特異性免疫功能。

桑黃；復(fù)方桑黃口服液；粗多糖；小鼠；免疫功能the self-developed compound P.igniarius oral liquid’s crude polysaccharide and determine its application prospect,the effects of compound P. igniarius oral liquid’s polysaccharide on the immunologic function from the following aspects were studied,such as weight of immune organs,charcoal clearance rate,macrophage phagocytosis,the determination of natural killer (NK)cell activity,etc.

國家級大學(xué)生創(chuàng)新訓(xùn)練計劃項目“以藥用菌桑黃多糖為主要成分的增強免疫功能食品的研制”（201314046008）；校級教改項目：食品質(zhì)量與安全專業(yè)綜合改革（0137Z5）。

傅海慶（1973-），男，福建上杭人，副教授，碩士，主要從事食品科學(xué)技術(shù)研究，E-mail：627511829@qq.com。*通訊作者。

2015-03-20

修回日期 2015-05-24

Supported by National-level Innovative Training Program for Undergraduate Students "On the Preparation of Immunity Enhancement Food TakingPhellinus igniariusPolysaccharide from Medicinal Fungus as Main Component"(201314046008);the Project of Unversity Teaching Reform:On Comprehensive Reform of Food Quality and Safety Specialty(0137Z5).

*Corresponding author.E-mail:627511829@qq.com

Received:March 20,2015 Accepted:May 24,2015