CN107011393A - A kind of arginine monoglycosides（AF）Novel synthesis and its purposes in terms of medicine - Google Patents

Abstract

The present invention provides a kind of arginine monoglycosides (AF) artificial synthesis, and this synthetic method synthetic ratio is high, environmentally friendly, while suitable for industrialized production.That is the combination of arginine and one or two kinds of above monose is reacted in one or two kinds of above solvent, this reaction system can add following material, one or more kinds of mixtures in vitamin C, sodium sulfite, edible organic acid, heat time is 120 DEG C of 200min, optimal reaction temperature is 80 DEG C, the double monoglycosides of generation arginine.It can be good at removing hydroxyl radical free radical and superoxide anion present invention is disclosed the arginine monoglycosides after synthesis, while H can be suppressed₂₂The growth of tumor-bearing mice tumour, improves the immunity of immunosuppressed mice.

Description

A kind of arginine monoglycosides（AF）Novel synthesis and its purposes in terms of medicine

Technical field

The present invention relates to a kind of arginine monoglycosides（AF）Novel synthesis and its purposes in terms of medical treatment.

Background technology

Arginine monoglycosides English entitled Arginyl-Fructose, abbreviation AF, also known as arginine fructoside, molecular weight For 337.3, chemical name is 1-（Arginine-N^αBase）- 1- deoxidation-D fructose.AF monomers after purification are white powder, AF poles It is soluble in water, the organic solvents such as methanol are practically insoluble in, with hygroscopicity, to thermally labile.

Arginine monoglycosides（AF）, be present in many plants of nature, Zheng Yinan it is fresh ginseng be processed into red ginseng, it is sun-dried During ginseng, it was found that AF presence.Contents of the AF in nature is very low, but research is found in processing of Panax ginseng into sun-dried AF contents are higher when ginseng, red ginseng.After AF is the heated working process of fresh ginseng, it is anti-that Mei Lade occurs for sugar and amino acid in ginseng It should generate^[1,2].Research finds that AF has very strong pharmacological activity, positive role is played in disease treatment.Grind at present Study carefully and show, AF is in anti-diabetic^[3,4], it is anti-oxidant^[5,6], it is antitumor^[7], play a role in the treatment such as liver protection.

The method of current arginine monoglycosides synthesis is mainly patent " arginine fructoside (AF) preparation method and its medical Purposes, application number：201410305612.1 " are reported, but its method solvent selected in building-up process is not only dirty Environment is contaminated, is hazardous to the human body, synthetic ratio is relatively low, process is also complex for follow-up isolating and purifying, and the present invention is on overcoming State and a kind of new synthesis technique is developed in synthesis defect, and disclose some biological activity of arginine glucoside after synthesis.

The content of the invention

The purpose of the present invention is the synthetic method and its medical application for disclosing a kind of arginine monoglycosides.

The synthetic method synthetic ratio of arginine monoglycosides disclosed by the invention is more than 63%, and the proportioning of arginine and monose is 1：0.5-2.

Arginine required in the synthetic method of arginine monoglycosides disclosed by the invention can be L-arginine, D- essences The mixture of propylhomoserin one or two kinds of therein.

Monose required in the synthetic method of arginine monoglycosides disclosed by the invention can be D-Glucose, D- fruits One or more kinds of mixtures in sugar, D- galactolipins, D- xyloses.

Generated time required in the synthetic method of arginine monoglycosides disclosed by the invention is 120-300min.

Synthesis temperature required in the synthetic method of arginine monoglycosides disclosed by the invention is 80 DEG C.

In the synthetic method of arginine monoglycosides disclosed by the invention required for solvent for propane diols, glycerine, propyl alcohol, One or more kinds of mixed solvents in isopropanol.

Sodium sulfite, vitamin C, vitamin can also be added in the synthetic method of arginine monoglycosides disclosed by the invention One or more kinds of mixtures in E.

It is edible that citric acid, malic acid etc. can also be added in the synthetic method of arginine monoglycosides disclosed by the invention One or more kinds of mixtures in organic solid acid.

The arginine monoglycosides of synthesis disclosed by the invention, which have, to be removed hydroxyl radical free radical and ultra-oxygen anion free radical, resists Tumour, the purposes for improving immunity.

The positive effect of the present invention is：AF synthetic method in the present invention, synthetic ratio is greatly improved, and reachable 63%, together When reduce AF production cost, and thick synthetic has no irritating odor, and belongs to the synthetic method of green environment friendly.

Experiments show that, the AF of synthesis has removing free radical, work(that is antitumor and improving immunity of organisms Effect.

The arginine monoglycosides of experimental example 1.（AF）Synthesis technique

1.1 experimental method

1.1.1 the drafting of standard curve

Precision weighs the mg of AF reference substances 5, with water constant volume in 25 mL volumetric flasks, 2,4,6,8 mL reference substances is pipetted respectively molten Liquid is settled to scale in 10 mL volumetric flasks with water, obtains the control that mass concentration is respectively 0.1,0.2,0.3,0.4 mg/mL Product solution.

HPLC conditions：Venusil-AA amino acid analysises dedicated columns (5 μm, the mm of 4.6 mm × 250)；Mobile phase：A phases- Sodium acetate buffer solution（925 mL pure water, 7.6 g sodium acetates, 2 mL phosphoric acid, 2-3 d glacial acetic acids, 75 mL acetonitriles）, B phases- Organic phase（Acetonitrile: water=4: 1）.The mL/min of flow velocity 1.0；The nm of Detection wavelength 254；40 DEG C of column temperature；The μ L of sample size 20.

1.1.2 single factor experiment

1.1.2.1 optimum reacting time is screened

Fixed 85 DEG C of reaction temperature, solid-liquid ratio 1:20, the g of malic acid addition 0.75, investigate the reaction time be 60,90,120, 150th, influences of 180 min to AF synthetic ratios.

1.1.2.2 optimal reaction temperature is screened

The min of fixation response time 150, solid-liquid ratio 1:20, the g of malic acid addition 0.75, investigate reaction temperature be 70,75,80, 85th, 90 DEG C of influences to AF synthetic ratios.

1.1.2.3 screen optimal solid-liquid ratio

The min of fixation response time 150,85 DEG C of reaction temperature, the g of malic acid addition 0.75 investigates solid-liquid ratio 1:10、1:15、 1:20、1:25、1:Influences of 30 g/mL to AF synthetic ratios.

1.2.2.4 optimal malic acid addition is screened

The min of fixation response time 150,85 DEG C of reaction temperature, solid-liquid ratio 1:20, malic acid addition 0.5,0.75,1.0 is investigated, Influences of 1.25 g to AF synthetic ratios.

1.1.3 response phase method is tested

On the basis of single factor test, using statistical analysis software DesignExpert 8.0.6, according to Box-Behnken center Combination experiment design principle, with the reaction time（X₁）, reaction temperature（X₂）, solid-liquid ratio（X₃）, malic acid addition（X₄）To become certainly Amount, AF synthetic ratios（Y）For response, optimize, experimental factor is as shown in table 1 with level code.

The response surface optimization experimental factor level of table 1

1.2 result of the test

1.2.1 the drafting of standard curve

Peak area of the AF reference substance solutions of various concentrations under 254 nm is determined, with mass concentration X (mg/mL) to peak area Y draws standard curve, and obtaining regression equation is：Y=298449.88X, R²=0.9991, in the mg/ of mass concentration 0.1 ~ 0.5 It is in good linear relation in mL.

1.2.2 single factor experiment

1.2.2.1 influence of the reaction time to AF synthetic ratios

In the case where other experimental conditions are constant, with the extension of time, the acid catalyzed AF synthetic ratios of apple gradually rise, When the time reaching 150 min, synthetic ratio peaks, as reaction continues, and reacts to AF and reduces direction progress.Therefore, The scope in the reaction time that Box-Behnken experiments are chosen is 120-180 min（Accompanying drawing 1）.

1.2.2.2 influence of the reaction temperature to AF synthetic ratios

Test of many times finds that when between 70-90 DEG C of temperature, AF synthetic reaction occurs rapidly, meets what AF was formed in red ginseng Temperature conditionss, when temperature reaches 85 DEG C, AF synthetic ratios peak, as temperature continues to raise, synthetic ratio reduction.Cause This, the scope for the reaction temperature that Box-Behnken experiments are chosen is 80-90 DEG C（Accompanying drawing 2）.

1.2.2.3 influence of the solid-liquid ratio to AF synthetic ratios

In the case where other experimental conditions are constant, during using solid-liquid ratio as variable, when glycerine dosage is too low, reaction is difficult to mix It is even, and react not thorough, synthetic ratio is relatively low, with the increase of solvent volume, reaches 1:During 20 g/mL, AF synthetic ratio highests, with Glycerine volume to continue to increase, synthetic ratio declines.Therefore, the scope for the solid-liquid ratio that Box-Behnken experiments are chosen is 1:15- 1:25 g/mL（Accompanying drawing 3）.

1.2.2.4 influence of the malic acid addition to AF synthetic ratios

In the case where other experimental conditions are constant, with the increase of malic acid addition, AF synthetic ratio gradually rises, apple When sour addition reaches 0.75g, synthetic ratio reaches maximum, is then decreased with the increase of malic acid content.Therefore, The scope for the malic acid addition that Box-Behnken experiments are chosen is 0.5-1.0 g（Accompanying drawing 4）.

1.2.3 response phase method optimum synthesis test results and analysis

1.2.3.1 result of the test

Box-Behnken designs are carried out with the analysis softwares of Design Expert 8.0, design and its result of the test are shown in Table 2,1-24 factorial experiments of tested number, 25-29 multi-center trials of tested number.

1.2.3.2 interpretation of result

Result of the test to table 2 is analyzed, and obtains AF synthetic ratios（Y）To reaction temperature（X₁）, the reaction time（X₂）, solid-liquid ratio （X₃）, acid adding amount（X₄）Secondary multinomial regression equation be：

Y=67.79+2.96X₁+1.74X₂-1.77X₃+1.05X₄₊1.24X₁X₂+1.53X₁X₃+1.72X₂X₃-1.55X₂X₄- 2.01X₃X₄-5.15X₁ ²-6.09X₂ ²-5.49X₃ ²-6.62X₄ ²。

Table 3 is the results of analysis of variance of regression model, and model F values are 53.18, R2=0.9815, P<0.0001, explanation You and degree of regression equation are preferable, and experiment is with very high credibility and accuracy.The amendment coefficient R of model²Adj= 0.9631, show that the equation preferably reflects generated time, synthesis temperature, solid-liquid ratio and malic acid addition and AF synthetic ratios Relation, illustrate that the model can explain 96.31% response value changes, therefore available synthesis of this model to AF is analyzed And prediction.From the analysis result of F values（A=95.2、B=32.83、C=34.03、D=11.89）As can be seen that in selected each factor In horizontal extent, the order of the influence size to AF synthetic ratios is：Reaction temperature ＞ solid-liquid ratio ＞ reaction time ＞ malic acid adds Enter amount.

The Box-Behnken experimental designs scheme of table 2 and result

The regression model the results of analysis of variance of table 3

R²=0.9815, R²Adj=0.9631, R²Pred=0.9159, CV=1.81

1.2.3.3 the response surface analysis of AF synthesis techniques and optimization

Accompanying drawing 6,10 curved surfaces are precipitous, and contour is oval, illustrate reaction temperature and solid-liquid ratio, solid-liquid ratio and malic acid addition There is very strong reciprocation, in pole conspicuousness；The curved surface of accompanying drawing 5,6,8 is relatively gentle, illustrates reaction temperature and reaction time, reaction Temperature has certain reciprocation with solid-liquid ratio, reaction time and solid-liquid ratio, but contour is almost circular, and reciprocation is not It is very strong；The curve of accompanying drawing 7, contour is circle, illustrates that reaction temperature and malic acid addition reciprocation be not notable.Accompanying drawing There is extreme value in 9, illustrate that the selected scope of experiment is more reasonable.

1.2.3.4 the determination of AF synthetic technological conditions

Understood with reference to analysis of regression model result, the optimum process condition of AF synthesis is 85 DEG C of reaction temperature, reaction time 150 Min, liquid ratio 1:20（g/mL）, the g of malic acid addition 0.75.AF theoretical synthetic ratio is 67.79% on this condition, actual Synthetic ratio is 68.92%, and theoretical value is coincide substantially with actual value.As can be seen here, the optimal synthesis work that Responds Surface Methodology is optimized Skill data are true and reliable.

1.3 discuss

AF synthesis techniques are optimized using Response Surface Method, by rational experimental design, so as to draw each factor to AF synthetic ratios Influence size is followed successively by：Reaction temperature>Solid-liquid ratio>Reaction time>Malic acid addition.Solid-liquid ratio and malic acid addition are to AF The reciprocal effect of synthesis is the most notable.Its optimum synthesis condition is 85 DEG C of reaction temperature, the min of reaction time 150, solid-liquid ratio 1: 20（g/mL）, malic acid addition 0.75 g, AF actual synthetic ratio be 68.92%.Test the Responsive surface model used notable Property is preferable so that accurately and reliably, the result of study is to the further perfect of artificial synthesized AF methods, with certain reason to result By reference value and actual application prospect.

AF monomers content in nature is low, isolates and purifies difficulty, therefore uses artificial synthesized method, to reduce AF synthesis Cost, improves value.Zhao Ting, Zheng Yinan etc. using glacial acetic acid as catalyst AF synthetic ratio are 20-30% or so.Glacial acetic acid It is a kind of organic acid, and has strong impulse smell, its steam is to eye and the irritant effect of nose.Herein with malic acid It is general AF preferably one of the selections of synthesis food medicine for catalyst.The use of malic acid, solves glacial acetic acid penetrating odor Shortcoming, and optimize AF synthesis techniques using response phase method, it preferably handles discrete levels value, experimental period is short, precision Height, can study factor interaction, filter out optimum synthesis condition, significantly improve AF synthetic ratios, be next step Research lays the foundation, and also makes it possible a large amount of production and applications of AF.

The arginine monoglycosides of experimental example 2（AF）Scavenging action to hydroxyl radical free radical and ultra-oxygen anion free radical

The preparation of 2.1 AF sample solutions

L-arginine 10g, glucose 10g, the g of malic acid 15 are taken, is dissolved in 200 mL glycerine, shakes up, in 85 DEG C of water-bath bars 90 min are reacted under part, the total synthetics of AF are obtained, high-effective cationic post is splined on, 1.5 % ammoniacal liquor elution, part receiver connects sample, After batch processing, sample measures purity by HPLC, and vacuum freeze drier is freezed as white powder.Cation seperation column is repeatedly pure Change, its purity is surveyed through efficient liquid phase, more than 95%.AF after purification is taken, the sample that mass concentration is 1 mg/mL is configured to water Solution, based on this concentration, is progressively diluted to 0.5,0.4,0.1,0.08,0.05 mg/mL sample solutions, stand-by.

2.2 Antioxidative Activity Determination

2.2.1 scavenging actions of the AF to hydroxyl radical free radical

2.2.1.1 hydroxyl radical free radical generation structure

This experiment uses Fenton methods, sets up OH radical reaction system models.Its reactional equation is：H₂O₂+ Fe²⁺ → OH^-+ OH+Fe³⁺, the time of OH survivals is short, but has very high reactivity.And salicylic acid can effectively be caught OH, produces the complex compound of certain color, and the color product has strong absorption at 500 nm.If in this experiment, AF has clear Except OH functions, then OH is fought for salicylic acid, so that colored complex growing amount is reduced, color mitigates.So, utilize this Method, is contrasted with blank control pipe, so that it may the removing OH free radical effects of determination sample.Clearance rate calculation formula is：

E（·OH）/ %=1-（A _i-A _j ）×100%

A _o

EFor the clearance rate of hydroxyl radical free radical；A _oFor blank control pipe absorbance；A _iFor reaction solution absorbance；A _jNot add salicylic acid The absorbance of extract solution itself.

2.2.1.2 the preparation of solution

FeSO₄Solution（6 mmol/L）:Weigh FeSO₄The g of crystal 0.0456, with water constant volume in 50 mL volumetric flasks, is configured to 6 Mmol/L solution, it is stand-by.

H₂O₂(30%):Take 30% H₂O₂13.6 μ L constant volumes are configured to 2.4 mmol/L solution, treated in 50 mL volumetric flasks With.

Salicylic acid-ethanol solution（6 mmol/L）：The g of salicylic acid solid 0.0414 is weighed, with absolute ethyl alcohol constant volume in tin In the 50 mL volumetric flasks that paper bag is wrapped up in, 6 mmol/L solution are configured to, it is stand-by.

2.2.1.3 the measure of hydroxyl radical free radical

A _oThe measure of value：2 mL water are added in test tube, 6 mmol/LFeSO are added₄Solution 2 mL, 2.4 mmol/LH₂O₂It is molten The mL of liquid 2, shakes up, at room temperature static 10 min, afterwards, adds 6 mmol/L salicylic acids-mL of ethanol solution 2,37 DEG C of water-baths 30 Min, centrifugation（3000 r/min,10 min）, abandoning supernatant, 500 nm determine its absorbance.

A _iThe measure of value：The mL of 0.5,0.4,0.1,0.08,0.05 mg/mL AF solution 2 is separately added into test tube, then Add 6 mmol/LFeSO₄Solution 2 mL, 2.4 mmol/LH₂O₂The mL of solution 2, shakes up, at room temperature static 10 min, afterwards, plus Enter 6 mmol/L salicylic acids-ethanol solution 2 mL, 37 DEG C of min of water-bath 30, centrifuge（3000 r/min,10 min）, discard Clear liquid, 500 nm determine its absorbance.

A _jThe measure of value：The mL of 0.5,0.4,0.1,0.08,0.05 mg/mL AF solution 2 is separately added into test tube, then Add 6 mmol/LFeSO₄Solution 2 mL, 2.4 mmol/LH₂O₂The mL of solution 2, shakes up, at room temperature static 10 min, afterwards, and 37 DEG C min of water-bath 30, centrifugation（3000 r/min,10 min）, abandoning supernatant, 500 nm determine its absorbance.

2.2.2 scavenging actions of the AF to ultra-oxygen anion free radical

2.2.2.1 ultra-oxygen anion free radical generation structure

This experiment determines AF to O using assay NBT photoreduction₂ ^-The Scavenging activity of free radical.Pyrogallol is in alkalescence condition Lower autoxidation, generation ultra-oxygen anion free radical O₂ ^-With autoxidation intermediate product, the product has absworption peak at 420 nm, its Oxidation process is：The first step, mouse thymus cells are into Semiquinone Radicals；Second step, is oxidized to quinone, adjoint in the process The generation of superoxide radical.

If AF oxidation resistances are stronger, mouse thymus cells process will be hindered, O₂ ^-The generation of free radical is just It can be suppressed, absworption peak weakens, determination sample is come with absorbance and removes O₂ ^-The ability of free radical.Clearance rate calculation formula It is as follows：

E（O₂ ^-）/ %=1-（A _i-A _j ）×100%

A _o

E For the clearance rate of ultra-oxygen anion free radical；A _oFor blank control pipe absorbance；A _iFor reaction solution absorbance；A _jFor not Plus the absorbance of pyrogallol itself.

2.2.2.2 the preparation of solution

Pyrogallol solution（25 mmol/L）：The g of pyrogallol crystal 0.158 is weighed, with water constant volume in 50 mL volumetric flasks, 25 mmol/L solution are configured to, it is stand-by.

Hydrochloric acid（80 mmol/L）：Pipette draws 2 mL hydrochloric acid, and constant volume is configured to 80 in 25 mL volumetric flasks Mmol/L hydrochloric acid solutions, it is stand-by.

Phosphate buffer（PH=8,0.05 mol/L）：The g of disodium hydrogen phosphate 7.164 is weighed, with water constant volume in 100 mL In volumetric flask, fully dissolving；The g of sodium dihydrogen phosphate 3.121 is weighed, with water constant volume in 100 mL volumetric flasks, fully dissolving；Point Not Xi Qu the disodium hydrogen phosphate aqueous solution 22.88 mL, the mL of biphosphate sodium water solution 2.125 in 100 mL volumetric flasks, constant volume, Fully mix, be configured to PH=8,0.05 mol/L phosphate buffers, it is stand-by.

2.2.2.3 the measure of superoxide anion

A _oThe measure of value：The mL of PH=8,0.05 mol/L phosphate buffers 4.5 is added in test tube, is added in 25 DEG C of water-baths 20 min of heat, afterwards, add 1 mL water and 25 mmol/L pyrogallol solution, after mixing, 25 DEG C of 10 min of reaction, add 80 The mL terminating reactions of mmol/L hydrochloric acid solutions 1, are shaken up, and react 3 min, and its absorbance is surveyed at 420 nm.

A _iThe measure of value：The mL of PH=8,0.05 mol/L phosphate buffers 4.5 is added in test tube, in 25 DEG C of water-baths 20 min of middle heating, afterwards, are separately added into the mL of 0.5,0.4,0.1,0.08,0.05 mg/mL AF sample solutions 1 and 25 Mmol/L pyrogallol solution, after mixing, 25 DEG C of 10 min of reaction add the mL terminating reactions of 80 mmol/L hydrochloric acid solutions 1, Shake up, react 3 min, its absorbance is surveyed at 420 nm.

A _jThe measure of value：The mL of PH=8,0.05 mol/L phosphate buffers 4.5 is added in test tube, in 25 DEG C of water-baths 20 min of middle heating, afterwards, are separately added into the mL of 0.5,0.4,0.1,0.08,0.05 mg/mL AF sample solutions 1, after mixing, 25 DEG C of 10 min of reaction, add the mL terminating reactions of 80 mmol/L hydrochloric acid solutions 1, shake up, react 3 min, surveyed at 420 nm Its absorbance.

2.3 result

2.3.1 AF is to hydroxyl radical free radical Scavenging activity

Experimental result is shown, with the increase of AF sample quality concentration, the Scavenging activity of hydroxyl radical free radical is gradually strengthened, works as AF During 0.5 mg/mL, clearance rate reaches 78.14 %.This time result shows, AF can play removing hydroxyl radical free radical effect, and effect Really obvious, concrete numerical value is shown in Table 4.

The AF of table 4 is to hydroxyl radical free radical Scavenging activity

AF mass concentrations（mg/mL）	Ai	Aj	Ai- Aj	E（·OH）/%
					Blank	0.837	0.256	0.581	—
0.5	0.782	0.236	0.546	78.14
					0.4	0.816	0.246	0.570	72.18
0.1	0.851	0.256	0.595	69.70
					0.08	0.911	0.274	0.637	62.47
0.05	0.979	0.284	0.685	52.49

2.3.2 AF is to ultra-oxygen anion free radical Scavenging activity

Experimental result is shown, with the increase of AF sample quality concentration, the Scavenging activity of ultra-oxygen anion free radical is gradually increased By force, as 0.5 mg/mL of AF, clearance rate reaches 78.94 %.This time result shows, AF can play removing superoxide anion certainly Acted on by base, and effect is obvious, concrete numerical value is shown in Table 5.

The AF of table 5 is to ultra-oxygen anion free radical Scavenging activity

AF mass concentrations（mg/mL）	Ai	Aj	Ai- Aj	E（·OH）/%
					Blank	0.314	0.029	0.285	—
0.5	0.090	0.030	0.060	78.94
					0.4	0.119	0.033	0.086	69.82
0.1	0.149	0.030	0.119	58.24
					0.08	0.174	0.029	0.145	49.12
0.05	0.189	0.031	0.158	44.56

2.4 brief summary

The result of this experiment shows that AF can preferably remove superoxide anion and hydroxyl radical free radical, and with the increase of AF concentration, Scavenging activity is also in enhancing, and when AF concentration is 0.5 mg/mL, hydroxyl radical free radical clearance rate reaches 78.14%, superoxide anion Free radical scavenging activity reaches 78.94%, shows that AF has stronger oxidation resistance, its specific mechanism also needs to further research.

The arginine monoglycosides of experimental example 3（AF）H22 tumor-bearing mices Tumor growth inhibition is acted on

3.1 test method

3.1.1 modeling method

Take H₂₂Oncocyte liquid（Special product research institute provides）, it is inoculated in after mouse peritoneal, 7-9 d, dislocated execution, 75% alcohol Pasteurised completely, aseptically extracts mouse ascites, is placed in the test tube with ground stopper for filling physiological saline, mixes, centrifugation（1000 R/min, 3 min）, abandoning supernatant, and brine is used, it is repeated 3 times, finally adjusts tumor cell suspension with physiological saline Cell number is 1.0 × 10⁷Individual/mL, subcutaneous vaccination is in ICR mouse right fores, and 0.2mL/ is only.

3.1.2 animal packet and administration

After the h of mouse inoculation 24, success Mice Inoculated is randomly divided into 5 groups, every group 10, i.e. model group（M, 0.9% physiology salt Water）, positive controls（P, endoxan, 25 mg/kg）, AF low dose groups（AF-L,12.5 mg/kg）, AF middle dose groups （AF-M,25 mg/kg）AF high dose groups（AF-H,50 mg/kg）, each group is administered by 10 mL/kg mouse weights daily, positive Control group intraperitoneal injection, remaining each group gavage gives corresponding medicine, and (blank control group and model group give 0.9% physiology Salt solution), it is administered once a day, the d of successive administration 21.

3.1.3 tumour inhibiting rate is determined

After the h of last dose 12, eyeball takes blood and after the execution mouse that dislocates, clamps mouse tumor area skin with tweezers on the other hand, separately One hand that operating scissors cuts off skin, and exposure tumour, with operating scissors along tumor's profiles, is peeled off tumour, carried out with 9% physiological saline Rinsing, filter paper is weighed after blotting, and calculates tumour inhibiting rate.

Tumour inhibiting rate（%）=（The average knurl weight of the average knurl weight-experimental group of model group）The % of the average knurl weight of ÷ model groups × 100.

3.1.4 organ index

After the h of last dose 12, eyeball takes blood, the r of centrifuge 4500 separation serum, the serum keeping after processing in -20 DEG C of refrigerators, It is stand-by.The dissection of system is carried out after mouse is put to death, spleen and thymus gland is taken out, is rinsed with 9% physiological saline, filter paper is blotted After weigh, calculate spleen index and thymus index.

The body weight (g) of organ index=organ weights (g) × 100/

3.1.5 biochemical indicator is detected

3.1.5.1 liver function and renal function index are determined

By determining serum Glutamic Acid aminopherase（ALT）And aspartate amino transferase（AST）, urea nitrogen (BUN) and creatinine (CRE) content, to reflect influence of the medicine to liver, renal function.

3.1.5.2 in hepatic tissue SOD vigor and MDA contents measure

SOD vigor is detected with xanthine oxidase, thiobarbituricacidα- is used（TBA）Method detects MDA contents, in strict accordance with examination Agent box specification is operated.

3.1.5.3 TNF-α, IL-2 assays in serum

The mice serum dispensed is taken, normal temperature unfreezing is operated in strict accordance with ELISA specifications, clicks and enters 96 orifice plates, and ELIASA is determined Afterwards, Data Management Analysis is carried out.

3.1.6 tumor tissue pathology checks

Take the tumor tissues after dissection to be fixed in 10% formaldehyde fixer, be dehydrated, transparent, waxdip, finally embedded.With cutting Tumor tissues after embedding are cut into 4 ~ 5 μm by piece machine, are then extended piece, are dragged for piece, piece dried, by the section handled well, through H＆E After decoration method dyeing, the structure of tissues observed and the form of cell, are finally analysed and compared under the microscope.

3.1.7 statistical analysis

Analyzed using SPSS17.0 application software, data with mean+SD (± s) represent.Multigroup is compared and adopts With one-way analysis of variance method, withP<0.05 represents that difference is statistically significant.

3.2 experimental result

3.2.1 AF is to H₂₂The tumor-inhibiting action of tumor-bearing mice

As shown in table 6, AF-L, AF-M dosage group tumour is played must inhibitory action, compared with model group, existed extremely notable Difference（p<0.01）, but tumour inhibiting rate is less than positive controls.There was no significant difference with model group for AF-H groups.Three agent of AF administrations Amount foot, is compared, and AF-L groups tumor killing effect is best, inhibitory rate to 47.65%.

The AF of table 6 is to H₂₂Tumor-bearing mice tumor-inhibiting action (±s, n=10)

Groups	Dosage（mg/kg）	Tumor weight （g）	Inhibitory rate（%）
				Model	—	1.82	—
Positive	25	0.85**	53.37
				AF-L	12.5	0.95**	47.65
AF-M	25	1.24**	31.81
				AF-H	50	1.67	9.08

*p<0.05, ** p<0.01, vs. model group

3.2.2 AF is to H₂₂The influence of tumor-bearing mice immune organ

As shown in table 7, the raising that AF can be different degrees of to the thymus index and renal index of mouse.With blank control group ratio Compared with model group has significant difference with positive controls（p<0.01）, there is significant difference in AF-H group thymus indexs（p< 0.05）.Compared with model group, AF-L, AF-M, AF-H dosage group have pole significant difference（p<0.01）.

The AF of table 7 is to H₂₂Tumor-bearing mice immune organ influence (±s, n=10)

Group	Dosage（mg/kg）	Spleen index（mg/g）	Thymus index（mg/g）
				Normal	—	0.57±0.04**	0.23±0.01**
Model	—	0.39±0.02##	0.16±0.02##
				Positive	25	0.47±0.05**##	0.19±0.02*##
AF-L	12.5	0.530±0.02**	0.21±0.03**
				AF-M	25	0.55±0.03**	0.22±0.02**
AF-H	50	0.54±0.05**	0.20±0.01**

*p<0.05, ** p<0. 01, vs. model group； #p<0. 05, ## p<0. 01, vs. normal control group

3.2.3AF to H₂₂The influence of tumor-bearing mice cell factor

As shown in table 8, with blank control group there is pole significant difference in model group.Compared with model group, positive controls, AF- L, AF-M group Serum IL-2 significantly rise, and there is pole significant difference（p<0.01）；There is significant difference in AF-H groups （p<0.05）.Compared with M groups, P, AF-L, AF-M, AF-H group TNF-α content are significantly improved, pole conspicuousness is presented（p< 0.01）.

The AF of table 8 to cytokine TNF-α in mice serum, IL-2 influence (±s, n=10)

Groups	Dosage（mg/kg）	IL-2 (pg/mL)	TNF-α（pg/mL）
				Normal	—	71.08±10.76	148.21±28.03
Model	—	39.27±5.95##	110.39±16.45##
				Positive	25	65.56±7.23**	174.05±41.35**
AF-L	12.5	56.53±6.68**	161.29±21.55**
				AF-M	25	80.17±5.64**	145.82±25.35**
AF-H	50	51.21±7.28*	144.61±16.26**

*p<0.05, ** p<0.01, vs. model group；#p<0.05, ## p<0.01, vs. normal control group

3.2.4 AF is to H₂₂The influence of tumor-bearing mice hepatic and renal function

Compared with blank control group, model group CRE, BUN content has pole significant difference（p<0.01）.With model group ratio Compared with positive controls CRE contents have significant difference（p<0.05）；There is pole significant difference in AF-L, AF-M group CRE contents （p<0.01）；AF-H groups are with model group without significant difference.BUN contents in serum, compared with model group, positive controls, AF-L Pole significant difference is presented in group（p<0.01）；There is significant difference in AF-M, AF-H group（p<0.05）.

Each dosage groups of AF can significantly reduce serum alt, AST contents.Model group exists extremely notable with blank control group Sex differernce（p<0.01）；Compared with model group, positive controls, AF-L, AF-M, AF-H group serum alt content have pole Significant difference；In positive controls, AF-L, AF-H group serum there is pole significant difference in AST contents（p<0.01）, AF-M groups There is significant difference（p<0.05）, as shown in table 9.

The AF of table 9 is to H₂₂Tumor-bearing mice hepatic and renal function influence (±s, n=10)

*p<0.05, ** p<0.01, vs. model group； #p<0. 05, ## p<0. 01, vs. normal control group

3.2.5 AF is to H₂₂The influence of tumor-bearing mice SOD and MDA content

As shown in table 10, with blank control group there is pole significant difference in model group.Compared with model group, administration group mouse liver SOD contents are significantly increased in tissue, and positive controls, AF-L, AF-M, AF-H group have significant difference（p<0.01）. Compared with model group, MDA contents are significantly raised in positive controls, AF-L, AF-M liver organization（p<0.01）, AF-H groups and mould Type group no significant difference.

The AF of table 10 is to H₂₂Tumor-bearing mice SOD and MDA content influence (±s, n=10)

Groups	Dosag（mg/kg）	SOD(U/mgprot)	MDA(nmol/mgprot)
				Normal	—	216.95±17.57	10.06±2.08
Model	—	124.78±15.06##	14.88±0.92##
				Positive	25	210.98±15.64**	10.79±2.06**
AF-L	12.5	251.26±30.88**	8.17±2.24**
				AF-M	25	166.70±29.47**	8.79±2.43**
AF-H	50	163.24±34.17**	13.70±1.85

*p<0.05, ** p<0. 01, vs. model group； #p<0.05, ## p<0.01, vs. normal control group

3.3 brief summaries are with discussing

H₂₂Tumor cell line, rate of vaccination is higher, and tumour is complete after being inoculated with successfully, and is not susceptible to transfer, convenient to tumour growth Observation directly perceived, the measurement of gross tumor volume and weight the features such as.At present, in domestic and international Tumor Assays research, mostly with H₂₂Tumour Cell line is model.This experiment is with H₂₂Tumor-bearing mice is test model, studies AF antitumor activities, as a result shows, AF-L groups are made With obvious, inhibitory rate to 47.65%, less than endoxan positive controls inhibiting rate（53.37%）, but both are not present Significant difference.

The generation of tumour is contacted with immune system there is also larger, and the two complements each other.Development, transfer, the leaching of tumour Profit, deterioration etc. are all along with the decline of immunologic function^[8].So during oncotherapy, how to improve the anti-of body itself Anti-tumor immunity, which is caused, widely to be probed into^[9-10].Two important immune organs of body, thymus gland and spleen, its internal organs refer to Number is often used in the immunocompetent height of evaluation^[11].This paper results show, AF-L, AF-M, AF-H group thymus index and spleen Index is significantly higher than model control group, and there is significant difference（p<0.01）, thus illustrate, AF can delay tumor-bearing mice to exempt from Epidemic disease organ failure.IL-2, TNF-α play vital effect in anti-tumor-bearing mice is immune.IL-2 is that T cell growth must The growth factor of palpus, while the immunologic function of B cell, NK cells, macrophage etc. can be raised, can also stimulate TNF, interference The secretion of the others cell factor such as element^[12].TNF-α be the activity most strong antitumor cell that finds both at home and abroad at present because Son^[13], it is produced by the mononuclear macrophage activated, can directly dissolve tumour, can also be resisted microorganism invasion and be adjusted The function of immunity of organism.This experimental study shows that AF-L, AF-M group can improve IL-2 in mice serum, TNF-α content, with Be present pole significant difference in model group, you can illustrate, AF-L, AF-M group can strengthen the body's immunity of tumor-bearing mice, from And suppress the growth of tumour.

Glutamic-pyruvic transaminase（ALT）And glutamic-oxalacetic transaminease（AST）In main enrichment liver organization, so liver organization is damaged Wound or during severe necrosis, ALT and AST are largely leaked into serum, therefore the two content is significantly raised in serum, so, often with Whether serum alt, AST contents height damage or downright bad important indicator to monitor liver.In this experiment, in administration group serum ALT, AST content are below model group, and there is significant difference, with blank control group without significant change, illustrate AF to mouse Liver not damaged.BUN, CRE are one of clinically conventional leading indicators of detection renal function.When glomerular filtration rate(GFR declines During to normal less than 50%, BUN concentration is raised rapidly.When with diseases such as acute and chronic glomerulonephritis, filter glomerulus Cross hypofunction, CRE rises.This time test, BUN, CRE content are substantially less than model group in serum（p<0.01）, with blank There was no significant difference for control group, illustrates AF to tumor-bearing mice renal function not damaged.

There is certain relation in generation, the development of free radical and tumour^[14-15].SOD is a kind of antioxidase, is antioxygen The first line of defence of change, can be disproportionated into H by super positive anion₂O₂, and the change of MDA contents can oxygen in response organization indirectly The change of free-radical contents.Therefore the oxidation resistant ability of body can be reflected by determining SOD, MDA content^[16].Result of the test It has been shown that, AF can significantly improve SOD value（p<0.01）, AF-L, AF-M group can significantly reduce MDA value（p<0.01）, explanation AF can improve the oxidation resistance of body, remove interior free yl.

In summary, AF have must antitumous effect, especially AF low dose groups, performance the most substantially, its energy It is enough substantially to suppress the growth of tumour, and immunity of organism activity can be improved.But the thorough treatment of tumour, is a long process, also Need further exploratory development.

The arginine monoglycosides of experimental example 4（AF）Influence to immunosuppressed mice immunologic function

4.1 experimental method

4.1.1 animal packet and dosage

100 ICR mouse, after adaptability is raised one week, are randomly divided into 5 groups, suppression is immunized in every group 20, i.e. Normal group (N) Simulation group (M), immunosupress AF low dose groups（M-L, 20 mg/kg）, immunosupress AF middle dose groups（M-M, 40 mg/kg）, immunosupress AF high dose groups（M-H, 80 mg/kg）.Each group is given by 10 mL/kg body weight to mouse stomach daily Medicine, is administered once a day, the d of successive administration 28.M, M-L, M-M and M-H group, weekly first three day, intraperitoneal injection of cyclophosphamide （CTX, 80 mg/kg）, form immunosupress.

4.1.2 mouse immune organ organ index is determined

After the h of last dose 12, eyeball takes blood, the r of centrifuge 4500 separation serum, the serum keeping after processing in -20 DEG C of refrigerators, It is stand-by.The dissection of system is carried out after mouse is put to death, spleen and thymus gland is taken out, is rinsed with 9% physiological saline, filter paper is blotted After weigh, calculate spleen index and thymus index.

The body weight (g) of organ index=organ weights (g) × 100/

4.1.3 external Splenic vein hemodynamics experiment

It is administered after 21d, every group is randomly selected 10 mouse, and dislocation is put to death, puts in 75 % alcohol and soak 5 min, sterile to take spleen It is dirty, RPMI-1640 is added, the grinding of 300 mesh steel meshes draws splenocyte suspension in 1.5 mL centrifuge tubes, centrifuges 10 min （15000 rpm, 4 DEG C）, abandon supernatant.ACK Lysis Buffer are added in above-mentioned centrifuge tube, 10 min are centrifuged again （15000 rpm, 4 DEG C）, abandon supernatant.RPMI-1640 is added, cell count is made into 2 × 10⁶/ mL splenocyte suspensions.Examination Test and be divided into three groups, first group is that control group is spleen lymph Natural Transformation group；Second group of addition ConA (5 μ g/ml, 100 μ L), T cell is stimulated to convert；3rd group of addition LPS（10 μg/ml,100 μL）, stimulate B cell to convert.Each group cell is sequentially added In 96 orifice plates, 100 μ L splenocyte suspensions are added per hole, 3 multiple holes are set up per hole.Cell culture condition is：5 %CO2 are cultivated Case, 37 DEG C, 48 h.Cultivate after 48 h, MTT is added in every hole（5 mg/mL）20 μ L, continue to cultivate after 4 h, centrifugation（2000 Rpm, 10 min）, Aspirate supernatant, add 150 μ L DMSO fully dissolve purple crystal, the nm of ELIASA 490 under the conditions of, survey Fixed each hole OD values.

4.1.4 TNF-α, IL-2 assays

The mice serum dispensed is taken, normal temperature unfreezing is operated in strict accordance with ELISA specifications, clicks and enters 96 orifice plates, and ELIASA is determined Afterwards, Data Management Analysis is carried out.

4.1.5 the detection of specific IgG antibodies

The mice serum dispensed is taken, normal temperature unfreezing, ELISA method determines mouse specific IgG antibodies level.

4.1.6 fluorescence quantitative RT-RCR is determined

4.1.6.1 RNA is extracted in organizing

Take out in the spleen tissue that each group mouse is stored in liquid nitrogen, the mortar after DEPC water process and be fully ground to powder Shape.Plus 1 mL Trizol in 1.5 mL centrifuge tubes, add a little spleen powder, after oscillator is fully mixed, stand 5 min.Plus 200 μ L chloroforms in above-mentioned centrifuge tube, cover tightly, after being mixed with hand is reverse, room temperature places 10 min, 12000 g from The min of the heart 15（In 4 DEG C of centrifuges）.4. supernatant is suctioned out in 1.5 new mL centrifuge tubes, plus 500 μ L isopropanols, mix, It is stored at room temperature after 10 min, 12000 g centrifuge 10 min（In 4 DEG C of centrifuges）, retain precipitation.5. plus the cold mL of ethanol 1 of 75 % in In above-mentioned pelleting centrifugation pipe, fully washing is precipitated, and 7500 g centrifuge 5 min（In 4 DEG C of centrifuges）, abandon supernatant.6. drying at room temperature RNA is to translucent.7. 20 μ L DNase/RNase-Free Deionized Water dissolving precipitations are added into centrifuge tube ,- 80 DEG C freeze.RNA extracts figure, sees accompanying drawing 11.

4.1.6.2 reverse transcription synthesizes cDNA

TNF-α, IL-2 gene order are obtained from database NCBI, and Primer5.0 softwares are used with reference to the gene order of inquiry The quantitative primer of TNF-α, IL-2 is designed, reverse transcription primer uses the random primer in kit, and fluorescent quantitation primer sequence is shown in Table 11。

The fluorescent quantitation primer sequence of table 11

Kit specification step is spun according to Japan, reverse transcription experiment is carried out, obtains cDNA products, in -80 DEG C of preservations.Reverse transcription Sample-adding system is shown in Table 12.

The reverse transcription of table 12 is loaded system

Reagent	Sample-adding amount
		Total RNA	7 μL (1ug)
primer mix	0.5 μL
		RT Enzyme Mix	0.5 μL
5×RT Buffer	2 μL
		Total	10 μL

4.1.6.3 real time fluorescent quantitative reacts

Using GAPDH as internal control primer, detect TNF-α, IL-2 in mouse spleen tissue using fluorescent dye SYBR-Green I Middle relative expression quantity, each three repetitions of sample.Amplification reaction condition is as follows：95 DEG C of min of pre-degeneration 5；95 DEG C are reacted 10 seconds, 60 DEG C are reacted 30 seconds, 40 circulations.After reaction terminates, record solubility curve and amplification curve, per the Ct values of hole sample, use 2^-△△CTMethod is calculated.Reaction system is shown in Table 13.

The RT-PCR reaction systems of table 13

4.1.7 statistical procedures

Analyzed using SPSS17.0 application software, data with mean+SD (± s) represent.Multigroup is compared and adopts With one-way analysis of variance method, withP<0.05 represents that difference is statistically significant.

4.2 experimental result

4.2.1 influences of the AF to mouse immune organ organ index

Compared with M groups, N, M-L, M-M group thymus index, index and spleen index have significant difference（P<0.01）, M-H groups are without bright Aobvious change, as shown in table 14.

Influences of the AF of table 14 to mouse immune organ organ index

Note:*P ＜0.05,** P ＜0.01,compared with model

4.2.2 influences of the AF to Splenic vein hemodynamics

Compared with M groups, M-L, M-M group Splenic vein hemodynamics rate substantially increase, and pole conspicuousness, such as accompanying drawing 12 is presented in M-L groups It is shown.

4.2.3 influences of the AF to cytokine TNF-α, IL-2 in mice serum

As shown in Figure 13, compared with M groups, TNF-α content significantly rises in N group serum（P<0.01）, in pole conspicuousness, M- TNF-α content is raised in L, M-M group serum（P<0.05）, M-H group TNF-α contents and M group no significant differences.Compared with M groups, M, M-L group Serum IL-2 are significantly raised（P<0.01）, the rise of M-M group IL-2 contents（P<0.05）, M-H groups IL-2 contains There was no significant difference with M groups for amount.

4.2.4 influences of the AF to mouse specific IgG antibodies

As a result show, the immune response that M, M-L, M-M group mouse obtain is good, is compared with M group mouse, antibody level is extremely notable Property improve（P<0.01）, M-H groups are shown in accompanying drawing 14 without significant change.

4.2.5 AF is to TNF-α in mouse spleen tissue, the influence of IL-2 mRNA expression

Fluorescent quantitative PCR result shows that TNF-α, IL-2 melting curves are simple spike, and amplification curve is typical S types, baseline It is smooth.M-L groups TNF-α, IL-2mRNA expression are respectively 7.73 ± 0.16,10.63 ± 0.34, M-M groups TNF-α, IL- 2mRNA expression is respectively 2.6 ± 0.03,5.43 ± 0.23, is significantly higher than M groups（1.83±0.72、2.45±0.02）（P ＜ 0.01、P ＜0.05）, see accompanying drawing 15.

4.3 discuss

This studies have shown that ICR mouse are administered after 28 d, significantly improve immunosuppressed mice spleen and thymus index；M-L group spleens Lymphocyte proliferation test is significantly improved compared with M groups；M-L, M-M group mouse specific IgG antibodies are apparently higher than M groups；Immunosupress is small In mouse serum, tumor necrosis factor TNF-alpha and interleukin 2 IL-2 content are significantly improved.And it is fixed by real-time fluorescence PCR detections are measured, TNF-α and IL-2 mRNA level in-sites substantially increase.

This experiment is studied with vitro tests such as vivo medicine-feeding research mode combination real-time fluorescence quantitative PCRs, as a result table Bright, AF can mitigate the atrophy of immune organ phenomenon that CTX is caused.From the point of view of immune response, immunosupress AF low dosages in this experiment Group, immunosupress AF middle dose groups mouse specific IgG antibodies level are apparently higher than immunosuppression model group mouse, Chen Bo^[17], Guy etc.^[18]Research shows that specific IgG antibodies are the main antibody components of serum, can remove harmful complement fragment, suppresses scorching Disease reaction shields in immunity of organism.The IgG levels of this research show that AF can improve immunosuppressed mice immune response Ability.Liu Jia etc.^[19], Zhao Dingliang etc.^[20]Report, IL-2 can promote the increment and differentiation of T, bone-marrow-derived lymphocyte, can induce killing thin Born of the same parents（LAK）Generation.Immunity of organism function for monitoring can be strengthened.Tan Bing etc.^[21], Li Wei etc.^[22]Report, TNF-α can be resisted micro- The function of biotic intrusion and regulation immunity of organism.This time test, with ELISA reagent methods, detect AF immunosupress group mouse blood IL-2, TNF-α content are significantly improved in clear.Meanwhile, using quantitative fluorescent PCR, detection TNF-α and IL-2 mRNA level in-sites.More than Result of the test shows that AF has stronger immunopotentiating ability, and the immunosupress of energy antagonism CTX immunologic hypofunction mouse is made With.But this is tested, and simply the part in immune Research, should further inquire into research, with abundant verification test result.

Brief description of the drawings

Influence of Fig. 1 reaction time to AF synthetic ratios

Influence of Fig. 2 reaction temperatures to AF synthetic ratios

Influence of Fig. 3 solid-liquid ratios to AF synthetic ratios

Influence of Fig. 4 malic acid addition to AF synthetic ratios

Fig. 5 reaction temperatures and the influence of reaction time reciprocation AF synthetic ratio

Fig. 6 reaction temperatures and the influence of solid-liquid ratio reciprocation AF synthetic ratios

Fig. 7 reaction temperatures and the influence of malic acid addition reciprocation AF synthetic ratios

Fig. 8 reaction time and the influence of solid-liquid ratio reciprocation AF synthetic ratios

Fig. 9 reaction time and the influence of malic acid addition reciprocation AF synthetic ratios

The influence of Figure 10 solid-liquid ratios and malic acid addition reciprocation AF synthetic ratios

Figure 11 .RNA extract result figure

Influences of Figure 12 .AF to Splenic vein hemodynamics rate

Influences of Figure 13 AF to TNF-α, IL- alpha contents in mice serum

Influences of Figure 14 .AF to IgG content in mice serum

Figure 15 AF is to TNF-α in mouse spleen tissue, the influence of IL-2 mRNA expression.

Specific embodiment

Embodiment 1

Accurately weigh arginine：Glucose=1g:1g, adds glycerine 10ml and citric acid 0.1g, and shaking table 160rpm is mixed, 120min is reacted under the conditions of 85 DEG C, column front derivation efficient liquid phase detects AF contents, and synthetic ratio is 55.52%.

Embodiment 2

Accurately weigh arginine：Fructose=1g:1g, adds glycerine 10ml and citric acid 0.1g, and shaking table 160rpm is mixed, and 85 120min is reacted under the conditions of DEG C, column front derivation efficient liquid phase detects AF contents, and synthetic ratio is 58.52%.

Embodiment 3

Accurately weigh arginine：Fructose：Xylose=1g:0.5g：0.5g, adds glycerine 10ml and citric acid 0.1, shaking table 160rpm is mixed, and 180min is reacted under the conditions of 85 DEG C, and column front derivation efficient liquid phase detects AF contents, and synthetic ratio is 60.52%.

Embodiment 4

Accurately weigh arginine：Xylose=1g:1g, adds glycerine 10ml and citric acid 0.1g, and shaking table 160rpm is mixed, and 85 120min is reacted under the conditions of DEG C, column front derivation efficient liquid phase detects AF contents, and synthetic ratio is 65.52%.

Embodiment 5

Accurately weigh arginine：Xylose=1g:1g, adds propane diols 10ml and vitamin C 0.1g, and shaking table 160rpm is mixed, 120min is reacted under the conditions of 85 DEG C, column front derivation efficient liquid phase detects AF contents, and synthetic ratio is 45.52%.

Embodiment 6

Accurately weigh arginine：Xylose=1g:1g, adds isopropanol 10ml and vitamin C 0.1g, sodium sulfite 0.05g shaking tables 160rpm is mixed, and 120min is reacted under the conditions of 85 DEG C, and column front derivation efficient liquid phase detects AF contents, and synthetic ratio is 45.52%.

Embodiment 7

Accurately weigh L-arginine：D-Arg：Xylose=0.5g:0.5g：1g, adds isopropanol 10ml and vitamin C 0.1g, sodium sulfite 0.05g shaking tables 160rpm are mixed, and 120min, column front derivation efficient liquid phase detection AF are reacted under the conditions of 85 DEG C Content, synthetic ratio is 65.52%.

Embodiment 8

Anti-senility oral liquid

The AF powder of Example 1 after purification, is fitted into vial after being configured to the 35mg/ml aqueous solution, sterilizing, is made every Branch 10ml oral liquid.Other requirements meet《Pharmacopoeia of People's Republic of China》2010 editions relevant regulations about oral liquid.

Embodiment 9

Beauty and skin care water

The AF powder purified in Example 1, is added in surfactant matrix liquid, and AF ultimately joins concentration for 100mg/100mL.Its He requires to meet China cosmetic production requirement specification.

Bibliography

[1] Takaku, T., Han, L.K., Kameda K., et al. Production of arginyl- frictosyl glucose during processing of red ginseng. J. Tradit. Med. 1996, 13, 118-123.

[2] Suzuki ,Y., Choi, K.J., Uchida,K., et al. Arginyl-fructosyl-glucose and arginyl-fructose, compoundsrelated to browning reaction in the model system of steaming and heat-drying processes for the preparation of red ginseng. J. Ginseng Res. 2004, 28, 143-148.

[3] the artificial synthesized L-arginine monoglycosides of Zhao Ting（AF）And its anti-diabetic pharmacology activity research [D] Changchun：Jilin Agriculture university, 2013.

[4] Kwang-Hyoung Lee, Kyoung-Soo Ha, Sung-Hoon Jo, et al. Effect of Long- Term Dietary Arginyl-Fru-ctose (AF) on Hyperglycemia and HbA1c in Diabetic db/db Mice. J. Mol. Sci. 2014, 15, 8352-8359.

[5] Cho, E.J.;Piao, X.L.; Jang, M.H. et al. The effect of steaming on the free amino acid contents and antioxidant activity of Panax ginseng. J. Food Chem. 2008,107: 876–882.

[6] By Lee, Jung-Sook; Kim, Gyo-Nam; Lee, Sang-Hyun, et al. In vitro and cellular antioxidant activity of arginyl-fructose and arginyl-fructosyl- glucose. J. Food Science and Biotechnology 2009, 18(6), 1505-1510.

[7] Keum,Y.S; Park,K.K; Lee,J.M., et al. Antioxidant and anti-tumor promoting activities of the methanol extract of heat-processed ginseng. J. Cancer Lett. 2000, 150,41-48.

[8] the practical medicine of Cheng Lichun, progress [J] of model green grass or young crops active targeting liposomes in antineoplaston and clinic, 2011,14 (5):426-429.

[9] Dan Zhang, Yuhong Sun, Mao Li, Qin Sun, Minghua Liu. Effects ofPeriplaneta americana polypeptideextracts on tumor growth and immune function in tumor-bearing mice [J].Chinese Journal of New Drugs ,2015,24(6):681-686.

[10] Xinrui Yang, Gang Wang, Libin Ji, Haiyan Wang. Effects of Arenaria Kansuensis Aqueous Extract on Immune Function of H₂₂ Tumor-bearing Mice[J] .Cancer Res Prev Treat ,2015, 42(7):662-665.

[11] Xiong Q, Jiao Y, Zhao X, et al. Purification, characterization and immunostimul-atory activity of polysaccharide from Cipangopaludina chinensis [J] Carbohydrate Polymers, 2013,98 (1): 217- 223.

[12] Tang Yan, Zhang Dan, Meng Xianglin, wait influence of the TGPs liposome to tumor-bearing mice tumour growth and immunologic function [J] Chinese Journal of New Drugs, 2014,23（21）：2547-2551.

[13] Li Molin, Li Zhuangang, the grand melphalans of Shu Xiao cure the process of tumor-bearing mice and relation [J] the cells of TNF-α with Molecular immunology magazine, 2007,23 (4):320.

[14] Jakhar R, Paul S, Pard YR, el al.3,5,7,3’,4’-Pentamethoxyflavone, a quercetin derivative protects DNA from oxidative challenges: potential mechanism of action[J].Photochem Photobiol B, 2014, 131: 96-103.

[15] Fuhua Li, Dong Liu, Jian Ming. Cellular Antioxidant and Antiproliferative Activities of Flavonoids Extracted from Tartary Buckwheat (Fagopyrum tartaricum (L.) Gaertn) Bran [J].Food Science,2014.35(7):58-63.

[16] Esterbauer H, Schaur R J, Zolner H. Chemistry and biochemistry of 4- hydroxynonenal, malonaldehyde and related aldehydes [J]. Free Radic Biol Med, 1991, 11(1): 81-128.

[17] Chen Bo .H-IgG are damaged to Neonatal Rat Brain ischemic hypoxia protective effect and Mechanism Study [D] Changchun：It is lucky Woods university, 2013,16-22.

[18] Guy B, Hessler C, Fourage S, et al.Systemic immunization with urease protects mice against

Helicobacter pylori infection J. Vaccine,1998,16(8):850-856.

[19] Liu Jia, Dan Anshan, grandson enter the progress of magnificent interleukin 2s with applying [J] HEILONGJIANG ANIMAL SCIENCE AND VETERINARY MEDICINEs, 2009（21）：57-61.

[20] Zhao Dingliang, single flat cytokine -2s latest Progress [J] JOURNAL OF MICROBIOLOGYs of wind, 2013,5（4）：77- 79.

[21] Tan Bing, Li Yuyuan, the international clinical practice magazines of progress [J] of Nie Yuqiang tumor necrosis factor-alphas, 2007,5 （3）：129-133.

[22] progress [J] the animal medicines progress of Li Wei, Liu Jia, Bai Jiayuan cachectins, 2010,6（12）： 12-14。

Claims (10)

1. under certain temperature, time, solvent, utilize synthesis of glycoside under the conditions of amino acid and monose, it is characterised in that amino The ratio of acid and monose is 1：0.5-2, synthetic ratio is more than 63%.

2. the glucosides synthesized according to claim 1, it is characterised in that the amino acid required in building-up process can be L- The mixture of arginine, D-Arg one or two kinds of therein.

3. the glucosides synthesized according to claim 1, it is characterised in that the monose required in building-up process can be D- Portugals One or more kinds of mixtures in grape sugar, D-Fructose, D- galactolipins, D- xyloses.

4. the glucosides synthesized according to claim 1, it is characterised in that the generated time required in building-up process is 120- 300min。

5. the glucosides synthesized according to claim 1, it is characterised in that required optimum synthesising temperature is in building-up process 80℃。

6. the glucosides synthesized according to claim 1, it is characterised in that the solvent required in building-up process is propane diols, third One or more kinds of mixed solvents in triol, propyl alcohol, isopropanol.

7. the glucosides synthesized according to claim 1, it is characterised in that the sodium sulfite that can also be added in building-up process, dimension One or more kinds of mixtures in raw element C, vitamin E.

8. the glucosides synthesized according to claim 1, it is characterised in that citric acid, malic acid can also be added in building-up process Etc. one or more kinds of mixtures in edible organic solid acid.

9. the glucosides synthesized according to claim 1, it is characterised in that with removing hydroxyl radical free radical and superoxide anion certainly By base, improve the immunity of immunosuppressed mice, suppress H₂₂The purposes of tumor-bearing mice tumour growth.

10. the glucosides synthesized according to claim 1, it is characterised in that can be used for adjuvant therapy of tumors, carry and exempting from In the medicine of epidemic disease power, food, health products.