CN109879980A - A kind of preparation method and antioxidant of rice bran polysaccharide metal complex - Google Patents
A kind of preparation method and antioxidant of rice bran polysaccharide metal complex Download PDFInfo
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Abstract
The present invention discloses the preparation method and antioxidant of a kind of rice bran polysaccharide metal complex, is related to antioxidant technology field.The preparation method of the rice bran polysaccharide metal complex obtains mixed solution the following steps are included: rice bran polysaccharide and trisodium citrate is soluble in water；Under alkaline condition, Xiang Suoshu mixed solution and dripping metal salt solution forms reaction solution；The reaction solution is centrifuged, supernatant is obtained, the supernatant obtains rice bran polysaccharide metal complex crude product after alcohol precipitation, centrifugation；By the rice bran polysaccharide metal complex crude product it is soluble in water after dialyse, obtain reservation liquid, by the reservation liquid concentration, alcohol precipitation, drying, obtain rice bran polysaccharide metal complex.Rice bran polysaccharide metal complex produced by the present invention not only has anti-oxidation function, but also can directly be absorbed to supplement the microelement of body without generating toxic side effect to body；And property is stablized, and is able to play the antioxidation to body for a long time, can be used for preparing antioxidant.
Description
Technical field
The present invention relates to antioxidant technology field, in particular to the preparation method of a kind of rice bran polysaccharide metal complex and
Antioxidant.
Background technique
Active oxygen is the by-product of aerobic metabolism, and the strong oxidizing property having is that phagocyte eliminates the micro- of invasion body
The important weapon of biology.But activity in vivo oxygen generates excessive, antioxidant ability of organism decline or generation and purge mechanism is unbalance,
It will be made to accumulate in vivo and cause cell oxidative damage.Active oxygen and its oxidative damage are related to a variety of diseases, including prestige
Coerce the major disease of human health such as: cancer, cardiovascular disease, diabetes and neurodegenerative disease.
Excessive active oxygen can be offset to the oxidative attacks of body using anti-oxidant substance.Anti-oxidant substance includes day
Right antioxidant (such as vitamin C, vitamin E) and artificial synthesized antioxidant (such as 2,6- di-t-butyl -4- methylbenzene
Phenol, butylated hydroxy anisole, tert-butylhydroquinone and propylgallate etc.).The former is fast in body intracellular metabolite speed, is difficult
Reach the elimination effect to the active oxygen long period；The latter easily causes certain diseases or causes to damage to body, and enters
Metabolism after human body in vivo is more difficult.
Summary of the invention
The main object of the present invention is to propose the preparation method and antioxidant of a kind of rice bran polysaccharide metal complex, it is intended to
The elimination effect that a kind of pair of active oxygen has the long period is prepared, and the substance of toxic side effect will not be generated to body.
To achieve the above object, the present invention proposes a kind of preparation method of rice bran polysaccharide metal complex, and the rice bran is more
The preparation method of sugared metal complex, comprising the following steps:
Rice bran polysaccharide and trisodium citrate is soluble in water, obtain mixed solution；
Under alkaline condition, Xiang Suoshu mixed solution and dripping metal salt solution forms reaction solution；
The reaction solution is centrifuged, supernatant is obtained, the supernatant obtains rice bran polysaccharide metal complex after alcohol precipitation, centrifugation
Object crude product；
By the rice bran polysaccharide metal complex crude product it is soluble in water after dialyse, reservation liquid is obtained, by the reservation liquid
Concentration, alcohol precipitation, drying obtain rice bran polysaccharide metal complex.
Preferably, described that rice bran polysaccharide and trisodium citrate is soluble in water, in the step of obtaining mixed solution, the rice bran
The mass ratio of polysaccharide and trisodium citrate is 2:(0.5~1).
Preferably, it is described under alkaline condition, Xiang Suoshu mixed solution and dripping metal salt solution forms reaction solution
Step specifically includes:
To the mixed solution and dripping alkaline solution to adjust the mixed solution pH as 8.0~9.0；
Under hot bath, while stirring to the mixed solution and dripping metal salt solution to form reaction solution, simultaneously
To alkaline solution described in the mixed solution and dripping so that the mixed solution pH is maintained in 8.0~9.0 ranges；
When precipitating in the reaction solution, stop that the metal salt solution is added dropwise.
Preferably, it is described to the mixed solution and dripping alkaline solution with adjust the mixed solution pH be 8.0~9.0
The step of in, the alkaline solution be sodium hydroxide solution.
Preferably, described under hot bath, it is anti-to be formed to the mixed solution and dripping metal salt solution while stirring
Solution is answered, while the alkaline solution is added dropwise so that the reaction solution pH is maintained at 8.0~9.0 models into the reaction solution
In step in enclosing, the metal salt solution is the salting liquid of alkali metal or the salting liquid of alkaline-earth metal.
Preferably, the metal salt solution is the soluble salt solutions of metallic iron, the soluble salt solutions of metallic copper, metal
Any one in the soluble salt solutions of calcium and the soluble salt solutions of metallic zinc.
Preferably, described under hot bath, it is anti-to be formed to the mixed solution and dripping metal salt solution while stirring
Solution is answered, while the alkaline solution is added dropwise so that the reaction solution pH is maintained at 8.0~9.0 models into the reaction solution
In step in enclosing, the temperature of the hot bath is 65 DEG C~75 DEG C.
Preferably, the supernatant after alcohol precipitation, centrifugation rice bran polysaccharide metal complex crude product the step of specifically include:
Dehydrated alcohol is added into the supernatant, is centrifuged after standing 10~12h, obtains sediment；
By the sediment acetone washing, rice bran polysaccharide metal complex crude product is obtained after dry.
Preferably, it is described by the rice bran polysaccharide metal complex crude product it is soluble in water after dialyse, obtain reservation liquid, will
The reservation liquid concentration, alcohol precipitation, drying, in the step of obtaining rice bran polysaccharide metal complex,
The dialyzate used of dialysing is water；And/or
The dialysis time is 24~48h.
The present invention also proposes that a kind of antioxidant, the antioxidant include according to above-mentioned rice bran polysaccharide metal complex
The rice bran polysaccharide metal complex of preparation method preparation.
It in technical solution of the present invention, uses native compound rice bran polysaccharide for raw material, passes through the hydroxyl on rice bran polysaccharide surface
Each metal ion species are complexed with carboxyl, obtained rice bran polysaccharide metal complex not only has the anti-oxidant function of rice bran polysaccharide itself
Can, and due to rice bran polysaccharide and complexing of metal ion, obtained rice bran polysaccharide metal complex property is stablized, and is sent out for a long time
The antioxidation to body is waved, therefore, the rice bran polysaccharide metal complex of the method that provides through the invention preparation can also be with
As antioxidant, the antioxidant good in oxidation resistance, and due to each metal ion species of rice bran polysaccharide metal complex complexing
It can also be directly absorbed and utilized, and have no toxic side effect by body, to have both the work of some minor metallic elements of supplement body
With.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is the flow diagram of an embodiment of the preparation method of rice bran polysaccharide metal complex provided by the invention；
Fig. 2 is the infrared spectrogram of product made from Examples 1 to 4 and rice bran polysaccharide；
Fig. 3 is clearance rate of the rice bran polysaccharide metal complex made from the Examples 1 to 4 of various concentration to DPPH free radical
Variation diagram；
Fig. 4 is that rice bran polysaccharide metal complex made from the Examples 1 to 4 of various concentration becomes the clearance rate of OH free radical
Change figure；
Fig. 5 is clearance rate of the rice bran polysaccharide metal complex made from the Examples 1 to 4 of various concentration to superoxide radical
Variation diagram；
Fig. 6 is that rice bran polysaccharide metal complex made from the Examples 1 to 4 of various concentration imitates the removing of activity in vivo oxygen
Fruit variation diagram；
Fig. 7 is the cytotoxicity comparison diagram of rice bran polysaccharide metal complex made from rice bran polysaccharide and Examples 1 to 4.
The embodiments will be further described with reference to the accompanying drawings for the realization, the function and the advantages of the object of the present invention.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Currently, common antioxidant includes natural and artificial synthesized antioxidant.The former is in body
Accretion rate is fast, is extremely difficult to the elimination effect to the active oxygen long period；The latter easily causes certain diseases or makes to body
At damage, and the metabolism entered after human body in vivo is more difficult.
In consideration of it, the present invention proposes a kind of preparation method of rice bran polysaccharide metal complex, rice bran as shown in connection with fig. 1 is more
The flow diagram of one embodiment of the preparation method of sugared metal complex, the preparation method of the rice bran polysaccharide metal complex
The following steps are included:
Step S10, rice bran polysaccharide and trisodium citrate is soluble in water, obtain mixed solution.
Wherein, the mass ratio of the rice bran polysaccharide and trisodium citrate is 2:(0.5~1).
Rice bran polysaccharide has certain reducing power, all has to DPPH free radical, hydroxyl radical free radical and superoxide radical
Preferable Scavenging activity, the present invention develop rice bran polysaccharide in the further application in antioxidant field, more as rice bran is prepared
The raw material of sugared metal complex.Trisodium citrate is added in rice bran polysaccharide solution, slow setting and stablizing solution system can be played
Effect, additional amount be subject to can stablizing solution system, through inventor the study found that rice bran polysaccharide and trisodium citrate plus
Entering amount is 2:(0.5~1) when, solution system tends towards stability, so as to save the dosage of trisodium citrate.
Step S20, under alkaline condition, Xiang Suoshu mixed solution and dripping metal salt solution forms reaction solution.
Rice bran polysaccharide surface is rich in hydroxyl and carboxyl, and when rice bran polysaccharide solution alkaline, these hydroxyls and carboxyl will
Complex reaction occurs with the metal ion in solution and forms rice bran polysaccharide metal complex.This complex compound has rice bran polysaccharide sheet
The inoxidizability of body, and since property is stablized, it can be stabilized in body, to be able to play the antioxygen to body for a long time
Change effect avoids fast, short to the body antioxidation time problem of current natural metabolism.
In order to preferably reach complexing effect, in the specific implementation, step S20 the following steps are included:
Step S201, Xiang Suoshu mixed solution and dripping alkaline solution is to adjust the mixed solution pH as 8.0~9.0；
Step S202, under hot bath, while stirring to the mixed solution and dripping metal salt solution to form reaction
Solution, while to alkaline solution described in the mixed solution and dripping so that the mixed solution pH is maintained at 8.0~9.0 ranges
It is interior；
Step S203, when precipitating in the reaction solution, stop that the metal salt solution is added dropwise.
Wherein, the metal salt solution is the salting liquid of alkali metal or the salting liquid of alkaline-earth metal.Any one is soluble
Alkali metal salt or alkali salt made of solution can be complexed with rice bran polysaccharide, be made property it is stable rice bran polysaccharide gold
Belong to complex compound, there is inoxidizability and can be directly absorbed and utilized by body, avoids antioxidant generation artificial synthesized at present
Thank to difficult problem.
And since metal ion can be directly absorbed and utilized by body together with rice bran polysaccharide and will not generate poison to body
Side effect, therefore, this rice bran polysaccharide metal complex can also be used as the replenishers of microelement (metallic element).For into one
Step improves practical value, can choose more universal several metallic elements in the metallic element of human body shortage, for example, iron, calcium,
Zinc, copper etc., as complexing object.In the present embodiment, the metal salt solution is preferably the soluble salt solutions of metallic iron, gold
Belong to any one in the soluble salt solutions, the soluble salt solutions of calcium metal and the soluble salt solutions of metallic zinc of copper, example
Such as, ferric chloride solution, copper chloride solution, calcium chloride solution or solution of zinc sulfate.
In addition, in order to ensure reaction progress, the alkaline environment for needing that pH is maintained to be 8.0~9.0, that is to say, that need anti-
In the range of alkaline solution being added dropwise constantly to ensure that the pH of reaction system is in 8.0~9.0 always before answering and in reaction process,
Here the alkaline solution used can be the solution of any one meta-alkalescence, in the present embodiment, preferably common and inexpensive
Sodium hydroxide solution.
To promote reaction forward to carry out, reaction process is preferably carried out in hot bath, in the present embodiment, the hot water
The temperature of bath is preferably 65 DEG C~75 DEG C.
Step S30, the reaction solution is centrifuged, obtains supernatant, the supernatant obtains rice bran polysaccharide after alcohol precipitation, centrifugation
Metal complex crude product.
In the specific implementation, step S30 the following steps are included:
Step S301, the reaction solution is centrifuged, obtains supernatant；
Step S302, dehydrated alcohol is added in Xiang Suoshu supernatant, is centrifuged after standing 10~12h, obtains sediment；
Step S303, by the sediment acetone washing, rice bran polysaccharide metal complex crude product is obtained after dry.
Step S40, by the rice bran polysaccharide metal complex crude product it is soluble in water after dialyse, reservation liquid is obtained, by institute
It states and retains liquid concentration, alcohol precipitation, drying, obtain rice bran polysaccharide metal complex.
It is therein therefore, it is necessary to be removed by dialysis due to containing in rice bran polysaccharide metal complex crude product there are many impurity
Small molecular weight impurity, in the present embodiment, the mode of dialysis are preferably flowing water dialysis, i.e., preferred water, will be equipped with thick as dialyzate
The bag filter of the aqueous solution of product, which is placed in deionized water, dialyses, and dialysis time is preferably 24~48h.
In addition, the antioxidant includes according to above-mentioned rice bran polysaccharide metal network the invention also provides a kind of antioxidant
Close the rice bran polysaccharide metal complex of the preparation method preparation of object.
Since rice bran polysaccharide metal complex property is stable and has good anti-oxidation function, it can be used as anti-
Oxidant uses, or as the ingredient of antioxidant.Antioxidant comprising the rice bran polysaccharide metal complex not only has
There is good antioxidation, and has both the effect of some minor metallic elements of supplement body.Certainly, the antioxidant
It may include other additives, all includes being existed according to the antioxidant of the rice bran polysaccharide metal complex of above method preparation
In the scope of the present invention.
Technical solution of the present invention is described in further detail below in conjunction with specific embodiments and the drawings, it should be understood that
Following embodiment is only used to explain the present invention, is not intended to limit the present invention.
Embodiment 1
1g rice bran polysaccharide, 0.5g trisodium citrate are weighed, is dissolved in 60ml water, 70 DEG C of stirred in water bath is placed in, is allowed to molten
Solution, obtains mixed solution.
In 70 DEG C of water-baths, under stirring to 10% sodium hydroxide solution of mixed solution and dripping to pH value be 9.0,
2mol/L ferric chloride solution is added dropwise simultaneously and carries out complex reaction, continues to be added dropwise 10% sodium hydroxide solution during the reaction to protect
PH value is held in 8.0~9.0 ranges.When occurring insoluble red-brown precipitation in reaction solution, stop that sodium hydroxide solution is added dropwise
And ferric chloride solution.
It is centrifuged 10min (revolving speed 8000r/min) after reaction solution is stood cooling, takes supernatant.It is added into supernatant
Dehydrated alcohol carries out alcohol precipitation, after standing 12h, is centrifuged 5min (revolving speed 8000r/min) again, takes lower sediment thing.It will precipitating
Object is washed with ethyl alcohol and acetone, obtains rice bran polysaccharide iron crude product after dry.
Crude product solution is made by rice bran polysaccharide iron crude product is soluble in water, it is for 24 hours to crude product solution dialysis, molten after must dialysing
Liquid.Solution after dialysis is rotated to 20ml, dehydrated alcohol is added and carries out alcohol precipitation, after taking precipitating dry, obtains the rice bran of purification
Polyferose (RBP-Fe).
Embodiment 2
2g rice bran polysaccharide, 0.5g trisodium citrate are weighed, is dissolved in 60ml water, 65 DEG C of stirred in water bath is placed in, is allowed to molten
Solution, obtains mixed solution.
In 65 DEG C of water-baths, under stirring to 10% sodium hydroxide solution of mixed solution and dripping to pH value be 8.0,
0.45mol/L solution of zinc sulfate is added dropwise simultaneously and carries out complex reaction, continues that 10% sodium hydroxide solution is added dropwise during the reaction
To keep pH value in 8.0~9.0 ranges.When occurring white precipitate in reaction solution, stop that sodium hydroxide solution and sulphur is added dropwise
Sour zinc solution.
It is centrifuged 10min (revolving speed 8000r/min) after reaction solution is stood cooling, takes supernatant.It is added into supernatant
Dehydrated alcohol carries out alcohol precipitation, after standing 10h, is centrifuged 5min (revolving speed 8000r/min) again, takes lower sediment thing.It will precipitating
Object is washed with ethyl alcohol and acetone, obtains rice bran polysaccharide zinc crude product after dry.
Crude product solution is made by rice bran polysaccharide zinc crude product is soluble in water, dialyses 36h to crude product solution, it is molten after must dialysing
Liquid.Solution after dialysis is rotated to 20ml, dehydrated alcohol is added and carries out alcohol precipitation, after taking precipitating dry, obtains the rice bran of purification
Polysaccharide zinc (RBP-Zn).
Embodiment 3
2g rice bran polysaccharide, 0.8g trisodium citrate are weighed, is dissolved in 60ml water, 70 DEG C of stirred in water bath is placed in, is allowed to molten
Solution, obtains mixed solution.
In 70 DEG C of water-baths, under stirring to 10% sodium hydroxide solution of mixed solution and dripping to pH value be 8.5,
2mol/L calcium chloride solution is added dropwise simultaneously and carries out complex reaction, continues to be added dropwise 10% sodium hydroxide solution during the reaction to protect
PH value is held in 8.0~9.0 ranges.When occurring white precipitate in reaction solution, stop that sodium hydroxide solution and calcium chloride is added dropwise
Solution.
It is centrifuged 10min (revolving speed 8000r/min) after reaction solution is stood cooling, takes supernatant.It is added into supernatant
Dehydrated alcohol carries out alcohol precipitation, after standing 11h, is centrifuged 5min (revolving speed 8000r/min) again, takes lower sediment thing.It will precipitating
Object is washed with ethyl alcohol and acetone, obtains rice bran polysaccharide calcium crude product after dry.
Crude product solution is made by rice bran polysaccharide calcium crude product is soluble in water, dialyses 48h to crude product solution, it is molten after must dialysing
Liquid.Solution after dialysis is rotated to 20ml, dehydrated alcohol is added and carries out alcohol precipitation, after taking precipitating dry, obtains the rice bran of purification
Polysaccharide calcium (RBP-Ca).
Embodiment 4
1g rice bran polysaccharide, 0.4g trisodium citrate are weighed, is dissolved in 60ml water, 75 DEG C of stirred in water bath is placed in, is allowed to molten
Solution, obtains mixed solution.
In 75 DEG C of water-baths, under stirring to 10% sodium hydroxide solution of mixed solution and dripping to pH value be 8.5,
2mol/L copper chloride solution is added dropwise simultaneously and carries out complex reaction, continues to be added dropwise 10% sodium hydroxide solution during the reaction to protect
PH value is held in 8.0~9.0 ranges.When occurring blue flocculent deposit in reaction solution, stop that sodium hydroxide solution and chlorine is added dropwise
Change copper solution.
It is centrifuged 10min (revolving speed 8000r/min) after reaction solution is stood cooling, takes supernatant.It is added into supernatant
Dehydrated alcohol carries out alcohol precipitation, after standing 12h, is centrifuged 5min (revolving speed 8000r/min) again, takes lower sediment thing.It will precipitating
Object is washed with ethyl alcohol and acetone, obtains rice bran polysaccharide copper crude product after dry.
Crude product solution is made by rice bran polysaccharide copper crude product is soluble in water, dialyses 30h to crude product solution, it is molten after must dialysing
Liquid.Solution after dialysis is rotated to 20ml, dehydrated alcohol is added and carries out alcohol precipitation, after taking precipitating dry, obtains the rice bran of purification
Polysaccharide copper (RBP-Cu).
Product made from Examples 1 to 4 is verified.
(1) it characterizes
Infrared detection is carried out to product made from rice bran polysaccharide and Examples 1 to 4 respectively, records infared spectrum such as Fig. 2 institute
Show.
As shown in Figure 2 ,-OH the stretching vibration absworption peak of RBP-Fe, RBP-Cu, RBP-Zn and RBP-Ca move to respectively
3271cm-1、3257.14cm-1、3279.40cm-1And 3295.42cm-1Place, explanation-OH take part in the coordination with each metal ion；
- COO- the stretching vibration absworption peak of RBP-Fe, RBP-Cu, RBP-Zn and RBP-Ca move to 1582.56cm respectively-1、
1633.61cm-1、1644.68cm-1And 1634cm-1Place, explanation-COO- take part in the coordination with each metal ion.
(2) tenor is tested
Rice bran polysaccharide metal complex made from Examples 1 to 4 is detected respectively using Atomic Absorption Spectrometer, is surveyed
It is as follows to obtain result: iron content is 1.29x 10 in RBP-Fe5Copper content is 1.22x 10 in mg/kg, RBP-Cu5Mg/kg, RBP-
Zn content is 4.05x 10 in Zn3Calcium content is 5.66x 10 in mg/kg, RBP-Ca4mg/kg。
Experiment (one) and experiment (two) are identified that the product of Examples 1 to 4, qualification result proves that the present invention provides
Technical solution make metal ion and rice bran polysaccharide surface hydroxyl and carbonyl complexing, rice bran polysaccharide metal complex can be made
Object.
(3) to the scavenging effect of DPPH free radical
Comparison liquid: it with the DPPH solution of dehydrated alcohol configuration 0.1mol/L, is kept in dark place.
Test liquid: taking rice bran polysaccharide metal complex to be dissolved in water, compound concentration be respectively 0.125mg/ml, 0.25mg/ml,
The solution of 0.5mg/ml, 1mg/ml, 2mg/ml, 2.5mg/ml, 3mg/ml and 4mg/ml.Each 2ml of the solution of above-mentioned concentration is taken, point
Not Jia Ru 2mL comparison liquid, after being protected from light 30min, as test liquid.
The absorbance value of above-mentioned comparison liquid and test liquid at 517nm is measured using ultraviolet specrophotometer, record is for examination
Liquid absorbance is A1, comparison liquid absorbance is A0.The rice bran polysaccharide metal complex of Examples 1 to 4 preparation is examined according to the above method
It surveys.
Using rice bran polysaccharide (RBP), vitamin C (VC) as positive control.The clearance rate to DPPH is calculated by following equation 1
And it is as shown in Figure 3 to draw line chart.
Formula 1:DPPH clearance rate (%)=(A0-A1)/A0x 100
(4) to the scavenging effect of OH free radical
The phosphate buffer of 1ml 0.1mol/L pH 7.4, the salicylic acid of 1ml 9mmol/L are sequentially added into test tube
Solution, 1ml (concentration is respectively 0.25mg/ml, 0.5mg/ml, 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml, 5mg/ml) rice bran
The aqueous solution of polyose metal complex compound, the ferrous sulfate solution of 1ml 9mmol/L and the hydrogen peroxide solution of 1ml 9mmol/L, 37
With the absorbance at ultraviolet specrophotometer measurement 510nm after DEG C water-bath 1h, it is denoted as A0；Rice bran polysaccharide metal complex is not added
The control group 1 of aqueous solution measures absorbance and is denoted as A1；The aqueous solution and hydrogen peroxide solution of rice bran polysaccharide metal complex is not added
Control group 2 measures absorbance and is denoted as A2.The rice bran polysaccharide metal complex of Examples 1 to 4 preparation detects according to the above method.With
Rice bran polysaccharide (RBP), vitamin C (VC) are used as positive control.
It calculates by following equation 2 to the clearance rate of OH free radical and to draw line chart as shown in Figure 4.
Formula 2:OH free radical scavenging activity (%)=[(A0-A1)/(A2-A1)]x 100
(5) to the scavenging effect of superoxide radical
Sequentially added into test tube the rice bran polysaccharide metal complex of 1ml aqueous solution (concentration be followed successively by 0.125mg/ml,
0.25mg/ml, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml, 3mg/ml, 4mg/ml), 2mL 50mmol/L pH8.2
The pyrogallol solution (above-mentioned all reagents are preheated in 25 DEG C) of Tris-HCl buffer, 0.1mL 3mmol/L, with ultraviolet survey
Determine the absorbance at 320nm, is denoted as A0, and every 30s measurement is primary, METHOD FOR CONTINUOUS DETERMINATION 5min；Rice bran polysaccharide metal complex is not added
Aqueous solution control group measurement absorbance be denoted as A1.The rice bran polysaccharide metal complex of Examples 1 to 4 preparation presses above-mentioned side
Method detection.Using rice bran polysaccharide (RBP), vitamin C (VC) as positive control.
The clearance rate to superoxide radical is calculated by following equation 3 and draws line chart as shown in Figure 5.
Formula 3: superoxide radical clearance rate (%)=[(Δ A1-ΔA0)/A1]x 100
(6) to the scavenging effect of reactive oxygen species (ROS)
Taking Hela cell, (plating density in 96 orifice plates is 1x104/ hole) 37 DEG C are placed in, 5%CO2Under the conditions of cultivate
For 24 hours, when cell reaches 70-80% convergence degree, be separately added into concentration be 0.25mg/ml, 0.5mg/ml, 0.75mg/ml,
100 μ L of 1.0mg/ml, 1.25mg/ml and 1.5mg/ml sample solution is added 100 μ L, 5 μm of ol/L DCFH-DA solution, uses enzyme
The fluorescence intensity at instrument detection excitation wavelength 485nm, launch wavelength 538nm is marked, primary, METHOD FOR CONTINUOUS DETERMINATION is measured at interval of 4min
1h.Sample solution is not added as blank control, using the hydrogen peroxide of 120 μm of ol/L as positive control.Sample solution is to implement
Rice bran polysaccharide metal complex solution or rice bran polysaccharide (RBP) solution prepared by example 1~4, record result are as shown in Figure 6.
In Fig. 6, A is the curve graph of intracellular reactive oxygen level variation after the RBP of various concentration is added；
B is the curve graph of intracellular reactive oxygen level variation after the RBP-Fe of various concentration is added；
C is the curve graph of intracellular reactive oxygen level variation after the RBP-Cu of various concentration is added；
D is the curve graph of intracellular reactive oxygen level variation after the RBP-Zn of various concentration is added；
E is the curve graph of intracellular reactive oxygen level variation after the RBP-Ca of various concentration is added；
F is reactive oxygen species after RBP, RBP-Fe, RBP-Cu, RBP-Zn and RBP-Ca of 1.0mg/ml concentration is added
The curve graph of level variation.
Test (three) to experiment (six) to the anti-oxidation function of rice bran polysaccharide metal complex made from Examples 1 to 4 into
It has gone assessment, and has been compared using rice bran polysaccharide, vitamin C as positive control.
From the figure 3, it may be seen that rice bran polysaccharide metal complex made from Examples 1 to 4 (RBP-Fe, RBP-Cu, RBP-Zn and
RBP-Ca) there is preferable elimination effect to DPPH free radical；
As shown in Figure 4, rice bran polysaccharide metal complex made from Examples 1 to 4 (RBP-Fe, RBP-Cu, RBP-Zn and
RBP-Ca) there is preferable elimination effect to OH free radical；
As shown in Figure 5, rice bran polysaccharide metal complex made from Examples 1 to 4 (RBP-Fe, RBP-Cu, RBP-Zn and
RBP-Ca) there is preferable elimination effect to superoxide radical；
It will be appreciated from fig. 6 that rice bran polysaccharide metal complex made from Examples 1 to 4 (RBP-Fe, RBP-Cu, RBP-Zn and
RBP-Ca after) having preferable elimination effect, especially addition rice bran polysaccharide metal complex 1h to reactive oxygen species, into the cell
Active o content has been significantly lower than the active o content of blank control.
In conclusion rice bran polysaccharide metal complex prepared by the present invention has good antioxidation.
(7) cytotoxicity assay
Taking Hela cell, (plating density in 96 orifice plates is 1x104/ hole) in 37 DEG C, 5%CO2Under conditions of cultivate
For 24 hours, when cell reaches 70-80% convergence degree, 100 μ of rice bran polysaccharide metal complex solution that concentration is 0.25mg/ml is added
After cultivating for 24 hours under the same terms, the MTT solution of 10 μ L 5mg/ml is added in l, continues to cultivate 4h.Culture medium and sample are drawn,
The Formazan crystal generated into the cell is dissolved with DMSO and is denoted as A with the absorbance at ultraviolet determination 570nm1.Not add rice
Chaff polyose metal complex compound hole (cell survival rate 100%) is denoted as A as the absorbance at blank control group measurement 570nm0。
The rice bran polysaccharide metal complex of Examples 1 to 4 preparation detects according to the above method.It is right using rice bran polysaccharide (RBP) as the positive
According to.
Cell survival rate is calculated by formula 4 and is drawn as shown in Figure 7.
Formula 4: cell survival rate (%)=(A0-A1)/A0x 100
As shown in Figure 7, when solution concentration is 0.25mg/ml, the sample sets inner cell survival rate of RBP-Fe solution is added
It is 99.1%；The sample sets inner cell survival rate that RBP-Cu solution is added is 83.3%；It is added in the sample sets of RBP-Zn solution
Cell survival rate is 96.0%；The sample sets inner cell survival rate that RBP-Ca solution is added is 97.8%, illustrates preparation of the present invention
Rice bran polysaccharide metal complex not will cause damage to body using rear.
The above is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, for this field
For technical staff, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any
Modification, equivalent replacement, improvement etc. should all be included within the scope of the present invention.
Claims (10)
1. a kind of preparation method of rice bran polysaccharide metal complex, which comprises the following steps:
Rice bran polysaccharide and trisodium citrate is soluble in water, obtain mixed solution；
Under alkaline condition, Xiang Suoshu mixed solution and dripping metal salt solution forms reaction solution；
The reaction solution is centrifuged, obtains supernatant, it is thick that the supernatant obtains rice bran polysaccharide metal complex after alcohol precipitation, centrifugation
Product；
By the rice bran polysaccharide metal complex crude product it is soluble in water after dialyse, obtain reservation liquid, by the reservation liquid concentration,
Alcohol precipitation, drying obtain rice bran polysaccharide metal complex.
2. the preparation method of rice bran polysaccharide metal complex as described in claim 1, which is characterized in that described by rice bran polysaccharide
Soluble in water with trisodium citrate, in the step of obtaining mixed solution, the mass ratio of the rice bran polysaccharide and trisodium citrate is 2:
(0.5~1).
3. the preparation method of rice bran polysaccharide metal complex as described in claim 1, which is characterized in that described in alkaline condition
Under, Xiang Suoshu mixed solution and dripping metal salt solution formed reaction solution the step of specifically include:
To the mixed solution and dripping alkaline solution to adjust the mixed solution pH as 8.0~9.0；
Under hot bath, while stirring to the mixed solution and dripping metal salt solution to form reaction solution, while to institute
It states and the alkaline solution is added dropwise in reaction solution so that the reaction solution pH is maintained in 8.0~9.0 ranges；
When precipitating in the reaction solution, stop that the metal salt solution is added dropwise.
4. the preparation method of rice bran polysaccharide metal complex as claimed in claim 3, which is characterized in that described to the mixing
Alkaline solution is added dropwise in solution to adjust in the step of mixed solution pH is 8.0~9.0, the alkaline solution is hydroxide
Sodium solution.
5. the preparation method of rice bran polysaccharide metal complex as claimed in claim 3, which is characterized in that described in hot bath
Under, while stirring to the mixed solution and dripping metal salt solution to form reaction solution, while into the reaction solution
The alkaline solution is added dropwise so that the reaction solution pH is maintained in the step in 8.0~9.0 ranges, the metal salt solution
For the salting liquid of alkali metal or the salting liquid of alkaline-earth metal.
6. the preparation method of rice bran polysaccharide metal complex as claimed in claim 5, which is characterized in that the metal salt solution
For the soluble salt solutions of metallic iron, the soluble salt solutions of metallic copper, the soluble salt solutions of calcium metal and metallic zinc can
Any one in dissolubility salting liquid.
7. the preparation method of rice bran polysaccharide metal complex as claimed in claim 3, which is characterized in that described in hot bath
Under, while stirring to the mixed solution and dripping metal salt solution to form reaction solution, while into the reaction solution
The alkaline solution is added dropwise so that the reaction solution pH is maintained in the step in 8.0~9.0 ranges, the temperature of the hot bath
Degree is 65 DEG C~75 DEG C.
8. the preparation method of rice bran polysaccharide metal complex as described in claim 1, which is characterized in that the supernatant is through alcohol
The step of obtaining rice bran polysaccharide metal complex crude product after heavy, centrifugation specifically includes:
Dehydrated alcohol is added into the supernatant, is centrifuged after standing 10~12h, obtains sediment；
By the sediment acetone washing, rice bran polysaccharide metal complex crude product is obtained after dry.
9. the preparation method of rice bran polysaccharide metal complex as described in claim 1, which is characterized in that described by the rice bran
It dialyses after polyose metal complex compound crude product is soluble in water, obtains reservation liquid, by the reservation liquid concentration, alcohol precipitation, drying, obtain rice
In the step of chaff polyose metal complex compound,
The dialyzate used of dialysing is water；And/or
The dialysis time is 24~48h.
10. a kind of antioxidant, which is characterized in that including described in any item rice bran polysaccharide metal networks according to claim 1~9
Close the rice bran polysaccharide metal complex of the preparation method preparation of object.
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