A kind of antioxidant and preparation method thereof having both antitumor action
Technical field
The present invention relates to a kind of deep processed products of edible mushroom, in particular to the polysaccharide extracted using T.lobayense Heim as raw material, warp
The antioxidant for having both antitumor action that oversulfated modification generates.
Background technique
T.lobayense Heim (Tricholoma lobayense Heim) is also known as Tricholoma Giganteum Massee, big spoken parts in traditional operas mushroom, Tricholoma lobayense,
Japan claims white matsutake, is that a kind of natural health care of preciousness and high-grade edible mushroom, polysaccharide and proteoglycans have
Immunological regulation and antitumor activity.
Chinese invention patent ZL201210014537.4 shows that T.lobayense Heim polysaccharide TL-3 is the one kind extracted from T.lobayense Heim
The acidic polysaccharose of hyperbranched small molecule, molecular weight are 4.22 × 103Da, have with the comparable antioxidant activity of Vc, can
As a kind of Nantural non-toxic antioxidant, it is applied to the fields such as food, medicine and cosmetics.But the compound still has stabilization
The disadvantages of property is poor, oxidation resistance is to be improved.
Currently, polysaccharide due to can improve its intrinsic bioactivity after chemical modification or obtain new functional characteristic by
Extensive concern.Common method of modifying has sulphation, acetylation, carboxy methylation etc., wherein sulphation is the biology for enhancing polysaccharide
Active classical way.Research shows that sulfated polysaccharides is the potent inhibitor of human immunodeficiency virus (HIV).Ganoderma lucidum polysaccharide
More effectively inhibit the growth of S-180 cell after sulphation than before.The polysaccharide sulfated anti-oxidant work that they not only can be improved
Property, also them is made to have effects that antibacterial.The method of sulphation modification has very much, such as chlorosulfonic acid method, sulphate method, wherein chlorine sulphur
Acid meets water capacity explosive, and concentrated sulfuric acid corrosivity is too strong.
In view of reagent cost and the yield of the convenient of method, safety and product, to realize industrialized production, the present invention
Sulfur trioxide-pyridine method is applied in T.lobayense Heim polysaccharide for the first time in the sulphation modification of the highest component TL-3 of activity, is obtained
A kind of sulphation modification method simple, mild, product yield is high and active and strong stability.
Summary of the invention
It is an object of that present invention to provide a kind of novel antioxidant for having both antitumor action and preparation method thereof namely sulphur
It is acidified the T.lobayense Heim polysaccharide STL-3 of modification, has both antitumor and antioxidation, and STL-3 is to HELF cytotoxic pair
Effect has huge application potential in fields such as health cares;The present invention is for the first time by sulfur trioxide-pyridine (SO3- Py) method fortune
It is a kind of simple, mild, product yield height and active and stability in the sulphation modification for using T.lobayense Heim polysaccharide TL-3
Strong sulphation modification method.
The present invention is to realize goal of the invention, is adopted the following technical scheme that
A kind of novel antioxidant having both antitumor action, it is characterised in that: the antioxidant is sulphation modification
T.lobayense Heim polysaccharide STL-3, be using T.lobayense Heim polysaccharide TL-3 as raw material, using sulfur trioxide-pyridine method carry out sulphation repair
Decorations obtain.The T.lobayense Heim polysaccharide STL-3 molecular weight of the sulphation modification is 8.12 × 103Da, sulfate radical content are
13.60%, degree of substitution 1.22, by rhamnose, glucose, galactolipin, glucuronic acid, arabinose, mannose and gala
7 kinds of monosaccharide compositions of uronic acid.
The preparation method of the above-mentioned novel antioxidant for having both antitumor action is: being not less than 95% T.lobayense Heim with purity
Polysaccharide TL-3 is raw material, 35~50 times of material quality of solvent formamide (PF), 25 DEG C of water-bath 30min is added, then press raw material matter
1~2 times of addition sulfur trioxide-pyridine (SO of amount3- Py), magnetic agitation 3h under 60~80 DEG C of oil baths, finally with 4mol/L's
NaOH tune pH dialyses, concentration, alcohol precipitation is overnight, and precipitating is freeze-dried to obtain T.lobayense Heim sulfated polysaccharides STL-3, as to neutrality
Have both the novel antioxidant of antitumor action.
The beneficial effects of the present invention are embodied in:
The present invention utilizes sulfur trioxide-pyridine (SO using T.lobayense Heim polysaccharide TL-3 as raw material3- Py) method carry out sulphation repair
Decorations, have obtained the T.lobayense Heim polysaccharide STL-3 of sulphation modification.This method step is easy, easily operated, and the conversion ratio of TL-3 is
30%, in product sulfate radical content be 13.60%, degree of substitution 1.22, final products purity is up to 99% or more.
Gained STL-3 of the invention is removing DPPH and O2 -The IC of free radical50Value has been over positive control Vitamin C
Acid;4 times of TL-3 high of inhibiting rate ratio to HeLa cell of STL-3;STL-3 is to the inhibiting rate of MCF-7 cell almost 5 more than TL-3
Times;In the concentration of 10 μ g/mL, (a famous anticancer drug can largely make cancer cell lethal to 5-Fu, but simultaneously
Also have huge side effect to normal cell) it is only 50% or so to the survival rate of HELF cell, but in 400 μ g/mL, STL-
4 times of 5-Fu high of survival rate ratio or more of 3, and have certain promotion growth to cell after modifying;STL-3 has both antitumor function
Energy and oxidation resistance, obvious effect, market application prospect are very wide.
Detailed description of the invention
Fig. 1 is the T.lobayense Heim of the raw materials used T.lobayense Heim polysaccharide TL-3 of the embodiment of the present invention 1 and products obtained therefrom sulphation modification
High performance liquid chromatography (HPLC) test map of polysaccharide STL-3;
Fig. 2 is the T.lobayense Heim of the raw materials used T.lobayense Heim polysaccharide TL-3 of the embodiment of the present invention 1 and products obtained therefrom sulphation modification
The infared spectrum of polysaccharide STL-3;
Fig. 3 is sample in the embodiment of the present invention 4 to the test result of the Scavenging activity of DPPH free radical;
Fig. 4 is sample in the embodiment of the present invention 4 to superoxide anion (O2 -) Scavenging activity test result;
Fig. 5 is sample in the embodiment of the present invention 4 to the test result of the Scavenging activity of hydroxyl radical free radical (OH);
Fig. 6 is the test result of sample reducing power in the embodiment of the present invention 4;
Fig. 7 is sample in the embodiment of the present invention 4 to the test result of the rejection ability of HeLa and MCF-7 cell;
Fig. 8 is sample in the embodiment of the present invention 4 to the test result of the toxic effect to HELF cell.
Specific embodiment
Embodiment 1
The novel antioxidant that the present embodiment has both antitumor action is the T.lobayense Heim polysaccharide STL-3 of sulphation modification,
Molecular weight is 8.12 × 103Da, sulfate radical content 13.60%, degree of substitution 1.22, by rhamnose, glucose, galactolipin,
7 kinds of glucuronic acid, arabinose, mannose, galacturonic acid monosaccharide compositions;Its preparation step is as follows:
It takes purity to be not less than 95% T.lobayense Heim polysaccharide TL-3 100mg, the solvent formamide (PF) of 5g, 25 DEG C of water is added
30min is bathed, the SO of 200mg is added3- Py, magnetic agitation 3h, NaOH (4mol/L) adjusts pH to neutrality under 70 DEG C of oil baths, dialysis,
Concentration, alcohol precipitation is overnight, precipitates the freeze-dried T.lobayense Heim polysaccharide STL-3 for obtaining sulphation modification.
Fig. 1 is the T.lobayense Heim polysaccharide of the raw materials used T.lobayense Heim polysaccharide TL-3 of the present embodiment and products obtained therefrom sulphation modification
High performance liquid chromatography (HPLC) test map of STL-3;Fig. 2 is the raw materials used T.lobayense Heim polysaccharide TL-3 of the present embodiment and gained produces
The infared spectrum of the T.lobayense Heim polysaccharide STL-3 of product sulphation modification.
Embodiment 2
The novel antioxidant that the present embodiment has both antitumor action is the T.lobayense Heim polysaccharide STL-3 of sulphation modification,
Molecular weight is 8.12 × 103Da, sulfate radical content 13.60%, degree of substitution 1.22, by rhamnose, glucose, galactolipin,
7 kinds of glucuronic acid, arabinose, mannose, galacturonic acid monosaccharide compositions;Its preparation step is as follows:
It takes purity to be not less than 95% T.lobayense Heim polysaccharide TL-3 100mg, the solvent formamide (PF) of 4g, 25 DEG C of water is added
30min is bathed, the SO of 100mg is added3- Py, magnetic agitation 3h, NaOH (4mol/L) adjusts pH to neutrality under 80 DEG C of oil baths, dialysis,
Concentration, alcohol precipitation is overnight, precipitates the freeze-dried T.lobayense Heim polysaccharide STL-3 for obtaining sulphation modification.
The HPLC map and infared spectrum of the present embodiment products obtained therefrom are similar to Example 1.
Embodiment 3
The novel antioxidant that the present embodiment has both antitumor action is the T.lobayense Heim polysaccharide STL-3 of sulphation modification,
Molecular weight is 8.12 × 103Da, sulfate radical content 13.60%, degree of substitution 1.22, by rhamnose, glucose, galactolipin,
7 kinds of glucuronic acid, arabinose, mannose, galacturonic acid monosaccharide compositions;Its preparation step is as follows:
It takes purity to be not less than 95% T.lobayense Heim polysaccharide TL-3 100mg, is added the solvent formamide (PF) of 4.5g, 25 DEG C
Water-bath 30min adds the SO of 200mg3- Py, magnetic agitation 3h, NaOH (4mol/L) adjusts pH to neutrality under 60 DEG C of oil baths, thoroughly
Analysis, concentration, alcohol precipitation is overnight, precipitates the freeze-dried T.lobayense Heim polysaccharide STL-3 for obtaining sulphation modification.
The HPLC map and infared spectrum of the present embodiment products obtained therefrom are similar to Example 1.
Embodiment 4
The novel antioxidant that the present embodiment has both antitumor action is the T.lobayense Heim polysaccharide STL-3 of sulphation modification,
Molecular weight is 8.12 × 103Da, sulfate radical content 13.60%, degree of substitution 1.22, by rhamnose, glucose, galactolipin,
7 kinds of glucuronic acid, arabinose, mannose, galacturonic acid monosaccharide compositions;Its preparation step is as follows:
It takes purity to be not less than 95% T.lobayense Heim polysaccharide TL-3 100mg, is added the solvent formamide (PF) of 3.5g, 25 DEG C
Water-bath 30min adds the SO of 200mg3- Py, magnetic agitation 3h, NaOH (4mol/L) adjusts pH to neutrality under 72 DEG C of oil baths, thoroughly
Analysis, concentration, alcohol precipitation is overnight, precipitates the freeze-dried T.lobayense Heim polysaccharide STL-3 for obtaining sulphation modification.
The HPLC map and infared spectrum of the present embodiment products obtained therefrom are similar to Example 1.
The antioxidant activity of 4 products obtained therefrom of embodiment is tested:
(1) to the scavenging effect of DPPH free radical
Prepare the gold of various concentration (25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 300 μ g/mL, 400 μ g/mL)
The aqueous solution of the T.lobayense Heim polysaccharide STL-3 of good fortune mushroom polysaccharide TL-3 and sulphation modification, takes the methanol of 2mL and 2mL DPPH molten respectively
Liquid (0.13mmol/L) mixes.Reaction mixture is uniformly mixed with quick vortex mixer and is incubated for 30min at 25 DEG C of room temperature, it is quiet
It sets, measures light absorption value with ultra-violet and visible spectrophotometer at 517nm;Every group experimental setup three parallel, takes light absorption value
Average value.Make positive control with Vc to operate instead of T.lobayense Heim polysaccharide by above-mentioned steps;
The Scavenging activity of DPPH free radical is calculated according to following formula:
Wherein, A0The absorbance value in 2mL methanol is added for 2mLDPPH solution;A1It is that 2mL methanol adds 2mL various concentration
The absorbance value of the mixture of sample;A2For 2mL DPPH methanol solution add 2mL various concentration sample absorbance value.
Test results are shown in figure 3, it can be seen from the figure that all samples remove DPPH within the scope of test concentrations
Ability is in apparent concentration dependent.Under same concentrations, the Scavenging activity ratio TL-3 of sulfated polysaccharides STL-3 is significantly increased.Value
It obtains one to be mentioned that, activity of the STL-3 in 25 μ g/mL of low dosage is almost twice higher than TL-3, or even than positive controls (Vc)
It is higher.The IC of TLH-3, STLH-3 and Vc50Value is 69.3 μ g/mL, 9.0 μ g/mL and 21.2 μ g/mL respectively.According to it has been reported that
The removing DPPH ability of few polysaccharide and sulfated polysaccharides from edible mushroom can exceed that STL-3.
(2) superoxide anion (O is removed2 -) ability
Prepare various concentration (25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 300 μ g/mL, 400 μ g/mL, 500 μ
G/mL the aqueous solution of the T.lobayense Heim polysaccharide STL-3 of T.lobayense Heim polysaccharide TL-3 and sulphation modification).
Prepare the sample aqueous solution mixing of the Tris-HCl buffer (50mM, pH value 8.3) and 0.4mL of 0.5mL, room temperature
Concussion shakes up at 25 DEG C, stands 20min, is added 0.1mL pyrogallol (3mmol), is sufficiently mixed;Every 30s measurement at 325nm
Light absorption value continues 5min, measures final light absorption value.Sample is replaced to compare group with distilled water, Vc makees positive control progress
Above-mentioned experimental implementation.Every group experimental setup three parallel, is averaged.
·O2 -Scavenging activity calculated according to following formula:
Wherein, Δ A1For the final light absorption value of the polysaccharide solution of each concentration, Δ A0For pair for replacing sample with distilled water
According to the light absorption value of group.
Test results are shown in figure 4, it can be seen from the figure that the IC of TL-3, STL-3 and Vc50Value is 111.6 μ g/ respectively
ML, 27.1 μ g/mL and 29.9 μ g/mL.STL-3 shows the removing energy more taller than positive control within the scope of test concentrations
Power.
(3) to the scavenging effect of hydroxyl radical free radical (OH)
Accurate formulation various concentration (10 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 400 μ g/ respectively
ML, 500 μ g/mL) T.lobayense Heim polysaccharide TL-3, sulphation modification T.lobayense Heim polysaccharide STL-3 and Vc sample aqueous solution, 6mM
FeSO4Solution, the H of 2.4mM2O2Solution, the alcohol-water poplar acid solution of 6mM.
Respectively take 1mL FeSO4Solution, 1mL alcohol-water poplar acid solution are added 1mL sample solution, reaction mixture are being tried
It shakes and mixes in pipe, stand 10min.Add 1mL H2O2Solution reaction 30min.Using Vc as positive control, with masking foil point
It Bao Guo not positive control, TL-3 and STL-3 water bath with thermostatic control 30min under the conditions of 37 DEG C.React the 2,3- dihydroxy benzenes first generated
Acid has light absorption value at 510nm.It replaces polysaccharide solution as blank group using distilled water, replaces salicylic acid as control using distilled water
Group, every group of experiment set three groups it is parallel.
The Scavenging activity of OH is calculated according to following formula:
Wherein, A0For the light absorption value of blank group, A2For the light absorption value of various concentration polysaccharide sample, A1For the extinction of control group
Value.
Test results are shown in figure 5, it can be seen from the figure that the OH Scavenging activity ratio TL-3 of STL-3 and Vc is strong very much.
All samples have obvious concentration dependent to the scavenging capacity of OH.The IC of TL-3, STL-3 and Vc50Value is respectively 173.8 μ
G/mL, 73.5 μ g/mL and 50.1 μ g/mL.It will be apparent that sulfated polysaccharide STL-3 removing OH ability ratio TL-3 is more effective.
(4) reducing power
Prepare various concentration (10 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 400 μ g/mL, 500 μ
G/mL the aqueous solution of the T.lobayense Heim polysaccharide STL-3 of T.lobayense Heim polysaccharide TL-3, sulphation modification);Preparing 0.2mol/L pH is
The K that mass concentration is 1% is added after taking 0.2mL in 6.6 PBS buffer solution3Fe(CN)6Solution 1.0mL adds the sample of 1mL,
Mixture is placed in 50 DEG C of water-bath 20min.Then, trichloroacetic acid (TCA) solution that 1.0mL mass concentration is 10% is added,
10min is centrifuged under 3000 revs/min.1.5mL supernatant is taken, 1% iron chloride (FeCl of 1.5mL distilled water and 0.2mL is added3)
Solution.Mixture is protected from light 30min at 25 DEG C.Light absorption value is detected at 700nm.Make positive control with ascorbic acid (Vc),
Sample is replaced to make blank with distilled water.The reducing power of sample is calculated according to the following formula:
Wherein A0Indicate the blank group light absorption value of not sample, A2Indicate the light absorption value of sample, A1Indicate no K3Fe(CN)6
The light absorption value of solution.
Test results are shown in figure 6, it can be seen from the figure that all samples show a degree of reducing power.TL-3,
The IC of STL-3 and Vc50Value is respectively 274.2 μ g/mL, 90.5 μ g/mL and 64.8 μ g/mL.The reducing power of STL-3 compares TL-3
It significantly improves, and close to Vc.
The anti tumor activity in vitro of 4 products obtained therefrom of embodiment is tested:
(1) cell culture
HELF cell, HeLa cell and MCF-7 cell are cultivated in DMEM high glycoform culture medium.In DMEM added with
100mL/L fetal calf serum, 100U/mL penicillin and 100 μ g/mL streptomysins, in 37 DEG C, 5%CO2Incubator wet atmosphere
Middle incubation.
(2) MTT is detected
The T.lobayense Heim polysaccharide STL-3 of T.lobayense Heim polysaccharide TL-3 and sulphation modification is to HeLa cell, MCF-7 cell and HELF
The detection method for cytotoxicity of gas-liquid of cell: the 10 of 0.1mL are separately added into 96 orifice plates5Cell/mL density HeLa cell, MCF-
7 cells and after HELF cell culture 24 hours, by 5-Fu, TL-3 and STL-3 of various concentration (100 μ g/mL, 200 μ g/mL,
300 μ g/mL, 400 μ g/mL, 500 μ g/mL, 600 μ g/mL, 700 μ g/mL;100 μ L) be added separately to each culture medium, 37 DEG C
CO2It is incubated for 24 hours in incubator.MTT solution (5mg/mL, 20 μ L) is added in each hole, is incubated 4 hours again.Remove training
Support the DMSO standing 10min that 100 μ L are added in base.Absorbance is detected with microtiter plate reader at 570nm.Tumour cell
The inhibiting rate of HeLa and MCF-7 and the survival rate formula of normal cell HELF are as follows:
Wherein A and B is the mean light absorbency of negative control and test sample respectively.
Test result is as shown in Figure 7 and Figure 8, it can be seen from figure 7 that the concentration with sample increases, the inhibition of sample
Ability is all in dose dependent.To the inhibition growth ability aspect of HeLa cell, polysaccharide STL-3 significantly has than TL-3 stronger
Rejection ability.In 200 μ g/mL, STL-3 is 4 times high to the inhibiting rate ratio TL-3's of HeLa cell.In addition, STL-3 is also shown
Have apparent cytotoxicity to MCF-7 cell, 400 μ g/mL, STL-3 inhibiting rate almost 5 times more than TL-3.STL-3 pairs
The IC of HeLa cell50Value is 216.7 μ g/mL, to the IC of MCF-7 cell50Value is 233.9 μ g/mL.By with other edible mushrooms
Polysaccharide is compared, and STL-3 has fairly good advantage in terms of having tumor cytotoxic activity.As can be seen from Figure 8, with polysaccharide culture
Cell still maintains very high survival rate after 24 hours, both TL-3 and STL-3 was without toxic side effect.The cell for reviewing 5-Fu is deposited
Vigor gradually decreases, and in the concentration of 10 μ g/mL, 5-Fu is only 50% or so to the survival rate of HELF cell.In 400 μ g/mL,
Influence of the 5-Fu to cell viability is 5 times of STL-3 or more.