CN104116023A - Application of gastrodia tuber polysaccharide in preparation of functional food with effect of helping inhibit colon cancer - Google Patents
Application of gastrodia tuber polysaccharide in preparation of functional food with effect of helping inhibit colon cancer Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses application of gastrodia tuber polysaccharide in preparation of functional food with the effect of helping inhibit colon cancer. The gastrodia tuber polysaccharide is preferably prepared by the following method: extracting gastrodia tuber taken as a raw material with boiling water, removing protein, dialyzing and carrying out ethanol treatment. The gastrodia tuber polysaccharide has the function of adjusting the tumor microenvironment. The gastrodia tuber polysaccharide can adjust the tumor microenvironment through multiple ways such as inhabitation of tumor angiogenesis, activation of immunity and inhabitation of fibroblast growth of the colon cancer, so that the gastrodia tuber polysaccharide has a good effect of helping inhibit colon tumors.
Description
Technical field
The invention belongs to polysaccharide technical field, be specifically related to a kind of gastrodia elata polysaccharide and there is the application in the auxiliary functional food that suppresses colon cancer effect in preparation.
Background technology
Immunity negative regulation research provides New Policy for oncotherapy.In tumor microenvironment, exist a large amount of negative regulation cells, they interact, and influence each other, and are accompanied by the coevolution of tumour cell and microenvironment.Because the immunogenicity of tumour cell changes and improper growth, stimulate fibroblast to discharge inflammatory factor, antigen presenting cell transmits tumour antigen signal, the common immunocyte of recruiting infiltrates tumor by local, and the abnormality proliferation of tumour cell causes the compressing to adjacent cells, cause the tissue damages such as ischemic necrosis, produce a large amount of inflammatory factors, therefore tumor by local is in non-controllability inflammatory conditions, cause the formation of microenvironment immunity negative regulation, the formation of induced tumor associated fibroblast cell (CAF) and marrow sample SC (MDSC), IL10, the beta induced macrophage of TGF-is changed into the M2 type of inhibitive ability of immunity by anti-personnel M1 type, regulatory T cells significantly increases at tumor by local.Consequently suppress the activity of cytotoxic T cell (CTL), NK (NK), maintain the immune tolerance state of tumor by local.Taking the immune negative regulation cell in tumor microenvironment as target spot, suppress its growth, induce its differentiation, thereby the immune negative regulation state in releasing tumor microenvironment, just can maintain tumour cell and come back to resting state in the tumour cell of resting state or reverse state of activation, just can reach the tumorigenic object of prevention.
Research discovery in recent years, polysaccharide just has antitumor activity and relatively little to the toxic and side effect of human body significantly, the bioactivity research of polysaccharide has been become to the focus of domestic and international experts and scholars' research at medical domain.In recent years find that some polysaccharide and compound thereof are to various diseases, as immunologic derangement, cancer, diabetes, hypertension, hepatitis, thrombus, pneumonia, virus etc. all have significant curative effect, and the adjusting of the various biological phenomenas of participation cell.
Rhizoma Gastrodiae is herbaceos perennial, is distributed in national most areas.Its dry tuber also claims rhizoma Gastrodiae, is to commonly use and famousr and precious Chinese medicine simply, the disease such as clinical dizzy, the extremity numbness of being used for having a headache, child convulsion, epilepsy, tic, lockjaw.There is no at present taking rhizoma Gastrodiae as raw material and prepare polysaccharide component, exploitation has auxiliary colon cancer effect application that suppresses.
Summary of the invention
The object of the present invention is to provide gastrodia elata polysaccharide to there is the application in the auxiliary functional food that suppresses colon cancer effect in preparation.
Wherein said auxiliary inhibition colon cancer effect refers to by suppressing colon cancer tumor angiogenesis, immune activation and suppressing colon cancer fibroblastic growth effect, regulates colon cancer tumor microenvironment, thereby plays the auxiliary colon cancer effect that suppresses.
As one embodiment of the present invention, gastrodia elata polysaccharide in the present invention preferably prepares by the following method: taking rhizoma Gastrodiae as raw material, through preparing containing extracting in boiling water, deproteinized, dialysis and Ethanol Treatment operation, described gastrodia elata polysaccharide has the effect that regulates tumor microenvironment.
As one of the present invention preferred embodiment, gastrodia elata polysaccharide of the present invention specifically prepares by the following method: taking rhizoma Gastrodiae as raw material, after repeatedly extracting with boiling water, merge extract, extract is concentrated to obtain to concentrate, in concentrate, add trichloroacetic acid solution to carry out deproteinized processing, after deproteinized, reactant liquor is neutralized, is pricked a bag dialysis treatment, by reactant liquor in bag filter concentrate, centrifugal treating, get supernatant and add ethanol to carry out alcohol precipitation, dry processing, obtain gastrodia elata polysaccharide.
The present invention is in the preparation process of gastrodia elata polysaccharide, and the design parameter of each operation is preferably as follows:
When extracting in boiling water, extraction time is 1 ~ 5 time, and while extraction, the consumption of boiling water is 5 ~ 20 times of rhizoma Gastrodiae gross mass at every turn, and each extraction time is 1.5 ~ 5 h.
When extracting in boiling water, the consumption of boiling water is preferably 5 ~ 20 times of rhizoma Gastrodiae gross mass, and extraction time is preferably 3 times, and while wherein extraction for the first time, the consumption of boiling water is preferably 10 ~ 20 times of raw material rhizoma Gastrodiae gross mass, extracts 2 ~ 5 h; While extraction for the second time, the consumption of boiling water is preferably 10 ~ 20 times of raw material rhizoma Gastrodiae gross mass, extracts 1.5 ~ 5h; While extraction for the third time, the consumption of boiling water is preferably 10 ~ 20 times of raw material rhizoma Gastrodiae gross mass, extracts 1.5 ~ 5 h.
Extract is preferably concentrated into approximately 1/10 of former extract cumulative volume and obtains concentrate.
The quality percentage composition of trichloroacetic acid solution of the present invention is preferably 35%, and the deproteinized processing time is preferably 3 ~ 6 h, and the volume of trichloroacetic acid solution is preferably identical with the volume of concentrate.
When neutralisation treatment, can adopt quality percentage composition is 10% sodium hydrate aqueous solution.
While pricking bag dialysis treatment, preferably adopt the bag filter of molecular cut off 3500Da to carry out dialysis treatment.
Ethanol carries out when alcohol precipitation is processed preferably adopting volumn concentration to be preferably 70 ~ 95% ethanol water.
Concentrate and can adopt multiple concentrated mode of the prior art, wherein with Vacuum Concentration the best.When Vacuum Concentration, temperature is preferably 40 DEG C.
Gastrodia elata polysaccharide in the present invention, can be directly oral, also can make said multiple formulation in pharmacy, as capsule-type, tablet, pulvis, granule or oral liquid etc., has auxiliary effect that suppresses colon cancer tumour after eating.
Therefore, the formulation of functional food of the present invention is preferably capsule-type, tablet, pulvis, granule or oral liquid etc.
Compared with prior art, tool of the present invention has the following advantages:
(1) gastrodia elata polysaccharide in the present invention, its purity and active high, can be by suppressing tumor angiogenesis, immune activation and suppress the multipaths such as colon cancer fibroblastic growth to regulate tumor microenvironment, it has good auxiliary inhibition colon cancer function of tumor;
(2) preparation method of the gastrodia elata polysaccharide in the present invention, technique simple and stable is efficient, is applicable to suitability for industrialized production, and cost is low;
(3) gastrodia elata polysaccharide in the present invention, can be for the preparation of having the auxiliary functional food that suppresses colon cancer tumor function.
Brief description of the drawings
Fig. 1 is the activity that in the embodiment of the present invention 4, gastrodia elata polysaccharide can suppress the luciferase with BRE reporter gene of BMP2 induction;
Fig. 2 is that in the embodiment of the present invention 5, gastrodia elata polysaccharide can activate the activity with the luciferase of NF-к B reporter gene;
Fig. 3 is that in the embodiment of the present invention 6, gastrodia elata polysaccharide can suppress the fibroblastic growth of colon cancer.
Detailed description of the invention
Below in conjunction with drawings and Examples, the present invention will be further described, but do not limit in any form the present invention.
The each raw material adopting in following examples, if no special instructions, is commercially available prod.
embodiment 1
Gastrodia elata polysaccharide in the present embodiment, prepares by the following method: get rhizoma Gastrodiae raw material, adopt extracting in boiling water, while extraction for the first time, the consumption of water is 20 times of raw material gross mass, boils 5 h and filters; While extraction for the second time, the consumption of water is 20 times of raw material gross mass, boils 3 h and filters; While extraction for the third time, the consumption of water is 10 times of raw material gross mass, boil 2 h and filter, merging filtrate, Vacuum Concentration is about 1/10 to original volume, lower with.Concentrate is carried out to trichloroacetic acid except albumen processing, add 30% trichloroacetic acid (mass percent) of same volume (lower same), after processing time is 4 h, by 10% NaOH(mass percent) neutralization, lower same, after neutralization, reactant liquor is placed in to bag filter and pricks bag dialysis, when dialysis, the molecular cut off of bag filter is about 3500Da conventionally, lower same, then liquid in bag filter is concentrated, centrifugal, getting supernatant adds 85% ethanol water (volumn concentration) of 3 ~ 4 times of volumes to carry out alcohol precipitation process, taking precipitate is through containing (approximately 40 DEG C of vacuum drying, process to obtain gastrodia elata polysaccharide component down together).
embodiment 2
Gastrodia elata polysaccharide in the present embodiment, prepares by the following method: get rhizoma Gastrodiae raw material, adopt extracting in boiling water, while extraction for the first time, the consumption of water is 15 times of rhizoma Gastrodiae raw material gross mass, boils 5 h and filters; While extraction for the second time, the consumption of water is 15 times of rhizoma Gastrodiae raw material gross mass, boils 3 h and filters; While extraction for the third time, the consumption of water is 10 times of rhizoma Gastrodiae raw material gross mass, boils 3 h and filters, merging filtrate, Vacuum Concentration.Concentrate is carried out to trichloroacetic acid except albumen processing, add 25% trichloroacetic acid (mass percent) of same volume, after processing time is 3 h, by 10% NaOH(mass percent) neutralization, after neutralization, reactant liquor is placed in to bag filter and pricks bag dialysis, then liquid in bag filter is concentrated, centrifugal, get supernatant and add 70% ethanol water (volumn concentration) of 3 ~ 4 times of volumes to carry out alcohol precipitation process, taking precipitate is through obtaining gastrodia elata polysaccharide component containing vacuum drying treatment.
embodiment 3
Gastrodia elata polysaccharide in the present embodiment, prepares by the following method: get rhizoma Gastrodiae raw material, adopt extracting in boiling water, while extraction for the first time, the consumption of water is 10 times of rhizoma Gastrodiae raw material gross mass, boils 3 h and filters, while extraction for the second time, the consumption of water is 20 times of rhizoma Gastrodiae raw material gross mass, boils 3 filtration h, while extraction for the third time, the consumption of water is 10 times of rhizoma Gastrodiae raw material gross mass, boiling 5 h filters, merging filtrate, Vacuum Concentration, concentrate is carried out to trichloroacetic acid except albumen processing, add 15% trichloroacetic acid (mass percent) of same volume, after processing time is 5 h, by 10% NaOH(mass percent) neutralization, after neutralization, reactant liquor is placed in to bag filter and pricks bag dialysis, then liquid in bag filter is concentrated, centrifugal, getting supernatant adds 95% ethanol water (volumn concentration) of 3 ~ 4 times of volumes to carry out alcohol precipitation process, taking precipitate is through obtaining gastrodia elata polysaccharide component containing vacuum drying treatment.
Make said multiple formulation in pharmacy, as capsule, tablet, pulvis, particle or oral liquid etc.
embodiment 4 gastrodia elata polysaccharides can suppress the activity of the luciferase with BRE reporter gene of BMP2 induction
C2C12 cell line is purchased from Chinese Academy of Sciences's cell bank, and C2C12-pGF1-BRE cell line is built by Shanghai Pharmaceutical Inst., Chinese Academy of Sciences's fourth laboratory of chatting about, and is stored in liquid nitrogen.C2C12-pGF1-BRE cell, with containing 10 % Gibco hyclone DMEM medium culture, is placed in 37 DEG C of saturated humidities, contains 5% CO
2insulating box in cultivate, with 1 × 10
4the density of cells/well is inoculated in 96 orifice plates, every hole 100 μ L.After adherent 24 h, suck 60 μ L culture mediums, add sample (the gastrodia elata polysaccharide sample in embodiment 1) the 50 μ L that configure respective concentration, final concentration is respectively 0.5 mg/mL, 0.1 mg/mL, 0.02 mg/mL, and 10 μ LBMP2 are adjusted to final concentration 200 ng/mL.Establish blank group and the contrast of BMP2 group simultaneously.After administration 16 h, suck culture medium, add 20 μ LReporter Lysis 1X Buffer, the cell pyrolysis liquid of dissolving 20 μ L are transferred to blank, add 40 μ L luciferase substrates, in 3 minutes, read plate, read RLU value.Adopt SPSS statistical software to carry out statistical procedures.Data all with mean ± standard deviation (.
x±
s) represent, between two groups, relatively adopt independent sample
tinspection, relatively adopts one-way analysis of variance between many groups.
Experimental result as shown in Figure 1, gastrodia elata polysaccharide has the activity of the luciferase with BRE reporter gene that suppresses BMP2 induction under 0.02 mg/mL low concentration, and this activity increases and significantly increases with polysaccharide concentration, shows that this polysaccharide has dose dependent aspect this activity.Known cancer new vessels is created in the generation evolution of tumour and plays key effect, and BMP2 signal path plays a crucial role in tumor angiogenesis process.This polysaccharide can significantly suppress BMP2 signal path, shows that this polysaccharide has potential inhibition tumor promotion.
embodiment 5 gastrodia elata polysaccharides can activate the activity with the luciferase of NF-к B reporter gene
THP-1 cell line is purchased from Chinese Academy of Sciences's cell bank, and THP-1/pGF1-NF-к B cell line is built by Shanghai Pharmaceutical Inst., Chinese Academy of Sciences's fourth laboratory of chatting about, and is stored in liquid nitrogen.THP-1/pGF1-NF-к B cell is with containing 10% hyclone RPMI-1640 medium culture.Be placed in 37 DEG C of saturated humidities, contain 5% CO
2insulating box in cultivate, with 5 × 10
4the density of cells/well is inoculated in 96 orifice plates, every hole 50 μ L volumes.Add given the test agent 50 μ L (the gastrodia elata polysaccharide sample in embodiment 1), final concentration is respectively 0.5 mg/mL, 0.1 mg/mL, 0.02 mg/mL, sets up blank group and LPS control group simultaneously.Add LPS solution 10 μ L, final concentration is 1 μ g/mL, is placed in 37 DEG C of saturated humidities, contains 5% CO
2insulating box in overnight incubation.After overnight incubation, every sky adds 100 μ L Bright-Glo
tMluciferase Assay System substrate, reads plate in ELIASA, reads RLU value.
Adopt SPSS statistical software to carry out statistical procedures.Data all with mean ± standard deviation (.
x±
s) represent, between two groups, relatively adopt independent sample
tinspection, relatively adopts one-way analysis of variance between many groups.
As shown in Figure 2, this polysaccharide has the activity of good activation NF-к B signal path to experimental result under 0.5 mg/mL low concentration.NF-к B can strengthen the immunity of body after activating, indirectly bring into play antitumor action.
embodiment 6 gastrodia elata polysaccharides can suppress the growth of colon cancer CT26-CAF
Colon cancer associated fibroblast clone CT26-CAF is built by Institute of Biophysics, Academia Sinica.Cell is inoculated in to 96 orifice plates, and cell density is 1 × 10
4/ hole.Gastrodia elata polysaccharide in embodiment 1 is dissolved in DMEM+ /+culture medium, and making its concentration is 1.0 mg/mL.Cultivate 4 days, adopt CCK detection kit (Beijing Zhuan Meng biotech firm) to measure cell growth state.Every kind of polysaccharide sample size is 3.
Statistical analysis: relatively adopt variance analysis between many groups of continuous datas, first data are carried out to homogeneity test of variance, calculate Levene statistical value, if variance is neat, between organizing,, method is LSD and Dunnett to multiple ratio, if heterogeneity of variance, adopts bearing calibration Dunnett t.Statistical software is SPSS17.0, and significance is α=0.05.
As shown in Figure 3, this gastrodia elata polysaccharide can significantly suppress the growth of colon cancer CT26-CAF to experimental result under the concentration of 0.5 mg/mL.CT26-CAF plays important regulating and controlling effect in colon cancer, can promote the growth of colon cancer cell.This polysaccharide can, by suppressing the growth of colon cancer CT26-CAF, play the effect that regulates colon cancer microenvironment.
To sum up experimental results show that this gastrodia elata polysaccharide can regulate tumor microenvironment by suppressing the multipaths such as tumor angiogenesis, immune activation and immune negative regulator, and further bring into play potential inhibitor against colon carcinoma cells tumor promotion, thereby gastrodia elata polysaccharide can be passed through to common process, make said multiple formulation in pharmacy, for the preparation of having the auxiliary functional food that suppresses colon cancer effect, the formulation of functional food can be capsule-type, tablet, pulvis, granule or oral liquid etc.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, be included in protection scope of the present invention.
Claims (9)
1. gastrodia elata polysaccharide has the application in the auxiliary functional food that suppresses colon cancer effect in preparation.
2. gastrodia elata polysaccharide according to claim 1 has the application in the auxiliary functional food that suppresses colon cancer effect in preparation, it is characterized in that: described auxiliary inhibition colon cancer effect refers to by suppressing colon cancer tumor angiogenesis, immune activation and suppressing colon cancer fibroblastic growth effect, regulate colon cancer tumor microenvironment, thereby play the auxiliary colon cancer effect that suppresses.
3. gastrodia elata polysaccharide according to claim 1 has the application in the auxiliary functional food that suppresses colon cancer effect in preparation, it is characterized in that: described gastrodia elata polysaccharide prepares by the following method: taking rhizoma Gastrodiae as raw material, through preparing containing extracting in boiling water, deproteinized, dialysis and Ethanol Treatment operation, described gastrodia elata polysaccharide has the effect that regulates tumor microenvironment.
4. gastrodia elata polysaccharide according to claim 3 has the application in the auxiliary functional food that suppresses colon cancer effect in preparation, it is characterized in that: described gastrodia elata polysaccharide specifically prepares by the following method: taking rhizoma Gastrodiae as raw material, after repeatedly extracting with boiling water, merge extract, extract is concentrated to obtain to concentrate, in concentrate, add trichloroacetic acid solution to carry out deproteinized processing, after deproteinized, reactant liquor is neutralized, prick bag dialysis treatment, reactant liquor in bag filter is concentrated, centrifugal treating, getting supernatant adds ethanol to carry out alcohol precipitation, dry processing, obtain gastrodia elata polysaccharide.
5. gastrodia elata polysaccharide according to claim 4 has the application in the auxiliary functional food that suppresses colon cancer effect in preparation, it is characterized in that: when extracting in boiling water, extraction time is 1 ~ 5 time, when each extraction, the consumption of boiling water is 5 ~ 20 times of rhizoma Gastrodiae gross mass, and each extraction time is 1.5 ~ 5 h.
6. gastrodia elata polysaccharide according to claim 4 has the application in the auxiliary functional food that suppresses colon cancer effect in preparation, it is characterized in that: when deproteinized, adopt trichloroacetic acid solution, the quality percentage composition of described trichloroacetic acid solution is 35%, and the processing time is 3 ~ 6 h.
7. gastrodia elata polysaccharide according to claim 4 has the application in the auxiliary functional food that suppresses colon cancer effect in preparation, it is characterized in that: when Ethanol Treatment, adopting volumn concentration is 70 ~ 95% ethanol water.
8. gastrodia elata polysaccharide according to claim 4 has the application in the auxiliary functional food that suppresses colon cancer effect in preparation, it is characterized in that: when dialysis, the molecular cut off of bag filter is 3500Da.
9. gastrodia elata polysaccharide according to claim 1 has the application in the auxiliary functional food that suppresses colon cancer effect in preparation, it is characterized in that: the formulation of described functional food is capsule-type, tablet, pulvis, granule or oral liquid.
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