CN101724089B - Acidic aloe polysaccharide as well as preparation and purification method and application thereof - Google Patents
Acidic aloe polysaccharide as well as preparation and purification method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines Containing Plant Substances (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention belongs to the technical field of biological pharmacy, in particular to a preparation and purification method of acidic aloe polysaccharide and application thereof in a helicobacter pylori and escherichia coli resistant medicament. A test proves that the method for obtaining acidic aloe polysaccharide by adopting Chinese aloe as a raw material, extracting by hot water, obtaining a rough extract by an ethanol precipitation method, dissolving by hot water, removing protein by trichloroacetic acid, obtaining rough polysaccharide after ethanol precipitation and purifying by a DEAE-52 ion-exchange column chromatography has the characteristics of high product purity, higher yield, simple equipment requirement and more favorable repeatability (shown as a summary attached figure). The acidic aloe polysaccharide can effectively reduce the sticking rate of helicobacter pylori on an MKN 45 cell. In addition, the acidic aloe polysaccharide also has the obvious inhibiting function onsticking an LOVO cell by escherichia coli. The acidic aloe polysaccharide is a broad-spectrum sticking resistant agent and can be used for preparing a helicobacter pylori sticking resistant medicament and applied to treating relevant diseases caused by the infection of the helicobacter pylori.
Description
One, technical field
The invention belongs to biological pharmacy technical field, be specifically related to the preparation, purifying of acidic aloe polysaccharide and stick application in the relative disease that medicine and treatment Hp cause at the anti-Hp of preparation.
Two, background technology
Aloe is the widespread use with regard to being used as a kind of medicine food dual purpose plant since ancient times.Modern pharmacological research shows that aloe has sterilization, anti-inflammatory, rushes down following, anti-oxidant, radioprotective, antiulcer agent and effect such as antitumor.According to domestic and international research, the contained activeconstituents of aloe has two big classes, one class is to be present in aloe leaf bottom and near the anthraquinones such as aloin of epidermis, another kind of is isolated Aloe gel from the Fresh leaf of aloe parenchyma tracheid, wherein about 98.5% is natural moisture, and carbohydrate---mucopolysaccharide then accounts for more than 60% of total solids.As the Aloe polysaccharide of one of main effective constituent of Aloe gel, its pharmaceutical research is also active gradually.Multiple pharmacological effect such as that studies show that in recent years, Aloe polysaccharide have is anti-ageing, radioprotective, anti-gastric-ulcer, antitumor, immunomodulatory.Therefore, Aloe polysaccharide has potential prevention or therapeutic action for these relative diseases.But have not yet to see its research report aspect bacterial adhesions such as disinfecting helicobacter pylori.
Helicobacter pylori (Helicobacter pylori) is the bacterium of a kind of Gram-negative, S shape or arc-shaped bend.Be closely connected between many evidences demonstration gastroduodenal diseases and the helicobacter pylori, can bring out human chronic gastritis, peptide ulceration, stomach mucous membrane dependency lymphoid tissue lymphoma, intestines cancer of the stomach.The World Health Organization has classified helicobacter pylori as I class carcinogens, and it is the main incitant of cancer of the stomach.The treatment plan of Hp mainly is to adopt triple therapy at present, but along with antibiotic widespread use, the incidence of Resistant strain increases to cause treating gradually fails.
Studies have shown that bacterium is the committed step that causes a series of pathological changes of later stage and cause relative disease to sticking of host cell.Anti-binder this committed step capable of blocking, thereby the disease that prevention and treatment infectation of bacteria cause.Compare with traditional sterilant, the not direct killing bacteria of anti-binder has so just effectively prevented the generation of Resistant strain.The process of sticking of acidic aloe polysaccharide among the present invention blocking-up Hp and host cell is compared with traditional antibacterial therapy and to be had the advantages that being difficult for producing Resistant strain, is a kind of treatment or the new tool of preventing the relative disease that Hp causes.Simultaneously, it also can suppress colibacillary sticking, and demonstrates the anti-effect of sticking of certain wide spectrum.
Three, summary of the invention
The problem that the present invention need solve:
The present invention endeavours to solve the preparation, purifying of acidic aloe polysaccharide and sticks application in the relative disease that medicine and treatment Helicobacter pylori infection cause at the anti-Hp of preparation.
Technical scheme of the present invention:
(1) acidic aloe polysaccharide of the present invention, be to adopt water extraction and alcohol precipitation method from the Chinese aloe aloe, to extract and prepare through the method for DEAE-52 anionite-exchange resin purifying, its molecular weight is about 29.39KDa, the monose composition mainly contains 37% semi-lactosi, 15.3% pectinose, 9% rhamnosyl, 7.1% seminose, 3.7% glucose and 1.4% wood sugar, and contains 25.6% uronic acid;
(2) to prepare purification process be to be raw material with the Chinese aloe aloe to acidic aloe polysaccharide, adopts hot water extraction, and the method for ethanol sedimentation obtains crude extract, and by the hot water dissolving, trichoroacetic acid(TCA) removes albumen, obtains Crude polysaccharides behind the ethanol sedimentation.Obtain acidic aloe polysaccharide through DEAE-52 ion-exchange chromatography purifying again;
(3) acidic aloe polysaccharide among the present invention can significantly reduce the adherence rate of Hp to MKN 45 cells, and it is relevant to be certain dosage, can stick in the relative disease that medicine and treatment Helicobacter pylori infection cause at the anti-Hp of exploitation to use.
(4) acidic aloe polysaccharide among the present invention can also significantly reduce the adherence rate of intestinal bacteria to the LOVO cell, shows that the polysaccharide among the present invention has the anti-effect of sticking of wide spectrum, can be in preparation at using anti-the sticking in the medicine of other bacteriums.
The present invention compared with prior art, its beneficial effect is:
(1) the present invention is raw material with the Chinese aloe aloe, adopts hot water extraction, and the method for ethanol sedimentation obtains crude extract, and by the hot water dissolving, trichoroacetic acid(TCA) removes albumen, obtains Crude polysaccharides behind the ethanol sedimentation.Obtain acidic aloe polysaccharide through DEAE-52 ion-exchange chromatography purifying again, this method has that product purity height, productive rate are big, equipment requirements is simple, circulation ratio characteristics preferably, for the further application of acidic aloe polysaccharide provides the foundation and foundation;
(2) the present invention is a research object with the acidic aloe polysaccharide of preparation, integrated use pharmacology, biological method, disclosed acidic aloe polysaccharide and can significantly reduce the adherence rate of Hp MKN 45 cells, and it is relevant to be certain dosage, can stick in the medicine the anti-Hp of exploitation and intestinal bacteria to use.And can become a kind of potential antibacterium and stick medicine.
(3) in addition, the acidic aloe polysaccharide among the present invention can also significantly reduce the adherence rate of intestinal bacteria to the LOVO cell, shows that the polysaccharide among the present invention has the anti-effect of sticking of wide spectrum.And can become the anti-medicine that sticks of a kind of potential broad spectrum.
Four, description of drawings
Fig. 1: acidic aloe polysaccharide extraction, separation and purifying schema.
Fig. 2: acidic aloe polysaccharide sticks MKN45 cell inhibiting effect to Hp.Control group is handled without acidic aloe polysaccharide, and its Hp adherence rate is as 100%.Other groups are compared with control group respectively, obtain relative adherence rate (%).Data are compared with control group through the T check, and p＜0.05 is considered to exist significant difference.Wherein,
*P＜0.05,
*P＜0.01,
* *P＜0.001.
Fig. 3: acidic aloe polysaccharide sticks LOVO cell inhibiting effect to intestinal bacteria.Control group is handled without acidic aloe polysaccharide, and its intestinal bacteria adherence rate is as 100%.Other groups are compared with control group respectively, obtain relative adherence rate (%).Data are compared with control group through the T check, and p＜0.05 is considered to exist significant difference.Wherein,
* *P＜0.001.
Five, embodiment
Further describe the present invention by following examples, but should notice that scope of the present invention is not subjected to any restriction of these embodiment.
Material and source:
Reagent: ethanol (Nanjing chemical reagents corporation); Phenol (Tianjin Chemical Reagents Factory No.1); Trichoroacetic acid(TCA) (Shanghai Ling Feng chemical reagent company limited); Sulfuric acid (Nanjing Chemistry Reagent Co., Ltd.); FITC (SIGMA company); Sodium-chlor (Nanjing chemical reagents corporation); 1640 substratum (Gibco company); The Columbia substratum (biom é rieux, France); Agar powder (Shanghai medication chemistry group); Yeast extract (Nanjing great order Bioisystech Co., Ltd).
Material: Hp (U.S.'s bacterial classification is preserved the center); Intestinal bacteria (functional biological molecular studies institute of Nanjing University).
Equipment: UV-2201 ultraviolet-visible pectrophotometer (Japanese Shimadzu company); High speed freezing centrifuge (Japanese Hitachi company); Rotatory evaporator (Shanghai Yarong Biochemical Instrument Plant); BSZ-100 auto partial sampler (Shanghai Hu Xi analytical instrument Co., Ltd., Factory); Microplate reader (TECAN company).
The preparation and the purifying of embodiment 1 acidic aloe polysaccharide
(1) after the chopping of fresh aloe leaf,, obtains crude extract behind the ethanol sedimentation through hot water extraction.Take by weighing an amount of crude extract, with 50～70% alcohol immersion twice, each 24～48h, after residue volatilized solvent, stirring was dissolved in an amount of distilled water, the centrifugal insoluble substance of removing.Add trichoroacetic acid(TCA) in the supernatant liquor and remove albumen, centrifuging and taking supernatant after the standing over night repeats aforesaid operations.Centrifugal after the adding 95% ethanol standing over night behind the supernatant concentrating under reduced pressure, get white or light yellow blocks of solid after the precipitation oven dry, be aloe coarse polysaccharid;
(2) aloe coarse polysaccharid obtains acidic aloe polysaccharide through the ion-exchange chromatography purifying.Adopt DEAE-52 anionite-exchange resin, at first use the distilled water wash-out, take 0.1～0.4M NaCl wash-out afterwards, collect elutriant, the phenol sulfuric acid process is measured polysaccharide content in every pipe elutriant, merges elutriant, concentrates, dialysis, freeze-drying promptly get refining acidic aloe polysaccharide of the present invention.This method favorable reproducibility, productive rate are stablized, and can be the follow-up test sampling.Schema is seen accompanying drawing 1.
Embodiment 2 acidic aloe polysaccharides stick MKN 45 cell inhibiting effects to Hp
(1) the Hp method of scoring is seeded on Colombia's nutrient agar and (contains 7% fresh sheep blood), cultivates 3 days down in 37 ℃ of little aerobic environments.Scrape afterwards and get the physiological saline that bacterium colony places sterilization, transferring bacterium liquid is proper concn.In the bacterium liquid that modulates concentration, add FITC-DMSO solution, incubated at room 1～3h.Then that mark is good bacteria suspension is centrifugal, pours out supernatant, adds the D-Hanks solution contain tween 20 and blows and beats repeatedly for several times, continues centrifugally, repeats abovely to operate the FITC on flush away is unmarked 2 times.
(2) the MKN45 cell original fluid that will cultivate in 96 orifice plates discards, and changes the RPMI-1640 that contains 2% calf serum and different concns acidic aloe polysaccharide.One group in per 12 holes, the content of acidic aloe polysaccharide are respectively 0,0.01,0.1,0.5,1.0mg/ml, incubate 1h in advance in 37 ℃.Afterwards, jiggle mixing at the bacterium liquid that every hole adds the mark of 50 μ l in the hole of correspondence under the lucifuge condition, 37 ℃ of lucifuges are hatched 1h.Then, discard the nutrient solution that contains bacterium and medicine, flush away is the adherent bacterium not, measures the fluorescence volume in every hole in fluorescence microplate reader, and wherein excitation wavelength is 485nm, and emission wavelength is 528nm.Experimental result is seen Fig. 2.
Fig. 2 result shows, compare with control group, acidic aloe polysaccharide concentration be 0.1,0.5 and during 1.0mg/ml Hp the adherence rate of MKN45 cell is reduced to 88% (p＜0.05) respectively, 76% (p＜0.01) and 64% (p＜0.001).
Embodiment 3 acidic aloe polysaccharides stick the effect of LOVO cell to intestinal bacteria
The LOVO cell is planted 96 orifice plates and is cultivated with after containing 1640 culture medium culturing 48h of 10% calf serum.Intestinal bacteria are scraped after with LB culture medium culturing 24h and get bacterium colony, use the FITC mark according to the method described above.The LOVO cell original fluid that to cultivate in 96 orifice plates behind the 24h discards, and one group in per 12 holes add respectively that acidic aloe polysaccharide content is respectively 0,0.01,0.1,0.5,1.0mg/ml and contain 1640 substratum of 2% calf serum, hatch 1h.After the intestinal bacteria that add the FITC mark afterwards hatched 1h, flush away is the adherent bacterium not, and microplate reader is measured fluorescent value.The results are shown in Figure 3.
Fig. 3 result shows, compare with control group, acidic aloe polysaccharide concentration be 0.5 and during 1.0mg/ml intestinal bacteria the adherence rate of LOVO cell is reduced to 78% (p＜0.001) and 68% (p＜0.001) respectively.
Claims (4)
1. acidic aloe polysaccharide, the acidic aloe polysaccharide that it is characterized in that adopting water extraction and alcohol precipitation method from the Chinese aloe aloe, to extract and prepare through the method for DEAE-52 anionite-exchange resin purifying, molecular weight is 29.39KDa, the monose composition mainly contains 37% semi-lactosi, 15.3% pectinose, 9% rhamnosyl, 7.1% seminose, 3.7% glucose and 1.4% wood sugar, and contains 25.6% uronic acid.
2. according to the preparation purification process of the described acidic aloe polysaccharide of claim 1, it is characterized in that constituting by following steps:
(1) after the chopping of fresh aloe leaf, through hot water extraction, obtain crude extract behind the ethanol sedimentation, the Aloe polysaccharide crude extract, with 50～70% alcohol immersion 2 times, each 24～48h, after residue volatilizes solvent, be dissolved in the distilled water the centrifugal insoluble substance of removing, add trichoroacetic acid(TCA) in the supernatant liquor and remove albumen, centrifuging and taking supernatant liquor after the standing over night, and repeat aforesaid operations, centrifugal after the adding 95% ethanol standing over night behind the supernatant liquor concentrating under reduced pressure, get white or light yellow blocks of solid after the precipitation oven dry, be aloe coarse polysaccharid;
(2) aloe coarse polysaccharid obtains acidic aloe polysaccharide through the ion-exchange chromatography purifying, adopt DEAE-52 anionite-exchange resin, at first use the distilled water wash-out, take 0.1～0.4M NaCl eluant solution afterwards, after collecting elutriant, the phenol sulfuric acid process is measured polysaccharide content in every pipe elutriant, merges elutriant, concentrates, dialysis, freeze-drying promptly get refining acidic aloe polysaccharide of the present invention.
3. acidic aloe polysaccharide according to claim 1 sticks application in medicine and the relative disease medicine that causes of treatment Helicobacter pylori infection at the anti-Hp of preparation.
4. acidic aloe polysaccharide according to claim 1 is in the anti-application of sticking in medicine of preparation at intestinal bacteria and other Gram-negative bacterias.
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| CN2009101835290A CN101724089B (en) | 2009-09-23 | 2009-09-23 | Acidic aloe polysaccharide as well as preparation and purification method and application thereof |
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| MX2016013475A (en) * | 2014-04-17 | 2017-01-23 | Unilever Nv | Aloe vera extract for personal care compositions. |
| CN105535925A (en) * | 2016-01-13 | 2016-05-04 | 安徽生物肽产业研究院有限公司 | Components and preparation method of leech biological activity small peptide medicine composition |
| CN108619273B (en) * | 2018-06-11 | 2021-05-28 | 天津允新科技有限公司 | Preparation method of plant fruit core oral liquid for treating helicobacter pylori |
| EP4232056A1 (en) * | 2020-10-21 | 2023-08-30 | 2QR Research BV | Aloe extracts for microbial neutralisation |
| CN112852704B (en) * | 2021-02-05 | 2023-04-04 | 安徽大学 | Application of black skin termitomyces albuminosus polysaccharide ORP-1 in proliferation and adhesion of intestinal bacteria |
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