CN106632710A - Radix codonopsis homogeneous polysaccharide with anti-gastric ulcer effect and preparation method and application thereof - Google Patents
Radix codonopsis homogeneous polysaccharide with anti-gastric ulcer effect and preparation method and application thereof Download PDFInfo
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- CN106632710A CN106632710A CN201610889548.5A CN201610889548A CN106632710A CN 106632710 A CN106632710 A CN 106632710A CN 201610889548 A CN201610889548 A CN 201610889548A CN 106632710 A CN106632710 A CN 106632710A
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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
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0051—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Fructofuranans, e.g. beta-2,6-D-fructofuranan, i.e. levan; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/34—Campanulaceae (Bellflower family)
- A61K36/344—Codonopsis
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Abstract
The invention discloses a radix codonopsis homogeneous polysaccharide with an anti-gastric ulcer effect. The polysaccharide has a molecular weight of 3000, and is a linear homogeneous polysaccharide formed by beta-D-fructofuranose residues through beta-(2->1)-glucosidic bonds. The separation and extraction method comprises the following steps: (1) extracting total radix codonopsis polysaccharides; and (2) performing ultrafiltration separation on the codonopsis polysaccharides. The invention further discloses an application of the radix codonopsis homogeneous polysaccharide in medicines for treating gastric ulcer.
Description
Technical field
The present invention relates to the preparation method of active ingredient of Chinese herbs, the Radix Codonopsis homogeneous polysaccharide specially with anti-ulcer effect
Isolation and purification method and its anti-ulcer effect.
Background technology
Radix Codonopsis（Radix Codonopsis）For campanulaceae of the genus codonopsis, there is tonifying middle-Jiao and Qi, strengthening spleen and tonifying lung,
It is clinically used for spleen and lung weakness, shortness of breath palpitations, anorexia and loose stool, virtual asthma cough, interior heat disappears and the disease such as coughs.Radix Codonopsis mainly contains polysaccharide, soap
The compositions such as glycosides, triterpene, alkaloid, wherein polyoses content are maximum, are one of important component of Radix Codonopsis.Modern pharmacological research shows,
Codonopsis pilosula polysaccharide has preferably biological in many aspects such as anti-aging, regulation immunity of organisms, anti anoxia, resisting stress, anti-oxidant
Activity.
But, the pharmacological research of document report is all directed to Radix Codonopsis total starches mostly, clear and definite to constituting homogeneous, structure at present
The pharmacologically active and its Mechanism Study of Codonopsis pilosula polysaccharide is almost without relevant report.
The content of the invention
The invention mainly solves the technical problem of by being classified by molecular weight to Codonopsis pilosula polysaccharide, then carrying out drug effect
Screening active ingredients are learned, obtains a kind of with the active polysaccharide for constituting homogeneous anti-gastric-ulcer.
The present invention adopts the following technical scheme that realization：
A kind of Radix Codonopsis homogeneous polysaccharide with anti-gastric-ulcer, general structure is as shown in Figure 6.
The polysaccharide molecular weight is 3000, is the straight chain that beta-D-fructofuranose residue is formed by connecting with β-(2 → 1)-glycosidic bond
Homogeneous polysaccharide.
The separating and extracting process of the above-mentioned Radix Codonopsis homogeneous polysaccharide with anti-gastric-ulcer, comprises the steps：
（1）, extract Radix Codonopsis total starches；
According to the patent that early stage is obtained（200710062520.5）, extract Radix Codonopsis total starches.
（2）, Codonopsis pilosula polysaccharide ultra-filtration and separation
By 10～30 times of distilled water diluting of Radix Codonopsis total starches, heating water bath to 20 DEG C～90 DEG C fully dissolvings, in less than 10 DEG C
12～72 hours are stood, 1～3g/mL is concentrated into, with 60%～95% ethanol alcohol precipitation, 4～24 hours is stood, centrifugation, precipitation is dry
It is dry；Repeat aforesaid operations 1-3 time；Radix Codonopsis total starches solution is successively by the filter membrane and 1000-10000 molecules of the molecular weight of 1-10 ten thousand
Amount filter membrane, obtains Radix Codonopsis homogeneous polysaccharide, is denoted as CPS-A.
The Structural Identification of Codonopsis pilosula polysaccharide CPS-A is as follows：
Purity and molecular weight determination are carried out using High Performance Gel Permeation Chromatography to the Codonopsis pilosula polysaccharide CPS-A that the present invention is obtained：
Chromatographic condition：Shodex OHpak SB-804 chromatographic columns, mobile phase is ultra-pure water, flow velocity 0.3mL/min, and detector is
Differential refraction detector；By the appropriate water dissolves of Codonopsis pilosula polysaccharide CPS-A, then sample introduction analysis, analysis result is in single symmetrical peak,
The component is illustrated for the homogeneous polysaccharide of molecular weight（As shown in Figure 1）.
Structure determination is carried out to the Codonopsis pilosula polysaccharide CPS-A that the present invention is obtained：
CPS-A Jing purity differentiates, it was demonstrated that has been single composition, can carry out structural analysis.
CPS-A Jing complete hydrolysis, gas chromatography-mass spectrography analysis, it is known that it is fructose, with1H and13CNMR is further
Analysis.
Above-mentioned analytical proof：CPS-A molecular weight is 3000, is that beta-D-fructofuranose residue is bonded with β-(2 → 1)-glucosides
The straight chain homogeneous polysaccharide for connecing.
Application of the Radix Codonopsis homogeneous polysaccharide in treatment Gastric Ulcer Treatment, following pharmacodynamic experiments confirm that CPS-A has anti-stomach
The pharmacologically active of ulcer.
Codonopsis pilosula polysaccharide CP-A is as follows to the inhibitory action of alcohol induced acute gastric ulcer rat in the present invention：
SD rats are randomly divided into 6 groups, per group of 10 rats：Blank group, model group, the high, medium and low dosage group of Codonopsis pilosula polysaccharide, sun
Property control（CBS）Group.Blank group is according to 10mL/kg gavage physiological saline, model group gavage distilled water 10mL/kg, party
The high, medium and low dosage group of gracilis polysaccharide is according to Codonopsis pilosula polysaccharide 0.8g/kg, 0.4g/kg, 0.2g/kg gavage.Positive drug group is according to Chinese holly
Rafter acid bismuth potassium 100mg/kg gavages.One time a day, and after continuous 7 days, animal fasting can't help water 24 hours, and the 8th day starts that front taboo is administered
Water.After administration 3 hours, in addition to blank group, each group gives the ethanol gavages of 1mL 70%.Blank group gives 1mL distilled water.Modeling 1 hour
After, animal is put to death in lumbar injection yellow Jackets 45mg/kg anesthetized rats, abdominal aorta blood drawing after 5 minutes.Take out stomach,
Coat of the stomach is cut off along greater curvature, stomach lining is observed, the measure of ulcer index is carried out, gastric mucosal damage index is calculated, then half
Gastric tissue is put into 10% neutral formalin solution fixed, and half prepares 10% tissue homogenate, for detecting gastric tissue in MPO,
SOD, GSH-Px activity and MDA, NO content.All data represent that the significance test of multigroup data difference is adopted with ± s
One-way analysis of variance.
As a result such as Fig. 7, Fig. 8, Tables 1 and 2.
Fig. 7 shows that normal rats stomach lining is smooth, without bleeding.Model group rats stomach lining has serious bar
Strand bleeding lesions, CPS-A high dose groups and middle dose group bleeding it is less.
Fig. 8 shows A under light microscopic（Normal group）Gastric Mucosal Cells marshalling, fine and close, rule, has no that meronecrosis comes off
It is abnormal, 0 grade of degree of injury（Mucosal lesion grading is carried out according to Lacy methods）.B（Model group）Gastric Mucosal Cells are extensively bad
Extremely come off, karyopyknosis, cytoplasm cracking, cell arrangement is disorderly, and interstitial is loose, and damage is invaded and mucosa cells lower floor, damages
Degree III level.C（CPS-A high dose groups）Stomach lining cells of superficial layer irregular arrangement, submucosa cell arrangement is neat, damages
Degree I level.D（CPS-A middle dose groups）Gastric Mucosal Cells extensive necrosis come off, but do not damage to body of gland.Degree of injury II levels.
E（CPS-A low dose groups）Gastric Mucosal Cells layer is thinning, and cell arrangement is loose and extensive necrosis come off, karyopyknosis, cell
Matter is cracked, and damage is invaded and body of gland, degree of injury III level.F（Positive drug CBS group）Gastric Mucosal Cells are normal,
The arrangement of mucous membrane upper cell is loose irregular, and karyopyknosis does not extend to lower confluent monolayer cells, degree of injury I levels.
Impacts of the CPS-A of table 1 to alcohol induced rat gastric ulcer gastric tissue oxidative stress
It is active that table 1 shows that CPS-A can in various degree improve SOD and GSH-Px in rat gastric tissue, reduce MPO activity and NO,
MDA contents.And rat stomach tissue GSH-Px activity increases with the increase of CPSa dosage, and MDA contents are with CPSa dosage
Increase and reduce, MPO activity reduces with the increase of CPSa dosage.
The rat gastric ulcer index of table 2 and ulcer inhibition rate（±s）
Table 2 shows that CBS group, CPS-A high doses and middle dose group can significantly reduce rat gastric ulcer index（According to
Guth methods carry out ulcer index measure）.
Description of the drawings
Fig. 1 represents CPS-A graph of molecular weight distribution.
Fig. 2 represents the gas-chromatography and standard monose compares figure of CPS-A.
Fig. 3 represents the infrared chromatogram of CPS-A.
Fig. 4 represents CPS-A nuclear magnetic resonance1H-NMR spectrum.
Fig. 5 represents CPS-A's13C-NMR spectrograms.
Fig. 6 represents the structural formula of CPS-A.
Fig. 7 represents impacts of the CPS-A to alcohol induced rat gastric ulcer gastric tissue form, wherein, A represents normal group, B tables
Representation model group, C represents Radix Codonopsis CPS-A high dose groups（50mg/kg）, D represents Radix Codonopsis CPS-A middle dose groups（25mg/kg）, E tables
Show Radix Codonopsis CPS-A low dose groups（12.5mg/kg）, F represents positive drug CBS group（100mg/kg）.
Fig. 8 represents CPS-A to alcohol induced rat gastric ulcer gastric tissue section light microscopic result（10×10）, wherein, A is represented
Normal group, B represents model group, and C represents Radix Codonopsis CPS-A high dose groups（50mg/kg）, D represents Radix Codonopsis CPS-A middle dose groups
（25mg/kg）, E represents Radix Codonopsis CPS-A low dose groups（12.5mg/kg）, F represents positive drug CBS group（100mg/
kg）.
Specific embodiment
The specific embodiment of the present invention is described in detail below.
Embodiment 1
The preparation of Codonopsis pilosula polysaccharide CPS-A, comprises the steps：
Radix Codonopsis total starches 100g, plus 2000mL distilled water are taken, 80 DEG C dissolve 1 hour, and period is stirred continuously.4 DEG C stand 12 hours,
Centrifugation.Supernatant concentration to 100mL, 90% ethanol alcohol precipitation, 4 DEG C stand 12 hours, centrifugation.Behaviour after precipitation is dried, before repeating
Make, obtain polysaccharide precipitation, with 3000mL distilled water, 80 DEG C dissolve 1 hour, and period is stirred continuously, and 4 DEG C stand 12 hours, centrifugation,
Successively with 100000 molecular weight films, 5000 molecular weight film ultrafiltration, pressure is 1.5 atmospheric pressure to supernatant, will be finally by 5000
The filtrate concentration of molecular weight film, is dried, and obtains 36.45g CPS-A.
Embodiment 2
The preparation of Codonopsis pilosula polysaccharide CPS-A, comprises the steps：
Radix Codonopsis total starches 100g, plus 3000mL distilled water are taken, 85 DEG C dissolve 1.5 hours, and period is stirred continuously.4 DEG C of standings 24 are little
When, centrifugation.Supernatant concentration to 100mL, 95% ethanol alcohol precipitation, 4 DEG C stand 24 hours, centrifugation.After precipitation is dried, before repeating
Operation, obtain polysaccharide precipitation, with 4000mL distilled water, 85 DEG C dissolve 1.5 hours, and period is stirred continuously, and 4 DEG C to stand 24 little
When, centrifugation, successively with 100000 molecular weight films, 5000 molecular weight film ultrafiltration, pressure is 2.0 atmospheric pressure to supernatant, will be last
Concentrated by the filtrate of 5000 molecular weight films, be dried, obtain 40.30g CPS-A.
Embodiment 3
The preparation of Codonopsis pilosula polysaccharide CPS-A, comprises the steps：
Radix Codonopsis total starches 100g, plus 2500mL distilled water are taken, 85 DEG C dissolve 1 hour, and period is stirred continuously.4 DEG C stand 24 hours,
Centrifugation.Supernatant concentration to 100mL, 95% ethanol alcohol precipitation, 4 DEG C stand 24 hours, centrifugation.Behaviour after precipitation is dried, before repeating
Make, obtain polysaccharide precipitation, with 3500mL distilled water, 85 DEG C dissolve 1 hour, and period is stirred continuously, and 4 DEG C stand 24 hours, centrifugation,
Successively with 100000 molecular weight films, 5000 molecular weight film ultrafiltration, pressure is 2.5 atmospheric pressure to supernatant, will be finally by 5000
The filtrate concentration of molecular weight film, is dried, and obtains 41.50g CPS-A.
The Structural Identification of the Codonopsis pilosula polysaccharide CPS-A of the present embodiment is as follows：
1st, saccharic composition decomposes and gas chromatographic analysis
30mg CPS-A are taken, tool plug test tube is put into, adds the trifluoroacetic acid 2mL of 2mol/L, tube sealing, 100 DEG C of hydrolysis 12h to take out
N afterwards2Dry up, it is standby.Derivative is prepared using sugared nitrile acetyl method：Standard monose and the sugar-like after polysaccharide sample hydrolysis are weighed respectively
Each 10mg, adds hydroxylamine hydrochloride 10mg, internal standard inositol 7mg, pyridine 0.5mL, in 90 DEG C of heating water bath 30min and vibrates, and takes out
Room temperature is cooled to, acetic anhydride 0.5mL is added, heating 30min is continued at 85 DEG C carries out acetylation, and product is concentrated to dryness, and adds
0.5mL chloroforms dissolve, and Jing after 0.45 membrane filtration, 0.2 μ L sample introductions, gas chromatograph is analyzed solution.As a result such as Fig. 2 institutes
Show, compare with standard monose, CPS-A hydrolysates correspond to fructose.
2nd, infrared absorption spectroscopy
Sample is determined using KBr pressed disc methods, as a result as shown in figure 3, the characteristic absorption of typical polysaccharide is presented from CPS-A in IR figures
Peak, in wave number 840cm-1Place is illustrated without α-glycosidic bond without absorption, and contains β-glycosidic bond.
3rd, NMR spectrum
1H and13The analysis of CNMR spectrums
Sample is dissolved in D2In O, determine under normal temperature, as a result see Fig. 4 and Fig. 5.Reference literature data, CPS-A's13CNMR spectrum signals with
The C signal of Beta-D-Fructopyranose polysaccharide is similar, with reference to document glycosidation displacement law, it is known that the fructose in CPS-A is with-β（1, 2）-
It is bonded.It is as follows to each signals assignment：62.0 （C-1）, 103.4（C-2）, 77.0（C-3）, 74.5（C-4）, 81.5（C-
5）, 60.8（C-6）.
It should be noted last that, above example is only unrestricted to illustrate technical scheme, although ginseng
It has been described in detail according to the embodiment of the present invention, it will be understood by those within the art that, to technical scheme
Modify or equivalent, without departure from the spirit and scope of technical scheme, it all should cover claim
In protection domain.
Claims (4)
1. a kind of Radix Codonopsis homogeneous polysaccharide with anti-gastric-ulcer, it is characterised in that：General structure is as follows：
。
2. the Radix Codonopsis homogeneous polysaccharide with anti-gastric-ulcer according to claim 1, it is characterised in that：The polysaccharide molecular weight is
3000, it is straight chain homogeneous polysaccharide that beta-D-fructofuranose residue is formed by connecting with β-(2 → 1)-glycosidic bond.
3. a kind of separating and extracting process of the Radix Codonopsis homogeneous polysaccharide with anti-gastric-ulcer described in claim 1 or 2, its feature exists
In：Comprise the steps：
（1）, extract Radix Codonopsis total starches；
（2）, Codonopsis pilosula polysaccharide ultra-filtration and separation
By 10～30 times of distilled water diluting of Radix Codonopsis total starches, heating water bath to 20 DEG C～90 DEG C fully dissolvings, in less than 10 DEG C
12～72 hours are stood, 1～3g/mL is concentrated into, with 60%～95% ethanol alcohol precipitation, 4～24 hours is stood, centrifugation, precipitation is dry
It is dry；Repeat aforesaid operations 1-3 time；Radix Codonopsis total starches solution is successively by the filter membrane and 1000-10000 molecules of the molecular weight of 1-10 ten thousand
Amount filter membrane, obtains Radix Codonopsis homogeneous polysaccharide.
4. application of the Radix Codonopsis homogeneous polysaccharide described in a kind of claim 1 or 2 in treatment Gastric Ulcer Treatment.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112375157A (en) * | 2020-12-04 | 2021-02-19 | 山西医科大学 | Codonopsis pilosula glucan with immunoregulation effect and preparation method and application thereof |
| CN112625305A (en) * | 2020-12-29 | 2021-04-09 | 山西医科大学 | Codonopsis pilosula fructan compound capable of promoting growth of pediococcus pentosaceus and preparation method and application thereof |
| CN115286721A (en) * | 2022-07-28 | 2022-11-04 | 深圳海创生物科技有限公司 | Active polysaccharide, active polysaccharide composition and application of active polysaccharide composition in preparation of product with effect of preventing or treating gastric injury |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112375157A (en) * | 2020-12-04 | 2021-02-19 | 山西医科大学 | Codonopsis pilosula glucan with immunoregulation effect and preparation method and application thereof |
| CN112625305A (en) * | 2020-12-29 | 2021-04-09 | 山西医科大学 | Codonopsis pilosula fructan compound capable of promoting growth of pediococcus pentosaceus and preparation method and application thereof |
| CN112625305B (en) * | 2020-12-29 | 2022-06-14 | 山西医科大学 | Codonopsis pilosula fructan compound capable of promoting growth of pediococcus pentosaceus and preparation method and application thereof |
| CN115286721A (en) * | 2022-07-28 | 2022-11-04 | 深圳海创生物科技有限公司 | Active polysaccharide, active polysaccharide composition and application of active polysaccharide composition in preparation of product with effect of preventing or treating gastric injury |
| CN115286721B (en) * | 2022-07-28 | 2023-08-25 | 深圳海创生物科技有限公司 | Active polysaccharide, active polysaccharide composition and application thereof in preparation of products with effect of preventing or treating gastric injury |
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Application publication date: 20170510 Assignee: Shanxi Medical University Asset Management Co., Ltd Assignor: Shanxi Medial University Contract record no.: X2019980000611 Denomination of invention: Radix codonopsis homogeneous polysaccharide with anti-gastric ulcer effect and preparation method and application thereof Granted publication date: 20181113 License type: Common License Record date: 20191115 |
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Assignee: Shanxi Medical University Asset Management Co.,Ltd. Assignor: SHANXI MEDICAL University Contract record no.: X2019980000611 Date of cancellation: 20210422 |
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