CN107236050A - Glycopolymers and its preparation method and application in scythian lamb rhizome - Google Patents
Glycopolymers and its preparation method and application in scythian lamb rhizome Download PDFInfo
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- CN107236050A CN107236050A CN201710386921.XA CN201710386921A CN107236050A CN 107236050 A CN107236050 A CN 107236050A CN 201710386921 A CN201710386921 A CN 201710386921A CN 107236050 A CN107236050 A CN 107236050A
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- China
- Prior art keywords
- glycopolymers
- scythian lamb
- lamb rhizome
- rhizome
- water
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Classifications
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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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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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
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Abstract
The invention discloses glycopolymers in scythian lamb rhizome and its preparation method and application, the preparation method is using scythian lamb rhizome dry rhizome as raw material, the combination of alcohol deposition method and alkali carries alcohol deposition method is classified using water extraction, concentration of alcohol carries out classification alcohol precipitation from low to high, obtains scythian lamb rhizome glycopolymers.The preparation technology of the present invention is simple, easy to operate, and can mass produce.The present invention prepares the sugared polymers CBP 1 and CBP 2 of two kinds of scythian lamb rhizomes first using ion-exchange chromatography and molecular sieve gel column chromatography method purifying scythian lamb rhizome glycopolymers.The invention provides the activity research of raw sugar polymer, the preparation method of refined sugar polymer and scythian lamb rhizome glycopolymers in terms of anti-osteoporosis in scythian lamb rhizome, the application for scythian lamb rhizome glycopolymers in fields such as medicine, health products, food provides foundation.
Description
Technical field
The invention belongs to medicine and technical field of health care food, and in particular to scythian lamb rhizome raw sugar polymer (including oligosaccharides
And polysaccharide), refined sugar polymer (including oligosaccharides and polysaccharide) and preparation method thereof, and further relate to scythian lamb rhizome glycopolymers (bag
Include oligosaccharides and polysaccharide) preparing the application in preventing and/or treating the medicine or health food or functional food of osteoporosis.
Background technology
Osteoporosis is a kind of degenerative bone lesion, is reduced with bone mineral quantity, bone micro-structure is destroyed, bone strength is reduced,
So as to cause the fragility of bone to increase and the disease being characterized that is prone to fracture.Extension and social senilization with human longevity
Development, the fracture caused by osteoporosis is while the elderly's disability rate and case fatality rate is greatly increased, hence it is evident that aggravated society
Can sanitarian financial burden.The drug therapy to osteoporosis is mainly calcium agent, vitamin D and bone information suppression at this stage
The three major types medicines such as medicine (including estrogen, SERM, diphosphonate etc.).Studies have shown that, for a long time
Can be increased using HRT suffer from breast cancer, the probability of the disease such as coronary heart disease, other chemical synthetic drugs clinically used
There is certain side effect in thing.Therefore, using Chinese medicine, especially nourishing disney and strengthening bone class Chinese herb prevention osteoporosis has become
The focus of the world of medicine's research in recent years.
Scythian lamb rhizome is Dicksoniaceae plant scythian lamb rhizome Cibotium barometz rhizome, originates in the torrid zone and sub-
Torrid areas, China East China, south China and southwest are distributed.Go through version《Chinese Pharmacopoeia》It is recorded for conventional Chinese medicine dog
Ridge, it is its bitter, sweet, it is warm-natured.Function with filling liver kidney, strengthening the bones and muscles, the channels and collaterals that relax, dehumidifying pain and diuresis, for waist and leg ache, hand
The diseases such as foot numbness, hemiplegia, leukorrhea seminal emission, metrorrhagia.
Chinese patent application CN103301168A discloses a kind of scythian lamb rhizome processing method, it is therefore intended that from scythian lamb rhizome
The a variety of active principles of middle separation, the invention adopt the technical scheme that according to sorting, clean, grind, rinse, air dry, precipitation,
Concentration, drying, packaging process are processed, and the technical scheme that the invention is taken is easy to operate, can be obtained with the technical scheme
The materials such as purity high fiber, starch, tannin and aspidinol are obtained, raw material availability is high.
Chinese patent application CN104435615A provides a kind of healthy medicated wine containing scythian lamb rhizome, the active component contained by it
It is prepared from by following raw materials in part by weight medicine proportioning：Scythian lamb rhizome, foliosous parnassia herb, fleece-flower root etc. and 30 ° of -50 ° of white wine 5000-
10000 parts, removal of impurities, crushing are crossed after 60 mesh sieves, are put into 30 ° of -50 ° of white wine and are soaked 10-20 days, are filtered off the dregs of a decoction and are produced medicinal liquor.
Chinese patent application CN106190716A discloses a kind of plateau scythian lamb rhizome kidney-nourishing health tonic wine and its preparation
Method, the health liquor being particularly made using scythian lamb rhizome, cynomorium songaricum, safflower, kansu sandwort herb as primary raw material.Its active raw materials
Including scythian lamb rhizome, cynomorium songaricum, safflower, kansu sandwort herb, lanatechead saussurea herb with flower, cordyceps sinensis, truffle, Gymnadenia conopsea, panax ginseng fruit, flat peach, epipremnum aureum
Flower, maca, raw cultivated land, the root of Chinese clematis, Chinese cassia tree, tortoiseshell, testis et penis canitis, gekko, Fructus Corni, Semen Cuscutae, the fruit of Cherokee rose, the fleece-flower root, glossy privet
Son, Radix Angelicae Sinensis, reticulate millettia, radix glycyrrhizae, rock sugar, pure grain distilled spirit.
At present, have been reported and the chemical composition at the positions such as the ground of scythian lamb rhizome and rhizome is studied, find gold
Hair rhizoma cibotii contains starch, tannin, the material such as volatile oil, water-soluble phenolic compounds, sugar and glycosides compound, also contains amino
The chemical compositions such as acid.However, to scythian lamb rhizome raw sugar polymer, refined sugar polymer and its grinding in terms of anti-curing osteoporosis
Study carefully that there is not been reported.
The content of the invention
It is an object of the invention to provide scythian lamb rhizome glycopolymers.
Another object of the present invention is to provide the preparation method of scythian lamb rhizome glycopolymers.
The technical solution used in the present invention is：
Scythian lamb rhizome glycopolymers, the scythian lamb rhizome glycopolymers include scythian lamb rhizome glycopolymers CBP-1 and Jin Mao
Rhizoma cibotii glycopolymers CBP-2, the sugared polymers that the scythian lamb rhizome glycopolymers CBP-1 is made up of glucose and mannose, its
Structure is：
Wherein n scope is 1~30；
The scythian lamb rhizome glycopolymers CBP-2 be by mannose, rhamnose, galacturonic acid, glucose, galactolipin,
The sugared polymers of arabinose composition, its structure is：
Wherein x, y, z, m scope are respectively 1~30.
The preparation method of scythian lamb rhizome glycopolymers, comprises the following steps：
S1 strippings and slicings：Scythian lamb rhizome dry rhizome is cut into block, scythian lamb rhizome block is obtained, by above-mentioned scythian lamb rhizome block
It is eluted with water, then soaks 5~15h, scythian lamb rhizome block after must soaking；
S2 water extractions：Scythian lamb rhizome block obtained by step S1 is subjected to water extraction, the addition of water is above-mentioned golden hair during water extraction
5~20 times of rhizoma cibotii block weight, the temperature of water is 60~100 DEG C during water extraction, and the water extraction time is 1~10h, collects and extracts
Liquid, the dregs of a decoction dry；
S3 alkali carries：The dregs of a decoction obtained by step S2 are immersed in the NaOH solution that concentration is 0.1~1M, the amount of NaOH solution is
10~20 times of above-mentioned dregs of a decoction weight, room temperature places 2~20h, takes supernatant, the HCl solution for being 0.5M with concentration by supernatant
Neutralized, it is 5~9 to make its pH, takes supernatant to be centrifuged, centrifugal speed is 4500~5600r/min, and centrifugation time is 8
~13min, takes supernatant, and 60 DEG C are concentrated under reduced pressure, and then adding ethanol makes ethanol volumetric concentration be 60~85%, stands after 24h
Centrifugation, collects precipitation, obtains alkali carries raw sugar polymer CB4；
S4 is purified：Removing protein is carried out to alkali carries raw sugar polymer CB4 obtained by step S3 using Sevag methods, after removing protein
Alkali carries raw sugar polymer CB4 dialysed, freezed with bag filter, produce scythian lamb rhizome glycopolymers CB4；
S5 ion-exchange chromatographies：Scythian lamb rhizome glycopolymers CB4 obtained by step S4 is dissolved in deionized water, is splined on
, there are 2 peaks, wherein peak one is water (0M NaCl) under the conditions of the eluent of different salinity in the posts of DEAE-Cellulose 52
Elution fraction, peak two is 0.05M NaCl elution fractions, tracks elution curve using phend-sulphuric acid in elution process, according to
Elution curve collects sugar moieties respectively, respectively by after the concentration of gained eluent, freeze-drying, obtains the glycopolymers of peak one and peak disaccharides
Polymer；
S6 molecular sieve gels are chromatographed：By the glycopolymers sample of peak one obtained by step S5, dissolved with water, centrifuge, take
Clear liquid, upper Sephadex G-75 posts, is eluted with water, and elution curve is tracked using phend-sulphuric acid, and appearance one is single
Symmetrical peak, collects main peak, concentrates, and freeze-drying obtains scythian lamb rhizome glycopolymers CBP-1；Similarly, by peak two obtained by step S5
Glycopolymers sample, is dissolved with water, centrifugation, takes supernatant, upper Sephadex G-75 posts are eluted with water, using benzene
Phenol-sulfuric acid method tracks elution curve, a single symmetrical peak occurs, collects main peak, concentrates, freeze-drying, obtains scythian lamb rhizome sugar
Polymer CBP-2.
Further, the step S1 immersions 12h.
Further, the addition of water is 10 times of above-mentioned scythian lamb rhizome block weight, water during the step S2 water extractions
The temperature of water is 85 DEG C when carrying, and the water extraction time is 2h.
Further, the dregs of a decoction obtained by step S2 are immersed in the NaOH solution that concentration is 0.3M in the step S3,
The amount of NaOH solution is 15 times of above-mentioned dregs of a decoction weight, and room temperature places 3h, takes supernatant, with concentration is 0.5M's by supernatant
HCl solution is neutralized, and it is 7 to make its pH, takes supernatant to be centrifuged, centrifugal speed is 5000r/min, centrifugation time is
10min, takes supernatant, and 60 DEG C are concentrated under reduced pressure, and then adding ethanol makes ethanol volumetric concentration be 75%.
Further, bag filter described in the step S4 obtains molecular cut off for 1000Da.
In addition, present invention also offers the preparation method of raw sugar polymer in scythian lamb rhizome, comprising the following steps：
(1) stripping and slicing：Scythian lamb rhizome dry rhizome is cut into block, scythian lamb rhizome block is obtained, above-mentioned scythian lamb rhizome is block
Thing is eluted with water, and then soaks 5~15h, scythian lamb rhizome block after must soaking；
(2) water extraction：Scythian lamb rhizome block obtained by step (1) is subjected to water extraction, the addition of water is above-mentioned gold during water extraction
5~20 times of hair rhizoma cibotii block weight, the temperature of water is 60~100 DEG C during water extraction, and the water extraction time is 1~10h, collects and extracts
Liquid, the dregs of a decoction dry；
(3) it is classified alcohol precipitation：After extract solution concentration obtained by step (2), adding ethanol makes ethanol volumetric concentration be a%, quiet
Put, collect precipitation, obtain raw sugar polymer CB1；Supernatant is concentrated again, adding ethanol makes ethanol volumetric concentration be b%, quiet
Put, collect precipitation, obtain raw sugar polymer CB2；Supernatant is concentrated again, adding ethanol makes ethanol volumetric concentration be c%, quiet
Put, collect precipitation, obtain raw sugar polymer CB3；Wherein 10≤a ＜ b ＜ c ＜ 100；
(4) alkali carries：The dregs of a decoction obtained by step (2) are soaked in the NaOH solution that concentration is 0.1~1M, stand 2~20h,
Supernatant is neutralized with concentration for 0.1~1M HCl, and the pH for making supernatant is 5~9, and centrifugation takes supernatant, supernatant is concentrated,
Adding ethanol makes ethanol volumetric concentration be 50~90%, stands, collects precipitation, obtain alkali carries raw sugar polymer CB4；
(5) purify：To raw sugar polymer CB1, raw sugar polymer CB2, raw sugar polymer CB3 and step obtained by step (3)
(4) gained alkali carries raw sugar polymer CB4 is purified, and produces scythian lamb rhizome raw sugar polymer.
Further, step (1) the immersion 12h.
Further, the addition of water is 10 times of above-mentioned scythian lamb rhizome block weight, water during step (2) water extraction
The temperature of water is 85 DEG C when carrying, and the water extraction time is 2h.
Further, 10≤a ＜ b ＜ c ＜ 100 in the step (3), and the ＜ c ＜ of 10≤a ＜, 60,60 ＜ b ＜ 80,80
100。
Further, all concentrations are concentrated under reduced pressure for 40~70 DEG C in the step (3), all standings when
Between be 10~28h.
Further, the dregs of a decoction obtained by step (2) are immersed in the NaOH solution that concentration is 0.3M in the step (4),
The amount of NaOH solution is 15 times of above-mentioned dregs of a decoction weight, and room temperature places 3h, takes supernatant, with concentration is 0.5M's by supernatant
HCl solution is neutralized, and it is 7 to make its pH, takes supernatant to be centrifuged, centrifugal speed is 5000r/min, centrifugation time is
10min, takes supernatant, and 60 DEG C are concentrated under reduced pressure, and then adding ethanol makes ethanol volumetric concentration be 75%.
Further, it is respectively to raw sugar polymer using Sevag methods that concrete operations are purified described in the step (5)
Dialysed, freezed through bag filter again after CB1, CB2, CB3, CB4 removing protein, removing protein.
Further, the molecular cut off of bag filter described in the step (5) is 1000Da.
It is still another object of the present invention to provide above-mentioned scythian lamb rhizome glycopolymers and scythian lamb rhizome raw sugar polymer in system
Application in the standby medicine or health food or functional food for preventing and treating osteoporosis.
Compared with prior art, present invention employs above technical scheme, have the following advantages and advantages：
(1) compared with traditional water-boiling method extracts glycopolymers, the present invention is classified alcohol deposition method and alkali carries alcohol deposition method using water extraction
Combination, concentration of alcohol carries out classification alcohol precipitation from low to high, initial gross separation is carried out to scythian lamb rhizome glycopolymers, while high concentration
Ethanol can by polarity big, good water solubility glycopolymers are small with polarity, poorly water-soluble glycopolymers are separated, make extracted sugar
Polymer composition contains a greater variety of glycopolymers compositions.It is easy to operate and the preparation technology of the present invention is simple, and can be with
Large-scale production.
(2) present invention utilizes ion-exchange chromatography and the purifying scythian lamb rhizome sugar polymerization of molecular sieve gel column chromatography method
Thing, prepares two kinds of scythian lamb rhizome refined sugar polymers, and be to its physicochemical property, molecular weight, monose composition etc. first
That unites analyzes and identifies, and has successfully drawn the feature structure of two kinds of glycopolymers.The invention provides raw sugar in scythian lamb rhizome
The activity research of polymer, the preparation method of refined sugar polymer and scythian lamb rhizome glycopolymers in terms of anti-osteoporosis, is gold
Application of the hair rhizoma cibotii glycopolymers in fields such as medicine, health products, food provides foundation.
(3) scythian lamb rhizome raw sugar polymer is isolated and purified by column chromatography in the present invention, effect is notable, first
Prepare the sugared polymers CBP-1 and CBP-2 of two kinds of scythian lamb rhizomes.
(4) present invention is identified the two kinds of scythian lamb rhizome glycopolymers structures prepared, and specify that its physics and chemistry
Matter and structure, structure foundation is provided to probe into its pharmacological activity mechanism.
(5) the invention provides the preparation method of the composition with anti-osteoporosis activity in scythian lamb rhizome, and screen
The glycopolymers position that content is high, activity is strong is gone out.
Brief description of the drawings
Fig. 1 is CBP-1 high-efficient liquid phase chromatogram；
Fig. 2 is CBP-1 infared spectrum；
Fig. 3 is CBP-1's13C NMR spectras；
Fig. 4 is CBP-1's1H NMR spectras；
Fig. 5 is CBP-1's1H-1H COSY collection of illustrative plates；
Fig. 6 is CBP-1 HSQC collection of illustrative plates；
Fig. 7 is CBP-1 HMBC collection of illustrative plates；
Fig. 8 is CBP-2 high-efficient liquid phase chromatogram；
Fig. 9 is CBP-2 infared spectrum；
Figure 10 is CBP-2's13C NMR spectras；
Figure 11 is CBP-2's1H NMR spectras；
Figure 12 is CBP-2 HSQC collection of illustrative plates；
Figure 13 is CBP-2 HMBC collection of illustrative plates；
Figure 14 is influence of the scythian lamb rhizome glycopolymers to femur of mature ovariectomized rats bone density；
Figure 15 is influence of the scythian lamb rhizome glycopolymers to ovariectomized female rats lumbar vertebra bone density；
Figure 16 is influence of the scythian lamb rhizome glycopolymers to femur of mature ovariectomized rats bone mineral quantity；
Figure 17 is influence of the scythian lamb rhizome glycopolymers to ovariectomized female rats lumbar vertebra bone mineral quantity.
Embodiment
Below by way of the description of embodiment, the invention will be further described, but this is not the limit to the present invention
System, for those skilled in the art, that is made under other any Spirit Essences and principle without departing from the present invention changes
Become, modification, instead of, simplify, improve, but without departing from the present invention basic thought, should be equivalent substitute mode, all
Within protection scope of the present invention.
Embodiment 1, scythian lamb rhizome glycopolymers
The scythian lamb rhizome glycopolymers include scythian lamb rhizome glycopolymers CBP-1 and scythian lamb rhizome glycopolymers CBP-2；
The preparation method of described scythian lamb rhizome glycopolymers, comprises the following steps：
S1 strippings and slicings：10kg scythian lamb rhizome dry rhizomes are cut into block, scythian lamb rhizome block is obtained, by above-mentioned scythian lamb rhizome block
Shape thing is eluted with water, and then soaks 12h, scythian lamb rhizome block after must soaking；
S2 water extractions：Scythian lamb rhizome block obtained by step S1 is subjected to water extraction, the addition of water is above-mentioned golden hair during water extraction
10 times of rhizoma cibotii block weight, the temperature of water is 85 DEG C during water extraction, and the water extraction time is 2h, collects extract solution, and the dregs of a decoction dry；
S3 alkali carries：The dregs of a decoction obtained by step S2 are immersed in the NaOH solution that concentration is 0.3M, the amount of NaOH solution is upper
State dregs of a decoction weight 15 times, room temperature places 3h, takes supernatant, the HCl solution for being 0.5M with concentration by supernatant is neutralized, and is made
Its pH is 7, takes supernatant to be centrifuged, centrifugal speed is 5000r/min, centrifugation time is 10min, takes supernatant, 60 DEG C subtract
Pressure concentration, then adding ethanol makes ethanol volumetric concentration be 75%, stands and is centrifuged after 24h, collects precipitation, obtains the polymerization of alkali carries raw sugar
Thing CB4；
S4 is purified：Removing protein is carried out to alkali carries raw sugar polymer CB4 obtained by step S3 using Sevag methods, after removing protein
Alkali carries raw sugar polymer CB4 dialysed, freezed with bag filter, the molecular cut off of the bag filter is 1000Da, is produced
Scythian lamb rhizome glycopolymers CB4；
S5 ion-exchange chromatographies：Scythian lamb rhizome glycopolymers CB4 obtained by step S4 is dissolved in deionized water, is splined on
, there are 2 peaks, wherein peak one is water (0M NaCl) under the conditions of the eluent of different salinity in the posts of DEAE-Cellulose 52
Elution fraction, peak two is 0.05M NaCl elution fractions, tracks elution curve using phend-sulphuric acid in elution process, according to
Elution curve collects sugar moieties respectively, respectively by after the concentration of gained eluent, freeze-drying, obtains the glycopolymers of peak one and peak disaccharides
Polymer；
S6 molecular sieve gels are chromatographed：By the glycopolymers sample of peak one obtained by step S5, dissolved with water, centrifuge, take
Clear liquid, upper Sephadex G-75 posts, is eluted with water, and elution curve is tracked using phend-sulphuric acid, and appearance one is single
Symmetrical peak, collects main peak, concentrates, and freeze-drying obtains scythian lamb rhizome glycopolymers CBP-1；Similarly, by peak two obtained by step S5
Glycopolymers sample, is dissolved with water, centrifugation, takes supernatant, upper Sephadex G-75 posts are eluted with water, using benzene
Phenol-sulfuric acid method tracks elution curve, a single symmetrical peak occurs, collects main peak, concentrates, freeze-drying, obtains scythian lamb rhizome sugar
Polymer CBP-2.
Purity detecting：The glycopolymers CBP-1 of above-mentioned acquisition is made into 2% concentration (W/V) aqueous solution, HPGPC methods are surveyed
Obtain retention time.
The component CBP-1 obtained after being isolated and purified through ion exchange and gel filtration is in simple spike, and it is equal to illustrate CBP-1
One glycopolymers.
Further structural analysis is made with CBP-1, CBP-2 for being extracted in above-described embodiment below.
The structural analysis of embodiment 2, scythian lamb rhizome glycopolymers
(1) monosaccharide composition analysis
Collection of illustrative plates is measured using PMP- column front derivation high performance liquid chromatographies by complete acid hydrolysis products, as a result as shown in Figure 1.
Figure 1A is mixing monose standard items collection of illustrative plates, and wherein 1-9 is respectively mannose, rhamnose, glucuronic acid, galacturonic acid, Portugal
Grape sugar, galactolipin, xylose, arabinose, fucose.Figure 1B is that CBP-1 monose constitutes collection of illustrative plates, and control understands that CBP-1 is by grape
Sugar and mannose composition.
(2) infrared spectrum is detected
CBP-1 infrared spectrum testing result is as shown in Fig. 2 testing result is shown, CBP-1 contains the characteristic absorption peak of sugar
For：3390.82cm-1For the characteristic absorption peak of O-H stretching vibrations in sugar, 2925.97cm-1For the feature of C-H stretching vibrations in sugar
Absworption peak, 1637.92cm-1For O-H flexural vibrations absworption peaks.In 1740cm-1Interval has no absworption peak, illustrates CBP-1 without sugar
Aldehydic acid.1000-1200cm-1Characteristic absorption peak be due to ehter bond on sugared ring asymmetric stretching vibration produce.934.30cm-1
And 763.07cm-1Asymmetric stretching vibration and symmetrical stretching vibration for pyranose.
(3) methylation analysis
Sample is hydrolyzed, reduced through methylating, and GC-MS is analyzed after acetylation, is as a result shown, and CBP-1 contains → 4)-α-D-
Glc- (1 →, → 1)-β-D-Glc- (6 → and → 3,6)-α-D-Man- (1 → and α-D-Glc- (1 → saccharide residues.
(4) nuclear magnetic resonance spectroscopy of glycopolymers
Homogeneous glycopolymers CBP-1 samples are placed in nuclear magnetic tube, D is used2Spectrum, acquired results are surveyed after O dissolvings
As shown in figure 3 to figure 7.
The ownership of each carbon and hydrogen is understood according to the nuclear magnetic spectrum of above-mentioned Fig. 3~7, as a result as shown in table 1.
Table 1：CBP-1 nuclear magnetic resonance spectroscopy results
Through above-mentioned complete sour water solution, methylation analysis, infrared spectrum detection and nmr analysis, as a result show, CBP-1 is one
The sugared polymers kind being made up of glucose and mannose, from methylation analysis illustrate its contain → 4)-α-D-Glc- (1 →, → 1)-
β-D-Glc- (6 → and → 3,6)-α-D-Man- (1 → (connection between 1 → saccharide residue, different saccharide residues is suitable with α-D-Glc-
Sequence is shown that analysis show that CBP-1 structure is more than by two-dimentional nuclear-magnetism HMBC spectrum analysis：
Wherein n scope is 1~30.
Similarly, the structure to glycopolymers CBP-2 carries out above-mentioned same analysis：Complete sour water solution-PMP column front derivations-
High performance liquid chromatography (Fig. 8), methylation analysis, infrared spectrum detection (Fig. 9) and nmr analysis (shown in Figure 10~13), are obtained
Following information：
Glycopolymers CBP-2：Monosaccharide composition analysis CBP-2 is by mannose, rhamnose, galacturonic acid, glucose, gala
Sugar, arabinose composition.Methylation analysis show be by → 4)-α-D-Glc- (1 →, → 1)-β-D-Gal- (6 →, → 3,6)-
α-D-Man- (1 →, → 3)-α-L-Ara- (1 →, → 3) ((1 → saccharide residue is constituted 1 → and α-D-GlcA--α-L-Rha-, no
Drawn with the order of connection between saccharide residue by two-dimentional nuclear-magnetism HMBC spectrum analysis, analysis show that CBP-2 structure is more than：
Wherein x, y, z, m scope are respectively 1~30.
The function of resisting osteoporosis research of test example one, rhizoma cibotii glycopolymers
1st, experiment material：Glycopolymers CB1, glycopolymers CB2, glycopolymers CB3 and glycopolymers CB4.
2nd, experimental subjects：3 monthly ages did not mated female sd inbred rats 70, and SPF grades (in Traditional Chinese Medicine University Of Guangzhou experimental animal
The heart is provided).
3rd, experimental method：
3.1st, experimental design and packet：
3 monthly ages did not mated female sd inbred rats 70, and SPF grades (by Traditional Chinese Medicine University Of Guangzhou, Experimental Animal Center is provided) presses
Body weight principle of reciprocity is randomly divided into 7 groups, and each group number of animals and medication are shown in Table 2, wherein, positive controls E2 is 17 β-female two
Alcohol, model group OVX and sham-operation group SHAM is distilled water gastric infusion.The daily gastric infusion of Post operation, weighs weekly once, presses
Changes of weight adjusts corresponding dosage, continuous gavage 90d.
Table 2：Experimental design and packet
3.2nd, ovariectomized rats operation method：
By rat with Nembutal sodium solution (4%) intraperitoneal injection of anesthesia of 45mg/kg dosage, abdomen position is fixed.At it most
Under last rib, midaxillary line and the infall away from about 1cm on the outside of backbone, unhairing exposure surgical field of view.First appointing takes side to be performed the operation,
Sterilized with Betagen Solution, cut skin, muscle of back and sarolemma, gently white shiny cellulite is pulled out with pincet and cut
Mouth is outer, fractionation of fatty group, just visible ovary.Ovary lower end fallopian tubal is ligatured with operation suture thread, ovary is then extractd.Cut
After mouth suture, outside is coated with antiphlogistic powder (Benzylpenicillin sodium salt), and opposite side ovary is extractd with method.The processing method of sham-operation group (SHAM)
Ibid, fritter adipose tissue is simply cut off, ovary is not extractd.It is postoperative to be placed in warm environment, nurse observation.
3.3rd, materials are with preserving：
Concrete operations are as follows：
(1) weigh, anaesthetize；
(2) from belly dissection rat exposure internal organ, press close to clamp artery with tweezers at muscle of back and take 2mL blood in heparin
In sodium pipe, change commonly take blood vessel to take whole blood (to stand after lh, serum is taken after centrifuging 10min through 3000rmp, -80 DEG C of frosts are protected immediately
Deposit standby).
3) right side shin bone, femur and fifth lumbar vertebra are taken, is put in the urine cup equipped with 4%PBS paraformaldehyde solutions, after after 24h
The ethanol solution of replacing 70%.- 20 DEG C of freezen protectives.
4) left femur, shin bone and third and fourth lumbar vertebrae are taken, physiological saline gauze wrapped simultaneously uses masking foil outer wrapping, Yu Mi
In envelope, packing is placed in -80 DEG C of stored frozens.
3.4th, rat bone density is determined：
Using Dual-energy X-rays absorptionmetry Hologic Discovery WI 85003DXA detection fourth lumbar vertebra bone (L4) and
Full bone density (bone mineral density, BMD), front end femoral bmd (1cm), the distal femoral bone density of fl
(2cm) and bone mineral content (bone mineral content, BMC).
3.5th, bone biomechanical is tested：
During test, the femur and vertebra of -80 DEG C of preservations are placed in -20 DEG C of pre- defrostings, then normal temperature unfreezing, uses physiological saline
It is multiple wet.The biomechanical property of fl (three-point bending test) is analyzed using Mini858Bionix material testing systems.Test
When MTS testing machines on, with the pressure head of 1mm diameters, loading velocity is 6mm/min, and span (L) is 15mm, and sensor is 500N.Instrument
Device records the load and radial degree changing value of each moment point automatically, draws load-radial degree curve, and obtain corresponding parameter.
4th, experimental result：
4.1st, scythian lamb rhizome glycopolymers ring to removal ovary female rats ghost image：
Analyze influence of the scythian lamb rhizome glycopolymers to removal ovary female rats body weight.Compared with SHAM groups, OVX rats go
After ovary seven weeks, body weight substantially increases (P<0.01), prompting removal ovary causes rat body weight substantially to increase, modeling success.With OVX
Group is compared, and scythian lamb rhizome glycopolymers CB1, CB2, CB3 and CB4 administration group is from after the 7th week, and increased weight starts to mitigate (P<
0.05), positive controls after the tenth week the trend of increased weight also there is obvious suppression.Illustrate administration group and the positive group equal energy of E2
Suppress the increase of ovariectomized female rats body weight.
4.2nd, influence of the scythian lamb rhizome glycopolymers to removal ovary female rats Uterine coefficient：
Analyze influence of the scythian lamb rhizome glycopolymers to removal ovary female rats Uterine coefficient.Compared with SHAM groups, OVX is big
(P is obviously reduced in Uterine coefficient after mouse removal ovary<0.01), prompting removal ovary, which is performed the operation, causes rat uterus atrophy, weight saving.With
OVX groups are compared, and CB1, CB2, CB4 administration group can substantially increase ovariectomized female rats uterus weight with positive group E2.Illustrate these three
Administration group can be improved ovariectomized female rats metratrophia with positive group.
4.3rd, influence of the scythian lamb rhizome glycopolymers to removal ovary female rats femur and lumbar vertebra bone density：
Scythian lamb rhizome glycopolymers are to removal ovary female rats femur and lumbar vertebra bone density (BMD, bone mineral
Density Figure 14,15 are shown in influence), are compared with SHAM groups, femur (Figure 14) and the lumbar vertebra (Figure 15) of OVX ovariectomized female rats
Bone density occurs that (P is obviously reduced<0.01), prompting ovariectomized female rats are successfully, reproduced primary osteoporosis.With OVX group phases
It can only increase ovariectomized female rats distal femoral (Figure 14) bone density than, E2 groups, and substantially to increase removal ovary big for CB4 administration groups
Total femur, front end femur, distal femoral (Figure 14) and lumbar vertebra (Figure 15) bone density (P of mouse<0.05) CB4 administration groups, are illustrated
It is capable of the osteoporosis symptoms of effectively preventing rat, or even effect is better than E2 groups.CB1, CB2, CB3 and E2 group and OVX group phases
Although than not dramatically increasing femur and lumbar vertebra bone density, but it can be seen that being improved in varying degrees.
4.4th, influence of the scythian lamb rhizome glycopolymers to removal ovary female rats femur and lumbar vertebra bone mineral quantity：
Figure 16,17 are shown in influence of the scythian lamb rhizome glycopolymers to removal ovary female rats femur and lumbar vertebra mineral quantity, with
SHAM groups compare, and OVX rat femurs with lumbar vertebra mineral quantity occur that (P is obviously reduced<0.01), prompting ovariectomized female rats are successfully answered
Primary osteoporosis processed.Compared with OVX groups, CB4 administration groups can substantially increase femur of mature ovariectomized rats with positive group E2
Mineral quantity (P<0.01), and CB1, CB3 can only substantially increase front end femur mineral quantity (P<0.05) (see Figure 16)；CB4 administration groups
Also substantially increase ovariectomized female rats lumbar vertebra mineral quantity (P<0.01) (see Figure 17), the above results explanation, scythian lamb rhizome CB4 administrations
Group can substantially increase femur of mature ovariectomized rats and lumbar vertebra bone mineral quantity, and CB1, CB2, CB3 administration group have different degrees of
Improve.
4.5th, influence of the scythian lamb rhizome glycopolymers to biomechanical properties：
Influence of the scythian lamb rhizome glycopolymers to biomechanical properties is as shown in table 3.
Table 3：Influence of the scythian lamb rhizome glycopolymers to biomechanical properties
All numerical value are average value ± SD. in table*P<0.05,**P<0.01vs.OVX；#P<0.05,##P<
0.01vs.SHAM。
As can be seen from Table 3, compared with SHAM groups, OVX rat femur biomethanics relevant parameters reduce, and point out
Ovariectomized rats biomechanical property declines；Compared with OVX groups, administration group can effectively improve ovariectomized female rats biomethanics
Performance, wherein CB4 administration groups effect are the most notable.
In the scythian lamb rhizome glycopolymers that are extracted of the present invention, CB4 administration groups for anti-curing osteoporosis effect the most
Significantly, wherein refined sugar polymers CBP-1, CBP-2 for being isolated and purified in CB4 are probably to play above-mentioned function of resisting osteoporosis
Active ingredient, it may have dramatically increase the effect of femur of mature ovariectomized rats and lumbar spine bmd and bone mineral quantity, its is obvious
The mechanism of action for improving bone biomechanical property needs further research.
5th, conclusion：
After the ovary excision of female rats, estrogen level reduction, active Bone m etabolism, bone conversion enhancing, bone mineral quantity are lost
Lose, be the classical model of high conversion hysteria osteoporosis when imitating postmenopausal women.This experimental study shows, passes through the above method
The scythian lamb rhizome glycopolymers at each position extracted can dramatically increase femur of mature ovariectomized rats and lumbar spine bmd and bone
Mineral quantity, hence it is evident that improve bone biomechanical property, so as to play its effect for treating rat model osteoporosis.
The anti-osteoporosis mechanism of scythian lamb rhizome glycopolymers need to subsequently be studied, and the structure of homogeneous glycopolymers
Strong foundation will be provided for the mechanism for probing into scythian lamb rhizome glycopolymers anti-osteoporosis by identifying.
Claims (7)
1. scythian lamb rhizome glycopolymers, it is characterised in that the scythian lamb rhizome glycopolymers include scythian lamb rhizome glycopolymers
CBP-1 and scythian lamb rhizome glycopolymers CBP-2, what the scythian lamb rhizome glycopolymers CBP-1 was made up of glucose and mannose
Sugared polymers, its structure is：
Wherein n scope is 1~30；
The scythian lamb rhizome glycopolymers CBP-2 be by mannose, rhamnose, galacturonic acid, glucose, galactolipin, I
The sugared polymers of uncle's sugar composition, its structure is：
Wherein x, y, z, m scope are respectively 1~30.
2. the preparation method of scythian lamb rhizome glycopolymers as claimed in claim 1, it is characterised in that comprise the following steps：
S1 strippings and slicings：Scythian lamb rhizome dry rhizome is cut into block, scythian lamb rhizome block is obtained, by above-mentioned scythian lamb rhizome block water
Clean, then soak 5~15h, scythian lamb rhizome block after must soaking；
S2 water extractions：Scythian lamb rhizome block obtained by step S1 is subjected to water extraction, the amount of water is that above-mentioned scythian lamb rhizome is block during water extraction
5~20 times of thing weight, the temperature of water is 60~100 DEG C during water extraction, and the water extraction time is 1~10h, collects extract solution, and the dregs of a decoction dry in the air
It is dry；
S3 alkali carries：The dregs of a decoction obtained by step S2 are immersed in the NaOH solution that concentration is 0.1~1M, the amount of NaOH solution is above-mentioned
10~20 times of dregs of a decoction weight, room temperature places 2~20h, takes supernatant, supernatant is neutralized with HCl solution, makes its pH be
5~9, take supernatant to be centrifuged, centrifugal speed is 4500~5600r/min, centrifugation time is 8~13min, takes supernatant,
60 DEG C are concentrated under reduced pressure, and then adding ethanol makes ethanol volumetric concentration be 60~85%, stands and is centrifuged after 24h, collects precipitation, obtain alkali
Carry raw sugar polymer CB4；
S4 is purified：Removing protein is carried out to alkali carries raw sugar polymer CB4 obtained by step S3 using Sevag methods, by the alkali after removing protein
Carry raw sugar polymer CB4 to be dialysed, freezed with bag filter, produce scythian lamb rhizome glycopolymers CB4；
S5 ion-exchange chromatographies：Scythian lamb rhizome glycopolymers CB4 obtained by step S4 is dissolved in deionized water, is splined on
, there are 2 peaks, wherein peak one is water (0M NaCl) under the conditions of the eluent of different salinity in the posts of DEAE-Cellulose 52
Elution fraction, peak two is 0.05M NaCl elution fractions, tracks elution curve using phend-sulphuric acid in elution process, according to
Elution curve collects sugar moieties respectively, respectively by after the concentration of gained eluent, freeze-drying, obtains the glycopolymers of peak one and peak disaccharides
Polymer；
S6 molecular sieve gel column chromatographies：By the glycopolymers sample of peak one obtained by step S5, dissolved with water, centrifuge, take supernatant
Liquid, upper Sephadex G-75 posts, is eluted with water, and elution curve is tracked using phend-sulphuric acid, and appearance one is single right
Claim peak, collect main peak, concentrate, freeze-drying obtains scythian lamb rhizome glycopolymers CBP-1；Similarly, by peak disaccharides obtained by step S5
Polymer samples, are dissolved with water, centrifugation, take supernatant, upper Sephadex G-75 posts are eluted with water, using benzene
Phenol-sulfuric acid method tracks elution curve, a single symmetrical peak occurs, collects main peak, concentrates, freeze-drying, obtains scythian lamb rhizome sugar
Polymer CBP-2.
3. the preparation method of scythian lamb rhizome glycopolymers as claimed in claim 2, it is characterised in that the step S1 immersions
12h。
4. the preparation method of scythian lamb rhizome glycopolymers as claimed in claim 2, it is characterised in that during the step S2 water extractions
The addition of water is 10 times of above-mentioned scythian lamb rhizome block weight, and the temperature of water is 85 DEG C during water extraction, and the water extraction time is 2h.
5. the preparation method of scythian lamb rhizome glycopolymers as claimed in claim 2, it is characterised in that will step in the step S3
The dregs of a decoction obtained by rapid S2 are immersed in the NaOH solution that concentration is 0.3M, and the amount of NaOH solution is 15 times of above-mentioned dregs of a decoction weight, room
Temperature places 3h, takes supernatant, the HCl solution for being 0.5M with concentration by supernatant is neutralized, and it is 7 to make its pH, takes supernatant to enter
Row centrifugation, centrifugal speed is 5000r/min, and centrifugation time is 10min, takes supernatant, and 60 DEG C are concentrated under reduced pressure, and then add ethanol
It is 75% to make ethanol volumetric concentration.
6. the preparation method of scythian lamb rhizome glycopolymers as claimed in claim 2, it is characterised in that described in the step S4 thoroughly
The molecular cut off for analysing bag is 1000Da.
7. scythian lamb rhizome glycopolymers as claimed in claim 1 prevent and treat the medicine or health food or work(of osteoporosis preparing
Application in energy food.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109762075A (en) * | 2019-01-07 | 2019-05-17 | 广东药科大学 | A kind of snow chrysanthemum glycopolymers and its preparation method and application |
| CN110013566A (en) * | 2019-05-24 | 2019-07-16 | 大连医科大学附属第一医院 | A kind of preparation method of composite bone repairing material |
-
2017
- 2017-05-26 CN CN201710386921.XA patent/CN107236050A/en active Pending
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| Title |
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| 易盼: "狗脊多糖的分离纯化及生物活性研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 许重远等: "中药狗脊多糖的含量测定及高效毛细管电泳指纹图谱分析", 《中药材》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109762075A (en) * | 2019-01-07 | 2019-05-17 | 广东药科大学 | A kind of snow chrysanthemum glycopolymers and its preparation method and application |
| CN110013566A (en) * | 2019-05-24 | 2019-07-16 | 大连医科大学附属第一医院 | A kind of preparation method of composite bone repairing material |
| CN110013566B (en) * | 2019-05-24 | 2021-09-14 | 大连医科大学附属第一医院 | Preparation method of composite bone repair material |
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