CN105037447B - Root of bidentate achyranthes glycopolymers and its production and use - Google Patents
Root of bidentate achyranthes glycopolymers and its production and use Download PDFInfo
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Abstract
The invention discloses glycopolymers in the root of bidentate achyranthes and its production and use, this method is using the root of bidentate achyranthes as raw material, each position root of bidentate achyranthes polymer is obtained by the combination of water extract-alcohol precipitation and alkali carries alcohol precipitation, the extracting method is carried out under the conditions of comparatively gentle, the intact component for saving glycopolymers.The root of bidentate achyranthes glycopolymers have remarkable result in terms of anti-osteoporosis disease, in addition, the present invention takes off albumen, DEAE cellulose chromatographies, Sephacryl column chromatographies etc. by Sevag methods and isolated and purified, glycopolymers sterling is obtained, structural analysis shows that the root of bidentate achyranthes glycopolymers that this method obtains are polysaccharide and oligosaccharides.These can provide foundation for the application in the future in health products, medicine and other fields.
Description
Technical field
The invention belongs to medicine and technical field of health care food, and in particular to the preparation method of glycopolymers in plant, more
More particularly to the preparation method of radix achyranthis bidentatae raw sugar polymer, refined sugar polymer, the root of bidentate achyranthes raw sugar polymer is further related to pre-
Application in anti-and/or treatment osteoporosis.Glycopolymers in the present invention include polysaccharide and oligosaccharides.
Background technology
Osteoporosis (osteoporosis, OP) is a kind of with the reduction of bone mineral quantity, bone micro-structure destruction, bone strength
Reduce, so as to cause the fragility of bone to increase and be prone to the disease that fracture is characterized.Osteoporosis is a kind of multiple
Geriatric disease, it is on the rise with the aging of population, osteoporosis has seriously endangered senior health and fitness.Postmenopausal women
Because of the double influence declined by aging and hormone in vivo level, turn into the maximum aggrieved crowd of the disease, account for total number of patients
More than 70%.The medicine of current anti-curing osteoporosis is mainly bone resorption inhibitor, bone formation-promoter and mineralising medicine, this
A little medicines belong to replacement therapy, and during long-term use, big with toxic side effect, targeting is low, and compliance is poor, expense height etc.
Shortcoming.Therefore, it is necessary to a kind of medicine efficiently, less toxic is looked for treat osteoporosis.Chinese medicine is in treatment osteoporosis
There is long history in terms of disease, there is whole body to nurse one's health, toxic side effect is small, the advantage such as cheap, is increasingly closed by people
Note.And in numerous Chinese medicines, nourishing disney and strengthening bone class Chinese medicine due to it is good the effect of be constantly subjected to the favor of people.
The root of bidentate achyranthes（Achyranthes bidentataB.l）For the dry root of the amaranthaceous plant root of bidentate achyranthes, be " four-Huai medicine " it
One.With filling liver kidney, strengthening the bones and muscles, dispelling stasis of blood and stimulating the menstrual flow, the work(ensuringd proper downward flow of the blood, it is mainly used in that soreness of waist and knee joint, muscles and bones be powerless, Amenorrhea disease
The disease of lump in the abdomen, dizziness due to hyperactivity of liver-YANG.Treatment " heumatism ", the prescription totally 21 of " atrophic debility of bones " is expressly recited in history tree, is related to 77 kinds of medicine, its
The middle root of bidentate achyranthes occurs 11 times.One of the main active of root of bidentate achyranthes glycopolymers as the root of bidentate achyranthes, have been reported to have immunological regulation, resist
A variety of pharmacological activity such as tumour, hypoglycemic, protection liver kidney.
At present, the research both at home and abroad to root of bidentate achyranthes glycopolymers is concentrated mainly on its antitumor, anti-oxidant, immunological regulation, drop
The activity research of sugar, anti-aging etc., to its research in terms of anti-curing osteoporosis, there is not been reported.
The content of the invention
It is an object of the invention to provide a kind of root of bidentate achyranthes glycopolymers ABW3-1.
Another object of the present invention is to provide a kind of extracting method of glycopolymers in root of bidentate achyranthes.
It is still another object of the present invention to provide above-mentioned root of bidentate achyranthes glycopolymers to prepare preventing and treating medicine for treating osteoporosis or health care
Application in food or functional food.
The technical solution used in the present invention is：
A kind of root of bidentate achyranthes glycopolymers ABW3-1, the oligosaccharides that the glycopolymers are made up of glucose and fructose, its structure are：
。
The extracting method of glycopolymers in a kind of root of bidentate achyranthes, this method include following operating procedure：
1）Shearing：Root of bidentate achyranthes dry root is cut into segment, is cleaned up, dried with water；
2）Water extraction：The root of bidentate achyranthes sheared is subjected to water extraction, collects extract solution and residue respectively；
3）It is classified alcohol precipitation：After extract solution is concentrated, adding ethanol makes ethanol volumetric concentration be a%, stands, and collects precipitation, i.e.,
Obtain raw sugar polymer AB1；Supernatant is concentrated again, adding ethanol makes ethanol volumetric concentration be b%, stands, and collects precipitation, i.e.,
Obtain raw sugar polymer AB2；Supernatant is concentrated again, adding ethanol makes ethanol volumetric concentration be c%, stands, and collects precipitation, i.e.,
Obtain raw sugar polymer AB3；Wherein 10≤a ＜ b ＜ c ＜ 100；
4）Alkali carries：Residue after water extraction is soaked in 0.1~1M NaOH solutions, stands 1~4h, supernatant with 0.1~
1M HCl are neutralized, and the pH for making supernatant is 6~8, centrifuging and taking supernatant, concentrates supernatant, and adding ethanol makes ethanol volumetric concentration
For 50~90%, stand, collect precipitation, produce alkali carries raw sugar polymer ABB；
5）Purifying：Above-mentioned raw sugar polymer AB1, AB2, AB3, ABB are purified, produce root of bidentate achyranthes glycopolymers.
Further, step 2）Described in the concrete operations of water extraction be to cutting with 60 ~ 100 DEG C of hot water of 5~15 times of volumes
The good root of bidentate achyranthes is extracted, 1~4h of extraction time.
Further, step 3）In all concentrations be concentrated under reduced pressure for 40~70 DEG C, the time of all standings is
10~28h.
Further, step 3）In the ＜ c ＜ 100 of 10≤a ＜, 60,60 ＜ b ＜ 80,80.
Further, by step 5）In glycopolymers AB3 after purification carry out ion-exchange chromatography again, entered with distilled water
Row elution, elution curve is tracked using phend-sulphuric acid, is collected sugar moieties according to elution curve, is concentrated, freeze-drying；Then
Dissolved, centrifuged with water, taken supernatant to carry out molecular sieve gel column chromatography, eluted with water, washed using phend-sulphuric acid tracking
De- curve, sugar moieties are collected according to elution curve, concentration, root of bidentate achyranthes glycopolymers ABW3-1 are obtained after freeze-drying.
Root of bidentate achyranthes glycopolymers, the extracting method of the glycopolymers is extracting method described above.
Root of bidentate achyranthes glycopolymers ABW3-1 answering in the medicine of preventing and treating osteoporosis or health food or functional food is prepared
With.
Root of bidentate achyranthes glycopolymers AB1, AB2, AB3, ABB is preparing the medicine of preventing and treating osteoporosis or health food or function food
Application in product.
Application of the root of bidentate achyranthes glycopolymers in the medicine or health food or functional food for preparing preventing and treating osteoporosis.
The beneficial effects of the invention are as follows：
1. the present invention purifies root of bidentate achyranthes glycopolymers using ion-exchange chromatography and molecular sieve gel column chromatography method, first
A kind of root of bidentate achyranthes essence oligosaccharides is prepared, and the physicochemical property to the smart oligosaccharides, molecular weight, monose composition etc. has carried out point of system
Analysis identification, and successfully drawn the feature structure of the oligosaccharides.The invention provides raw sugar polymer in the root of bidentate achyranthes, refined sugar polymer
The activity research of preparation method and root of bidentate achyranthes glycopolymers in terms of anti-osteoporosis is root of bidentate achyranthes glycopolymers in medicine, health products
Application Deng field provides foundation.
2. compared with traditional water-boiling method extracts glycopolymers, the present invention is using the combination of water extraction and alcohol precipitation method and alkaline extraction, second
Determining alcohol carries out classification alcohol precipitation from low to high, carries out initial gross separation to root of bidentate achyranthes glycopolymers, while high concentration ethanol can be by polarity
Greatly, the glycopolymers of good water solubility are small with polarity, glycopolymers of poorly water-soluble separate, and make extracted glycopolymers composition
Contain a greater variety of glycopolymers compositions.It is easy to operate and this preparation technology is simple, and can mass produce.
3. being isolated and purified in the present invention by column chromatography to root of bidentate achyranthes raw sugar polymer, significant effect, prepare first
Go out a kind of root of bidentate achyranthes oligosaccharides sterling.
4. the present invention is identified a kind of root of bidentate achyranthes oligosaccharide structure prepared, the physicochemical property of the oligosaccharides specify that
And structure, provide structure foundation to probe into its pharmacological activity mechanism.
5. the invention provides the preparation method of the composition in the root of bidentate achyranthes with anti-osteoporosis activity, and filter out
The glycopolymers position that content is high, activity is strong.
Brief description of the drawings
Fig. 1 is ABW3-1 HPGPC chromatograms；
Fig. 2 is ABW3-1 infared spectrum；
Fig. 3 is ABW3-1's13C NMR spectras；
Fig. 4 is ABW3-1's1H NMR spectras；
Fig. 5 is ABW3-1 HSQC collection of illustrative plates；
Fig. 6 is ABW3-1 HMBC collection of illustrative plates；
Fig. 7 is influence of the root of bidentate achyranthes glycopolymers to ovariectomized female rats body weight；
Fig. 8 is influence of the root of bidentate achyranthes glycopolymers to ovariectomized female rats Uterine coefficient；
Fig. 9 is influence of the root of bidentate achyranthes glycopolymers to femur of mature ovariectomized rats bone density；
Figure 10 is influence of the root of bidentate achyranthes glycopolymers to ovariectomized female rats lumbar vertebra bone density；
Figure 11 is influence of the root of bidentate achyranthes glycopolymers to femur of mature ovariectomized rats bone mineral quantity；
Figure 12 is influence of the root of bidentate achyranthes glycopolymers to ovariectomized female rats lumbar vertebra bone mineral quantity.
Embodiment
1. the extracting method of glycopolymers in the root of bidentate achyranthes, this method includes following operating procedure：
1）Shearing：Root of bidentate achyranthes dry root is cut into segment, with cold water Rapid Cleaning, dried；
2）Water extraction：The root of bidentate achyranthes sheared is subjected to water extraction, collects extract solution and residue respectively；
3）It is classified alcohol precipitation：After extract solution is concentrated, adding ethanol makes ethanol volumetric concentration be a%, stands, and collects precipitation, i.e.,
Obtain raw sugar polymer AB1；Supernatant is concentrated again, adding ethanol makes ethanol volumetric concentration be b%, stands, and collects precipitation, i.e.,
Obtain raw sugar polymer AB2；Supernatant is concentrated again, adding ethanol makes ethanol volumetric concentration be c%, stands, and collects precipitation, i.e.,
Obtain raw sugar polymer AB3；Wherein 10≤a ＜ b ＜ c ＜ 100；Preferred 10≤a ＜ b ＜ c ＜ 100, and 10≤a ＜ 60,60
The ＜ c ＜ 100 of ＜ b ＜ 80,80；
4）Alkali carries：Residue after water extraction is soaked in 0.1~1M NaOH solutions, stands 1~4h, supernatant with 0.1~
1M HCl are neutralized, and the pH for making supernatant is 6~8, centrifuging and taking supernatant, concentrates supernatant, and adding ethanol makes ethanol volumetric concentration
For 50~90%, stand, collect precipitation, produce alkali carries raw sugar polymer ABB；
5）Purifying：Above-mentioned raw sugar polymer AB1, AB2, AB3, ABB are purified, produce root of bidentate achyranthes glycopolymers.
Preferably, step 1）Described in segment be 1~2cm segment.
Preferably, step 2）Described in the concrete operations of water extraction be to the root of bidentate achyranthes with 60 ~ 100 DEG C of hot water of 5~15 times of volumes
Extracted, 1~4h of extraction time.
Preferably, step 3）In all concentrations be concentrated under reduced pressure for 40~70 DEG C, the time that all alcohol precipitations are stood is 10
~28h.
Preferably, step 4）Middle NaOH solution dosage volume is 10~20 times of residue volume.
Preferably, step 5）Described in purify concrete operations be using Sevag methods respectively to raw sugar polymer AB1, AB2,
AB3, ABB removing protein, dialysed after removing protein, freezed through bag filter again.
Preferably, the molecular cut off of above-mentioned bag filter is 3000 Da.
Preferably, to step 5）In glycopolymers AB3 after purification carry out following chromatographic purifying again：
1）Ion-exchange chromatography：Glycopolymers AB3 after purification carries out ion-exchange chromatography, and ladder is carried out with distilled water
Degree elution, elution curve is tracked using phend-sulphuric acid, sugar moieties are collected according to elution curve respectively, then concentration, freezing are dry
It is dry；Dissolved respectively with water again, centrifuge, take supernatant；
2）Molecular sieve gel column chromatography：Supernatant obtained by previous step is subjected to molecular sieve gel column chromatography again, entered with water
Row elution, elution curve is tracked using phend-sulphuric acid, sugar moieties are collected according to elution curve, obtained after concentration, freeze-drying
Glycopolymers sterling, is named as ABW3-1.
Preferably, above-mentioned ion exchange column is DEAE ion exchange columns.
Preferably, above-mentioned molecular sieve gel chromatography selects Sephacryl molecular sieve chromatography posts.
Preferably, the oligosaccharides that above-mentioned root of bidentate achyranthes glycopolymers ABW3-1 is made up of glucose and fructose, mainly by → 2)-β-
D-Fru- (1 → and → 2)-α-D-Fru- (6 → composition, terminal sugar residues are made up of α-D-Glc- (1 → and → 2)-β-D-Fru,
ABW3-1 structure is：
。
Preferably, ABW3-1 molecular weight is 1074 Da.
Root of bidentate achyranthes glycopolymers, the preparation method of the glycopolymers are the extraction side of glycopolymers in the root of bidentate achyranthes described above
Method.
Root of bidentate achyranthes glycopolymers ABW3-1 answering in the medicine of preventing and treating osteoporosis or health food or functional food is prepared
With.
Root of bidentate achyranthes glycopolymers AB1, AB2, AB3, ABB is preparing the medicine of preventing and treating osteoporosis or health food or function food
Application in product.
With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.
The extracting method of glycopolymers in the root of bidentate achyranthes of embodiment 1
1）Shearing：10kg root of bidentate achyranthes dry root is cut to 1 ~ 2cm with scissors, with cold water Rapid Cleaning, dried；
2）Water extraction：The root of bidentate achyranthes that will have been sheared, with 10 times of volume hot water（60~100℃）Extraction, extraction time 2h, collect
Extract solution, residue dry.
3）It is classified alcohol precipitation：Extract solution is after 60 DEG C are concentrated under reduced pressure, and adding ethanol makes ethanol volumetric concentration be 50%, and room temperature is quiet
After putting 24h, centrifugation, precipitation is collected, weigh 323g, is raw sugar polymer AB1；Supernatant is collected after 60 DEG C are concentrated under reduced pressure, is added
Entering ethanol makes ethanol volumetric concentration be 70%, after being stored at room temperature 24h, centrifugation, collects precipitation, weigh 893g, is raw sugar polymer
Above-mentioned same operation is pressed in AB2, repetition（In addition to ethanol volumetric concentration is 90%, other operations are same as above）, obtain 671g raw sugar and gather
Compound AB3.
4）Alkali carries：The dregs of a decoction after water extraction being immersed in the 0.3M NaOH solutions of 15 times of volumes, room temperature places 2h, on
Clear liquid is neutralized with 0.5M HCl, and it is 6 ~ 8 to make its pH, takes supernatant to be centrifuged（5000r/min, 10min）, take supernatant
Liquid, 60 DEG C are concentrated under reduced pressure, and then adding ethanol makes ethanol volumetric concentration be 75%, is centrifuged after standing 24h, collects precipitation, be weighed as
410g, this is alkali carries raw sugar polymer ABB.
5）Purifying：Removing protein is carried out to each component raw sugar polymer respectively using Sevag methods, raw sugar polymer after removing protein
Use bag filter（Molecular cut off is 3000 Da）Dialysed, freezed, produce root of bidentate achyranthes glycopolymers.
The root of bidentate achyranthes glycopolymers ABW3-1 of embodiment 2 extracting method
1）Shearing：10kg root of bidentate achyranthes dry root is cut to 1 ~ 2cm with scissors, with cold water Rapid Cleaning, dried；
2）Water extraction：The root of bidentate achyranthes that will have been sheared, with 10 times of volume hot water（60~100℃）Extraction, extraction time 2h, collect
Extract solution, residue dry.
3）Alcohol precipitation：For extract solution after 60 DEG C are concentrated under reduced pressure, adding ethanol makes ethanol volumetric concentration be 50%, stands 24h, receives
Collect supernatant after 60 DEG C are concentrated under reduced pressure, adding ethanol makes ethanol volumetric concentration be 70%, stands 24h, repeats by above-mentioned same
Operation（Except ethanol volumetric concentration is 90%, other operations are same as above）, after being stored at room temperature 24h, centrifugation, precipitation is collected, is weighed
671g, it is raw sugar polymer AB3；
4）Purifying：Removing protein is carried out to raw sugar polymer AB3 using Sevag methods, raw sugar polymer is dialysed after removing protein
Bag（Molecular cut off is 3000 Da）Dialysed, freezed, produce root of bidentate achyranthes glycopolymers AB3.
5）Ion-exchange chromatography：The above-mentioned glycopolymers AB3 after purification of 100mg are taken, are dissolved in 5mL deionized water,
The posts of DEAE-Cellulose 52 are splined on, occur 2 peaks under the conditions of the eluent of different salinity, wherein peak one is water（0M
NaCl）Elution fraction, peak two are 0.1M NaCl elution fractions（Elution curve is tracked using phend-sulphuric acid in elution process,
Sugar moieties are collected according to elution curve respectively）, respectively by after the concentration of gained eluent, freeze-drying, obtain two kinds of glycopolymers
（The glycopolymers of peak one, the glycopolymers of peak two）.
6）Molecular sieve gel chromatographs：By the glycopolymers sample of peak one after above-mentioned freeze, dissolved with water, centrifuge, take
Supernatant, upper Sephacryl S-100 HR posts, is eluted with water, tracks elution curve using phend-sulphuric acid, occurs one
Individual single symmetrical peak, main peak is collected, concentration, root of bidentate achyranthes glycopolymers ABW3-1 is obtained after freeze-drying.
Purity and molecular weight detection：The glycopolymers ABW3-1 of above-mentioned acquisition is made into 2% concentration（W/V）The aqueous solution,
HPGPC methods measure retention time, and molecular weight is calculated to obtain according to standard curve.
As a result as shown in figure 1, the component ABW3-1 obtained after ion exchange and gel filtration isolate and purify is in single
Symmetrical peak, it is homogeneous glycopolymers to illustrate ABW3-1, and it is 1074 Da to measure molecular weight.
Stated below to more than and the ABW3-1 being separated to is extracted in embodiment make further structural analysis.
Root of bidentate achyranthes oligosaccharides ABW3-1 structural analysis
（1）Monosaccharide composition analysis
From complete acid hydrolysis products chromatography of ions HPAEC-PAD collection of illustrative plates, ABW3-1 monose composition is glucose and fruit
Sugar.
（2）Infrared spectrum detects
ABW3-1 infrared spectrum testing result is as shown in Fig. 2 there it can be seen that the feature that ABW3-1 contains oligosaccharides is inhaled
Receiving peak is：3368.98cm-1For O-H stretching vibrations, 2935.72cm-1For C-H stretching vibrations, 1643.45cm-1Inhaled for carbonyl
Receive peak.927.69cm-1With 813.60 cm-1For the characteristic absorption peak of furan nucleus；873.72cm-1There are absworption peak, explanation in place
ABW3-1 is mainly to be made up of β type saccharide residues.
（3）Methylation analysis
Sample is hydrolyzed, reduced, GC-MS is analyzed after acetylation, the results showed that ABW3-1 contains → 1 through methylating)-β-D-
Fruf- (2 → and → 2)-β-D-Fruf- (6 → saccharide residue.
（4）The nuclear magnetic resonance spectroscopy of glycopolymers
Homogeneous glycopolymers ABW3-1 samples are placed in nuclear magnetic tube, use D2Spectrum, acquired results such as Fig. 3~6 are surveyed after O dissolvings
It is shown.
The ownership of each carbon and hydrogen is understood according to the nuclear magnetic spectrum of above-mentioned Fig. 3~6, it is as shown in table 1 below.
The ABW3-1 nuclear magnetic resonance spectroscopy results of table 1
aUnresolved from other signals.
Through above-mentioned sour water solution completely, methylation analysis, infrared spectrum detection and nmr analysis, it is one as a result to show ABW3-1
Kind is by glucose and fructose with 1:The oligosaccharides of 5 ratios composition, illustrates that it contains → 1 from methylation analysis)-β-D-Fruf-(2→
With → 2)-β-D-Fruf- (6 → saccharide residue, the order of connection between different saccharide residues are obtained by two-dimentional nuclear-magnetism HMBC spectrum analysis
Go out, show that ABW3-1 structure is by analysis above:
, its molecular weight is 1074
Da。
In addition, we to be isolated to Inokopolyose ABW2-1, ABP2-2 structure early stage as follows.
Inokopolyose ABW2-1 structure：
Wherein m is that 0~19, n is 0~19, and m+n is 2~19.
Inokopolyose ABP2-2 structure：
Its mean molecule quantity is 3406 Da.
Above homogeneous polysaccharide, oligosaccharides are probably the active component of root of bidentate achyranthes glycopolymers anti-osteoporosis, thus, homogeneous sugar is poly-
The Structural Identification of compound will provide strong foundation subsequently to probe into the mechanism of root of bidentate achyranthes glycopolymers anti-osteoporosis.
The activity of the root of bidentate achyranthes glycopolymers extracted below to the present invention detects.
The function of resisting osteoporosis research of the root of bidentate achyranthes glycopolymers of embodiment 3
1 experimental method
1.1 experimental designs and packet：3 monthly ages did not mated female sd inbred rats 70, SPF levels（It is real by Traditional Chinese Medicine University Of Guangzhou
Animal center offer is provided）, 7 groups are randomly divided into by body weight principle of reciprocity, each group number of animals and medication are shown in Table 1, and its is positives
Control group E2 is 17 beta estradiols, and model group OVX and sham-operation group SHAM is distilled water.The daily gastric infusion of Post operation, weekly
Weigh once, corresponding dosage, continuous gavage 90d are adjusted by changes of weight.
The experimental design of table 1 and packet
1.2 ovariectomized rats operation methods：
By rat with the Nembutal sodium solution of 45mg/kg dosage（4%）Intraperitoneal injection of anesthesia, abdomen position are fixed.In its most end
Under 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, with
Betagen Solution is sterilized, and cuts skin, muscle of back and sarolemma, and white shiny cellulite gently is pulled out into otch with pincet
Outside, fractionation of fatty group, ovary can be seen.Ovary lower end fallopian tubal is ligatured with operation suture thread, then extracts ovary.Otch
After suture, outside is coated with antiphlogistic powder（Benzylpenicillin sodium salt）, opposite side ovary is extractd with method.Sham-operation group（SHAM）Processing method it is same
On, fritter adipose tissue is simply cut off, does not extract ovary.It is postoperative to be placed in warm environment, nurse observation.
1.3 materials and preservation
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 immediately（After standing lh, serum is taken after 3000rmp centrifuges 10min, -80 DEG C of frosts are protected
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 24h more
Change 70% ethanol solution.- 20 DEG C of freezen protectives.
4）Take left femur, shin bone and third and fourth lumbar vertebrae, physiological saline gauze wrapped and with masking foil outer wrapping, Yu Mi
In envelope, packing is placed in -80 DEG C of stored frozens.
1.4 rat bone densities determine
Using the DXA of Dual-energy X-rays absorptionmetry Hologic Discovery WI 85003 detection fourth lumbar vertebra bones (L4)
Full bone density (bone mineral density, BMD), front end femoral bmd (1cm) with fl, distal femoral bone
Density (2cm) and bone mineral content (bone mineral content, BMC).
1.5 bone biomechanicals are tested
During test, femur and vertebra that -80 DEG C are preserved are placed in -20 DEG C of pre- defrostings, and then normal temperature unfreezing, uses physiological saline
It is multiple wet.Fl is analyzed using Mini858 Bionix material testing systems（Three-point bending test）Biomechanical property.Survey
During examination on MTS testing machines, with the pressure head of 1mm diameters, loading velocity 6mm/min, span (L) is 15mm, sensor 500N.
Instrument records the load and radial degree changing value of each moment point automatically, draws load-radial degree curve, and obtain corresponding parameter.
Experimental result
2.1 root of bidentate achyranthes glycopolymers ring to removal ovary female rats ghost image
Root of bidentate achyranthes glycopolymers ring to removal ovary female rats ghost image sees Fig. 7, after the administration of each group animal compared with before administration,
Body weight substantially increases.Compared with SHAM groups, after ten weeks, body weight substantially increases OVX ovariectomized rats（P<0.01）, prompting goes
Ovary causes rat body weight substantially to increase.Compared with OVX groups, root of bidentate achyranthes glycopolymers AB1, AB2, AB3 and ABB administration group is from the 7th
Zhou Hou, increased weight start to mitigate (P<0.05), positive controls after the tenth week the trend of increased weight also occur substantially pressing down
System.Illustrate that administration group can suppress the increase of ovariectomized female rats body weight with positive group E2.
Influence of 2.2 root of bidentate achyranthes glycopolymers to removal ovary female rats Uterine coefficient
Fig. 8 is shown in influence of the root of bidentate achyranthes glycopolymers to removal ovary female rats Uterine coefficient, compared with SHAM groups, OVX rats
Uterine coefficient is obviously reduced after removal ovary（P<0.05）, prompt removal ovary to cause rat uterus atrophy, weight saving.With OVX groups
Compare, AB1, AB2, AB3 administration group can substantially increase ovariectomized female rats uterus weight with positive group E2.Illustrate these three administration groups
Can be improved ovariectomized female rats metratrophia with positive group.
Influence of 2.3 root of bidentate achyranthes glycopolymers to removal ovary female rats femur and lumbar vertebra bone density
Root of bidentate achyranthes glycopolymers are to removal ovary female rats femur and lumbar vertebra bone density（BMD, bone mineral
density）Influence see Fig. 9,10, compared with SHAM groups, the femur of OVX ovariectomized female rats（Fig. 9）With lumbar vertebra（Figure 10）Bone
Density is obviously reduced（P<0.01）, prompt ovariectomized female rats to be successfully, reproduced primary osteoporosis.Compared with OVX groups,
AB1, AB2, AB3 administration group can substantially increase femur of mature ovariectomized rats with positive group E2（Fig. 9）With lumbar vertebra（Figure 10）Bone it is close
Degree（P<0.05）.Illustrate that corresponding administration group is capable of the osteoporosis symptoms of effectively preventing rat with positive group E2.
Influence of 2.4 root of bidentate achyranthes glycopolymers to removal ovary female rats femur and lumbar vertebra bone mineral quantity
Figure 11,12 are shown in influence of the root of bidentate achyranthes glycopolymers to removal ovary female rats femur and lumbar vertebra mineral quantity, with SHAM
Group compares, and OVX rat femurs are obviously reduced with lumbar vertebra mineral quantity（P<0.01）, prompt ovariectomized female rats to be successfully, reproduced original
Hair property osteoporosis.Compared with OVX groups, AB1, AB2, AB3 administration group can substantially increase ovariectomized female rats with positive group E2
Femur mineral quantity（See Figure 11）；AB1, AB2 administration group substantially increase ovariectomized female rats lumbar vertebra mineral quantity（P<0.05）（See figure
12）, the above results illustrate that each root of bidentate achyranthes glycopolymers administration group plays the role of certain anti-curing osteoporosis.
Influence of 2.5 root of bidentate achyranthes glycopolymers to biomechanical properties
Influence of the root of bidentate achyranthes glycopolymers of table 2 to biomechanical properties
All numerical value are average value ± SD. * P in table< 0.05, **P < 0.01 vs. OVX; #P < 0.05,
##P < 0.01 vs. SHAM。
Influence of the root of bidentate achyranthes glycopolymers to biomechanical properties is shown in Table 2, compared with SHAM groups, OVX rat femur Biological Strengths
Learn relevant parameter to reduce, prompt ovariectomized rats biomechanical property to decline；Compared with OVX groups, the equal energy of administration group
Ovariectomized female rats biomechanical property is effectively improved, the above results illustrate that administration group has the work of certain anti-curing osteoporosis
With.
The root of bidentate achyranthes oligosaccharides ABW3-1 that is extracted of the present invention be probably play above-mentioned function of resisting osteoporosis active ingredient it
One, it may have dramatically increase femur of mature ovariectomized rats and the effect of lumbar spine bmd and bone mineral quantity, it is obviously improved bone life
The mechanism of action of thing mechanical property needs further to be studied.
Conclusion
After the ovary excision of female rats, estrogen level reduces, Bone m etabolism is active, bone conversion strengthens, bone mineral quantity is lost
Lose, be the classical model of high conversion hysteria osteoporosis when imitating postmenopausal women.This experimental study shows, passes through above-mentioned side
The root of bidentate achyranthes glycopolymers at each position of method extraction can dramatically increase femur of mature ovariectomized rats and lumbar spine bmd and bone ore deposit
Object amount, hence it is evident that improve bone biomechanical property, thus play it and treat the effect of rat model osteoporosis.
The anti-osteoporosis mechanism of root of bidentate achyranthes glycopolymers need to subsequently be studied, and the Structural Identification of homogeneous glycopolymers
Strong foundation will be provided to probe into the mechanism of root of bidentate achyranthes glycopolymers anti-osteoporosis.
Above-described is only some embodiments of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation.For the person of ordinary skill of the art, under other any Spirit Essences and principle without departing from the present invention
Change, modification, replacement, the simplification made, should be equivalent substitute mode, are included within protection scope of the present invention.
Claims (8)
- A kind of 1. root of bidentate achyranthes glycopolymers ABW3-1, it is characterised in that：The oligosaccharides that the glycopolymers are made up of glucose and fructose,Its structure is：
- A kind of 2. extracting method of glycopolymers in root of bidentate achyranthes, it is characterised in that：This method includes following operating procedure：1) shear：Root of bidentate achyranthes dry root is cut into segment, is cleaned up, dried with water；2) water extraction：The root of bidentate achyranthes sheared is subjected to water extraction, collects extract solution and residue respectively；3) it is classified alcohol precipitation：After extract solution is concentrated, adding ethanol makes ethanol volumetric concentration be a%, stands, and collects precipitation, produces Raw sugar polymer AB1；Supernatant is concentrated again, adding ethanol makes ethanol volumetric concentration be b%, stands, and collects precipitation, produces Raw sugar polymer AB2；Supernatant is concentrated again, adding ethanol makes ethanol volumetric concentration be c%, stands, and collects precipitation, produces Raw sugar polymer AB3；Wherein 10≤a ＜ b ＜ c ＜ 100；4) alkali carries：Residue after water extraction is soaked in 0.1~1M NaOH solutions, stands 1~4h, 0.1~1M of supernatant HCl is neutralized, and the pH for making supernatant is 6~8, centrifuging and taking supernatant, concentrates supernatant, and adding ethanol makes ethanol volumetric concentration be 50 ~90%, stand, collect precipitation, produce alkali carries raw sugar polymer ABB；5) purify：Above-mentioned raw sugar polymer AB1, AB2, AB3, ABB are purified, produce root of bidentate achyranthes glycopolymers；Glycopolymers AB3 in step 5) after purification is subjected to ion-exchange chromatography again, is eluted with distilled water, uses benzene Phenol-sulfuric acid method tracks elution curve, collects sugar moieties according to elution curve, concentrates, freeze-drying；Then dissolved, centrifuged with water, Take supernatant to carry out molecular sieve gel column chromatography, eluted with water, elution curve is tracked using phend-sulphuric acid, according to washing De- curve collects sugar moieties, concentration, the root of bidentate achyranthes glycopolymers ABW3-1 described in claim 1 is obtained after freeze-drying.
- 3. according to the method for claim 2, it is characterised in that：The concrete operations of water extraction described in step 2) are with 5~15 60~100 DEG C of hot water of times volume extract to the root of bidentate achyranthes cut, 1~4h of extraction time.
- 4. according to the method for claim 2, it is characterised in that：All concentrations are dense for 40~70 DEG C of decompressions in step 3) Contracting, the time of all standings is 10~28h.
- 5. according to the method for claim 2, it is characterised in that：The ＜ c ＜ of 10≤a ＜, 60,60 ＜ b ＜ 80,80 in step 3) 100。
- 6. the root of bidentate achyranthes glycopolymers ABW3-1 described in claim 1 is preparing the medicine or health food or work(of preventing and treating osteoporosis Application in energy food.
- 7. root of bidentate achyranthes glycopolymers AB1, AB2, AB3, ABB made from claim 2 methods described is preparing preventing and treating osteoporosis Application in medicine or health food or functional food.
- 8. root of bidentate achyranthes glycopolymers made from any methods described of claim 2~5 are preparing the medicine of preventing and treating osteoporosis or guarantor Application in health food or functional food.
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| JP2011519947A (en) * | 2008-05-06 | 2011-07-14 | バイオノボ・インコーポレーテッド | Estrogen-like extract used to treat vaginal and vulvar atrophy |
| KR101444760B1 (en) * | 2013-08-05 | 2014-09-26 | 동우당제약(주) | A composition for preventing bone metabolism-related diseases and increasing bone function comprising mix extracts of Achyranthes bidentata and Siegesbeckia glabrescens |
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Non-Patent Citations (3)
| Title |
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| Achyranthes bidentata root extract prevent OVX-induced osteoporosis in rats;Rong Zhang et al.;《Journal of Ethnopharmacology》;20110606;第139卷;第12-18页 * |
| Anti-inflammatory and anti-osteoarthritis effects of fermented Achyranthes japonica Nakai;Sung-Gyu Lee et al.;《Journal of Ethnopharmacology》;20120602;第142卷;第634-641页 * |
| Osteoprotective effect of extract from Achyranthes bidentata in ovariectomized rats;Cui-Cui He et al.;《Journal of Ethnopharmacology》;20091126;第127卷;第229-234页 * |
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